Effects of omega-3 fatty acid supplementation on the pattern of oxylipins: a short review about the modulation of hydroxy-, dihydroxy-, and epoxy-fatty acids

Annika I. Ostermann ab and Nils Helge Schebb *ab
aInstitute for Food Toxicology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany
bChair of Food Chemistry, Faculty of Mathematics and Natural Sciences, University of Wuppertal, Gaußstraße 20, Wuppertal, Germany. E-mail: nils@schebb-web.de; Tel: +49-202-439-3457

Received 14th March 2017 , Accepted 30th May 2017

First published on 2nd June 2017


A growing body of evidence suggests that the intake of the long chain omega-3 polyunsaturated fatty acids (n3-PUFA) eicosapentaenoic acid (C20:5 n3, EPA) and docosahexaenoic acid (C22:6 n3, DHA) is linked to beneficial health effects, particularly in the prevention of cardiovascular and inflammatory diseases. Although the molecular mode of action of n3-PUFA is still not fully understood, it is not controversial that a significant portion of the (patho)-physiological effects of PUFA are mediated by their oxidative metabolites, i.e. eicosanoids and other oxylipins. Quantitative targeted oxylipin methods allow the comprehensive monitoring of n3-PUFA supplementation induced changes in the pattern of oxylipins in order to understand their biology. In this short review, results from intervention studies are summarized analyzing >30 oxylipins from different PUFAs in response to n3-PUFA supplementation. The results are not only qualitatively compared with respect to the study design, n3-PUFA dose and trends in the lipid mediators, but also quantitatively based on the relative change in the oxylipin level induced by n3-PUFA. The evaluation of the data from the studies shows that the change in oxylipins generally corresponded to the observed changes in their precursor PUFA, i.e. the lower the individual n3-status at the baseline, the higher the increase in EPA and DHA derived oxylipins. The strongest relative increases were found for EPA derived oxylipins, while changes in arachidonic acid (C20:4 n6, ARA) derived eicosanoids were heterogeneous. After 3–12 weeks of supplementation, similar relative changes were observed in free and total (free + esterified) oxylipins in plasma and serum. Regarding EPA derived oxylipins, the results indicate a trend for a linear increase with dose. However, the interpretation of the quantitative oxylipin patterns between studies is hampered by strong inter-individual variances in oxylipin levels between and also within the studies. In the future, the reason for these varying oxylipin plasma concentrations needs to be clarified in order to understand oxylipin and n3-PUFA biology.


Introduction

In many epidemiological and intervention studies high endogenous levels of omega-3 polyunsaturated fatty acids (n3-PUFA), including eicosapentaenoic acid (C20:5 n3, EPA) and docosahexaenoic acid (C22:6 n3, DHA), have been correlated with beneficial effects on human health, e.g. in cardiovascular1 or inflammatory diseases.2 However, results are conflicting and in some studies these positive effects have not been observed, e.g. in intervention studies on the effectivity of n3-PUFAs on the relapse in Crohn's disease,3 or on major cardiovascular events (fatal and nonfatal cardiovascular events and cardiac interventions) in a population with previous myocardial infarction receiving medication.4

At the molecular level, a part of n3-PUFA related effects has been attributed to a shift in the pattern of lipid mediators formed in the arachidonic acid (C20:4 n6, ARA) cascade by competing with ARA for conversion in the three enzymatic pathways of this signaling cascade2,5 (Fig. 1). Conversion of ARA by cyclooxygenases (COX) leads to prostaglandin (PG) H2 which is further metabolized by downstream enzymes to series 2 prostanoids, such as PGE2, a potent mediator in the regulation of pain, fever and inflammation.2,6,7 By contrast, conversion of EPA is slower, thereby reducing the levels of formed mediators8 and leads to less potent series 3 PG.2,7 Similarly, leukotriene (LT) B4, formed by lipoxygenase (LOX) action on ARA, acts pro-inflammatory by neutrophil attraction,6 while its EPA derived counterpart (LTB5) shows much lower potency.2,7 Beyond this, EPA and DHA can also give rise to highly potent lipid mediators. For instance, in the third branch of the ARA cascade, enzymes of the cytochrome P450 (CYP) 2C and 2J family act as epoxygenases on PUFA. The main products of EPA and DHA, 17(18)-epoxy eicosatetraenoic acid (EpETE) and 19(20)-epoxy docosapentaenoic acid (EpDPE), are potent anti-arrhythmic acting mediators9 and 19(20)-EpDPE has been shown to inhibit angiogenesis, while Ep-FA deriving from ARA promote angiogenesis.10 Also, multiple hydroxylation of EPA and DHA leads to potent inflammation resolving oxylipins, termed specialized pro-resolving mediators (SPM), like resolvins or maresins.11


image file: c7fo00403f-f1.tif
Fig. 1 Simplified overview of selected CYP, LOX and COX pathways of LA, ALA, ARA, EPA and DHA in the ARA cascade. HpETE – hydroperoxy eicosatetraenoic acid; Lx – lipoxin; Mar – maresin, PD – protectin; RvD – D series resolvins; HpEPE – hydroperoxy eicosapentaenoic acid.

Overall, the influence of EPA and DHA on the profile of lipid mediators formed in the ARA cascade is diverse and biological effects of n3-PUFA seem to result more from a shift in the whole pattern of oxylipins rather than from changes of selected metabolites. For an interpretation of EPA and DHA derived effects and the characterization of the impact of individual metabolites, it is therefore important to monitor a comprehensive set of oxylipins from all branches of the ARA cascade and supplementation induced shifts therein.12,13 Oxylipin patterns in healthy subjects on different diets can be correlated with those during onset and progression of diseases allowing to evaluate the (patho-)physiological role of (n3-PUFA derived) mediators. However, many studies in this field analyze and/or report only selected (groups) of oxylipins of interest, e.g. SPM and epoxy-FA,14 SPM and precursor thereof,15,16 epoxy-FA17 or mainly ARA derived mediators.18

Nonetheless, several studies characterized n3-PUFA (EPA + DHA) supplementation induced changes in a comprehensive set of oxylipins derived from different n6- and n3-PUFA (ARA, EPA and DHA amongst others) and across the different branches of the ARA cascade (COX, LOX, CYP and autoxidation)12,19–28 and in one study29 the effects of individual EPA and n3 docosapentaenoic acid (n3-DPA) supplementation were compared. However, the outcome of these studies has not been systematically compared. The aim of the present work is therefore to summarize the results of n3-PUFA intervention, to evaluate if they lead to similar quantitative results and pinpoint differences which have to be addressed in future studies. In order to allow the quantitative comparison of the effects of n3-PUFA treatment on all three branches of the ARA cascade, only studies analyzing a comprehensive set of >30 oxylipins from different precursor FA and reporting mediators from different groups of oxylipins (e.g. hydroxy-, dihydroxy-, and epoxy-fatty acids) were included and focus was set on supplementation induced changes in healthy subjects.

Dose, intervention period and study population

EPA, DHA and other n3-PUFAs can be biosynthesized in humans from the essential n3-PUFA alpha linolenic acid (C18:3 n3, ALA) in multiple steps, involving conversion by delta-5 and delta-6 desaturases and elongases.30,31 However, conversion rates of ALA to EPA are low in populations on a western diet rich in n6-PUFA,31 resulting in low shares of EPA and DHA in blood (red blood cells (RBC) and plasma).32 The way to supply humans with EPA and DHA is consumption of long-chain n3-PUFA rich seafood, like oily fish,1,2 or by intake of dietary supplements.33 Dietary recommendations for the uptake of EPA + DHA range from ≥250 mg d−1 to ≥500 mg d−1 (e.g. Academy of Nutrition and Dietetics, AHA, EFSA, and WHO)34 and according to the German Society for Nutrition, primary prevention of coronary heart disease with a daily intake of up to 250 mg EPA and DHA seems probable.35 However, in order to achieve the triglyceride lowering effects, daily doses need to be higher (2–4 g d−1) and a higher intake is recommended for patients with coronary heart disease (∼1 g d−1).34 Moreover, special recommendations for pregnant or lactating women concerning the intake of DHA exist.34 However, it seems that high doses of n3-PUFA might cause adverse effects. While supplementation with low doses of DHA (200 mg DHA per day, administered in capsule form as triglyceride from algal oil) caused anti-oxidative effects (evaluated based on the concentration of platelet α-tocopherol and urinary isoprostane 8-iso-PGF (autoxidation product of ARA)), one study showed that higher concentrations (up to 1600 mg DHA per day) seemed to increase autoxidation/oxidative stress markers.36

In supplementation studies investigating the effect of n3-PUFA in healthy subjects,12,19,20,22–27 daily intakes of EPA ranged from 0.36–1.9 g d−1 and DHA doses ranged from 0.24–1.5 g d−1 (Table 1). With a sum of EPA + DHA from 0.6–3.4 g d−1, this is higher than the minimum dietary recommendations for the intake of n3-PUFA in healthy subjects. It should be noted that a linear relationship between the dose and endogenous n3-PUFA levels in different blood compartments was found at least up to 4 meals of oily fish per week, equaling 1.87 g EPA + DHA (1[thin space (1/6-em)]:[thin space (1/6-em)]1.2, w[thin space (1/6-em)]:[thin space (1/6-em)]w) per day.37 At a dose of 3 g EPA + DHA (3[thin space (1/6-em)]:[thin space (1/6-em)]2, w[thin space (1/6-em)]:[thin space (1/6-em)]w) per day, incorporation of EPA into plasma phospholipids was still linear, while DHA incorporation seemed to reach saturation.38

Table 1 Overview over the study designs
    Subjects Intervention Comment Ref.
  Health status Gender Age [years] BMI [kg m−2] N Dose [g d−1]   Duration [weeks]
  EPA DHA Supplement
If not indicated otherwise, oxylipin concentrations were analysed at baseline and at the end of the supplementation period.a No information provided.b Information was taken from the inclusion criteria of the study.c Oxylipins measured after 12 (n = 4), 15 (n = 2) or 24 (n = 1) months of supplementation.d Value refers to the total dose of the prescription n3-PUFA ethyl ester product.e Mean ± standard error of the mean.f Mean ± standard deviation.g Median (interquartile range).h Study participants received partially medication typical for elderly persons.
Supplementation with EPA + DHA
Fischer & Konkel et al. (2014) Healthy M + F M: 31.3 ± 2.5e M: 24.9 ± 1.0e 10 + 10 0.46 [week 1–4] 0.38 [week 1–4] n3-PUFA as ethyl ester in capsules 8 week 9–16 washout; +sampling in week 1, 4, 9 and 16 19
F: 38.0 ± 1.8e F: 25.5 ± 1.3e 0.98 [week 5–8] 0.76 [week 5–8]
Keenan et al. (2012) Healthy M + F 21–59 21–32 9 + 21 1.86 1.46 Prescription n3-PUFA ethyl ester, capsules 4 20
Nording et al. (2013) Healthy M + F 21–55 19–28 4 + 8 1.9 1.5 Fish oil in capsules 6 22
Schebb et al. (2014) Healthy M + F 46–70 21–35 5 + 5 1.1 0.74 Fish oil as ethyl ester, capsules 12 23
Schuchardt et al. (2014) (A) Healthy M 26–36 22–29 6 1.008 0.672 Fish oil as rTAG, capsules Single dose 25
Schuchardt et al. (2014) (B) Healthy M 38 ± 8f 24.2 ± 4.15f 10 1.56 1.14 Fish oil as rTAG, capsules 12 24
Shearer et al. (2010) Healthy M + F 33 (24, 51)[thin space (1/6-em)]g 25.5 (21, 29)[thin space (1/6-em)]g 4 + 6 4d; about 1.9 g d−1 EPA and 1.5 g per day DHA20 Prescription n3-PUFA ethyl ester, capsules 4 12
Zhang et al. (2015) Healthy M + F 24 ± 2 a 6 + 6 0.36–0.4 0.24–0.28 Fish oil in capsules 3 +Sampling on days 3, 7, 14 27
Watkins et al. (2016) Healthyh F 75 ± 6f 27.1 ± 6.0f 20 0.72 0.48 Fish oil in capsules 6 months +Placebo group (olive oil) 26
 
Lundström et al. (2013) Asthma M + F 20–54 19–39 14 + 11 4.0 2.0 Capsules, ethyl ester 3 Cross over study with placebo (50[thin space (1/6-em)]:[thin space (1/6-em)]50 soybean: corn oil); 3 weeks washout 21
Schuchardt et al. (2014) (B) Hyperlipidemia M 40 ± 8f 28.5 ± 3.28f 10 1.56 1.14 Fish oil as rTAG, capsules 12 24
Zivkovic et al. (2012) Nephropathy a ≤40b a 7 1.9 1.5 Concentrated fish oil, n3-PUFA as ethyl ester in capsules51 12 monthsc +Placebo group (corn oil); sampling at 6, 9, 12, 15, 18, 24 months 28
Supplementation with other n3-PUFA
EPA/n3-DPA
Markworth et al. (2016) Healthy F 25.5 ± 3.3 22.3 ± 1.6 10 Day 1: 2g; Day 2–7: 1g Day 1: 2 g purified FFA with 18 g olive oil in breakfast; Day 2–7: 2 g 1[thin space (1/6-em)]:[thin space (1/6-em)]1 FFA[thin space (1/6-em)]:[thin space (1/6-em)]olive oil with orange juice 7 days Cross-over study with EPA, n3-DPA and placebo (olive oil), 2 weeks washout 29


The effect of n3-PUFA supplementation on the oxylipin pattern in non-healthy subjects was investigated in patients with mild to moderate asthma,21 hyperlipidemia24 and IgA nephropathy28 and combined EPA and DHA doses ranged from 2.7–6.0 g d−1 (Table 1).

The dietary intervention lasted for 3–12 weeks in most studies investigating the effects of n3-PUFA on the oxylipin pattern, and one study monitored the effects up to 6 months and 12 months, respectively (Table 1). In Schuchardt et al. (A) only a single dose of omega-3 fatty acids was administered and oxylipin levels were analyzed up to 48 h after ingestion.25

In most studies, EPA and DHA were administered as capsules, dominantly containing refined fish oil (Table 1). However, binding forms differed which might have an impact on the bioavailability of the PUFAs.39 For instance, Schuchardt et al. used re-esterified triglycerides in their studies,24,25 while n3-PUFA ethyl esters were used by Fischer and Konkel et al., Keenan et al. and Schebb et al. amongst others.12,19–21,23,28 Indeed, in some studies a higher bioavailability of re-esterified triglycerides in comparison with ethyl esters39 was found which might influence the effect of n3-PUFA supplementation on the oxylipin pattern. However, not only the lipid binding form is crucial for bioavailability of the fatty acids, but also galenic or matrix.39 For example, pre-emulsified n3-PUFA ethyl esters show a significantly higher absorption and thus bioavailability.40 Given the importance of the formation of an emulsion, the bioavailability of n3-PUFA from a supplementation product is thus influenced if it is ingested together with a meal containing a substantial amount of fat (releasing bile acids and colipase).

In most studies, fasting blood samples were used for oxylipin analysis.12,19–24 However, in Schuchardt et al. (A), blood samples were collected in part postprandial or fastened over a period of 48 h after a single n3-PUFA dose25 and in several studies no information was provided.26–28

Heterogeneous study populations were used in studies with healthy study participants, comprising male and female subjects, aged roughly 21–70 years with a body mass index (BMI) of 19–35 kg m−2 (Table 1). In the study by Watkins et al., study participants were older compared to other studies (75 ± 6 years26), and received partially medication typical for elderly persons. These differences might have an additional influence on the outcome of the supplementation on the oxylipin profile. Caligiuri et al. have shown that the basal level of some of the investigated oxylipins in elderly people (average age of 53 years) was higher compared to the younger study participants (average of 22 years), e.g. 5-LOX products 5-HETE, 5-HEPE and 7-HDHA or 20-HETE. Supplementation with ALA (6 g ALA per day for four weeks in form of a muffin prepared with flaxseed oil (30 g per muffin)) affected oxylipin levels in elderly differently from in the young, resulting in comparable oxylipin concentrations in both groups post-intervention. Some EPA and DHA derived OH-FA were even lower in the older study population after supplementation.41

Methods for the quantitative comparison of results

For the quantitative comparison of the changes in the pattern of oxylipins and their precursors (linoleic acid (C18:2 n6, LA), ALA, ARA, EPA and DHA), fold changes between concentrations post- and pre-n3-PUFA-supplementation (post/pre) were calculated in the different studies, based on the means of the analytes pre- and post-supplementation provided in the respective study (Table 2). This relative comparison of study results was chosen to compensate for the differences in absolute concentrations between the studies, originating, e.g. from different standards used for calibration.42 Variances in the analytes’ concentrations were not considered in this calculation. If sampling was conducted at more than one time point, basal levels of oxylipins and FA were compared to the longest supplementation period. If an analyte was described in the analytical method, or if an analyte was graphically presented in the manuscript, but no concentration was shown, authors were asked to kindly provide the exact concentration of the respective analytes. In case raw data (or raw means) were provided, calculations of the post/pre-ratio for all analytes from the respective study were based on the raw data. Means from the raw data for the post/pre-ratios were calculated if the analyte was detectable in ≥50% of the samples in one group. For samples with the analyte below the limit of quantification (LOQ) the LOQ was used for calculation. For the quantitative comparison of n3-PUFA induced effects on the oxylipin profile, exemplary oxylipins of the precursor PUFAs LA, ARA, ALA, EPA and DHA from all three enzymatic branches of the ARA cascade were chosen. The analytes were selected based on two criteria: (i.) they were reported in most of the studies and (ii.) analogous biochemical formation route in the AA cascade: 15-LOX products, terminally epoxygenated PUFA and their hydrolyzation products. For oxylipins originating from the COX pathway, ARA derived products PGE2 and thromboxane (Tx) B2 were selected as well as their EPA derived counterparts (PGE3 and TxB3).
Table 2 Post/pre-supplementation fold changes of precursor fatty acids and selected oxylipins
  Freeg Totalg
  Schebb et al. (2014)23 Schuchardt et al. (2014)24 (B) Watkins et al. (2016)26 Zhang et al. (2015)27 Lundström et al. (2013)21[thin space (1/6-em)]f Schuchardt et al. (2014)24 (B) Zivkovic et al. (2012)28 Fischer & Konkel et al. (2014)19 Keenan et al. (2012)20[thin space (1/6-em)]e Schebb et al. (2014)23 Shearer et al. (2010)12
Health Status Healthy Healthy Healthy Healthy Asthma Hyperlipidemia Nephropathy Healthy Healthy Healthy Healthy
Oxylipins Plasma Serum Serum Plasma Serum Serum Plasma Plasma Plasma Plasma Plasma
FAh RBC RBC Plasma a Serum PL RBC a RBC RBC RBC Plasma
a No information provided. b Analyte was below the limit of quantification in at least one of the groups (pre- or post-supplementation). c Analyzed, but not shown. d Analysis not possible due to analyte degradation during alkaline hydrolysis. e Post/pre-ratios for FA calculated based on [mol%]; for all other studies, FA rations were calculated based on [% of total FA]. f Shown is comparison of supplementation vs. placebo group. g Free oxylipins are non-esterified oxylipins present in plasma/serum. Total oxylipins reflect the concentration determined after alkaline lipid hydrolysis yielding the sum of free and esterified oxylipins. h Total fatty acids were determined from plasma, serum or RBC following transesterification.
n3-PUFA
ALA 0.94 0.56 0.97 a 0.88 0.68 a 0.81 0.93 0.94 0.99
13-HOTrE 0.97 0.92 c a 0.69 0.94 0.88 a 0.82 0.9 0.86
15(16)-EpODE 0.53 0.69 c a 0.65 0.54 0.91 a 0.8 0.51 0.88
15,16-DiHODE 0.9 0.89 c a 0.71 0.76 0.66 a 0.77 0.63 1.2
 
EPA 1.3 4.5 2.4 a 5.7 3.5 a 3.4 4.9 1.3 8.5
PGE3 a a c b b a 3.7 d d d d
TxB3 a a a b a a a d d d d
15-HEPE 1.5 4.3 1.7 b 5.6 2.2 4.3 3.2 6.1 2.7 5.5
17(18)-EpETE 1.8 2.5 4.5 b b 1.4 1.2 5.0 7.1 2.2 4.8
17,18-DiHETE 2.5 3.4 1.5 7 3.9 3.0 5.6 3.0 2.0 1.7 4.1
 
DHA 1.2 1.6 1.5 a 1.7 1.7 a 1.4 1.4 1.2 2.7
17-HDHA a a 1.2 1.2 b a 1.2 2.0 2.4 a 2.1
19(20)-EpDPE 1.2 1.6 c b b 0.84 1.8 2.2 2.4 1.1 2.0
19,20-DiHDPE 1.4 1.9 1.3 a b 2 1.7 1.9 2.4 0.77 2.1
 
n6-PUFA
LA 0.91 0.72 1.0 a 0.81 0.74 a 0.94 0.92 0.91 0.98
13-HODE a 1.1 c a 0.74 0.67 0.75 a 0.86 a 0.87
12(13)-EpOME 0.98 0.53 c a 0.76 0.36 0.75 a 0.9 0.67 0.93
12,13-DiHOME 1.2 0.91 c a 0.66 0.83 1 a 1.1 0.83 0.92
 
ARA 0.93 0.81 0.94 a 0.86 0.93 a 0.95 0.89 0.93 0.88
PGE2 a a c b 2.7 a 1 d d d d
TxB2 a a c 0.7 1.3 a 0.68 d d d d
15-HETE 0.95 1.4 4.4 0.74 1 0.93 0.71 0.85 0.8 0.55 0.8
14(15)-EpETrE 0.92 0.58 1.1 b 1.2 0.38 0.65 0.92 0.81 0.61 0.92
14,15-DiHETrE 0.89 0.84 0.79 1.4 0.7 0.82 0.79 0.95 0.95 0.59 0.89


Modulation of the n6- and n3-oxylipin pattern following n3-PUFA supplementation in healthy subjects

In order to understand the oxylipin biology in the course of a disease or in response to a treatment, it would be most appropriate to analyze oxylipin concentrations in the tissue of interest. However, in humans this is rarely feasible. Therefore, plasma and serum are often used as a proxy for tissues to quantitatively evaluate changes in oxylipins, e.g. in response to a dietary intervention. Relative changes in exemplary plasma and serum oxylipins (post/pre-fold change) from all three branches of the ARA cascade induced by supplementation with n3-PUFAs in different studies are presented in Table 2 and highlighted in a heat map in Fig. 2. Based on the findings that low doses of EPA and DHA led to a maximum in the incorporation of EPA into plasma phosphatidylcholine after about 14 days of supplementation37 and that EPA metabolites were rapidly increased after a single dose of n3-PUFA,25 we assumed a maximum modulation of the oxylipin pattern after about 14 days. Thus, the oxylipin pattern obtained following 3–12 weeks of supplementation (Table 2) would be maximally modulated, allowing the direct comparison of changes between the studies.
image file: c7fo00403f-f2.tif
Fig. 2 Heatmap of post/pre-supplementation fold changes in precursor FA and oxylipins (see also Table 2).

The major portion of circulating oxylipins, esp. epoxy-FA and hydroxy-FA, is found esterified in lipids.23,43 While the role of esterified oxylipins in (patho-)physiology is not as well understood as the role of free oxylipins, esterified oxylipins have also been shown to be biologically active.44 Although both free and esterified oxylipins in plasma or serum are used as a proxy for tissues in human studies, it remains to be determined if and how they represent concentrations in the tissue of interest. An important question in this context is the origin of circulating free and esterified oxylipins. Are they released from the tissue, thus representing the tissue status, or are they formed in situ? To which extent and in which biological context are free oxylipins released from or incorporated into lipids? Is this regulation altered in diseases? Thus, the question of analyzing free or esterified oxylipins is of importance for the interpretation of the results in a biological context. Liberation of oxylipins from their esterified form is often achieved using base hydrolysis, leading to a sum parameter of free and esterified oxylipins (“total oxylipins”).42 In a direct comparison, relative changes induced by dietary n3-PUFA supplementation revealed similar trends in free and total oxylipins.23 This finding is further supported by a comparison of changes induced in free and total oxylipins across different studies (Table 2). Although some trends were slightly different between free and total oxylipins (see below), the overall data indicate that after 3–12 weeks of supplementation, shifts occurring in the oxylipin pattern are similarly represented by both free and total oxylipins.

In response to n3-PUFA supplementation, concentrations of free and total EPA and DHA metabolites in the plasma and serum of healthy subjects increased. Generally, relative increases in EPA metabolites were more pronounced than changes in DHA metabolites, a trend that was found in all studies, and elevation in EPA and DHA oxylipins roughly corresponded to changes in the respective precursor FAs. Free DHA metabolites increased up to twofold, while changes in total DHA derived docosanoids were slightly higher in most studies (1.9–2.4-fold increase). However, in the study by Schebb et al., total 19(20)-EpDPE was only slightly increased (post/pre-ratio of 1.1) and its hydrolysis product was decreased (post/pre-ratio of 0.77). EPA oxylipins increased up to sevenfold and no trend was observable discriminating free (post/pre-ratio of 1.5–7.0) and total oxylipins (post/pre-ratio of 1.7–7.1). Differences in the administered doses between the studies might be a variable leading to this variance (0.36 vs. 1.9 g EPA per day see below).

It should be noted that a high relative increase did not necessarily correspond to higher absolute concentration changes. For instance, in the study by Schuchardt et al. (B),24 relative changes in free 15-hydroxy eicosapentaenoic acid (HEPE) were more pronounced than changes in 17,18-dihydroxy eicosatetraenoic acid (DiHETE) (post/pre-ratio of 4.3 vs. 3.4), while the absolute increase in 17,18-DiHETE was much higher due to the higher concentrations of this analyte (post-pre15-HETE = 360 pM; post-pre17(18)-DiHETE = 1060 pM). Similarly, the relative increase in 19(20)-EpDPE was lower (post/pre = 1.6) compared to 15-HETE, while the absolute increase was higher (post-pre19(20)-EpDPE = 600 pM). Likewise, in the study by Keenan et al.20 the post/pre-ratio of total 17(18)-EpETE was 7.1 versus 2.0 for the hydrolyzation product 17,18-DiHETE, while the respective absolute differences (post-pre) were 4.4 and 7.0 nM. The relative modulation of 17-hydroxy docosahexaenoic acid (HDHA) was similar to 17(18)-DiHETE (2.4), while the concentration of the 17-HDHA was increased by 55 nM post-supplementation.20 In the other studies, similar trends can be found.12,19,23,26,27 Since biological effects depend on the absolute concentration of an active substance in the target tissue, it is thus crucial to not only consider relative changes, but also absolute changes for the interpretation of biological effects associated with n3-PUFA supplementation.

Regarding responses in the individual pathways of the ARA cascade to n3-PUFA supplementation, results between the studies are heterogenous. For instance, some studies indicated the highest changes in the CYP pathway.19,22 However, a comparison of terminally epoxy-EPA and epoxy-DHA (CYP) with 15-LOX products (15-HEPE and 17-HDHA) in healthy subjects across different studies led to different results. For instance, changes in free 17(18)-EpETE compared to 15-HEPE were higher in the studies by Schebb et al.23 and Watkins et al.,26 while changes in 15-HEPE were more pronounced in the study by Schuchardt et al. (B).24 The results from total oxylipins show similar outcomes compared to free oxylipins: Fischer and Konkel et al.19 as well as Keenan et al.20 found a higher modulation of 17(18)-EpETE and Schebb et al.23 as well as Shearer et al.12 found a higher modulation of 15-HEPE. A comparison of the changes in 17-HDHA to 19(20)-EpDPE in healthy subjects was only possible for few studies and total oxylipins; however, results suggest a comparable modulation of both pathways.12,19,20 In the study by Schuchardt et al. (B), a distinct decrease in epoxy-FA derived from ARA, LA and ALA as well as dihydroxy-FA from ARA (but not LA and ALA) was described, while changes in LOX products were inconsistent (Table 2 and ref. 24). Again, these trends were not found in all studies (Table 2).

Changes in the COX pathway could not be evaluated due to insufficient data availability (Table 2). Only in the study by Zhang et al. a decrease in TxB2 was found (post/pre-ratio of 0.7027), while other studies did not provide the information on PGE2 and TxB2. Since most prostaglandins and thromboxanes are unstable during alkaline hydrolysis,42 these analytes were not reported in the studies analyzing total oxylipin concentrations. However, due to their physiological function in platelet activation (TxA2) and regulation of inflammation (PGE2),6,7 low concentrations of these mediators would be expected in healthy subjects.

While ARA was slightly decreased in all studies, irrespective of the blood fraction analyzed (red blood cells, plasma, serum phospholipids (PL)), changes in free plasma ARA derived oxylipins in healthy subjects were inconsistent. For instance, while 14(15)-epoxy eicosatrienoic acid (EpETrE) was slightly increased in the studies by Watkins et al. (1.1 fold26), this analyte was slightly decreased in the study by Schebb et al. (fold change of 0.9223) and highly decreased in the study by Schuchardt et al. (B, fold change of 0.5824). 15-HETE and 14, 15-dihydroxy eicosatrienoic acid (DiHETrE) showed similar trends. By contrast, in the studies analyzing total oxylipins, ARA metabolites uniformly decreased (post/pre-ratio of 0.95–0.55). Indeed, when evaluating changes in free ARA metabolites for each individual, it was shown that they were decreased in some, while they increased in others.22 Thus, n3-PUFA induced modulation of ARA derived oxylipins detectable in plasma/serum seems to be highly variable between subjects. Changes in EPA and DHA oxylipins in response to n3-PUFA supplementation were also highly variable, but did show an increasing trend in most subjects.22 This inconsistent trend in ARA oxylipins might be explained by the dietary and endogenous n6-PUFA status of the study subjects, strongly influencing the individual response of n6-PUFA derived oxylipins to n3-PUFA supplementation. By contrast, variance in the study population's dietary uptake of EPA and DHA is low, due to the low background intake of EPA and DHA in comparison with the supplementation level as discussed by Keenan et al.20 In response to n3-PUFA supplementation, oxylipins from the LA and ALA metabolomes were mostly decreased, as were their precursor FA.

Overall, while EPA and DHA as well as their metabolites increased with n3-PUFA supplementation, the effects were more pronounced for EPA (evaluation based on relative changes). However, relative changes in the studies differed considerably, esp. for EPA, which might be explained by the different doses administered (see below). Notably, when evaluating changes in the oxylipin pattern both relative and absolute changes have to be considered since they do not necessarily show the same trend. In response to EPA and DHA supplementation, ARA and total ARA eicosanoids were decreased, while effects on free ARA metabolites were diverse.

Plasma and serum as a proxy for the analysis of oxylipins in tissues

Although serum and plasma are used in an analogous manner as a proxy to describe changes in tissue oxylipin concentrations, care must be taken when directly comparing the absolute oxylipin concentrations from plasma and serum due to the increased production of docosanoids and eicosanoids during coagulation. Regarding relative changes in oxylipins, the results from different studies (in healthy and diseased subjects) indicate that both plasma and serum might be comparable in identifying trends in eicosanoids and other oxylipins induced by dietary supplementation. However, neither plasma nor serum oxylipins reflect the actual situation in inflamed tissue. Serum can be regarded as “ex vivo” blood coagulation assay resulting in a massive activation of platelet COX-1 and 12-LOX. In order to induce an immunological response and thus to mimic inflammation, e.g., a septic shock, ex vivo assays with stimulation of whole blood using lipopolysaccharides (LPS) to induce monocyte activation45 or using the calcium ionophore A23187 to induce platelet activation46 are additionally used. Beyond increased TxB2 formation, the production of other oxylipins from the COX and LOX pathways, esp. 12-LOX, has been shown to be increased in A23187 activated whole blood.47

Fischer et al.19 treated the whole blood of subjects supplemented with n3-PUFA with the calcium ionophore A23187. Following supplementation with n3-PUFAs, the levels of LTB4, PGE2 and TxB2 were barely altered and concentrations of EPA derived counterparts LTB5 and PGE3 were low compared to ARA derived eicosanoids. Correlating LTB5/LTB4 and PGE3/PGE2 with the ratio of precursor FA revealed slopes <1, indicating a preference of ARA versus EPA for the formation of LTB and PGE. No change was found in TxB3 following dietary intervention, probably due to insufficient supplementation doses in order to induce changes in TxB3 formation as discussed by Fischer et al.19 Interestingly, while in non-activated plasma, LOX pathways did show a higher efficiency in converting EPA and DHA compared to ARA (esterified oxylipins, slope of the product ratio vs. parent FA ratio correlations was at least 1.9, except 7-HDHA19), ARA was more efficiently converted in Ca-ionophore activated whole blood (free oxylipins, slopes of the product ratio vs. parent FA ratio correlations 0.01–1.319). However, it should be noted that this effect might also be caused by different lipid incorporation rates of EPA and DHA oxylipins in comparison with ARA.

Dose and time dependent responses in the oxylipin pattern to dietary n3-PUFA supplementation

Previous studies suggest a dose and time dependent response for the incorporation of EPA and DHA, e.g. into plasma lipid classes or blood cells in low doses which equaled up to four meals of oily fish per week.37 Interestingly, the maximum level of incorporation which was reached in the different tissues and blood compartments showed a clear dose dependency.37 However, the extent of incorporation depended on the tissue and blood compartment. For instance, while the relative amount of EPA + DHA in plasma phosphatidylcholine was linear up to a dose corresponding to four meals of oily fish per week (1.87 g d−1), a saturation of EPA + DHA in red blood cells was found in the highest concentration.37 Similarly, Flock et al. found a saturation of %EPA + DHA of total fatty acids in red blood cells in the highest dose supplemented (1800 mg(EPA + DHA) per day, ∼5 months48). With regard to plasma phospholipids, a linear incorporation of EPA was found up to 3 g (EPA + DHA) per day (3[thin space (1/6-em)]:[thin space (1/6-em)]2, w[thin space (1/6-em)]:[thin space (1/6-em)]w) for 12 weeks, while DHA incorporation seemed to reach saturation at the same dose.38

By contrast, almost no information is available on dose and time dependent changes in the oxylipin pattern in response to dietary n3-PUFA supplementation. In a study by Schuchardt et al. (A) with a single dose of n3-PUFA (1.008 g EPA and 0.672 g DHA), most free EPA metabolites in plasma were significantly elevated six hours post-ingestion, indicating that the EPA oxylipin level can be readily modified in a short period of time.25 DHA and DHA oxylipin levels were not altered which is consistent with the overall lower relative modulation of DHA in response to n3-PUFA supplementation. In the study by Fischer and Konkel et al. the study participants received a daily dose of 460 mg EPA + 380 mg DHA for four weeks and twice as much in the following four weeks.19 The ratio of the sum of CYP derived EPA and DHA epoxy and dihydroxy-metabolites to the sum of ARA analogs increased approximately twofold in the first week of intervention. In the following weeks, only a slight increase occurred. With the doubled dose in week 4, the ratio was rapidly increased until week 8 by about 1.6-fold.19 These results indicate a distinct time- and dose dependency in the modulation of metabolites in the third branch of the ARA cascade. Given the biological activity of the epoxy-n3-PUFA,9,10 this warrants further investigation.

Doses supplied to healthy volunteers in studies investigating the effect of n3-PUFA supplementation on the oxylipin pattern were in the range of up to 3.4 g EPA + DHA and the intervention period in most studies was 3 to 12 weeks (Table 1). Based on the assumption that time dependent changes in EPA metabolites occur in a similar manner to changes in their precursor FA (see above), we compared the magnitude of the change in different EPA derived oxylipins with the supplemented dose in order to evaluate dose response relations.

Across the different studies, relative changes in total 15-HEPE seem to correlate linearly with the supplied dose (Fig. 3B I). However, total 17(18)-EpETE, 17,18-DiHETE and the sum of both do not show a consistent trend across the studies (Fig. 3B II–IV). For free 15-HEPE and 17(18)-EpETE, a linear increase could be deduced combining the data of the studies by Schuchardt et al. (B) with Schebb et al. or Watkins et al. However, the results from all three studies combined did not show a clear trend (Fig. 3A I–II). Interestingly, relative changes in 17,18-DiHETE and in the sum of 17(18)-EpETE + 17,18-DiHETE seem to increase linearly with the dose between the three studies. However, for 17,18-DiHETE, a lower dose of EPA led in the study by Zhang et al. to a higher change (Fig. 3A III).


image file: c7fo00403f-f3.tif
Fig. 3 Correlation of the post/pre-concentration ratio (Table 2) for selected free (A) and total (B) EPA derived eicosanoids with the supplemented dose (Table 1) across the different studies.

One should note that the correlations shown in Fig. 2 can only be a rough estimate, because of different human subjects and different study designs. Taking the conflicting results on the clinical efficacy of n3-PUFA in different diseases into account, further studies on the dose dependent modulations of the oxylipin profile are urgently needed. These studies should not only correlate changes in the overall oxylipin pattern with the n3-PUFA dose but also monitor the genetic variability, e.g. single-nucleotide polymorphism as well as expression levels of the enzymes from the pathways of the ARA cascade. This will not only allow deducing dose–response dependencies but also lead to a better understanding of the molecular mode of action of n3-PUFA and may explain why n3-PUFAs are effective in some subjects while others do not respond to n3-PUFAs.

Inter-individual variations in responses to n3-PUFA supplementation

Although the change of EPA and DHA derived oxylipins showed the same increasing trend, high variances were observed, esp. in the changes of EPA and its metabolites between the studies. This is in accordance with findings within the different studies, showing high variances in the individual responses of the study participants to dietary intervention with n3-PUFA although compliance was high in the studies.20,22,24,26

Schuchardt et al. found that increases in serum EPA metabolites correlated well with increases of EPA in erythrocytes24 and EPA metabolites in plasma correlated well with the EPA plasma phospholipid level after a single dose of n3-PUFA.25 Consistently, Nording et al. described a correlation of changes in EPA/DHA in specific lipid classes (e.g. triglycerides, cholesterol ester) with their oxidative metabolites in plasma.22 Moreover, it has been found that the study participants’ individual responses in EPA + DHA in red blood cells (omega-3 index) correlated well with the respective basal status, in a way that a lower basal omega-3 index was associated with higher supplementation induced increases.20 This was also found in a study by Flock et al., investigating the dose response of the omega-3 index with increasing doses of n3-PUFA supplementation. The authors found a positive correlation of changes with the initial omega-3 index.48 Furthermore, a lower bodyweight and a higher n3-PUFA dose were associated with a higher increase in the omega-3 index.48 Keenan et al. correlated individual baseline PUFA and oxylipin concentrations with the respective dose-dependent change. The x-intercept of the resulting inverse correlation was introduced as the “change threshold”. This concentration defines the threshold for supplementation induced changes by n3-PUFA. Study participants entering the study with this PUFA/oxylipin concentration will most likely show only little changes after supplementation. The larger the difference of a subject's basal PUFA/oxylipin status from the “change threshold” the higher the change that will be observed with supplementation.20

These results indicate that changes in PUFA as well as oxylipin concentrations are dependent parameters and correlate with the basal level of PUFA/oxylipins. Thus, variances in the basal n3-PUFA level might account for part of the observed differences in individual responses to n3-PUFA supplementation.

Fischer and Konkel et al. found a positive correlation between ‘dose [mg per kg bodyweight per d] × basal omega-3 index’ and the omega-3 index in week 8.19 By contrast, in an explorative study by Nording et al., no significant correlation between changes in EPA, DHA, ARA or their oxylipins and age, body weight or BMI was found.22 Similarly, Schebb et al. did not find statistically different concentrations in oxylipins before and after supplementation between men and women for most analytes.23

Schuchardt et al. (B) observed that variances between the study participants were much higher for oxylipins compared to fatty acids. While trends in fatty acids induced by n3-PUFA supplementation were similar between the individuals, trends in oxylipins differed.24 These results indicate inter-individual differences in the metabolism of fatty acids. Indeed, Stephenson et al. reported differences in the response to n3-PUFA supplementation which were correlated with variants in the ALOX5 genotype.49 In another study, it was shown that dietary intervention influenced the expression of different PUFA metabolizing enzymes, e.g. ALOX5 or CYP2J2, in study participants that did not show a response (in the form of lowered triglyceride levels) to n3-PUFA supplementation.50

Most oxylipins in plasma are found in the lipoprotein fraction.43 However, only little information exists on the profile of oxylipins in lipoproteins and their modulation, e.g. by n3-PUFA supplementation. In statin-treated, hypertriglyceridemic patients, the different lipoprotein fractions showed a distinct pattern of oxylipins, which shifted from dominantly ARA metabolites to EPA and DHA derived ones following supplementation with EPA and DHA.52 Thus, it seems likely that modulations in individual lipoprotein levels and distributions – e.g. dietary PUFA induced modulation of the LDL/HDL ratio – contribute to the varying plasma oxylipin levels observed in different subjects.

Overall, it seems crucial to address the high inter-individual variances in future studies. Although these variations in a population of healthy subjects might hamper interpreting the effects of supplementation, identifying underlying causes could be an important step in the process of biomarker evaluation. For instance, if e.g. altered enzyme activity is an early stage symptom associated with a certain disease, the resulting oxylipin concentrations might become a valid biomarker.

Important prerequisites in the evaluation of oxylipins as a biomarker for diseases or in monitoring effects of n3-PUFA supplementation are analytical methods allowing a reliable quantification of oxylipins in biological samples as summarized in ref. 42. In order to allow the comparison of results from different laboratories and from different studies, standardized procedures for all steps in oxylipin analysis, e.g. blood sampling, sample preparation or the use of certified standards for quantification have to be established.

Modulation of oxylipins by n3-suplementation in (the onset of) diseases

Few studies investigated the effects of n3-PUFA supplementation on the oxylipin pattern in non-healthy subjects, including mild to moderate asthma,21 hyperlipidemia24 and IgA nephropathy.28 The changes in the oxylipin pattern in these subjects depicted in Table 2 were comparable to healthy individuals. Indeed, Schuchardt et al. (B) directly compared normo- vs. hyperlipidemic subjects and concluded that no differences between the oxylipin profiles in both study groups were observable.24 Based on patients with n3-PUFA supplementation, Zivkovic et al. identified oxylipins which were changed the most between responders and non-responders in terms of n3-PUFA induced improved kidney function.28 Here, the highest differences between both groups were not found for EPA and DHA epoxy- and dihydroxy-metabolites, but for hydroxy-LA and hydroxy-ARA. Based on this finding, one could conclude that – at least plasma levels of – EPA and DHA oxylipins are not linked to the improvement of kidney function by n3-PUFA.28

Conclusion

In this short review, we compared the effects of supplementation with the n3-PUFAs EPA and DHA on the oxylipin pattern across different studies. However, due to restrictions in data availability only a few oxylipins could be quantitatively compared. In particular, it was not possible to evaluate changes in the COX branch of the ARA cascade and in bioactive SPMs.

Overall, EPA and DHA derived oxylipins were increased compared to baseline, while changes in ARA were less consistent with a trend to decreasing levels. However, interpretation of these results is hampered by high inter-individual variances in oxylipin concentrations in response to n3-PUFA supplementation, while changes in fatty acids were consistent. Nonetheless, the currently available data indicate that the response in the oxylipin profile to n3-PUFA supplementation correlates with the basal fatty acid status. However, only scarce information is available on dose- as well as time-responses.

Thus, in future studies it will be important to understand the underlying reasons for the high variability of inter-individual responses in oxylipins to n3-PUFA supplementation. Moreover, the investigation of time- and dose dependencies in n3-PUFA induced changes in the oxylipin pattern is still lacking. This information will help to understand n3-PUFA biology, especially with regard to recent studies finding no beneficial effect of n3-PUFAs on human health, and to identify and establish oxylipin (ratios) as a biomarker for disease status and the effectiveness of (pharmacological) treatment. Moreover, studies investigating the effect of the background diet (high n6, mediterranean) and supplementation with individual PUFAs29 will be helpful to understand the regulation of the endogenous oxylipin profile.

Abbreviations

ALAAlpha linolenic acid
ARAArachidonic acid
BMIBody mass index
COXCyclooxygenase
CYPCytochrome P450
DiHDPEDihydroxy docosapentaenoic acid
DiHETEDihydroxy eicosatetraenoic acid
DiHETrEDihydroxy eicosatrienoic acid
DHADocosahexaenoic acid
EpETEEpoxy eicosatetraenoic acid
EpETrEEpoxy eicosatrienoic acid
EPAEicosapentaenoic acid
EpDPEEpoxy docosapentaenoic acid
HDHAHydroxy docosahexaenoic acid
HEPEHydroxy eicosapentaenoic acid
HETEHydroxy eicosatetraenoic acid
LALinoleic acid
LOXLipoxygenase
LTLeukotriene
n3-DPAn3 docosapentaenoic acid
PGProstaglandin
PLPhospholipid
PUFAPolyunsaturated fatty acid
RBCRed blood cells
SPMSpecialized pro-resolving lipid mediator
TxThromboxane

Acknowledgements

We kindly thank the authors of manuscripts on omega-3 fatty acid supplementation induced changes in the oxylipin pattern for providing raw data of their analytes. This study was supported by a grant of the German Research Foundation (DFG) to NHS (SCHE 1801).

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