White adipose tissue (WAT) is a complex organ with both metabolic and endocrine functions. Dysregulation of all of these functions of WAT, together with low-grade inflammation of the tissue in obese individuals, contributes to the development of insulin resistance and type 2 diabetes. n-3 polyunsaturated fatty acids (PUFAs) of marine origin play an important role in the resolution of inflammation and exert beneficial metabolic effects. Using experiments in mice and overweight/obese patients with type 2 diabetes, we elucidated the structures of novel members of fatty acid esters of hydroxy fatty acids—lipokines derived from docosahexaenoic acid (DHA) and linoleic acid, which were present in serum and WAT after n-3 PUFA supplementation. These compounds contained DHA esterified to 9- and 13-hydroxyoctadecadienoic acid (HLA) or 14-hydroxydocosahexaenoic acid (HDHA), termed 9-DHAHLA, 13-DHAHLA, and 14-DHAHDHA, and were synthesized by adipocytes at concentrations comparable to those of protectins and resolvins derived from DHA in WAT. 13-DHAHLA exerted anti-inflammatory and proresolving properties while reducing macrophage activation by lipopolysaccharides and enhancing the phagocytosis of zymosan particles. Our results document the existence of novel lipid mediators, which are involved in the beneficial anti-inflammatory effects attributed to n-3 PUFAs, in both mice and humans.

White adipose tissue (WAT) is an extremely plastic organ with important roles in energy balance, whole-body glucose homeostasis, and the immune system (14). The systemic effects of WAT largely reflect its role in the control of blood lipid levels, as well as in the secretion of numerous bioactive peptides (e.g., adiponectin, leptin) from WAT cells (5,6). Adipocytes also release lipid-based mediators such as branched fatty acid esters of hydroxy fatty acids (FAHFAs) (7) and palmitoleate (8), which could improve local and whole-body glucose metabolism (9,10). Moreover, FAHFA administration also stimulated glucagon-like peptide 1 and insulin secretion, and reduced obesity-associated WAT inflammation in mice through cell surface G-protein–coupled receptor 120–dependent signaling (7). Both FAHFAs and palmitoleate are produced in adipocytes via the fatty acid (FA) synthesis pathway (de novo lipogenesis). The activity of this pathway is decreased in response to obesity-associated hyperinsulinemia and WAT inflammation (8,11,12), resulting in reduced levels of the beneficial lipid mediators. Although the biosynthetic enzymes of FAHFAs are unknown, two FAHFA-specific hydrolases, AIG1 and ADTRP, were recently identified (13).

These newly described FAHFAs are members of a lipid class called estolides (intermolecular esters of hydroxy FAs) serving mainly as biodegradable lubricants (14). Recently, the discovery of FA estolides in humans and mice (7) and triacylglycerol estolides in the brushtail possum (15) brought these lipids to mammalian physiology. The FAHFA nomenclature introduced by Yore et al. (7) combines abbreviations of esterified FAs and hydroxy FAs (e.g., the combination of esterified palmitic acid and hydroxy stearic acid [HSA] was abbreviated as PAHSA). Although a recently published in silico library of all potential FAHFAs uses a nomenclature based on chemical structure (16), for practical reasons, here we use the shorter abbreviations (e.g., PA for palmitic acid, LA for linoleic acid, and DHA for docosahexaenoic acid, with the “H” prefix for hydroxy FAs [HFAs] and the position of branching).

The obesity-associated WAT inflammation imposes adverse local as well as whole-body metabolic effects (1,2,11,1719). This inflammatory response is accompanied by macrophage repolarization to the proinflammatory (classically activated) M1 state, which negatively affects WAT functions (20) and could be counteracted using both dietary and pharmacological interventions (21,22). Regarding the dietary influence, namely n-3 polyunsaturated FAs (PUFAs) of marine origin, which are considered to be healthy dietary constituents in individuals with diabetes (23) (see discussion), play an important role in the resolution of WAT inflammation (2428) and probably potentiate beneficial functions of immune cells in WAT like efferocytosis or autophagy (17).

n-3 PUFAs exhibit their anti-inflammatory effects through G-protein–coupled receptor 120, as well as through several other signaling pathways, resulting in improved insulin sensitivity in obese mice (2931). In addition, DHA-derived lipid mediators such as resolvin D1 have been reported to decrease WAT inflammation, shifting macrophage polarization toward the M2 form and improving insulin sensitivity in obese mice (2427). A wide range of lipid mediators, including resolvins D1 and D2, protectin D1, lipoxin A4, 17-hydroxydocosahexaenoic acid (HDHA), 18-HEPE, and 14-HDHA were identified in human subcutaneous WAT, whereas the levels of protectin D1 and 17-HDHA decreased in the subcutaneous WAT of patients with peripheral vascular disease (32). Protectin DX, also known to be produced in WAT, alleviated insulin resistance in db/db mice, but did not resolve WAT inflammation (33).

Given the beneficial effects of n-3 PUFAs on WAT inflammatory status, we hypothesized that novel FAHFA structures derived from n-3 PUFAs, with possible anti-inflammatory properties, could be found. To test this hypothesis, we performed lipidomic analysis using human and murine serum and WAT samples collected from subjects supplemented or not supplemented with n-3 PUFAs.

Materials and Reagents

All chemicals were purchased from Sigma-Aldrich (Prague, Czech Republic), unless otherwise stated. FAHFA standards (5-, 9-, 12-, 13-PAHSA, respectively, and 5-PAHSA-2H31 and 9-PAHSA-13C4) were purchased from Cayman Pharma (Neratovice, Czech Republic).

Human Samples

Serum samples were acquired within the framework of a clinical trial focused on the combined effects of the antidiabetic drug pioglitazone and n-3 PUFAs (34). Briefly, overweight/obese patients 40–70 years of age, in whom type 2 diabetes had been diagnosed and who had already been treated with metformin, were given either 5 g/day corn oil (Placebo) or 5 g/day eicosapentaenoic acid (EPA) plus DHA concentrate (EPAX 1050TG, EPAX AS, containing about 15% EPA, 40% DHA, wt/wt [i.e., ∼2.8 g of EPA plus DHA]) for 24 weeks. The serum samples and biopsy samples of abdominal subcutaneous WAT collected during the final visit after an overnight fast were stored at −80°C until liquid chromatography (LC)–tandem mass spectrometry (MS/MS) analysis.

Murine Samples

Male mice (C57BL/6J; The Jackson Laboratory, ME) were maintained in a controlled environment (22°C, 12-h light/dark cycle, light from 6.00 a.m.) and were fed a corn oil–based high-fat (HF) diet (lipid content 35%, wt/wt) or an HF diet with EPA plus DHA concentrate (HFF) (EPAX 1050TG) for 8 weeks, as previously described (21). Epididymal WAT, subcutaneous WAT, liver and interscapular brown adipose tissue, and serum samples were collected after an overnight fast, and were stored in liquid nitrogen. All animal experiments were approved by the Animal Care and Use Committee of the Institute of Physiology of the Czech Academy of Sciences (Approval Number 172/2009) and followed the guidelines.

Cell Cultures

Murine adipocyte cell line 3T3-L1 and human multipotent adipose-derived stem (hMADS) cells (7) were grown according to standard protocols. Differentiated adipocytes were incubated with 100 μmol/L LA and 100 μmol/L DHA complexed to BSA 3:1 for 24 h and extracted for FAHFA analysis. Adipocytes and stromal-vascular cells (SVCs) were prepared as before (26). RAW 264.7 cells and murine bone marrow–derived macrophages (BMDMs) were grown and stimulated with lipopolysaccharide (LPS) as before (35,36). Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats and treated with lectin, as described previously (37).

FAHFA Extraction

FAHFA extraction was performed based on the published method (7). Murine tissue (∼300 mg) or cells were homogenized using a MM400 bead mill (Retsch GmbH, Haan, Germany) chilled to −20°C in a mixture of citric acid buffer and methanol, and further extracted with dichloromethane (1:1:2 final ratio). Internal standards of 5-PAHSA-2H31 and 9-PAHSA-13C4 were added to the homogenate (100 pg/sample). Serum samples were extracted according to the same protocol, apart from the homogenization. The organic phase was collected, dried in a Speed-vac (Savant SPD121P; ThermoFisher Scientific), resuspended in dichloromethane, and applied on Strata SI-1 Silica SPE columns (55 µm, 70 Å; Sigma-Aldrich). FAHFAs were eluted from the SPE columns with ethylacetate, concentrated in the Speed-vac, resuspended in methanol, and immediately measured using LC-MS as follows.

LC and MS

Chromatographic separation was performed in an ultra-performance LC UltiMate 3000 Rapid Separation LC System (Thermo Scientific) equipped with a Kinetex C18 1.7 µm 2.1 × 150 mm column (Phenomenex). The flow rate was 200 µL/min at 50°C. The gradient program used to separate FAHFA was as follows: solvent A (70% water, 30% acetonitrile, 0.01% acetic acid, pH 4), solvent B (50% acetonitrile, 50% isopropanol), for 1 min (100% solvent A), 5 min (20% solvent A), 18 min (10% solvent A), 20 min (100% solvent A), and 25 min (100% solvent A), while a linear gradient was maintained between the steps. Isocratic elution (20% solvent A, 80% solvent B, for 60 min) was used for structural studies. Ultra-performance LC was coupled to a QTRAP 5500/SelexION, a hybrid, triple-quadrupole, linear ion trap mass spectrometer equipped with an ion mobility cell (Sciex). FAHFAs were detected in negative electrospray ionization mode with the following parameters: declustering potential, −130; collision energy, −35; collision cell exit potential, −15; curtain gas, 25; collision gas, high; ionspray voltage, −4500; temperature, 400; ionsource gas 1, 40; and ionsource gas 2, 50. For ion mobility experiments, differential mobility spectrometry (DMS) settings were as follows: DMS temperature, high; DMS modifier, isopropanol/high; separation voltage, 3,800; DMS offset, −3; DMS resolution enhancement, low; and compensation voltage, −5.0 for 13-PAHSA and −1.2 for 12-PAHSA. Multiple reaction monitoring (MRM) mode with one quantifier and two qualifier transitions per FAHFA was used for quantitation (7) (Table 1). Quantifier ion MRM was used as a survey scan for information-dependent acquisition in the linear ion trap for enhanced-resolution MS/MS and 2nd generation (MS/MS/MS) product ion spectra (scan rate 1,000 Da/s, scan mode profile, step size 0.05 Da, linear ion trap fill time 200 ms). Pure standards of PAHSAs were used for quantitation. For DHAHLA and DHAHDHA compounds, the 5-PAHSA calibration curve was used as a surrogate.

Table 1

MRM transition list

IDQ1 [M-H]Q3 FAQ3 HFAQ3 HFA-H2O
PAHSA 537.5 255.2 299.3 281.3 
DHAHLA 605.4 327.2 295.2 277.2 
DHAHDHA 653.4 327.2 343.2 325.2 
IDQ1 [M-H]Q3 FAQ3 HFAQ3 HFA-H2O
PAHSA 537.5 255.2 299.3 281.3 
DHAHLA 605.4 327.2 295.2 277.2 
DHAHDHA 653.4 327.2 343.2 325.2 

Q1, precursor ion; Q3, product ion; FA, quantifier ion; HFA and HFA-H2O, qualifier ions.

Synthesis of DHAHLA Standard

Organic synthesis of 13-DHAHLA was performed according to Steglich esterification from DHA and 13-HODE (38,39). Details are provided in the Supplementary Data.

Markers of Inflammation

The anti-inflammatory properties of DHAHLA were assessed according to published methods (7,36,37,40). Briefly, murine BMDMs or RAW 264.7 macrophages were incubated in the presence of LPS (100 ng/mL; Escherichia coli 0111:B4; Sigma-Aldrich) or 13-DHAHLA (10 μmol/L) alone or in combination with 9-PAHSA (10 μmol/L) or 13-DHAHLA (10 μmol/L) for 18 h. Control cells were incubated with the vehicle alone. Murine IL-6 ELISA (Cayman Chemicals) and quantitative PCR (21) were used to measure the markers of macrophage activation; details are provided in the Supplementary Data. BMDMs were incubated in the presence of LPS alone or in combination with DHA (10 μmol/L), interferon-γ (IFN-γ) (50 ng/mL) or 13-DHAHLA (10 μmol/L) for 18 h. LC-MS/MS metabolipidomics (26,36,37,41) was used to measure macrophage metabolic activation and the levels of lipid mediators. PBMCs were pretreated with 1 µmol/L 13-DHAHLA for 30 min and stimulated with lectin (phytohemagglutitin 10 µg/mL) for 24 h, and the levels of tryptophan and kynurenine were measured in the media. RAW 264.7 macrophages were stimulated with LPS (10 ng/mL) and incubated in the presence of various 13-DHAHLA concentrations for 18 h to explore the dose-dependent inhibition of macrophage activation. The phagocytosis of fluorescein-labeled zymosan (Life Technologies) by BMDMs was measured using a Victor X4 plate reader (PerkinElmer) (40).

Statistics

Statistical analysis was performed with SigmaStat and P < 0.05 was considered significant.

Targeted Lipidomics of FAHFAs Using LC-MS/MS/MS

In view of the beneficial effects of PAHSAs (7), we developed a targeted lipidomic methodology using LC coupled to hybrid tandem mass linear ion trap spectrometry to identify and quantify FAHFAs in human and murine samples. We took advantage of the ability of MS to switch from sensitive triple-quadrupole scan modes to highly sensitive full-scan ion trap mode within one analysis to obtain both quantitative and qualitative (structural) information. This approach enabled us to precisely quantify FAHFA levels using MRM and to identify the branching position on the backbone HFA using MS/MS/MS. For instance, 9-PAHSA can be ionized in negative mode to [M-H] ion 537.488 mass-to-charge ratio (m/z). Fragmentation by collision-induced dissociation results in the following three daughter ions: 299.3, 281.2, and 255.2 m/z, identified as fragments of hydroxystearic, octadecenoic, and palmitic acid, respectively (Fig. 1A and B) (7). Further fragmentation of ion 299.3 m/z (hydroxystearic acid) in the linear ion trap gave rise to the ions 155.144 and 127.113 m/z, which are specific to the position of the hydroxyl group on the hydroxystearic acid backbone (Fig. 1A), thus enabling us to identify the branching carbon.

Figure 1

Analysis of PAHSA isomers. A: Fragmentation scheme of 9-PAHSA with 9-HSA–specific fragments. B: MS/MS spectrum of 9-PAHSA showing fragmentation into three major ions. C: Chromatographic profile of PAHSA isomers (MRM 537.5 > 255.2) detected in a human serum sample (blue line) overlaid with synthetic standards (red line). The inserted table summarizes MS/MS/MS (or MS^3; indicates three stages of fragmentation) fragments specific to individual positional isomers of hydroxy group on HSA. C and D: Combination of triple-quadrupole scan modes and highly sensitive full-scan ion trap mode within one analysis. MS/MS/MS spectra of PAHSA standards. Specific fragments are highlighted in magenta.

Figure 1

Analysis of PAHSA isomers. A: Fragmentation scheme of 9-PAHSA with 9-HSA–specific fragments. B: MS/MS spectrum of 9-PAHSA showing fragmentation into three major ions. C: Chromatographic profile of PAHSA isomers (MRM 537.5 > 255.2) detected in a human serum sample (blue line) overlaid with synthetic standards (red line). The inserted table summarizes MS/MS/MS (or MS^3; indicates three stages of fragmentation) fragments specific to individual positional isomers of hydroxy group on HSA. C and D: Combination of triple-quadrupole scan modes and highly sensitive full-scan ion trap mode within one analysis. MS/MS/MS spectra of PAHSA standards. Specific fragments are highlighted in magenta.

The quantities and structures of FAHFA isomers were analyzed in human serum samples, and with the PAHSA isomers, we were able to identify nine positional isomers (13-, 12-, 11-, 10-, 9-, 8-, 7-, 6-, and 5-PAHSA) (Fig. 1C) according to their MS/MS/MS spectra (Fig. 1D). Given the practical impossibility of separating the positional isomers of 12- and 13-PAHSA in human samples (7) (Supplementary Fig. 1), the 12- and 13-PAHSA levels are reported together.

PAHSA Levels Were Not Altered by n-3 PUFA Supplementation in Patients with Diabetes and Obese Mice

First, we investigated the possible effect of n-3 PUFA supplementation on the levels of the known FAHFAs (see Introduction) in humans with diabetes, and therefore serum samples of metformin-treated patients supplemented with either corn oil capsules (Placebo) or n-3 PUFA capsules (n-3 PUFA) for 24 weeks (34) were analyzed. Serum levels of 5-, 9-, and 12/13-PAHSA isomers were not altered by n-3 PUFA supplementation (Fig. 2A). This observation was also supported by animal experiments on dietary obese mice that were fed an n-3 PUFA diet for 8 weeks (21), where no differences in serum PAHSA levels were detected (Fig. 2B).

Figure 2

PAHSA levels in humans and mice. A: Levels of PAHSA isomers in human serum samples. Black bars, Placebo; empty bars, patients supplemented with n-3 PUFA capsules. Values are expressed as the mean ± SE, n = 13–16. B: Levels of PAHSA isomers in murine serum samples. Black bars, mice on HF diet; empty bars, mice on HFF diet. Values are expressed as the mean ± SE. n = 7, representative of three independent experiments.

Figure 2

PAHSA levels in humans and mice. A: Levels of PAHSA isomers in human serum samples. Black bars, Placebo; empty bars, patients supplemented with n-3 PUFA capsules. Values are expressed as the mean ± SE, n = 13–16. B: Levels of PAHSA isomers in murine serum samples. Black bars, mice on HF diet; empty bars, mice on HFF diet. Values are expressed as the mean ± SE. n = 7, representative of three independent experiments.

Identification of Novel FAHFAs Derived From LA and DHA

Using the same MS approach as in the identification of PAHSA isomers, any combination of FAs and the branching position theoretically could be detected. Therefore, we focused on alternative combinations of FAs besides PAHSAs, and were able to identify novel members of the FAHFA family derived from LA and DHA, specifically DHAHLA, LAHDHA, and DHAHDHA in human serum (Supplementary Fig. 2, structures).

With DHAHLA, DHA esterified to a hydroxy LA, the following two positional isomers of the hydroxy FA backbone were detected: 9- and 13-HLA (also known as HODE); and therefore 9- and 13-DHAHLA. This is in agreement with the high concentrations of 9(S)- and 13(S)-HODE, enzymatic products of 15-lipoxygenase in the organism. As shown in the 13-DHAHLA fragmentation scheme (Fig. 3A), the ion 605.457 m/z gave rise to the fragment 295.228 m/z (13-HODE), which was further fragmented to characteristic ions 179.144 and 195.139 m/z (for details, see Supplementary Fig. 3).

Figure 3

Analysis of DHAHLA isomers. A: A fragmentation scheme of 13-DHAHLA with 13-HLA–specific fragments. B: MS/MS spectrum of 13-DHAHLA. C: Chromatographic profile of DHAHLA isomers (MRM 605.4 > 327.2) detected in murine serum sample (blue line) overlaid with synthetic standard of 13-DHAHLA (red line). The inserted table summarizes MS/MS/MS fragments specific to individual positional isomers of the hydroxyl group on HLA and MS/MS/MS spectra of 13-DHAHLA. Specific fragments are highlighted in magenta. HcLA, hydroxy-conjugated-LA.

Figure 3

Analysis of DHAHLA isomers. A: A fragmentation scheme of 13-DHAHLA with 13-HLA–specific fragments. B: MS/MS spectrum of 13-DHAHLA. C: Chromatographic profile of DHAHLA isomers (MRM 605.4 > 327.2) detected in murine serum sample (blue line) overlaid with synthetic standard of 13-DHAHLA (red line). The inserted table summarizes MS/MS/MS fragments specific to individual positional isomers of the hydroxyl group on HLA and MS/MS/MS spectra of 13-DHAHLA. Specific fragments are highlighted in magenta. HcLA, hydroxy-conjugated-LA.

Chromatographic separation of the murine serum sample revealed additional complexity of DHAHLA isomers when four separated DHAHLA peaks were detected (Fig. 3C). Structural characterization in the linear ion trap revealed that two major peaks were 13-DHAHLA and two minor peaks were 9-DHAHLA cis-trans isomers of double bonds in HLA acyl chains. Identity of the backbone fragmentation was confirmed using synthetic standards for 9(S)-HODE and 13(S)-HODE (Fig. 3C and Supplementary Fig. 2). Of note, only two peaks corresponding to physiologically relevant (9Z,11E,13S)-13-hydroxy-9,11-octadecadienoic acid- and (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid-derivatives, 9(S)-HODE and 13(S)-HODE, respectively, were observed in human WAT and serum samples, as well as in cultured cells (hMADS cells; data not shown); therefore, only these were considered for further analyses.

Similar to PAHSA tissue distribution (7), 13-DHAHLA was detected in murine adipose tissue depots and was upregulated after n-3 PUFA supplementation (Fig. 4A). Levels of FAHFA-specific hydrolases (13), Aig1 and Adtrp, were downregulated in murine adipose tissue and upregulated in the liver after n-3 PUFA supplementation. Interestingly, although Adtrp was exclusively associated with adipocytes, Aig1 was present also in SVC (Supplementary Fig. 4). Very low levels of 13-DHAHLA were detected in human subcutaneous fat biopsy samples after n-3 PUFA supplementation (0.38 ± 0.06 pmol/g), but no DHAHLA was detected in placebo-treated patients. Cultured 3T3-L1 and hMADS adipocytes, when supplemented with DHA and LA, were able to synthesize DHAHLA isomers (Fig. 4B). Although no DHAHLA was detected in macrophages treated similarly, macrophages were able to synthesize DHAHLA, when exposed to high concentrations of DHA and LA (Supplementary Fig. 5).

Figure 4

13-DHAHLA in adipose tissue. A: Levels of 13-DHAHLA in murine epididymal WAT (eWAT), subcutaneous WAT (scWAT), and interscapular brown adipose tissue (BAT) with and without n-3 PUFA supplementation. Black bars, mice on HF diet; empty bars, mice on HFF diet. Values are expressed as the mean ± SE, n = 4–8. *P < 0.05. B: Chromatographic profile of 13-DHAHLA in naïve 3T3-L1 adipocytes, in adipocytes supplemented with 100 μmol/L DHA and LA for 24 h, and in adipocytes supplemented with deuterium-labeled DHA and cold LA for 24 h, as indicated.

Figure 4

13-DHAHLA in adipose tissue. A: Levels of 13-DHAHLA in murine epididymal WAT (eWAT), subcutaneous WAT (scWAT), and interscapular brown adipose tissue (BAT) with and without n-3 PUFA supplementation. Black bars, mice on HF diet; empty bars, mice on HFF diet. Values are expressed as the mean ± SE, n = 4–8. *P < 0.05. B: Chromatographic profile of 13-DHAHLA in naïve 3T3-L1 adipocytes, in adipocytes supplemented with 100 μmol/L DHA and LA for 24 h, and in adipocytes supplemented with deuterium-labeled DHA and cold LA for 24 h, as indicated.

Although several positional isomers of HDHA exist (42), only one peak for DHAHDHA was detected in human serum. MS/MS/MS analysis revealed 14-HDHA–specific fragments (205.2 and 161.2 m/z) (39,42) in the corresponding peak. Importantly, oxidized phospholipids containing predominantly the 14-HDHA positional isomer were identified in human platelets (39). Besides the 14-HDHA backbone, smaller peaks of DHAHDHA were detected in murine samples. Because of very low intensities, we were unable to identify their branching positions, and thus we cannot rule out that other isomers were also present in murine samples. Overall, DHAHLA and DHAHDHA levels were elevated in the experimental groups supplemented with n-3 PUFAs (Fig. 5A and B), and a positive correlation between the omega-3 index in serum phospholipids (34) and their levels of 13-DHAHLA was found (Fig. 5C).

Figure 5

Novel FAHFA members containing DHA and LA. A: Levels of FAHFAs derived from DHA in human serum samples. Black bars, placebo; empty bars, patients supplemented with n-3 PUFA capsules. Values are expressed as mean ± SE, n = 11–16. *P < 0.05. B: Levels of FAHFAs in murine serum samples. Black bars, mice on HF diet; empty bars, mice on HFF diet. Values are expressed as mean ± SE, n = 7, representative of 3 independent experiments. *P < 0.05. C: Correlation between omega-3 index in serum phospholipids (data are replotted with the authors’ permission from Fig. 2 of the study by Veleba et al. [34]) and serum levels of 13-DHAHLA in patients. Filled circles, placebo group; empty circles, n-3 PUFA–supplemented patients.

Figure 5

Novel FAHFA members containing DHA and LA. A: Levels of FAHFAs derived from DHA in human serum samples. Black bars, placebo; empty bars, patients supplemented with n-3 PUFA capsules. Values are expressed as mean ± SE, n = 11–16. *P < 0.05. B: Levels of FAHFAs in murine serum samples. Black bars, mice on HF diet; empty bars, mice on HFF diet. Values are expressed as mean ± SE, n = 7, representative of 3 independent experiments. *P < 0.05. C: Correlation between omega-3 index in serum phospholipids (data are replotted with the authors’ permission from Fig. 2 of the study by Veleba et al. [34]) and serum levels of 13-DHAHLA in patients. Filled circles, placebo group; empty circles, n-3 PUFA–supplemented patients.

Anti-Inflammatory and Proresolving Effects of DHAHLA

Isomers of PAHSA were shown to be able to decrease macrophage activation and the release of proinflammatory cytokines after LPS stimulation (7). Also, oxidized derivatives of DHA, especially resolvins, protectins, and maresins (31,43), possess potent anti-inflammatory and proresolving activities. Therefore, RAW macrophages were stimulated with LPS, and the effects of 9-PAHSA (7) and 13-DHAHLA on macrophage activation were analyzed. Both PAHSA and DHAHLA prevented the increase in proinflammatory interleukin (IL)-6 concentrations in media and also decreased mRNA levels of IL-6, tumor necrosis factor-α (TNF-α), IL-1β, and Ptgs2 in cells (Fig. 6A and Supplementary Fig. 6). Next, the model of mitogen-stimulated human PBMCs was used to test the immunomodulatory potential of 13-DHAHLA. The activation of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme of the essential amino acid tryptophan catabolism toward kynurenine, causes tryptophan depletion, reduced the growth of microbes (37), and serves as an immune checkpoint (44). Freshly isolated PBMCs were preincubated with 1 μmol/L 13-DHAHLA for 30 min and then exposed to lectin phytohemagglutinin to activate T-helper type 1 lymphocytes and also the IDO pathway in macrophages (37). Activation of the IDO enzyme was indicated by a strong increase in the kynurenine/tryptophan ratio, which was partially prevented by 13-DHAHLA preincubation (Fig. 6B).

Figure 6

Anti-inflammatory effects of 13-DHAHLA. A: RAW 264.7 macrophages were incubated in the absence (Unstimulated) or presence of LPS (100 ng/mL) for 18 h, and the effect of 9-PAHSA (10 μmol/L) and 13-DHAHLA (10 μmol/L) on macrophage activation was tested. IL-6 protein levels in the medium, and IL-6, TNF-α, IL-1β, and Ptgs2 mRNA in cells. Values are expressed as the mean ± SE. n = 6, representative of three experiments. B: Human PBMCs were freshly isolated from a buffy coat, preincubated with 1 μmol/L 13-DHAHLA, and stimulated with phytohemagglutinin (PHA; 10 µg/mL) for 24 h. Levels of tryptophan and kynurenine were measured in the medium. Values are expressed as the mean ± SE. n = 8, representative of 3 PBMC isolations. C: BMDMs were incubated in the absence (Unstimulated) or presence of LPS (100 ng/mL) for 18 h, and the effect of 13-DHAHLA (10 μmol/L) and DHA (10 μmol/L) on macrophage activation was tested. Cells were either extracted for metabolomics—(Cit+Orn)/(Arg+Asp) macrophage activation index (relative fold)—or processed for the metabolipidomics of lipid mediators. D: Levels of DHA-derived lipid mediators 17-HDHA, protectin D1, and resolvin D1. Values are expressed as the mean ± SE. n = 6, representative of three experiments. N.D., not detected. E: RAW 264.7 macrophages were stimulated with LPS (10 ng/mL) and incubated in the presence of various concentrations of 13-DHAHLA for 18 h to explore dose-dependent inhibition of macrophage activation. The LPS-stimulated state is set as a reference point. Values are expressed as the mean ± SE. n = 6, representative of two experiments. F: BMDMs were pretreated for 15 min with 13-DHAHLA, as indicated, and were incubated with fluorescein-labeled zymosan for 30 min. Extracellular fluorescence was quenched with trypan blue and fluorescence measured using a plate reader. Values are expressed as the mean ± SE. n = 8, representative of three experiments. G: BMDMs were incubated in the absence (Unstimulated) or presence of IFN-γ (50 ng/mL) for 18 h, and the effect of 13-DHAHLA (10 μmol/L) on macrophage activation (intracellular citrulline levels) was tested. Values are expressed as the mean ± SE. n = 6. *P < 0.05.

Figure 6

Anti-inflammatory effects of 13-DHAHLA. A: RAW 264.7 macrophages were incubated in the absence (Unstimulated) or presence of LPS (100 ng/mL) for 18 h, and the effect of 9-PAHSA (10 μmol/L) and 13-DHAHLA (10 μmol/L) on macrophage activation was tested. IL-6 protein levels in the medium, and IL-6, TNF-α, IL-1β, and Ptgs2 mRNA in cells. Values are expressed as the mean ± SE. n = 6, representative of three experiments. B: Human PBMCs were freshly isolated from a buffy coat, preincubated with 1 μmol/L 13-DHAHLA, and stimulated with phytohemagglutinin (PHA; 10 µg/mL) for 24 h. Levels of tryptophan and kynurenine were measured in the medium. Values are expressed as the mean ± SE. n = 8, representative of 3 PBMC isolations. C: BMDMs were incubated in the absence (Unstimulated) or presence of LPS (100 ng/mL) for 18 h, and the effect of 13-DHAHLA (10 μmol/L) and DHA (10 μmol/L) on macrophage activation was tested. Cells were either extracted for metabolomics—(Cit+Orn)/(Arg+Asp) macrophage activation index (relative fold)—or processed for the metabolipidomics of lipid mediators. D: Levels of DHA-derived lipid mediators 17-HDHA, protectin D1, and resolvin D1. Values are expressed as the mean ± SE. n = 6, representative of three experiments. N.D., not detected. E: RAW 264.7 macrophages were stimulated with LPS (10 ng/mL) and incubated in the presence of various concentrations of 13-DHAHLA for 18 h to explore dose-dependent inhibition of macrophage activation. The LPS-stimulated state is set as a reference point. Values are expressed as the mean ± SE. n = 6, representative of two experiments. F: BMDMs were pretreated for 15 min with 13-DHAHLA, as indicated, and were incubated with fluorescein-labeled zymosan for 30 min. Extracellular fluorescence was quenched with trypan blue and fluorescence measured using a plate reader. Values are expressed as the mean ± SE. n = 8, representative of three experiments. G: BMDMs were incubated in the absence (Unstimulated) or presence of IFN-γ (50 ng/mL) for 18 h, and the effect of 13-DHAHLA (10 μmol/L) on macrophage activation (intracellular citrulline levels) was tested. Values are expressed as the mean ± SE. n = 6. *P < 0.05.

FAHFA might also serve as a pool of FA, and the anti-inflammatory properties of 13-DHAHLA could be partially mediated by DHA released via DHAHLA hydrolysis. To test this hypothesis, murine BMDMs were stimulated with LPS, and the effects of 10 μmol/L 13-DHAHLA and 10 μmol/L DHA on macrophage activation were analyzed using metabolipidomics (26,36). The ratio of the intracellular concentrations of (citrulline plus ornithine)/(arginine plus aspartate), which summarizes the intermediates of the related metabolic pathways of nitric oxide and reactive oxygen species production, was the most sensitive early marker of macrophage activation (36), and the proinflammatory effect of LPS was significantly reduced by both 13-DHAHLA and DHA (Fig. 6C). However, a detailed analysis of proresolving lipid mediators revealed that the macrophages converted DHA into 17-HDHA, and further to protectin D1 or resolvin D1, with anti-inflammatory and proresolving properties; whereas, with 13-DHAHLA, only a small amount of DHA was converted to 17-HDHA and protectin D1, and no resolvin D1 was detected (Fig. 6D). To further explore the ability of 13-DHAHLA to reduce macrophage activation by LPS, a dose-dependent experiment was performed. The lowest effective concentration of 13-DHAHLA was 10 nmol/L (Fig. 6E). Because the resolution of inflammation also involves phagocytosis, 13-DHAHLA was tested for its ability to enhance phagocytic activity toward zymosan particles. BMDMs pretreated with 13-DHAHLA increased the phagocytosis of fluorescein-labeled zymosan in a dose-dependent manner with a peak value at 100 nmol/L (Fig. 6F). Finally, an effect of 13-DHAHLA on the stimulation by proinflammatory cytokine IFN-γ (50 ng/mL) was tested. 13-DHAHLA prevented macrophage activation measured as levels of citrulline, the byproduct of nitric oxide synthase (Fig. 6G). These results document that 13-DHAHLA itself exerts anti-inflammatory and proresolving properties.

We identified novel members of FAHFA lipid class derived from DHA and LA (i.e., DHAHLA and DHAHDHA) with anti-inflammatory properties in the serum and WAT of both mice and patients with diabetes supplemented with n-3 PUFAs. In addition to DHAHLA and DHAHDHA isomers, which were elevated after the supplementation, other members of the FAHFA family not described before (7,16) were also found using our novel LC-MS/MS/MS screening for all theoretical FA combinations (e.g., LAHDHA, DHAHSA, OAHDHA). However, these lipids were not altered by n-3 PUFAs supplementation in humans, and therefore we did not explore them in detail.

The levels of DHAHLA and DHAHDHA were comparable to the concentrations of DHA-derived docosanoids (protectins, resolvins) in human serum. Hydroperoxy/hydroxy DHA serves as an intermediate in enzymatic reactions, which produce dihydroxylated and trihydroxylated docosanoids (43). FAHFAs are also produced by enzymatic reaction (7), probably using monohydroxylated DHA or HLA as a substrate, and therefore the concentrations of DHAHLA and DHAHDHA were within the expected range. The levels of 13-DHAHLA also correlated with the omega-3 index in the serum of patients. Compared with 5-PAHSA, 13-DHAHLA concentrations were ∼100-fold lower in serum. Similar to PAHSA, the novel lipids were also synthesized in WAT and adipocytes, expanding the family of adipose-derived lipid mediators. In addition to 13- and 9-DHAHLA derived from 13(S)- and 9(S)-HODE, two related compounds were identified as DHAHLA cis/trans-isomers where the backbone was conjugated LA (e.g., rumenic acid). Because these compounds were detected only in mice fed a custom-made diet, and were found in neither human samples nor in cell cultures, casein in the diet was most likely the source of conjugated LA (21). Other derivatives of HDHA were detected, but only 14-DHAHDHA was upregulated in humans in response to n-3 PUFA supplementation. The exceptional role of DHA hydroxylated at position 14 was also observed in oxidized phospholipids (39). Due to the demanding organic synthesis of the 14-HDHA backbone, HDHA derivatives were not explored further.

The chemical stability of DHAHLA is similar to that of resolvins and protectins (45). The pure compound had to be stored in amber glass vials under an argon atmosphere at −80°C. Although the ester bond between DHA and HLA can be hydrolyzed, yielding 13-HODE and DHA, we did not observe any extensive DHAHLA decomposition during short-term cell preincubations. However, sample acquisition and storage were critical for the successful analysis of DHAHLA. Murine samples had to be processed immediately or stored in liquid nitrogen. The storage of human serum or WAT samples at −80°C for periods longer than ∼6 months resulted in FAHFA degradation. Therefore, low levels of 13-DHAHLA were detected in WAT from patients supplemented with n-3 PUFAs, whereas no DHAHLA was detected in the placebo group, as these samples were collected 1–3 years before the analysis (data not shown).

Reflecting the anti-inflammatory effects of PAHSAs, the already known members of the FAHFA lipid class, on adipose tissue macrophages from obese mice (7) and the beneficial effects of DHA and its metabolites on adipose tissue inflammation (2426), we hypothesized that DHAHLA could also have immunomodulatory properties. First, we tested 13-DHAHLA for the ability to alter macrophage activation triggered by LPS. Our results demonstrated that the activation of macrophages by LPS and IFN-γ, measured as a production of IL-6, expression of IL-6, TNF-α and IL-1β, and as an alteration in metabolic pathways (metabolomic marker) (36) was alleviated by both 9-PAHSA and 13-DHAHLA. When compared with stimulation with pure DHA, 13-DHAHLA exerted anti-inflammatory properties, whereas only a limited production of proresolving mediators derived from DHA was detected, probably because of nonspecific hydrolysis in the media. This suggests that 13-DHAHLA does not serve as a temporary storage location of DHA for the production of docosanoids. Also, 13-DHAHLA was able to partially prevent IDO activation in PBMCs stimulated with lectin, to limit immunosuppression caused by tryptophan depletion (37,44), and stimulated the clearance of zymosan particles by BMDMs in a higher nanomolar range. Our results document that 13-DHAHLA has the ability to affect immune cells, alleviate macrophage activation, and stimulate the proresolving processes. It is conceivable that DHAHLA may contribute to the anti-inflammatory and proresolving effects attributed to the DHA in human and murine WAT (lower density of crown-like structures, lower local concentration of proinflammatory cytokines), as was already documented for the other DHA metabolites, namely, docosanoids and related endocannabinoids (2430,32). The expression of FAHFA hydrolases in WAT, liver, isolated adipocytes, and SVCs revealed that FAHFA degradation is probably regulated on both local and systemic levels.

Importantly, our results also documented that serum PAHSA levels, either in mice or in humans, were not affected by n-3 PUFA supplementation, and, therefore, that PAHSA was not involved in the anti-inflammatory effects of DHA.

The detection of DHAHLA and DHAHDHA in both mice and humans, and the emerging concept that the immune and metabolic systems are interconnected (17,26), strongly suggest the involvement of the novel lipids in the broad beneficial effects of n-3 PUFAs on health. Thus, in humans, in addition to their anti-inflammatory effects in WAT (see above), n-3 PUFAs attenuate systemic inflammatory processes (46), help to prevent cardiovascular disease (47), ameliorate nonalcoholic fatty liver disease (48), lower hypertriacylglycerolemia (49), and increase circulating adiponectin levels (50). Although n-3 PUFAs could not reverse type 2 diabetes and their impact on insulin sensitivity is controversial (23,28,34), n-3 PUFAs exert anti-inflammatory effects in the WAT of human subjects with insulin resistance (28). As demonstrated by our clinical trial (34), which also served as a source of the samples for this study, n-3 PUFAs could improve postprandial lipid metabolism in overweight/obese patients with type 2 diabetes, even in the face of a combined pharmacotherapy.

The demanding organic synthesis of pure compounds in higher amounts will be needed to further explore the proresolving properties of DHAHLA and related molecules in vivo, their ability to interact with G-protein–coupled receptors, and the potential involvement of these novel lipids in various biological effects of DHA. Future studies focused on these metabolites of DHA may provide important insights into the anti-inflammatory properties of n-3 PUFAs and provide new tools for the treatment of diseases linked with inflammation.

Acknowledgments. The authors thank Professor Charles N. Serhan (Harvard Medical School, Boston, MA) for the PD1 standard. hMADS were provided by the laboratory of Christian Dani (University of Nice Sophia Antipolis) and Amri Ez-Zoubir (University of Nice Sophia Antipolis) under the conditions of a material transfer agreement (Centre National de la Recherche Scientifique).

Funding. This work was supported by the Czech Science Foundation (grant 14-3684G) and the Ministry of Education, Youth and Sports of the Czech Republic (grant LH14040).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. O.K. conceived of, designed, performed, and interpreted the LC-MS, animal, and cell culture studies; performed the biological studies; and wrote the manuscript. M.B. and M.R. performed the LC-MS and biological studies. B.S. and E.K. designed the organic synthesis of FAHFA standards. M.P. and P.B. performed the synthesis of FAHFA standards. P.J., J.V., and J.K. Jr. collected human samples. T.P. contributed reagents and designed the human study. J.K. contributed reagents, designed the human study, and contributed to the data discussion and the writing of the manuscript. J.K. and O.K. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

1.
Masoodi
M
,
Kuda
O
,
Rossmeisl
M
,
Flachs
P
,
Kopecky
J
.
Lipid signaling in adipose tissue: connecting inflammation and metabolism
.
Biochim Biophys Acta
2015
;
1851
:
503
518
2.
Flachs
P
,
Rossmeisl
M
,
Kuda
O
,
Kopecky
J
.
Stimulation of mitochondrial oxidative capacity in white fat independent of UCP1: a key to lean phenotype
.
Biochim Biophys Acta
2013
;
1831
:
986
1003
3.
Iyer
A
,
Fairlie
DP
,
Prins
JB
,
Hammock
BD
,
Brown
L
.
Inflammatory lipid mediators in adipocyte function and obesity
.
Nat Rev Endocrinol
2010
;
6
:
71
82
[PubMed]
4.
Wahli
W
,
Michalik
L
.
PPARs at the crossroads of lipid signaling and inflammation
.
Trends Endocrinol Metab
2012
;
23
:
351
363
[PubMed]
5.
Trujillo
ME
,
Scherer
PE
.
Adipose tissue-derived factors: impact on health and disease
.
Endocr Rev
2006
;
27
:
762
778
[PubMed]
6.
Sell
H
,
Habich
C
,
Eckel
J
.
Adaptive immunity in obesity and insulin resistance
.
Nat Rev Endocrinol
2012
;
8
:
709
716
[PubMed]
7.
Yore
MM
,
Syed
I
,
Moraes-Vieira
PM
, et al
.
Discovery of a class of endogenous mammalian lipids with anti-diabetic and anti-inflammatory effects
.
Cell
2014
;
159
:
318
332
[PubMed]
8.
Cao
H
,
Gerhold
K
,
Mayers
JR
,
Wiest
MM
,
Watkins
SM
,
Hotamisligil
GS
.
Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism
.
Cell
2008
;
134
:
933
944
[PubMed]
9.
Czech
MP
,
Tencerova
M
,
Pedersen
DJ
,
Aouadi
M
.
Insulin signalling mechanisms for triacylglycerol storage
.
Diabetologia
2013
;
56
:
949
964
[PubMed]
10.
Herman
MA
,
Peroni
OD
,
Villoria
J
, et al
.
A novel ChREBP isoform in adipose tissue regulates systemic glucose metabolism
.
Nature
2012
;
484
:
333
338
[PubMed]
11.
Pedersen
DJ
,
Guilherme
A
,
Danai
LV
, et al
.
A major role of insulin in promoting obesity-associated adipose tissue inflammation
.
Mol Metab
2015
;
4
:
507
518
[PubMed]
12.
Nadler
ST
,
Stoehr
JP
,
Schueler
KL
,
Tanimoto
G
,
Yandell
BS
,
Attie
AD
.
The expression of adipogenic genes is decreased in obesity and diabetes mellitus
.
Proc Natl Acad Sci USA
2000
;
97
:
11371
11376
[PubMed]
13.
Parsons
WH
,
Kolar
MJ
,
Kamat
SS
, et al
.
AIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs
.
Nat Chem Biol
2016
;
12
:
367
372
[PubMed]
14.
Zerkowski
JA
.
Estolides: from structure and function to structured and functionalized
.
Lipid Technol
2008
;
20
:
253
256
15.
McLean
S
,
Davies
NW
,
Nichols
DS
,
Mcleod
BJ
.
Triacylglycerol estolides, a new class of mammalian lipids, in the paracloacal gland of the brushtail possum (Trichosurus vulpecula)
.
Lipids
2015
;
50
:
591
604
[PubMed]
16.
Ma
Y
,
Kind
T
,
Vaniya
A
,
Gennity
I
,
Fahrmann
JF
,
Fiehn
O
.
An in silico MS/MS library for automatic annotation of novel FAHFA lipids
.
J Cheminform
2015
;
7
:
53
[PubMed]
17.
Fitzgibbons
TP
,
Czech
MP
.
Emerging evidence for beneficial macrophage functions in atherosclerosis and obesity-induced insulin resistance
.
J Mol Med (Berl)
2016
;
94
:
267
275
18.
Hotamisligil
GS
,
Shargill
NS
,
Spiegelman
BM
.
Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance
.
Science
1993
;
259
:
87
91
[PubMed]
19.
Shoelson
SE
,
Lee
J
,
Yuan
M
.
Inflammation and the IKK beta/I kappa B/NF-kappa B axis in obesity- and diet-induced insulin resistance
.
Int J Obes Relat Metab Disord
2003
;
27
(
Suppl 3
):
S49
S52
[PubMed]
20.
Weisberg
SP
,
McCann
D
,
Desai
M
,
Rosenbaum
M
,
Leibel
RL
,
Ferrante
AW
 Jr
.
Obesity is associated with macrophage accumulation in adipose tissue
.
J Clin Invest
2003
;
112
:
1796
1808
[PubMed]
21.
Kuda
O
,
Jelenik
T
,
Jilkova
Z
, et al
.
n-3 fatty acids and rosiglitazone improve insulin sensitivity through additive stimulatory effects on muscle glycogen synthesis in mice fed a high-fat diet
.
Diabetologia
2009
;
52
:
941
951
[PubMed]
22.
Calder
PC
.
Marine omega-3 fatty acids and inflammatory processes: effects, mechanisms and clinical relevance
.
Biochim Biophys Acta
2015
;
1851
:
469
484
23.
Flachs
P
,
Rossmeisl
M
,
Kopecky
J
.
The effect of n-3 fatty acids on glucose homeostasis and insulin sensitivity
.
Physiol Res
2014
;
63
(
Suppl. 1
):
S93
S118
[PubMed]
24.
Hellmann
J
,
Tang
Y
,
Kosuri
M
,
Bhatnagar
A
,
Spite
M
.
Resolvin D1 decreases adipose tissue macrophage accumulation and improves insulin sensitivity in obese-diabetic mice
.
FASEB J
2011
;
25
:
2399
2407
[PubMed]
25.
Titos
E
,
Rius
B
,
González-Périz
A
, et al
.
Resolvin D1 and its precursor docosahexaenoic acid promote resolution of adipose tissue inflammation by eliciting macrophage polarization toward an M2-like phenotype
.
J Immunol
2011
;
187
:
5408
5418
[PubMed]
26.
Kuda
O
,
Rombaldova
M
,
Janovska
P
,
Flachs
P
,
Kopecky
J
.
Cell type-specific modulation of lipid mediator’s formation in murine adipose tissue by omega-3 fatty acids
.
Biochem Biophys Res Commun
2016
;
469
:
731
736
[PubMed]
27.
Rossmeisl
M
,
Jilkova
ZM
,
Kuda
O
, et al
.
Metabolic effects of n-3 PUFA as phospholipids are superior to triglycerides in mice fed a high-fat diet: possible role of endocannabinoids
.
PLoS One
2012
;
7
:
e38834
[PubMed]
28.
Spencer
M
,
Finlin
BS
,
Unal
R
, et al
.
Omega-3 fatty acids reduce adipose tissue macrophages in human subjects with insulin resistance
.
Diabetes
2013
;
62
:
1709
1717
[PubMed]
29.
Oh
DY
,
Talukdar
S
,
Bae
EJ
, et al
.
GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects
.
Cell
2010
;
142
:
687
698
[PubMed]
30.
Oh
da Y
,
Walenta
E
,
Akiyama
TE
, et al
.
A Gpr120-selective agonist improves insulin resistance and chronic inflammation in obese mice
.
Nat Med
2014
;
20
:
942
947
[PubMed]
31.
Serhan
CN
,
Chiang
N
,
Dalli
J
.
The resolution code of acute inflammation: novel pro-resolving lipid mediators in resolution
.
Semin Immunol
2015
;
27
:
200
215
[PubMed]
32.
Clària
J
,
Nguyen
BT
,
Madenci
AL
,
Ozaki
CK
,
Serhan
CN
.
Diversity of lipid mediators in human adipose tissue depots
.
Am J Physiol Cell Physiol
2013
;
304
:
C1141
C1149
[PubMed]
33.
White
PJ
,
St-Pierre
P
,
Charbonneau
A
, et al
.
Protectin DX alleviates insulin resistance by activating a myokine-liver glucoregulatory axis
.
Nat Med
2014
;
20
:
664
669
[PubMed]
34.
Veleba
J
,
Kopecky
J
 Jr
,
Janovska
P
, et al
.
Combined intervention with pioglitazone and n-3 fatty acids in metformin-treated type 2 diabetic patients: improvement of lipid metabolism
.
Nutr Metab (Lond)
2015
;
12
:
52
[PubMed]
35.
Kuda
O
,
Jenkins
CM
,
Skinner
JR
, et al
.
CD36 protein is involved in store-operated calcium flux, phospholipase A2 activation, and production of prostaglandin E2
.
J Biol Chem
2011
;
286
:
17785
17795
[PubMed]
36.
Suh
JH
,
Kim
RY
,
Lee
DS
.
A new metabolomic assay to examine inflammation and redox pathways following LPS challenge
.
J Inflamm (Lond)
2012
;
9
:
37
[PubMed]
37.
Becker
K
,
Schroecksnadel
S
,
Gostner
J
, et al
.
Comparison of in vitro tests for antioxidant and immunomodulatory capacities of compounds
.
Phytomedicine
2014
;
21
:
164
171
[PubMed]
38.
Neises
B
,
Steglich
W
.
Simple method for the esterification of carboxylic acids
.
Angew Chem Int Ed Engl
1978
;
17
:
522
524
39.
Morgan
AH
,
Hammond
VJ
,
Morgan
L
, et al
.
Quantitative assays for esterified oxylipins generated by immune cells
.
Nat Protoc
2010
;
5
:
1919
1931
[PubMed]
40.
Krishnamoorthy
S
,
Recchiuti
A
,
Chiang
N
, et al
.
Resolvin D1 binds human phagocytes with evidence for proresolving receptors
.
Proc Natl Acad Sci USA
2010
;
107
:
1660
1665
[PubMed]
41.
Colas
RA
,
Shinohara
M
,
Dalli
J
,
Chiang
N
,
Serhan
CN
.
Identification and signature profiles for pro-resolving and inflammatory lipid mediators in human tissue
.
Am J Physiol Cell Physiol
2014
;
307
:
C39
C54
[PubMed]
42.
Derogis
PB
,
Freitas
FP
,
Marques
AS
, et al
.
The development of a specific and sensitive LC-MS-based method for the detection and quantification of hydroperoxy- and hydroxydocosahexaenoic acids as a tool for lipidomic analysis
.
PLoS One
2013
;
8
:
e77561
[PubMed]
43.
Serhan
CN
,
Petasis
NA
.
Resolvins and protectins in inflammation resolution
.
Chem Rev
2011
;
111
:
5922
5943
[PubMed]
44.
Soliman
H
,
Mediavilla-Varela
M
,
Antonia
S
.
Indoleamine 2,3-dioxygenase: is it an immune suppressor?
Cancer J
2010
;
16
:
354
359
[PubMed]
45.
Fitzgerald J, Colas R, Shinohara M, Dalli J, Serhan CN. Lipid Mediator Metabololipidomics LC‐MS-MS Spectra Book 2014. Available from https://research.bwhanesthesia.org/site_assets/51520d191eea6679ce000001/serhan-lab/spectra-book-cb460aacacb011b96d8215b8091c3b8e.pdf [Internet], 2014. Boston, MA, Center for Experimental Therapeutics and Reperfusion Injury, Harvard Institute of Medicine, Brigham and Women’s Hospital and Harvard Medical School. Accessed 1 June 2014
46.
Hung
AM
,
Booker
C
,
Ellis
CD
, et al
.
Omega-3 fatty acids inhibit the up-regulation of endothelial chemokines in maintenance hemodialysis patients
.
Nephrol Dial Transplant
2015
;
30
:
266
274
[PubMed]
47.
Mozaffarian
D
,
Lemaitre
RN
,
King
IB
, et al
.
Plasma phospholipid long-chain ω-3 fatty acids and total and cause-specific mortality in older adults: a cohort study
.
Ann Intern Med
2013
;
158
:
515
525
[PubMed]
48.
Scorletti
E
,
Bhatia
L
,
McCormick
KG
, et al.;
WELCOME Study
.
Effects of purified eicosapentaenoic and docosahexaenoic acids in nonalcoholic fatty liver disease: results from the Welcome* study
.
Hepatology
2014
;
60
:
1211
1221
[PubMed]
49.
Kris-Etherton
PM
,
Harris
WS
,
Appel
LJ
;
AHA Nutrition Committee. American Heart Association
.
Omega-3 fatty acids and cardiovascular disease: new recommendations from the American Heart Association
.
Arterioscler Thromb Vasc Biol
2003
;
23
:
151
152
[PubMed]
50.
Wu
JH
,
Cahill
LE
,
Mozaffarian
D
.
Effect of fish oil on circulating adiponectin: a systematic review and meta-analysis of randomized controlled trials
.
J Clin Endocrinol Metab
2013
;
98
:
2451
2459
[PubMed]
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at http://diabetesjournals.org/site/license.

Supplementary data