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The prolonged, postweaning fast of northern elephant seal ( Mirounga angustirostris ) pups is characterized by a reliance on lipid metabolism and reversible, fasting-induced insulin resistance, providing a unique model to examine the effects of insulin on lipid metabolism. We have previously shown that acute insulin infusion induced a shift in fatty acid metabolism dependent on fasting duration. This study complements the previous study by examining the effects of fasting duration and insulin infusion on circulating levels of oxylipins, bioactive metabolites derived from the oxygenation of polyunsaturated fatty acids. Northern elephant seal pups were studied at two postweaning periods ( n = 5/period): early fasting (1-2 wk postweaning; 127 ± 1 kg) and late fasting (6-7 wk postweaning; 93 ± 4 kg). Different cohorts of pups were weighed, sedated, and infused with 65 mU/kg of insulin. Plasma was collected prior to infusion (T0) and at 10, 30, 60, and 120 min postinfusion. A profile of ∼80 oxylipins was analyzed by UPLC-ESI-MS/MS. Nine oxylipins changed between early and late fasting and eight were altered in response to insulin infusion. Fasting decreased prostaglandin F2α (PGF2α) and increased 14,15-dihydroxyicosatrienoic acid (14,15-DiHETrE), 20-hydroxyeicosatetraenoic acid (20-HETE), and 4-hydroxy-docosahexaenoic acid (4-HDoHE) ( P < 0.03) in T0 samples, whereas insulin infusion resulted in an inverse change in area-under-the-curve (AUC) levels in these same metabolites ( P < 0.05). In addition, 12-12-hydroperoxyeicosatetraenoic acid (HpETE) and 12-HETE decreased with fasting and insulin infusion, respectively ( P < 0.04). The oxylipins altered during fasting and in response to insulin infusion may contribute to the manifestation of insulin resistance and participate in the metabolic regulation of associated cellular processes.

Oxylipins are metabolites of polyunsaturated fatty acids that mediate cardiovascular health by attenuation of inflammation, vascular tone, hemostasis, and thrombosis. Very low-density lipoproteins (VLDL) contain oxylipins, but it is unknown whether the liver regulates their concentrations. In this study, we used a perfused liver model to observe the effect of inflammatory lipopolysaccharide (LPS) challenge and soluble epoxide hydrolase inhibition (sEHi) on VLDL oxylipins. A compartmental model of deuterium-labeled linoleic acid and palmitic acid incorporation into VLDL was also developed to assess the dependence of VLDL oxylipins on fatty acid incorporation rates. LPS decreased the total fatty acid VLDL content by 30% [6%,47%], and decreased final concentration of several oxylipins by a similar amount (13-HOTrE, 35% [4%,55%], -1.3 nM; 9(10)-EpODE, 29% [3%,49%], -2.0 nM; 15(16)-EpODE, 29% [2%,49%], -1.6 nM; AA-derived diols, 32% [5%,52%], -2.4 nM; 19(20)-DiHDPA, 31% [7%,50%], -1.0 nM). However, the EPA-derived epoxide, 17(18)-EpETE, was decreased by 75% [49%,88%], (-0.52 nM) with LPS, double the suppression of other oxylipins. sEHi increased final concentration of DHA epoxide, 16(17)-EpDPE, by 99% [35%,193%], (2.0 nM). Final VLDL-oxylipin concentrations with LPS treatment were not correlated with linoleic acid kinetics, suggesting they were independently regulated under inflammatory conditions. We conclude that the liver regulates oxylipin incorporation into VLDL, and the oxylipin content is altered by LPS challenge and by inhibition of the epoxide hydrolase pathway. This provides evidence for delivery of systemic oxylipin signals by VLDL transport., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Oxylipins are potent lipid mediators involved in a variety of physiological processes. Their profiling has the potential to provide a wealth of information regarding human health and disease and is a promising technology for translation into clinical applications. However, results generated by independent groups are rarely comparable, which increases the need for the implementation of internationally agreed upon protocols. We performed an interlaboratory comparison for the MS-based quantitative analysis of total oxylipins. Five independent laboratories assessed the technical variability and comparability of 133 oxylipins using a harmonized and standardized protocol, common biological materials (i.e., seven quality control plasmas), standard calibration series, and analytical methods. The quantitative analysis was based on a standard calibration series with isotopically labeled internal standards. Using the standardized protocol, the technical variance was within ±15% for 73% of oxylipins; however, most epoxy fatty acids were identified as critical analytes due to high variabilities in concentrations. The comparability of concentrations determined by the laboratories was examined using consensus value estimates and unsupervised/supervised multivariate analysis (i.e., principal component analysis and partial least squares discriminant analysis). Interlaboratory variability was limited and did not interfere with our ability to distinguish the different plasmas. Moreover, all laboratories were able to identify similar differences between plasmas. In summary, we show that by using a standardized protocol for sample preparation, low technical variability can be achieved. Harmonization of all oxylipin extraction and analysis steps led to reliable, reproducible, and comparable oxylipin concentrations in independent laboratories, allowing the generation of biologically meaningful oxylipin patterns., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 Mainka et al.)

Very little is known about how metabolic health status, insulin resistance or metabolic challenges modulate the endocannabinoid (eCB) or polyunsaturated fatty acid (PUFA)-derived oxylipin (OxL) lipid classes. To address these questions, plasma eCB and OxL concentrations were determined at rest, 10 and 20 min during an acute exercise bout (30 min total, ~45% of preintervention V̇O 2peak , ~63 W), and following 20 min recovery in overnight-fasted sedentary, obese, insulin-resistant women under controlled diet conditions. We hypothesized that increased fitness and insulin sensitivity following a ~14-week training and weight loss intervention would lead to significant changes in lipid signatures using an identical acute exercise protocol to preintervention. In the first 10 min of exercise, concentrations of a suite of OxL diols and hydroxyeicosatetraenoic acid (HETE) metabolites dropped significantly. There was no increase in 12,13-DiHOME, previously reported to increase with exercise and proposed to activate muscle fatty acid uptake and tissue metabolism. Following weight loss intervention, exercise-associated reductions were more pronounced for several linoleate and alpha-linolenate metabolites including DiHOMEs, DiHODEs, KODEs, and EpODEs, and fasting concentrations of 9,10-DiHODE, 12,13-DiHODE, and 9,10-DiHOME were reduced. These findings suggest that improved metabolic health modifies soluble epoxide hydrolase, cytochrome P450 epoxygenase (CYP), and lipoxygenase (LOX) systems. Acute exercise led to reductions for most eCB metabolites, with no evidence for concentration increases even at recovery. It is proposed that during submaximal aerobic exercise, nonoxidative fates of long-chain saturated, monounsaturated, and PUFAs are attenuated in tissues that are important contributors to the blood OxL and eCB pools., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

α-Linolenic acid (ALA) is an essential fatty acid and the precursor for long-chain n-3 PUFA. However, biosynthesis of n-3 PUFA is limited in a Western diet likely due to an overabundance of n-6 PUFA. We hypothesized that dietary reduction of n-6/n-3 PUFA ratio is sufficient to promote the biosynthesis of long-chain n-3 PUFA, leading to an attenuation of high fat (HF) diet-induced obesity and inflammation. C57BL/6 J mice were fed a HF diet from ALA-enriched butter (n3Bu, n-6/n-3=1) in comparison with isocaloric HF diets from either conventional butter lacking both ALA and LA (Bu, n-6/n-3=6), or margarine containing a similar amount of ALA and abundant LA (Ma, n-6/n-3=6). Targeted lipidomic analyses revealed that n3Bu feeding promoted the bioconversion of long-chain n-3 PUFA and their oxygenated metabolites (oxylipins) derived from ALA and EPA. The n3Bu supplementation attenuated hepatic TG accumulation and adipose tissue inflammation, resulting in improved insulin sensitivity. Decreased inflammation by n3Bu feeding was attributed to the suppression of NF-κB activation and M1 macrophage polarization. Collectively, our work suggests that dietary reduction of the n-6/n-3 PUFA ratio, as well as total n-3 PUFA consumed, is a crucial determinant that facilitates n-3 PUFA biosynthesis and subsequent lipidomic modifications, thereby conferring metabolic benefits against obesity-induced inflammation and insulin resistance., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In the brain, approximately 90% of oxylipins are esterified to lipids. However, the significance of this esterification process is not known. In the present study, we (1) validated an aminopropyl solid phase extraction (SPE) method for separating esterified lipids using 100 and 500 mg columns and (2) applied the method to quantify the distribution of esterified oxylipins within phospholipids (PL) and neutral lipids (NL) (i.e. triacylglycerol and cholesteryl ester) in rats subjected to head-focused microwave fixation (controls) or CO 2 -induced hypercapnia/ischemia. We hypothesized that oxylipin esterification into these lipid pools will be altered following CO 2 -induced hypercapnia/ischemia. Lipids were extracted from control (n = 8) and CO 2 -asphyxiated (n = 8) rat brains and separated on aminopropyl cartridges to yield PL and NL. The separated lipid fractions were hydrolyzed, purified with hydrophobic-lipophilic-balanced SPE columns, and analyzed with ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry. Method validation showed that the 500 mg (vs 100 mg) aminopropyl columns yielded acceptable separation and recovery of esterified fatty acid epoxides but not other oxylipins. Two epoxides of arachidonic acid (ARA) were significantly increased, and three epoxides of docosahexaenoic acid (DHA) were significantly decreased in brain NL of CO 2 -asphyxiated rats compared to controls subjected to head-focused microwave fixation. PL-bound fatty acid epoxides were highly variable and did not differ significantly between the groups. This study demonstrates that hypercapnia/ischemia alters the concentration of ARA and DHA epoxides within NL, reflecting an active turnover process regulating brain fatty acid epoxide concentrations., (© 2019 AOCS.)

Oxylipins, including the well-known eicosanoids, are potent lipid mediators involved in numerous physiological and pathological processes. Therefore, their quantitative profiling has gained a lot of attention during the last years notably in the active field of health biomarker discovery. Oxylipins include hundreds of structurally and stereochemically distinct lipid species which today are most commonly analyzed by (ultra) high performance liquid chromatography-mass spectrometry based ((U)HPLC-MS) methods. To maximize the utility of oxylipin profiling in clinical research, it is crucial to understand and assess the factors contributing to the analytical and biological variability of oxylipin profiles in humans. In this review, these factors and their impacts are summarized and discussed, providing a framework for recommendations expected to enhance the interlaboratory comparability and biological interpretation of oxylipin profiling in clinical research., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Elevated triglyceride-rich lipoproteins (TGRL) in circulation is a risk factor for atherosclerosis. TGRL from subjects consuming a high saturated fat test meal elicited a variable inflammatory response in TNFα-stimulated endothelial cells (EC) that correlated strongly with the polyunsaturated fatty acid (PUFA) content. This study investigates how the relative abundance of oxygenated metabolites of PUFA, oxylipins, is altered in TGRL postprandially, and how these changes promote endothelial inflammation. Human aortic EC were stimulated with TNFα and treated with TGRL, isolated from subjects' plasma at fasting and 3.5 hrs postprandial to a test meal high in saturated fat. Endothelial VCAM-1 surface expression stimulated by TNFα provided a readout for atherogenic inflammation. Concentrations of esterified and non-esterified fatty acids and oxylipins in TGRL were quantified by mass spectrometry. Dyslipidemic subjects produced TGRL that increased endothelial VCAM-1 expression by ≥35%, and exhibited impaired fasting lipogenesis activity and a shift in soluble epoxide hydrolase and lipoxygenase activity. Pro-atherogenic TGRL were enriched in eicosapentaenoic acid metabolites and depleted in esterified C18-PUFA-derived diols. Abundance of these metabolites was strongly predictive of VCAM-1 expression. We conclude the altered metabolism in dyslipidemic subjects produces TGRL with a unique oxylipin signature that promotes a pro-atherogenic endothelial phenotype.

Metabolic syndrome (MetSyn) is characterized by chronic inflammation which mediates the associated high risk for cardiovascular and other diseases. Oxylipins are a superclass of lipid mediators with potent bioactivities in inflammation, vascular biology, and more. While their role as locally produced agents is appreciated, most oxylipins in plasma are found in lipoproteins suggesting defective regulation of inflammation could be mediated by the elevated VLDL and low HDL levels characteristic of MetSyn. Our objective was to compare the oxylipin composition of VLDL, LDL, and HDL in 14 optimally healthy individuals and 31 MetSyn patients, and then to determine the effects of treating MetSyn subjects with 4g/day of prescription omega-3 fatty acids (P-OM3) on lipoprotein oxylipin profiles. We compared oxylipin compositions of healthy (14) and MetSyn (31) subjects followed by randomization and assignment to 4g/d P-OM3 for 16 weeks using LC/MS/MS. Compared to healthy subjects, MetSyn is characterized by abnormalities of (1) pro-inflammatory, arachidonate-derived oxylipins from the lipoxygenase pathway in HDL; and (2) oxylipins mostly not derived from arachidonate in VLDL. P-OM3 treatment corrected many components of these abnormalities, reducing the burden of inflammatory mediators within peripherally circulating lipoproteins that could interfere with, or enhance, local effectors of inflammatory stress. We conclude that MetSyn is associated with a disruption of lipoprotein oxylipin patterns consistent with greater inflammatory stress, and the partial correction of these dysoxylipinemias by treatment with omega-3 fatty acids could explain some of their beneficial effects., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Diets containing high n-3 polyunsaturated fatty acids (PUFA) decrease inflammation and the incidence of chronic diseases including cardiovascular disease and nonalcoholic fatty liver disease while trans-fatty acids (TFA) intake increases the incidence of these conditions. Some health benefits of n-3 PUFA are mediated through the impact of their oxygenated metabolites, i.e. oxylipins. The TFA, trans-10, cis-12-conjugated linoleic acid (CLA; 18:2n-6) is associated with adipose tissue (AT) inflammation, oxidative stress, and wasting. We examined the impact of a 4-week feeding of 0, 0.5, and 1.5% docosahexaenoic acid (DHA; 22:6n-3) in the presence and absence of 0.5% CLA on AT oxylipin profiles in female C57BL/6N mice. Esterified oxylipins in AT derived from linoleic acid (LNA), alpha-linolenic acid (ALA), arachidonic acid (ARA), eicosapentaenoic acid (EPA), DHA, and putative from CLA were quantified. CLA containing diets reduced AT mass by ~62%. Compared with the control diet, the DHA diet elevated concentrations of EPA-and DHA-derived alcohols and epoxides and LNA-derived alcohols, reduced ARA-derived alcohols, ketones, epoxides, and 6-keto-prostaglandin (PG) F 1α (P < 0.05), and had mixed effects on ALA-derived alcohols. Dietary CLA lowered EPA-, DHA-, and ALA-derived epoxides, ARA-derived ketones and epoxides, and ALA-derived alcohols. While dietary CLA induced variable effects in EPA-, DHA-, and LNA-derived alcohols and LNA-derived ketones, it elevated ARA-derived alcohols and PGF 1α , PGF 2α , and F2-isoprostanes. DHA counteracted CLA-induced effects in 67, 57, 43, and 29% of total DHA-, ARA-, EPA-, and ALA-derived oxylipins, respectively. Thus, CLA elevated proinflammatory oxylipins while DHA increased anti-inflammatory oxylipins and diminished concentration of CLA-induced pro-inflammatory oxylipins in AT.

Postmenopausal women (PMW) report marginal n-3 PUFA intakes and are at risk of chronic diseases associated with the skeletal, muscular, neuroendocrine, and cardiovascular systems. How n-3 PUFA affect the amounts of endocannabinoids (ECs) and oxylipins (OLs) of metabolic and physiologic importance in PMW is not clear. Based on our recent findings that dietary n-3 PUFA alter gene targets of the EC system and lower pro-inflammatory OL we proceeded to characterize these actions in blood of PMW. Our aim was to determine levels of the ECs, OLs, and global metabolites (GM) in white PMW (75±7y), randomized in a double-masked manner, from baseline to 6mo after receiving a fish oil supplement of n-3 PUFA (720mg 20:5n3+480mg 22:6n3/d, n=20) or placebo (1.8g oleic acid/d, n=20). ECs and OLs in serum were determined by UPLC-MS/MS and GM by GC-MS and LC-MS/MS. Plasma 20:5n3 and 22:6n3 levels increased in PMW given fish oil. EC n-6 acyl-ethanolamides, arachidonate-derived diols were decreased and 20:5n3 and 22:6n3 diols, epoxides, and alcohols were increased in PMW given fish oil. GM analysis revealed that n-3 PUFA supplementation increased renal steroid hormone and proteolytic metabolite levels in PMW. Herein, we confirm that gene targets of the EC system, previously found as modifiable by n-3 PUFA result in changes in the levels of ECs and OLs in PMW. This study shows phenotypic responses (in levels) to n-3 PUFA supplementation in PMW and increases of n-3 acyl-ethanolamide and n-3-derived OL of clinical considerations in aging., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We aimed to examine the prospective association between plasma FAs, oxylipins, and risk of acute myocardial infarction (AMI) in a Singapore Chinese population. A nested case-control study with 744 incident AMI cases and 744 matched controls aged 47-83 years was conducted within the Singapore Chinese Health Study. Nineteen plasma FAs and 12 oxylipins were quantified using MS. These were grouped into 12 FA clusters and 5 oxylipin clusters using hierarchical clustering, and their associations with AMI risk were assessed. Long-chain n-3 FAs [odds ratio (OR) = 0.67 per SD increase, 95% confidence interval (CI): 0.53-0.84, P < 0.001] and stearic acid (OR = 0.65, 95% CI: 0.44-0.97, P = 0.03) were inversely associated with AMI risk, whereas arachidonic acid (AA) was positively associated with AMI risk (OR = 1.25, 95% CI: 1.03-1.52, P = 0.02) in the multivariable model with adjustment for other FAs. Further adjustment for oxylipins did not substantially change these associations. An inverse association was observed between AA-derived oxylipin, thromboxane (TX)B2, and AMI risk (OR = 0.81, 95% CI: 0.71-0.93, P = 0.003). Circulating long-chain n-3 FAs and stearic acid were associated with a lower and AA was associated with a higher AMI risk in this Chinese population. The association between the oxylipin TXB2 and AMI requires further research., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

Obesity-induced changes in lipid metabolism are mechanistically associated with the development of insulin resistance and prediabetes. Recent studies have focused on the extent to which obesity-induced insulin resistance is mediated through oxylipins, derived from enzymatic and nonenzymatic lipid peroxidation. Vitamin E and vitamin C are widely used antioxidant supplements, but conflicting data exist as to whether supplementation with vitamins E and C reduces insulin resistance. The purpose of this work is (1) to test the hypothesis that supplementation with vitamin E and vitamin C prevents the development of insulin resistance and (2) to determine the extent to which antioxidant supplementation modifies obesity-induced changes in hepatic oxylipins. Using obesity-prone Sprague-Dawley rats fed a high-fat, hypercaloric diet, we found that vitamin E and C supplementation did not block the development of insulin resistance, despite increased plasma levels of these antioxidants and decreased hepatic F2-isoprostane (F2-IsoP) concentrations. The obese phenotype was associated with increased hepatic concentrations of cytochrome P450 (CYP450)-dependent linoleic acid and α-linolenic acid-derived epoxides. Antioxidant supplementation, but not obesity, decreased levels of the lipoxygenase (LOX)-dependent, arachidonic acid-derived products lipoxin A4 (LXA4), 8,15-dihydroxtetraenoate (8,15-DiHETE), and 5,15-DiHETE. Our data demonstrate that antioxidant supplementation and obesity impact hepatic LOX- and CYP450-dependent oxylipin metabolism., (Published by Elsevier Inc.)

Introduction of vegetable ingredients in fish feed has affected the fatty acid composition in farmed Atlantic salmon (Salmo salar L). Here we investigated how changes in fish feed affected the metabolism of mice fed diets containing fillets from such farmed salmon. We demonstrate that replacement of fish oil with rapeseed oil or soybean oil in fish feed had distinct spillover effects in mice fed western diets containing the salmon. A reduced ratio of n-3/n-6 polyunsaturated fatty acids in the fish feed, reflected in the salmon, and hence also in the mice diets, led to a selectively increased abundance of arachidonic acid in the phospholipid pool in the livers of the mice. This was accompanied by increased levels of hepatic ceramides and arachidonic acid-derived pro-inflammatory mediators and a reduced abundance of oxylipins derived from eicosapentaenoic acid and docosahexaenoic acid. These changes were associated with increased whole body insulin resistance and hepatic steatosis. Our data suggest that an increased ratio between n-6 and n-3-derived oxylipins may underlie the observed marked metabolic differences between mice fed the different types of farmed salmon. These findings underpin the need for carefully considering the type of oil used for feed production in relation to salmon farming., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

An inherited deficiency of the mitochondrial protein frataxin causes Friedreich's ataxia (FRDA); the mechanism by which this deficiency triggers neuro- and cardio-degeneration is unclear. Microarrays of neural tissue of animal models of the disease showed decreases in antioxidant genes, and increases in inflammatory genes. Cyclooxygenase (COX)-derived oxylipins are important mediators of inflammation. We measured oxylipin levels using tandem mass spectrometry and ELISAs in multiple cell and animal models of FRDA. Mass spectrometry revealed increases in concentrations of prostaglandins, thromboxane B2, 15-HETE and 11-HETE in cerebellar samples of knockin knockout mice. One possible explanation for the elevated oxylipins is that frataxin deficiency results in increased COX activity. While constitutive COX1 was unchanged, inducible COX2 expression was elevated over 1.35-fold (P < 0.05) in two Friedreich's mouse models and Friedreich's lymphocytes. Consistent with higher COX2 expression, its activity was also increased by 58% over controls. COX2 expression is driven by multiple transcription factors, including activator protein 1 and cAMP response element-binding protein, both of which were elevated over 1.52-fold in cerebella. Taken together, the results support the hypothesis that reduced expression of frataxin leads to elevation of COX2-mediated oxylipin synthesis stimulated by increases in transcription factors that respond to increased reactive oxygen species. These findings support a neuroinflammatory mechanism in FRDA, which has both pathomechanistic and therapeutic implications., (© The Author 2014. Published by Oxford University Press.)

Background: Oxylipins mediate inflammation, vascular tension, and more. Their presence in lipoproteins could explain why lipoproteins mediate nearly identical activities., Methods: To determine how oxylipins are distributed in the lipoproteins of hypertriglyceridemic subjects, and whether omega-3 fatty acids alter them in a manner consistent with improved cardiovascular health, we recruited 15 dyslipidemic subjects whose levels of low density lipoprotein cholesterol (LDL-C) were at goal but who remained hypertriglyceridemic (200-499 mg/dL). They were treated them with the indicated dose of 4 g/d omega-3 acid ethyl esters (P-OM3) for 8 weeks. Measured oxylipins included mid-chain alcohols (HETEs, HEPEs and HDoHEs), ketones (KETEs), epoxides (as EpETrEs, EpETEs, and EpDPEs)., Results: At baseline, arachidonate-oxylipins (HETEs, KETEs, and EpETrEs) were most abundant in plasma with the greatest fraction of total abundance (mean |95% CI|) being carried in high density lipoproteins (HDL); 42% |31, 57| followed by very low density lipoproteins (VLDL); 27% |20, 36|; and LDL 21% |16, 28|. EPA- and DHA-derived oxylipins constituted less than 11% of total. HDL carried alcohols and epoxides but VLDL was also rich in ketones. Treatment decreased AA-derived oxylipins across lipoprotein classes (-23% |-33, -12|, p = 0.0003), and expanded EPA-(322% |241, 422|, p<0.0001) and DHA-derived oxylipins (123% |80, 176|, p<0.0001)., Conclusions: Each lipoprotein class carries a unique oxylipin complement. P-OM3 treatment alters the oxylipin content of all classes, reducing pro-inflammatory and increasing anti-inflammatory species, consistent with the improved inflammatory and vascular status associated with the treatment., Trial Registration: ClinicalTrials.gov NCT00959842.

The dose-responsiveness of plasma oxylipins to incremental dietary intake of arachidonic acid (20:4n-6; ARA) and docosahexaenoic acid (22:6n-3; DHA) was determined in piglets. Piglets randomly received one of six formulas (n = 8 per group) from days 3 to 27 postnatally. Diets contained incremental ARA or incremental DHA levels as follows (% fatty acid, ARA/DHA): (A1) 0.1/1.0; (A2) 0.53/1.0; (A3-D3) 0.69/1.0; (A4) 1.1/1.0; (D1) 0.66/0.33; and (D2) 0.67/0.62, resulting in incremental intake (g/kg BW/day) of ARA: 0.07 ± 0.01, 0.43 ± 0.03, 0.55 ± 0.03, and 0.82 ± 0.05 at constant DHA intake (0.82 ± 0.05), or incremental intake of DHA: 0.27 ± 0.02, 0.49 ± 0.03, and 0.81 ± 0.05 at constant ARA intake (0.54 ± 0.04). Plasma oxylipin concentrations and free plasma PUFA levels were determined at day 28 using LC-MS/MS. Incremental dietary ARA intake dose-dependently increased plasma ARA levels. In parallel, ARA intake dose-dependently increased ARA-derived diols 5,6- and 14,15-dihydroxyeicosatrienoic acid (DiHETrE) and linoleic acid-derived 12,13-dihydroxyoctadecenoic acid (DiHOME), downstream metabolites of cytochrome P450 expoxygenase (CYP). The ARA epoxide products from CYP are important in vascular homeostatic maintenance. Incremental DHA intake increased plasma DHA and most markedly raised the eicosapentaenoic acid (EPA) metabolite 17,18-dihydroxyeicosatetraenoic acid (DiHETE) and the DHA metabolite 19,20-dihydroxydocosapentaenoic acid (DiHDPE). In conclusion, increasing ARA and DHA intake dose-dependently influenced endogenous n-6 and n-3 oxylipin plasma concentrations in growing piglets, although the biological relevance of these findings remains to be determined.

Oxylipins, including eicosanoids, affect a broad range of biological processes, such as the initiation and resolution of inflammation. These compounds, also referred to as lipid mediators, are (non-) enzymatically generated by oxidation of polyunsaturated fatty acids such as arachidonic acid (AA). A plethora of lipid mediators exist which makes the development of generic analytical methods challenging. Here we developed a robust and sensitive targeted analysis platform for oxylipins and applied it in a biological setting, using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) operated in dynamic multiple reaction monitoring (dMRM). Besides the well-described AA metabolites, oxylipins derived from linoleic acid, dihomo-γ-linolenic acid, α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid were included. Our comprehensive platform allows the quantitative evaluation of approximately 100 oxylipins down to low nanomolar levels. Applicability of the analytical platform was demonstrated by analyzing plasma samples of patients undergoing cardiac surgery. Altered levels of some of the oxylipins, especially in certain monohydroxy fatty acids such as 12-HETE and 12-HEPE, were observed in samples collected before and 24 h after cardiac surgery. These findings indicate that this generic oxylipin profiling platform can be applied broadly to study these highly bioactive compounds in relation to human disease.

A subject's baseline FA composition may influence the ability of dietary highly unsaturated omega-3 FAs (n3-HUFA) to change circulating profiles of esterified FAs and their oxygenated metabolites. This study evaluates the influence of basal n3-HUFA and n3-oxylipin status on the magnitude of response to n3-HUFA consumption. Blood was collected from fasting subjects (n = 30) before and after treatment (4 weeks; 11 ± 2 mg/kg/day n3-HUFA ethyl esters). Esterified FAs were quantified in erythrocytes, platelets, and plasma by GC-MS. Esterified oxylipins were quantified in plasma by LC-MS/MS. Treatment with n3-HUFAs increased n3-HUFAs and decreased n6-HUFAs in all reservoirs and increased plasma n3-oxylipins without significantly changing n6-oxylipin concentrations. As subject basal n3-HUFAs increased, treatment-associated changes decreased, and this behavior was reflected in the percentage of 20:5n3 + 22:6n3 in red blood cell membrane FAs (i.e., the omega-3 index). To maintain an omega-3 index of 8% and thus reduce cardiovascular disease risk, our analyses suggest a maintenance dose of 7 mg/kg/day n3-HUFA ethyl esters for a 70-kg individual. These results suggest that the basal n3 index may have clinical utility to establish efficacious therapeutic experimental feeding regimens and to evaluate the USDA Dietary Guidelines recommendations for n3-HUFA consumption.

Type 2 diabetes has profound effects on metabolism that can be detected in plasma. While increases in circulating non-esterified fatty acids (NEFA) are well-described in diabetes, effects on signaling lipids have received little attention. Oxylipins and endocannabinoids are classes of bioactive fatty acid metabolites with many structural members that influence insulin signaling, adipose function and inflammation through autocrine, paracrine and endocrine mechanisms. To link diabetes-associated changes in plasma NEFA and signaling lipids, we quantitatively targeted >150 plasma lipidome components in age- and body mass index-matched, overweight to obese, non-diabetic (n = 12) and type 2 diabetic (n = 43) African-American women. Diabetes related NEFA patterns indicated ∼60% increase in steroyl-CoA desaturase activity and ∼40% decrease in very long chain polyunsaturated fatty acid chain shortening, patterns previously associated with the development of nonalcoholic fatty liver disease. Further, epoxides and ketones of eighteen carbon polyunsaturated fatty acids were elevated >80% in diabetes and strongly correlated with changes in NEFA, consistent with their liberation during adipose lipolysis. Endocannabinoid behavior differed by class with diabetes increasing an array of N-acylethanolamides which were positively correlated with pro-inflammatory 5-lipooxygenase-derived metabolites, while monoacylglycerols were negatively correlated with body mass. These results clearly show that diabetes not only results in an increase in plasma NEFA, but shifts the plasma lipidomic profiles in ways that reflect the biochemical and physiological changes of this pathological state which are independent of obesity associated changes.

The long-chain omega-3 fatty acids (n-3 FA) eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA) have beneficial health effects, but the molecular mediators of these effects are not well characterized. Oxygenated n-3 FAs (oxylipins) may be an important class of mediators. Members of this chemical class include epoxides, alcohols, diols, and ketones, many of which have bioactivity in vitro. Neither the presence of n-3 oxylipins in human plasma nor the effect of n-3 FA ingestion on their levels has been documented. We measured plasma oxylipins derived from both the n-3 and n-6 FA classes in healthy volunteers (n = 10) before and after 4 weeks of treatment with prescription n-3 FA ethyl esters (4 g/day). At baseline, EPA and DHA oxylipins were detected in low (1-50 nM) range, with alcohols > epoxides >or= diols. Treatment increased n-3 oxylipin levels 2- to 5-fold and reduced selected n-6 oxylipins by approximately 20%. This is the first documentation that endogenous n-3 oxylipin levels can be modulated by n-3 FA treatment in humans. The extent to which the beneficial cardiovascular effects of n-3 FAs are mediated by increased n-3 and/or reduced n-6 oxylipin levels remains to be explored.

Eicosanoids, including epoxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, and other oxylipins derived from polyunsaturated fatty acids, have emerging roles in endothelial inflammation and subsequent atherosclerosis. Unlike eicosanoids in the prostanoid series, they are known to be esterified in cell lipids such as phospholipids and triglycerides; however, our understanding of these reservoirs is in its infancy. This review focuses on recent work identifying circulating oxylipins, primarily esterified with lipoprotein lipids, and their effects on markers of endothelial dysfunction. These oxylipins are known to be released by at least one lipase (lipoprotein lipase) and to mediate increased expression of inflammatory markers in endothelial cells, which coincides with the known roles of lipoproteins in endothelial dysfunction. The implications of the lipolytic release of lipoprotein-bound oxylipins for the inflammatory response, challenges to analysis of this oxylipin compartment, and the potential importance of non-arachidonate-derived oxylipins are discussed.

We previously demonstrated that defects in lipoprotein metabolism alter the distribution of oxygenated polyunsaturated fatty acids (PUFAs) in lipoprotein particles. If these oxidation products are released by lipoprotein lipase (LpL), then their delivery to peripheral tissues with bulk lipids could influence cellular function. Using 26-week-old normolipidemic and hyperlipidemic Zucker rats, we measured PUFA alcohols, epoxides, diols, ketones, and triols (i.e. oxylipins) in esterified and non-esterified fractions of whole plasma, VLDL, and LpL-generated VLDL-lipolysates. Whole plasma, VLDL, and lipolysate oxylipin profiles were distinct and altered by hyperlipidemia. While >90% of the whole plasma oxylipins were esterified, the fraction of each oxylipin class in the VLDL varied: 46% of alcohols, 30% of epoxides, 19% of diols, <10% of ketones, and <1% triols. Whole plasma was dominated by arachidonate alcohols, while the linoleate alcohols, epoxides, and ketones showed an increased prevalence in VLDL. LpL-mediated VLDL lipolysis of PUFA alcohols, diols and ketones was detected and the relative abundance of oxygenated linoleates was enhanced in the lipolysates, relative to their corresponding VLDL. In summary esterified oxylipins were seen to be LpL substrates with heterogeneous distributions among lipoprotein classes. Moreover, oxylipin distributions are changes within the context of obesity-associated dyslipidemia. These results support the notion that the VLDL-LpL axis may facilitate the delivery of plasma oxylipins to the periphery. The physiological implications of these findings are yet to be elucidated; however, these molecules are plausible indicators of systemic oxidative stress, and could report this status to the peripheral tissues.

BACKGROUND: Tai Chi (TC) controls pain through mind-body exercise and appears to alter inflammatory mediators. TC actions on lipid biomarkers associated with inflammation and brain neural networks in women with knee osteoarthritic pain were investigated. METHODS: A single-center, pre- and post-TC group (baseline and 8 wk) exercise pilot study in postmenopausal women with knee osteoarthritic pain was performed. 12 eligible women participated in TC group exercise. The primary outcome was liquid chromatography tandem mass spectrometry determination of circulating endocannabinoids (eCB) and oxylipins (OxL). Secondary outcomes were correlations between eCB and OxL levels and clinical pain/limitation assessments, and brain resting-state function magnetic resonance imaging (rs-fMRI). RESULTS: Differences in circulating quantitative levels (nM) of pro-inflammatory OxL after TC were found in women. TC exercise resulted in lower OxL PGE1 and PGE2 and higher 12-HETE, LTB4, and 12-HEPE compared to baseline. Pain assessment and eCB and OxL levels suggest crucial relationships between TC exercise, inflammatory markers, and pain. Higher plasma levels of eCB AEA, and 1, 2-AG were found in subjects with increased pain. Several eCB and OxL levels were positively correlated with left and right brain amygdala-medial prefrontal cortex functional connectivity. CONCLUSION: TC exercise lowers pro-inflammatory OxL in women with knee osteoarthritic pain. Correlations between subject pain, functional limitations, and brain connectivity with levels of OxL and eCB showed significance. Findings indicate potential mechanisms for OxL and eCB and their biosynthetic endogenous PUFA precursors that alter brain connectivity, neuroinflammation, and pain. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT04046003.

Inflammatory bowel disease (IBD) is a multifactorial disease with increasing incidence in the U.S. suggesting that environmental factors, including diet, are involved. It has been suggested that excessive consumption of linoleic acid (LA, C18:2 omega-6), which must be obtained from the diet, may promote the development of IBD in humans. To demonstrate a causal link between LA and IBD, we show that a high fat diet (HFD) based on soybean oil (SO), which is comprised of ~55% LA, increases susceptibility to colitis in several models, including IBD-susceptible IL10 knockout mice. This effect was not observed with low-LA HFDs derived from genetically modified soybean oil or olive oil. The conventional SO HFD causes classical IBD symptoms including immune dysfunction, increased intestinal epithelial barrier permeability, and disruption of the balance of isoforms from the IBD susceptibility gene Hepatocyte Nuclear Factor 4α (HNF4α). The SO HFD causes gut dysbiosis, including increased abundance of an endogenous adherent invasive Escherichia coli (AIEC), which can use LA as a carbon source. Metabolomic analysis shows that in the mouse gut, even in the absence of bacteria, the presence of soybean oil increases levels of LA, oxylipins and prostaglandins. Many compounds in the endocannabinoid system, which are protective against IBD, are decreased by SO both in vivo and in vitro. These results indicate that a high LA diet increases susceptibility to colitis via microbial and host-initiated pathways involving alterations in the balance of bioactive metabolites of omega-6 and omega-3 polyunsaturated fatty acids, as well as HNF4α isoforms.

Hemodialysis patients (HDPs) have higher blood pressure, higher levels of inflammation, a higher risk of cardiovascular disease, and unusually low plasma n-3 polyunsaturated fatty acid (PUFA) levels compared to healthy subjects. The objective of our investigation was to examine the levels of endocannabinoids (eCBs) and oxylipins (OxLs) in female HDPs compared to healthy matched female controls, with the underlying hypothesis that differences in specific PUFA levels in hemodialysis patients would result in changes in eCBs and OxLs. Plasma phospholipid fatty acids were analyzed by gas chromatography. Plasma was extracted and analyzed using ultra-performance liquid chromatography followed by electrospray ionization and tandem MS for eCBs and OxLs. The global untargeted metabolite profiling of plasma was performed by GCTOF MS. Compared to the controls, HDPs showed lower levels of plasma EPA and the associated OxL metabolites 5- and 12-HEPE, 14,15-DiHETE, as well as DHA derived 19(20)-EpDPE. Meanwhile, no changes in arachidonylethanolamide or 2-arachidonylglycerol in the open circulation were detected. Higher levels of multiple N-acylethanolamides, monoacylglycerols, biomarkers of progressive kidney disease, the nitric oxide metabolism-linked citrulline, and the uremic toxins kynurenine and creatine were observed in HDP. These metabolic differences in cCBs and OxLs help explain the severe inflammatory and cardiovascular disease manifested by HDPs, and they should be explored in future studies.

Non-alcoholic fatty liver disease (NAFLD) is a common liver pathology that includes steatosis, or non-alcoholic fatty liver (NAFL), and non-alcoholic steatohepatitis (NASH). Without a clear pathophysiological mechanism, it affects Hispanics disproportionately compared to other ethnicities. Polyunsaturated fatty acids (PUFAs) and inflammatory lipid mediators including oxylipin (OXL) and endocannabinoid (eCB) are altered in NAFLD and thought to contribute to its pathogenesis. However, the existence of ethnicity-related differences is not clear. We employed targeted lipidomic profiling for plasma PUFAs, non-esterified OXLs and eCBs in White Hispanics (HIS, n = 10) and Caucasians (CAU, n = 8) with biopsy-confirmed NAFL, compared with healthy control subjects (HC; n = 14 HIS; n = 8 CAU). NAFLD was associated with diminished long chain PUFA in HIS, independent of histological severity. Differences in plasma OXLs and eCBs characterized ethnicities in NASH, with lower arachidonic acid derived OXLs observed in HIS. The secondary analysis comparing ethnicities within NASH (n = 12 HIS; n = 17 CAU), confirms these ethnicity-related differences and suggests lower lipoxygenase(s) and higher soluble epoxide hydrolase(s) activities in HIS compared to CAU. While causes are not clear, these lipidomic differences might be with implications for NAFLD severity and are worth further investigation. We provide preliminary data indicating ethnicity-specific lipidomic signature characterizes NASH which requires further validation.

Polyunsaturated fats are energy substrates and precursors to the biosynthesis of lipid mediators of cellular processes. Adipose tissue not only provides energy storage, but influences whole-body energy metabolism through endocrine functions. How diet influences adipose-lipid mediator balance may have broad impacts on energy metabolism. To determine how dietary lipid sources modulate brown and white adipose tissue and plasma lipid mediators, mice were fed low-fat (15% kcal fat) isocaloric diets, containing either palm oil (POLF) or linoleate-rich safflower oil (SOLF). Baseline and post body weight, adiposity, and 2-week and post fasting blood glucose were measured and lipid mediators were profiled in plasma, and inguinal white and interscapular brown adipose tissues. We identified over 30 species of altered lipid mediators between diets and found that these changes were unique to each tissue. We identified changes to lipid mediators with known functional roles in the regulation of adipose tissue expansion and function, and found that there was a relationship between the average fold difference in lipid mediators between brown adipose tissue and plasma in mice consuming the SOLF diet. Our findings emphasize that even with a low-fat diet, dietary fat quality has a profound effect on lipid mediator profiles in adipose tissues and plasma.

Metabolic syndrome (MetS) is a complex condition encompassing a constellation of cardiometabolic abnormalities. Oxylipins are a superfamily of lipid mediators regulating many cardiometabolic functions. Plasma oxylipin signature could provide a new clinical tool to enhance the phenotyping of MetS pathophysiology. A high-throughput validated mass spectrometry method, allowing for the quantitative profiling of over 130 oxylipins, was applied to identify and validate the oxylipin signature of MetS in two independent nested case/control studies involving 476 participants. We identified an oxylipin signature of MetS (coined OxyScore), including 23 oxylipins and having high performances in classification and replicability (cross-validated AUCROC of 89%, 95% CI: 85-93% and 78%, 95% CI: 72-85% in the Discovery and Replication studies, respectively). Correlation analysis and comparison with a classification model incorporating the MetS criteria showed that the oxylipin signature brings consistent and complementary information to the clinical criteria. Being linked with the regulation of various biological processes, the candidate oxylipins provide an integrative phenotyping of MetS regarding the activation and/or negative feedback regulation of crucial molecular pathways. This may help identify patients at higher risk of cardiometabolic diseases. The oxylipin signature of patients with metabolic syndrome enhances MetS phenotyping and may ultimately help to better stratify the risk of cardiometabolic diseases.

Many commonly used chemotherapies induce mitochondrial dysfunction in cardiac muscle, which leads to cardiotoxicity and heart failure later in life. Dietary long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) have demonstrated cardioprotective function in non-chemotherapy models of heart failure, potentially through the formation of LC n-3 PUFA-derived bioactive lipid metabolites. However, it is unknown whether dietary supplementation with LC n-3 PUFA can protect against chemotherapy-induced cardiotoxicity. To test this, 36 female ovariectomized C57BL/6J mice were randomized in a two-by-two factorial design to either a low (0 g/kg EPA + DHA) or high (12.2 g/kg EPA + DHA) LC n-3 PUFA diet, and received either two vehicle or two chemotherapy (9 mg/kg anthracycline + 90 mg/kg cyclophosphamide) tail vein injections separated by two weeks. Body weight and food intake were measured as well as heart gene expression and fatty acid composition. Heart mitochondria were isolated using differential centrifugation. Mitochondrial isolate oxylipin and N-acylethanolamide levels were measured by mass spectrometry after alkaline hydrolysis. LC n-3 PUFA supplementation attenuated some chemotherapy-induced differences (Myh7, Col3a1) in heart gene expression, and significantly altered various lipid species in cardiac mitochondrial preparations including several epoxy fatty acids [17(18)-EpETE] and N-acylethanolamines (arachidonoylethanolamine, AEA), suggesting a possible functional link between heart lipids and cardiotoxicity.

BackgroundAlzheimer's disease, cardiovascular disease, and other cardiometabolic disorders may share inflammatory origins. Lipid mediators, including oxylipins, endocannabinoids, bile acids, and steroids, regulate inflammation, energy metabolism, and cell proliferation with well-established involvement in cardiometabolic diseases. However, their role in Alzheimer's disease is poorly understood. Here, we describe the analysis of plasma and cerebrospinal fluid lipid mediators in a case-control comparison of ~150 individuals with Alzheimer's disease and ~135 healthy controls, to investigate this knowledge gap.MethodsLipid mediators were measured using targeted quantitative mass spectrometry. Data were analyzed using the analysis of covariates, adjusting for sex, age, and ethnicity. Partial least square discriminant analysis identified plasma and cerebrospinal fluid lipid mediator discriminates of Alzheimer's disease. Alzheimer's disease predictive models were constructed using machine learning combined with stepwise logistic regression.ResultsIn both plasma and cerebrospinal fluid, individuals with Alzheimer's disease had elevated cytochrome P450/soluble epoxide hydrolase pathway components and decreased fatty acid ethanolamides compared to healthy controls. Circulating metabolites of soluble epoxide hydrolase and ethanolamides provide Alzheimer's disease predictors with areas under receiver operator characteristic curves ranging from 0.82 to 0.92 for cerebrospinal fluid and plasma metabolites, respectively.ConclusionsPrevious studies report Alzheimer's disease-associated soluble epoxide hydrolase upregulation in the brain and that endocannabinoid metabolism provides an adaptive response to neuroinflammation. This study supports the involvement of P450-dependent and endocannabinoid metabolism in Alzheimer's disease. The results further suggest that combined pharmacological intervention targeting both metabolic pathways may have therapeutic benefits for Alzheimer's disease.

The prolonged, postweaning fast of northern elephant seal (Mirounga angustirostris) pups is characterized by a reliance on lipid metabolism and reversible, fasting-induced insulin resistance, providing a unique model to examine the effects of insulin on lipid metabolism. We have previously shown that acute insulin infusion induced a shift in fatty acid metabolism dependent on fasting duration. This study complements the previous study by examining the effects of fasting duration and insulin infusion on circulating levels of oxylipins, bioactive metabolites derived from the oxygenation of polyunsaturated fatty acids. Northern elephant seal pups were studied at two postweaning periods (n = 5/period): early fasting (1-2 wk postweaning; 127 ± 1 kg) and late fasting (6-7 wk postweaning; 93 ± 4 kg). Different cohorts of pups were weighed, sedated, and infused with 65 mU/kg of insulin. Plasma was collected prior to infusion (T0) and at 10, 30, 60, and 120 min postinfusion. A profile of ∼80 oxylipins was analyzed by UPLC-ESI-MS/MS. Nine oxylipins changed between early and late fasting and eight were altered in response to insulin infusion. Fasting decreased prostaglandin F2α (PGF2α) and increased 14,15-dihydroxyicosatrienoic acid (14,15-DiHETrE), 20-hydroxyeicosatetraenoic acid (20-HETE), and 4-hydroxy-docosahexaenoic acid (4-HDoHE) (P < 0.03) in T0 samples, whereas insulin infusion resulted in an inverse change in area-under-the-curve (AUC) levels in these same metabolites (P < 0.05). In addition, 12-12-hydroperoxyeicosatetraenoic acid (HpETE) and 12-HETE decreased with fasting and insulin infusion, respectively (P < 0.04). The oxylipins altered during fasting and in response to insulin infusion may contribute to the manifestation of insulin resistance and participate in the metabolic regulation of associated cellular processes.

Cognitive decline is associated with both normal aging and early pathologies leading to dementia. Here we used quantitative profiling of metabolites involved in the regulation of inflammation, vascular function, neuronal function and energy metabolism, including oxylipins, endocannabinoids, bile acids, and steroid hormones to identify metabolic biomarkers of mild cognitive impairment (MCI). Serum samples (n = 212) were obtained from subjects with or without MCI opportunistically collected with incomplete fasting state information. To maximize power and stratify the analysis of metabolite associations with MCI by the fasting state, we developed an algorithm to predict subject fasting state when unknown (n = 73). In non-fasted subjects, linoleic acid and palmitoleoyl ethanolamide levels were positively associated with perceptual speed. In fasted subjects, soluble epoxide hydrolase activity and tauro-alpha-muricholic acid levels were negatively associated with perceptual speed. Other cognitive domains showed associations with bile acid metabolism, but only in the non-fasted state. Importantly, this study shows unique associations between serum metabolites and cognitive function in the fasted and non-fasted states and provides a fasting state prediction algorithm based on measurable metabolites.

α-Linolenic acid (ALA) is an essential fatty acid and the precursor for long-chain n-3 PUFA. However, biosynthesis of n-3 PUFA is limited in a Western diet likely due to an overabundance of n-6 PUFA. We hypothesized that dietary reduction of n-6/n-3 PUFA ratio is sufficient to promote the biosynthesis of long-chain n-3 PUFA, leading to an attenuation of high fat (HF) diet-induced obesity and inflammation. C57BL/6 J mice were fed a HF diet from ALA-enriched butter (n3Bu, n-6/n-3=1) in comparison with isocaloric HF diets from either conventional butter lacking both ALA and LA (Bu, n-6/n-3=6), or margarine containing a similar amount of ALA and abundant LA (Ma, n-6/n-3=6). Targeted lipidomic analyses revealed that n3Bu feeding promoted the bioconversion of long-chain n-3 PUFA and their oxygenated metabolites (oxylipins) derived from ALA and EPA. The n3Bu supplementation attenuated hepatic TG accumulation and adipose tissue inflammation, resulting in improved insulin sensitivity. Decreased inflammation by n3Bu feeding was attributed to the suppression of NF-κB activation and M1 macrophage polarization. Collectively, our work suggests that dietary reduction of the n-6/n-3 PUFA ratio, as well as total n-3 PUFA consumed, is a crucial determinant that facilitates n-3 PUFA biosynthesis and subsequent lipidomic modifications, thereby conferring metabolic benefits against obesity-induced inflammation and insulin resistance.

Walnut consumption can provide both vascular and metabolic health benefits, and walnut-induced changes in lipoprotein particle chemical payloads may be responsible for these health benefits. To explore this possibility with a focus on metabolic health, this study investigated the impact of walnut consumption on lipoprotein lipid composition and changes in LDL anti-inflammatory properties, as reported by inflamed adipocyte. Hypercholesterolemic, postmenopausal females were treated with 40 g/day (i.e., 1.6 servings/day; n=15) of walnuts for 4 weeks. Fatty acids and their oxygenated metabolites, i.e., oxylipins, were quantified in isolated lipoproteins. Human primary adipocytes were exposed to LDL and TNFα-stimulated adipokine production was measured. Walnut treatment elevated α-linolenic acid and its epoxides in all lipoproteins and depleted mid-chain alcohols in VLDL and LDL, but not HDL. Walnuts also reduced TNFα-induced diabetic adipocyte production of IL-6 (-48%, P=.0006) and IL-8 (-30%, P=.01), changes inversely correlated with levels of α-linolenic acid-derived epoxides but not α-linolenic acid itself. In conclusion, modest walnut consumption can alter lipoprotein lipid profiles and enhance their ability to inhibit TNFα-dependent pro-inflammatory responses in human diabetic primary adipocytes. Moreover, this study suggests the oxylipins, rather than the parent fatty acids, mediate LDL action of adipocytes.

Skin disease alters cutaneous lipid mediator metabolism, and if skin secretions contain evidence of these changes, they may constitute useful clinical matrices with low associated subject burden. The influences of skin diseases on sebum lipid mediators are understudied. Here, sebum oxylipins, endocannabinoids, sphingolipids, and fatty acids were quantified from the non-lesional bilateral cheeks of subjects with and without quiescent atopic dermatitis (AD) using LC-MS/MS and GC-MS. AD decreased C36 [NS] and [NdS] ceramide concentrations. Compared to males, females demonstrated increased concentrations of oxylipin alcohols and ketones, and saturated and monounsaturated non-esterified fatty acids, as well as decreased concentrations of C42 [NS] and [NdS] ceramides. Additionally, contemporaneously collected sweat lipid mediator profiles were distinct, with sebum showing higher concentrations of most targets, but fewer highly polar lipids. Therefore, AD and gender appear to alter sebum lipid metabolism even in non-lesional skin of quiescent subjects.

The phenolic profiles of extra virgin olive oils (EVOOs) may influence their cardiovascular benefits. In a randomized crossover of acute EVOO intake on platelet function, participants (n=9) consumed 40 mL of EVOO weekly. EVOOs were matched for total phenolic content and were either tyrosol-poor with 1:2 oleacein/oleocanthal (D2i0.5), or 2:1 oleacein/oleocanthal (D2i2), or predominantly tyrosol (D2i0). Ibuprofen provided a platelet inhibition control. Blood was collected pre- and 2 hr post-EVOO intake. D2i0.5 and D2i2 reduced 1 µg/mL collagen-stimulated maximum platelet aggregation (Pmax), with effects best correlated to oleocanthal intake (R=0.56, P=0.002). Total phenolic intake was independently correlated to eicosanoid production inhibition, suggesting that cyclooxygenase blockade was not responsible for the Pmax inhibition. Five participants exhibited >25% ΔPmax declines with D2i0.5 and D2i2 intake and plasma metabolomic profiles discriminated subjects by oil responsivity. Platelet responses to acute EVOO intake are associated with oil phenolic composition and may be influenced by diet.

Chronic low grade inflammation is closely linked to obesity-associated insulin resistance. To examine how administration of the anti-inflammatory compound indomethacin, a general cyclooxygenase inhibitor, affected obesity development and insulin sensitivity, we fed obesity-prone male C57BL/6J mice a high fat/high sucrose (HF/HS) diet or a regular diet supplemented or not with indomethacin (±INDO) for 7 weeks. Development of obesity, insulin resistance, and glucose intolerance was monitored, and the effect of indomethacin on glucose-stimulated insulin secretion (GSIS) was measured in vivo and in vitro using MIN6 β-cells. We found that supplementation with indomethacin prevented HF/HS-induced obesity and diet-induced changes in systemic insulin sensitivity. Thus, HF/HS+INDO-fed mice remained insulin-sensitive. However, mice fed HF/HS+INDO exhibited pronounced glucose intolerance. Hepatic glucose output was significantly increased. Indomethacin had no effect on adipose tissue mass, glucose tolerance, or GSIS when included in a regular diet. Indomethacin administration to obese mice did not reduce adipose tissue mass, and the compensatory increase in GSIS observed in obese mice was not affected by treatment with indomethacin. We demonstrate that indomethacin did not inhibit GSIS per se, but activation of GPR40 in the presence of indomethacin inhibited glucose-dependent insulin secretion in MIN6 cells. We conclude that constitutive high hepatic glucose output combined with impaired GSIS in response to activation of GPR40-dependent signaling in the HF/HS+INDO-fed mice contributed to the impaired glucose clearance during a glucose challenge and that the resulting lower levels of plasma insulin prevented the obesogenic action of the HF/HS diet.

Although the effects of fish oil supplements on airway inflammation in asthma have been studied with varying results, the independent effects of the fish oil components, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), administered separately, are untested. Here, we investigated airway inflammation and hyperresponsiveness using a mouse ovalbumin exposure model of asthma assessing the effects of consuming EPA (1.5% wt/wt), DHA (1.5% wt/wt), EPA plus DHA (0.75% each), or a control diet with no added omega-3 polyunsaturated fatty acids. Consuming these diets for 6 weeks resulted in erythrocyte membrane EPA contents (molar %) of 9.0 (± 0.6), 3.2 (± 0.2), 6.8 (± 0.5), and 0.01 (± 0.0)%; DHA contents were 6.8 (± 0.1), 15.6 (± 0.5), 12.3 (± 0.3), and 3.8 (± 0.2)%, respectively. The DHA group had the highest bronchoalveolar lavage (BAL) fluid eosinophil and IL-6 levels (P < 0.05). Similar trends were seen for macrophages, IL-4, and IL-13, whereas TNF-α was lower in omega-3 polyunsaturated fatty acid groups than the control (P < 0.05). The DHA group also had the highest airway resistance, which differed significantly from the EPA plus DHA group (P < 0.05), which had the lowest. Oxylipins were measured in plasma and BAL fluid, with DHA and EPA suppressing arachidonic acid-derived oxylipin production. DHA-derived oxylipins from the cytochrome P450 and 15-lipoxygenase pathways correlated significantly with BAL eosinophil levels. The proinflammatory effects of DHA suggest that the adverse effects of individual fatty acid formulations should be thoroughly considered before any use as therapeutic agents in asthma.

Gestational long-term hypoxia increases the risk of myriad diseases in infants including persistent pulmonary hypertension. Similar to humans, fetal lamb lung development is susceptible to long-term intrauterine hypoxia, with structural and functional changes associated with the development of pulmonary hypertension including pulmonary arterial medial wall thickening and dysregulation of arterial reactivity, which culminates in decreased right ventricular output. To further explore the mechanisms associated with hypoxia-induced aberrations in the fetal sheep lung, we examined the premise that metabolomic changes and functional phenotypic transformations occur due to intrauterine, long-term hypoxia. To address this, we performed electron microscopy, Western immunoblotting, calcium imaging, and metabolomic analyses on pulmonary arteries isolated from near-term fetal lambs that had been exposed to low- or high-altitude (3,801 m) hypoxia for the latter 110+ days of gestation. Our results demonstrate that the sarcoplasmic reticulum was swollen with high luminal width and distances to the plasma membrane in the hypoxic group. Hypoxic animals were presented with higher endoplasmic reticulum stress and suppressed calcium storage. Metabolically, hypoxia was associated with lower levels of multiple omega-3 polyunsaturated fatty acids and derived lipid mediators (e.g., eicosapentaenoic acid, docosahexaenoic acid, α-linolenic acid, 5- hydroxyeicosapentaenoic acid (5-HEPE), 12-HEPE, 15-HEPE, prostaglandin E3, and 19(20)-epoxy docosapentaenoic acid) and higher levels of some omega-6 metabolites (P < 0.02) including 15-keto prostaglandin E2 and linoleoylglycerol. Collectively, the results reveal broad evidence for long-term hypoxia-induced metabolic reprogramming and phenotypic transformations in the pulmonary arteries of fetal sheep, conditions that likely contribute to the development of persistent pulmonary hypertension. [ABSTRACT FROM AUTHOR]

Background The onset of menopause increases the risk of metabolic syndrome (MetS). Adiponectin is an adipokine associated with insulin sensitivity that is lower in people with MetS. Supplementing diets with linoleic acid (LA)-rich oil increased adiponectin concentrations and improved glucose control in women with type 2 diabetes. The effect of LA on adipokines, especially total and the bioactive form of adiponectin, high-molecular-weight (HMW) adiponectin, in women with MetS is unknown. Objectives The aim of this study was to explore the effect of supplementation of the diet with an oil rich in LA on adipokines in women with MetS. The effect of the LA-rich oil (LA-oil) on oxylipins, key metabolites that may influence inflammation and metabolism, was also explored. Methods In this open-label single-arm pilot study, 18 postmenopausal nondiabetic women with MetS enrolled in a 2-phase study were instructed to consume LA-rich vegetable oil (10 mL/d) as part of their habitual diets. Women consumed an oleic acid–rich oil (OA-oil) for 4 wk followed by an LA-oil for 16 wk. Fasting concentrations of adipokines, fatty acids, oxylipins, and markers of glycemia and inflammation were measured. Results After 4 wk of OA-oil consumption, fasting glucose and total adiponectin concentrations decreased whereas fasting C-reactive protein increased. After 16 wk of LA-oil supplementation total and HMW adiponectin and plasma oxylipins increased. Markers of inflammation and glycemia were unchanged after LA-oil consumption. Conclusions Supplementation with LA-oil increased total and HMW adiponectin concentrations and altered plasma oxylipin profiles. Larger studies are needed to elucidate the links between these changes and MetS. This trial was registered at clinicaltrials.gov as NCT02063165. [ABSTRACT FROM AUTHOR]

Pulmonary hypertension (PH) is a common consequence of bronchopulmonary dysplasia (BPD) and remains a primary contributor to increased morbidity and mortality among preterm infants. Unfortunately, at the present time, there are no reliable early predictive markers for BPD-associated PH. Considering its health consequences, understanding in utero perturbations that lead to the development of BPD and BPD-associated PH and identifying early predictive markers is of utmost importance. As part of the discovery phase, we applied a multiplatform metabolomics approach consisting of untargeted and targeted methodologies to screen for metabolic perturbations in umbilical cord blood (UCB) plasma from preterm infants that did (n = 21; cases) or did not (n = 21; controls) develop subsequent PH. A total of 1,656 features were detected, of which 407 were annotated by metabolite structures. PH-associated metabolic perturbations were characterized by reductions in major choline-containing phospholipids, such as phosphatidylcholines and sphingomyelins, indicating altered lipid metabolism. The reduction in UCB abundances of major choline-containing phospholipids was confirmed in an independent validation cohort consisting of UCB plasmas from 10 cases and 10 controls matched for gestational age and BPD status. Subanalyses in the discovery cohort indicated that elevations in the oxylipins PGE1, PGE2, PGF2a, 9- and 13-HOTE, 9- and 13-HODE, and 9- and 13-KODE were positively associated with BPD presence and severity. This expansive evaluation of cord blood plasma identifies compounds reflecting dyslipidemia and suggests altered metabolite provision associated with metabolic immaturity that differentiate subjects, both by BPD severity and PH development. [ABSTRACT FROM AUTHOR]

Low vitamin A (VA) status is common among lactating women in low-income countries. Lactation has substantial effects on mother's metabolism and VA is required in multiple biological processes, including growth, vision, immunity, and reproduction. The objective of this pilot study was to use metabolomics profiling to conduct a broad, exploratory assessment of differences in plasma metabolites associated with low VA status versus VA adequacy in lactating women. Plasma samples from lactating women who participated in a survey in Samar, Philippines, were selected from a cross-sectional study based on plasma retinol concentrations indicating low (VA–; n = 5) or adequate (VA+; n = 5) VA status (plasma retinol <0.8 or >1.05 µmol/L). The plasma results collected from 6 metabolomics assays (oxylipins, endocannabinoids, bile acids, primary metabolomics, biogenic amines, and lipidomics) were compared by group using liquid chromatography mass spectrometry. Twenty-eight metabolites were altered in the VA– versus VA+ status groups, with 24 being lipid mediators (P <.05). These lipid mediators included lower concentrations of arachidonic acid- and eicosapentaenoic acid-derived oxylipins, as well as lysophospholipids and sphingolipids, in the VA– group (P <.05). Chemical similarity enrichment analysis identified hydroxy-eicosatetraenoic acids, hydroxy-eicosapentaenoic acids, and dihydroxy-eicosatetraenoic acids as significantly altered oxylipin clusters (P <.0001, false discovery rate [FDR] P <.0001), as well as sphingomyelins, saturated lysophosphatidylcholines, phosphatidylcholines, and phosphatidylethanolamines (P <.001, FDR P <.01). The multiassay nutritional metabolomics profiling of low VA status compared with adequacy in lactating women was characterized by reduced lipid mediator concentrations. Future studies with stronger study designs and larger sample size are needed to confirm and validate these preliminary results. Vitamin A (VA) deficiency is highly prevalent among lactating women in low-income countries. The objective of this study was to use metabolomics profiling to conduct a broad, exploratory assessment of differences in plasma metabolites associated with low VA status versus VA adequacy in lactating women from Samar, Philippines. Twenty-eight metabolites were significantly altered in the low VA versus adequate VA status groups, with 24 being lipid mediators [Display omitted]. [ABSTRACT FROM AUTHOR]

Lipoprotein metabolism is critical to inflammation. While the periphery and central nervous system (CNS) have separate yet connected lipoprotein systems, impaired lipoprotein metabolism is implicated in both cardiometabolic and neurological disorders. Despite the substantial investigation into the composition, structure and function of lipoproteins, the lipoprotein oxylipin profiles, their influence on lipoprotein functions, and their potential biological implications are unclear. Lipoproteins carry most of the circulating oxylipins. Importantly, lipoprotein-mediated oxylipin transport allows for endocrine signaling by these lipid mediators, long considered to have only autocrine and paracrine functions. Alterations in plasma lipoprotein oxylipin composition can directly impact inflammatory responses of lipoprotein metabolizing cells. Similar investigations of CNS lipoprotein oxylipins are non-existent to date. However, as APOE4 is associated with Alzheimer's disease-related microglia dysfunction and oxylipin dysregulation, ApoE4-dependent lipoprotein oxylipin modulation in neurological pathologies is suggested. Such investigations are crucial to bridge knowledge gaps linking oxylipin- and lipoprotein-related disorders in both periphery and CNS. Here, after providing a summary of existent literatures on lipoprotein oxylipin analysis methods, we emphasize the importance of lipoproteins in oxylipin transport and argue that understanding the compartmentalization and distribution of lipoprotein oxylipins may fundamentally alter our consideration of the roles of lipoprotein in cardiometabolic and neurological disorders. [ABSTRACT FROM AUTHOR]