Oxylipin profiling for clinical research: Current status and future perspectives.

Oxylipins are potent lipid mediators with increasing interest in clinical research. They are usually measured in systemic circulation and can provide a wealth of information regarding key biological processes such as inflammation, vascular tone, or blood coagulation. Although procedures still require harmonization to generate comparable oxylipin datasets, performing comprehensive profiling of circulating oxylipins in large studies is feasible and no longer restricted by technical barriers. However, it is essential to improve and facilitate the biological interpretation of complex oxylipin profiles to truly leverage their potential in clinical research. This requires regular updating of our knowledge about the metabolism and the mode of action of oxylipins, and consideration of all factors that may influence circulating oxylipin profiles independently of the studied disease or condition. This review aims to provide the readers with updated and necessary information regarding oxylipin metabolism, their different forms in systemic circulation, the current limitations in deducing oxylipin cellular effects from in vitro bioactivity studies, the biological and technical confounding factors needed to consider for a proper interpretation of oxylipin profiles.

Eicosanoids and Oxylipin Signature in Hereditary Hemochromatosis Patients Are Similar to Dysmetabolic Iron Overload Syndrome Patients but Are Impacted by Dietary Iron Absorption.

INTRODUCTION: Oxylipins are mediators of oxidative stress. To characterize the underlying inflammatory processes and phenotype effect of iron metabolism disorders, we investigated the oxylipin profile in hereditary hemochromatosis (HH) and dysmetabolic iron overload syndrome (DIOS) patients. METHODS: An LC-MS/MS-based method was performed to quantify plasma oxylipins in 20 HH and 20 DIOS patients in fasting conditions and 3 h after an iron-rich meal in HH patients. RESULTS: Principal component analysis showed no separation between HH and DIOS, suggesting that the clinical phenotype has no direct impact on oxylipin metabolism. 20-HETE was higher in DIOS and correlated with hypertension (p = 0.03). Different oxylipin signatures were observed in HH before and after the iron-rich meal. Discriminant oxylipins include epoxy fatty acids derived from docosahexaenoic acid and arachidonic acid as well as 13-HODE and 9-HODE. Mediation analysis found no major contribution of dietary iron absorption for 16/22 oxylipins significantly affected by the meal. DISCUSSION: The oxylipin profiles of HH and DIOS seemed similar except for 20-HETE, possibly reflecting different hypertension prevalence between the two groups. Oxylipins were significantly affected by the iron-rich meal, but the specific contribution of iron was not clear. Although iron may contribute to oxidative stress and inflammation in HH and DIOS, this does not seem to directly affect oxylipin metabolism.

Integrated analysis of whole blood oxylipin and cytokine responses after bacterial, viral, and T cell stimulation reveals new immune networks.

Oxylipins are major immunomodulating mediators, yet studies of inflammation focus mainly on cytokines. Here, using a standardized whole-blood stimulation system, we characterized the oxylipin-driven inflammatory responses to various stimuli and their relationships with cytokine responses. We performed a pilot study in 25 healthy individuals using 6 different stimuli: 2 bacterial stimuli (LPS and live BCG), 2 viral stimuli (vaccine-grade poly I:C and live H1N1 attenuated influenza), an enterotoxin superantigen and a Null control. All stimuli induced a strong production of oxylipins but most importantly, bacterial, viral, and T cell immune responses show distinct oxylipin signatures. Integration of the oxylipin and cytokine responses for each condition revealed new immune networks improving our understanding of inflammation regulation. Finally, the oxylipin responses and oxylipin-cytokine networks were compared in patients with active tuberculosis or with latent infection. This revealed different responses to BCG but not LPS stimulation highlighting new regulatory pathways for further investigations.

The Plasma Oxylipin Signature Provides a Deep Phenotyping of Metabolic Syndrome Complementary to the Clinical Criteria.

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.

Evaluating the role of orange juice, HESPERidin in vascular HEALTH benefits (HESPER-HEALTH study): protocol for a randomised controlled trial.

INTRODUCTION: Although epidemiological studies associate the consumption of sugary beverages with adverse health effects, human experimental studies have demonstrated substantially different metabolic responses when 100% fruit juices are compared with artificial beverages. Fruit juices do not just provide sugars and associated calories, but they are also rich in bioactive compounds. Flavanones are bioactives specifically and abundantly found in citrus foods, with hesperidin as the major representative in sweet oranges. Flavanone intake has been associated with a lower incidence of mortality from cardiovascular disease (CVD). However, clinical evidence are too scarce to confirm the vasculoprotective effects of 100% orange juice (OJ) presumably mediated by flavanones and thereby do not allow firm conclusions to be drawn about their efficacy. METHODS AND ANALYSIS: The HESPER-HEALTH study aims to assess the efficacy of OJ in improving vascular function and the contribution of hesperidin to these effects. This double-blind, randomised, controlled, crossover study will be carried out in 42 volunteers predisposed to CVD, based on age and on overweight. It includes three 6-week periods of consumption of 330 mL/d of OJ versus control drinks with and without hesperidin at a dose in agreement with a daily OJ serving (approx. 200-215 mg). The primary outcome is endothelial function, assessed by flow mediated dilation, with measurements performed at fasting and postprandially in response to a challenge meal. The secondary outcomes include bioavailability and metabolism of flavanones, changes in other markers of vascular function, systemic biomarkers of cardiovascular risk, endothelial dysfunction and inflammation, vitamin C and carotenoids status, anthropometry and body composition, gut microbiota composition, nutrigenomic response and in oxylipin profiling. ETHICS AND DISSEMINATION: This ongoing study was approved by the Ethics committee Sud-Est III, Bron, France on 17 November 2020. The trial is registered on ClinicalTrials.gov. The results will be disseminated in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04731987; Pre-results.

Does iron overload in metabolic syndrome affect macrophage profile? A case control study.

AIMS: Dysmetabolic iron overload syndrome (DIOS) is common but the clinical relevance of iron overload is not understood. Macrophages are central cells in iron homeostasis and inflammation. We hypothesized that iron overload in DIOS could affect the phenotype of monocytes and impair macrophage gene expression. METHODS: This study compared 20 subjects with DIOS to 20 subjects with metabolic syndrome (MetS) without iron overload, and 20 healthy controls. Monocytes were phenotyped by Fluorescence-Activated Cell Sorting (FACS) and differentiated into anti-inflammatory M2 macrophages in the presence of IL-4. The expression of 38 genes related to inflammation, iron metabolism and M2 phenotype was assessed by real-time PCR. RESULTS: FACS showed no difference between monocytes across the three groups. The macrophagic response to IL-4-driven differentiation was altered in four of the five genes of M2 phenotype (MRC1, F13A1, ABCA1, TGM2 but not FABP4), in DIOS vs Mets and controls demonstrating an impaired M2 polarization. The expression profile of inflammatory genes was not different in DIOS vs MetS. Several genes of iron metabolism presented a higher expression in DIOS vs MetS: SCL11A2 (a free iron transporter, +76 %, p = 0.04), SOD1 (an antioxidant enzyme, +27 %, p = 0.02), and TFRC (the receptor 1 of transferrin, +59 %, p = 0.003). CONCLUSIONS: In DIOS, macrophage polarization toward the M2 alternative phenotype is impaired but not associated with a pro-inflammatory profile. The up regulation of transferrin receptor 1 (TFRC) in DIOS macrophages suggests an adaptive role that may limit iron toxicity in DIOS.

Harmonized procedures lead to comparable quantification of total oxylipins across laboratories.

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.

Stability of oxylipins during plasma generation and long-term storage.

Oxidized unsaturated fatty acids - i.e. eicosanoids and other oxylipins - are lipid mediators involved in the regulation of numerous physiological functions such as inflammation, blood coagulation, vascular tone and endothelial permeability. They have raised strong interest in clinical lipidomics in order to understand their role in health and diseases and their use as biomarkers. However, before the clinical translation, it is crucial to validate the analytical reliability of oxylipins. This notably requires to assess the putative artificial formation or degradation of oxylipins by (unsuitable) blood handling during plasma generation, storage and sample preparation. Using a liquid chromatography-mass spectrometry method covering 133 oxylipins we comprehensively analyzed the total (free + esterified) oxylipin profile in plasma and investigated the influence of i) addition of additives during sample preparation, ii) different storage times and temperatures during the transitory stage of plasma generation and iii) long-term storage of plasma samples at -80 °C. Addition of radical scavenger butylated hydroxytoluene reduced the apparent concentrations of hydroxy-PUFA and thus should be added to the samples at the beginning of sample preparation. The concentrations of all oxylipin classes remained stable (within analytical variance of 20%) during the transitory stage of plasma generation up to 24 h at 4 °C or 4 h at 20 °C before centrifugation of EDTA-whole blood and up to 5 days at -20 °C after plasma separation. The variations in oxylipin concentrations did not correlate with storage time, storage temperature or stage of plasma generation. A significant increase of potentially lipoxygenase derived hydroxy-PUFA compared to immediate processing was only detected when samples were stored for longer times before centrifugation, plasma separation as well as freezing of plasma revealing residual enzymatic activity. Autoxidative rather than enzymatic processes led to a slightly increased concentration of 9-HETE when plasma samples were stored at -80 °C for 15 months. Overall, we demonstrate that total plasma oxylipins are robust regarding delays during plasma generation and long-term storage at -80 °C supporting the application of oxylipin profiling in clinical research.

Effect of procyanidin on dietary iron absorption in hereditary hemochromatosis and in dysmetabolic iron overload syndrome: A crossover double-blind randomized controlled trial.

BACKGROUND & AIMS: Type I hereditary hemochromatosis (HH) and dysmetabolic iron overload syndrome (DIOS) are the two most prevalent iron overload diseases. Although many food components, particularly polyphenols, reduce iron bioavailability, there is no clinically validated nutritional strategy to reduce food-iron absorption in patients with these diseases. We aimed to determine whether supplementation with 100 mg of procyanidins during a meal reduces dietary iron absorption in patients with HH or DIOS. METHODS: 20 HH and 20 DIOS patients were enrolled in a double-blind three-period crossover randomized study. Basal serum iron level was measured following an overnight fast. Each patient consumed a standardized test iron-rich meal containing 43 mg of iron with two capsules of placebo or procyanidin supplementation. Each period was separated by a 3-day wash-out period. The primary objective was a reduction of dietary iron absorption, assessed by a reduction of serum-iron area under the curve (AUC) corrected for baseline serum iron. RESULTS: All patients completed the study. The meal and the procyanidin supplements were well tolerated. In both HH and DIOS patients, the iron-rich meal induced a significant increase of serum iron compared with baseline at 120, 180, 240 min, from 8 to 9.1% (p = 0.002, 0.001 and 0.003, respectively) in DIOS and from 15.8 to 25.7% (p < 0.001) in HH. Iron absorption was 3.5-fold higher in HH than in DIOS (p < 0.001). Procyanidin supplementation did not significantly modify iron absorption in DIOS (AUC of added iron 332.87 ± 649.55 vs 312.61 ± 678.61 µmol.h/L, p = 0.916) or in HH (1168.62 ± 652.87 vs 1148.54 µmol.h/L ± 1290.05, p = 0.917). CONCLUSIONS: An iron-rich test meal led to a marked increase in iron absorption in HH but a mild increase in DIOS. Procyanidin supplementation does not significantly reduce dietary iron absorption in either disease. CLINICAL TRIAL REGISTRY: clinicaltrials.gov (NCT03453918).

MS-based targeted metabolomics of eicosanoids and other oxylipins: Analytical and inter-individual variabilities.

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.

Obesogenic diet in aging mice disrupts gut microbe composition and alters neutrophil:lymphocyte ratio, leading to inflamed milieu in acute heart failure.

Calorie-dense obesogenic diet (OBD) is a prime risk factor for cardiovascular disease in aging. However, increasing age coupled with changes in the diet can affect the interaction of intestinal microbiota influencing the immune system, which can lead to chronic inflammation. How age and calorie-enriched OBD interact with microbial flora and impact leukocyte profiling is currently under investigated. Here, we tested the interorgan hypothesis to determine whether OBD in young and aging mice alters the gut microbe composition and the splenic leukocyte profile in acute heart failure (HF). Young (2-mo-old) and aging (18-mo-old) mice were supplemented with standard diet (STD, ∼4% safflower oil diet) and OBD (10% safflower oil) for 2 mo and then subjected to coronary artery ligation to induce myocardial infarction. Fecal samples were collected pre- and post-diet intervention, and the microbial flora were analyzed using 16S variable region 4 rRNA gene DNA sequencing and Quantitative Insights Into Microbial Ecology informatics. The STD and OBD in aging mice resulted in an expansion of the genus Allobaculum in the fecal microbiota. However, we found a pathologic change in the neutrophil:lymphocyte ratio in aging mice in comparison with their young counterparts. Thus, calorie-enriched OBD dysregulated splenic leukocytes by decreasing immune-responsive F4/80+ and CD169+ macrophages in aging mice. OBD programmed neutrophil swarming with an increase in isoprostanoid levels, with dysregulation of lipoxygenases, cytokines, and metabolite-sensing receptor expression. In summary, calorie-dense OBD in aging mice disrupted the composition of the gut microbiome, which correlates with the development of integrative and system-wide nonresolving inflammation in acute HF.-Kain, V., Van Der Pol, W., Mariappan, N., Ahmad, A., Eipers, P., Gibson, D. L., Gladine, C., Vigor, C., Durand, T., Morrow, C., Halade, G. V. Obesogenic diet in aging mice disrupts gut microbe composition and alters neutrophil:lymphocyte ratio, leading to inflamed milieu in acute heart failure.

Muscle Loss Associated Changes of Oxylipin Signatures During Biological Aging: An Exploratory Study From the PROOF Cohort.

Characterizations of the multiple mechanisms determining biological aging are required to better understand the etiology and identify early biomarkers of sarcopenia. Oxylipins refer to a large family of signaling lipids involved in the regulation of various biological processes that become dysregulated during aging. To investigate whether comprehensive oxylipin profiling could provide an integrated and fine characterization of the early phases of sarcopenia, we performed a quantitative targeted metabolomics of oxylipins in plasma of 81-year-old subjects from the PROOF cohort with decreased (n = 12), stable (n = 16), or increased appendicular muscle mass (n = 14). Multivariate and univariate analyses identified significant and concordant changes of oxylipin profiles according to the muscle status. Of note, 90% of the most discriminant oxylipins were derived from EPA and DHA and were increased in the sarcopenic subjects. The oxylipins signatures of sarcopenic subjects revealed subtle activation of inflammatory resolution pathways, coagulation processes, and oxidative stress as well as the inhibition of angiogenesis. Heat maps highlighted relationships between oxylipins and the cardiometabolic health parameters which were mainly lost in sarcopenic subjects. This exploratory study supports that targeted metabolomics of oxylipins could provide relevant and subtle characterization of early disturbances associated with muscle loss during aging.

Insight into the contribution of isoprostanoids to the health effects of omega 3 PUFAs.

Omega 3 polyunsaturated fatty acids have been reported to confer beneficial health effects notably in the field of cardiovascular and inflammatory diseases. The current knowledge suggests a significant portion of the effects of omega 3 polyunsaturated fatty acids are mediated by their oxygenated metabolites. This review attempts to cover the current literature about the contribution of specific omega 3 oxygenated metabolites, namely omega 3 isoprostanoids, which are produced through free-radical mediated oxidation. A special emphasis has been given to the most biologically relevant omega 3 polyunsaturated fatty acids namely the α-linolenic, eicosapentaenoic and docosahexaenoic acids. The review includes a comprehensive description of the biosynthetic pathways, a summary of studies related to the biological significance of omega 3 isoprostanoids as well as a critical description of analytical development in the field of omega 3 isoprostanoids profiling in biological samples.

DHA-derived oxylipins, neuroprostanes and protectins, differentially and dose-dependently modulate the inflammatory response in human macrophages: Putative mechanisms through PPAR activation.

Whereas the anti-inflammatory properties and mechanisms of action of long chain ω3 PUFAs have been abundantly investigated, research gaps remain regarding the respective contribution and mechanisms of action of their oxygenated metabolites collectively known as oxylipins. We conducted a dose-dependent and comparative study in human primary macrophages aiming to compare the anti-inflammatory activity of two types of DHA-derived oxylipins including the well-described protectins (NPD1 and PDX), formed through lipoxygenase pathway and the neuroprostanes (14-A4t- and 4-F4t-NeuroP) formed through free-radical mediated oxygenation and expected to be new anti-inflammatory mediators. Considering the potential ability of these DHA-derived oxylipins to bind PPARs and knowing the central role of these transcription factors in the regulation of macrophage inflammatory response, we performed transactivation assays to compare the ability of protectins and neuroprostanes to activate PPARs. All molecules significantly reduced mRNA levels of cytokines such as IL-6 and TNF-α, however not at the same doses. NPD1 showed the most effect at 0.1µM (-14.9%, p<0.05 for IL-6 and -26.7%, p<0.05 for TNF-α) while the three other molecules had greater effects at 10µM, with the strongest result due to the cyclopentenone neuroprostane, 14-A4t-NeuroP (-49.8%, p<0.001 and -40.8%, p<0.001, respectively). Part of the anti-inflammatory properties of the DHA-derived oxylipins investigated could be linked to their activation of PPARs. Indeed, all tested oxylipins significantly activated PPARγ, with 14-A4t-NeuroP leading to the strongest activation, and NPD1 and PDX also activated PPARα. In conclusion, our results show that neuroprostanes and more especially cyclopentenone neuroprostanes have potent anti-inflammatory activities similar or even more pronounced than protectins supporting that neuroprostanes should be considered as important contributors to the anti-inflammatory effects of DHA.

Quantification of 4-hydroxy-2-nonenal-protein adducts in the in vivo gastric digesta of mini-pigs using a GC-MS/MS method with accuracy profile validation.

Hydroxyalkenals are lipid oxidation end-products resulting from the oxidation of polyunsaturated fatty acids (PUFA). This study aimed at quantifying the production of 4-hydroxy-2-nonenal-protein adducts (HNE-P) via Michael addition from n-6 PUFA oxidation in the gastric digesta of mini-pigs after the consumption of meat-based meals with different plant antioxidant contents. Using the accuracy profile procedure, we validated an extraction protocol for the quantification of HNE-P by GC-MS/MS in gastric contents. The formation of HNE-P in the gastric compartment was observed for the first time, with concentrations ranging from less than 0.52 to 1.33 nmol HNE-P per 500 mg digesta. Nevertheless, most gastric HNE-P levels were below the limit of quantification of 0.52 nmol HNE-P per 500 mg digesta. In this animal study, the protective effect of plant antioxidant sources on HNE-P formation was not evidenced contrasting with the results using TBARS as markers.

Simultaneous quantitative profiling of 20 isoprostanoids from omega-3 and omega-6 polyunsaturated fatty acids by LC-MS/MS in various biological samples.

Isoprostanoids are a group of non-enzymatic oxygenated metabolites of polyunsaturated fatty acids. It belongs to oxylipins group, which are important lipid mediators in biological processes, such as tissue repair, blood clotting, blood vessel permeability, inflammation and immunity regulation. Recently, isoprostanoids from eicosapentaenoic, docosahexaenoic, adrenic and α-linolenic namely F3-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes and F1-phytoprostanes, respectively have attracted attention because of their putative contribution to health. Since isoprostanoids are derived from different substrate of PUFAs and can have similar or opposing biological consequences, a total isoprostanoids profile is essential to understand the overall effect in the testing model. However, the concentration of most isoprostanoids range from picogram to nanogram, therefore a sensitive method to quantify 20 isoprostanoids simultaneously was formulated and measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The lipid portion from various biological samples was extracted prior to LC-MS/MS evaluation. For all the isoprostanoids LOD and LOQ, and the method was validated on plasma samples for matrix effect, yield of extraction and reproducibility were determined. The methodology was further tested for the isoprostanoids profiles in brain and liver of LDLR(-/-) mice with and without docosahexaenoic acid (DHA) supplementation. Our analysis showed similar levels of total F2-isoprostanes and F4-neuroprostanes in the liver and brain of non-supplemented LDLR(-/-) mice. The distribution of different F2-isoprostane isomers varied between tissues but not for F4-neuroprostanes which were predominated by the 4(RS)-4-F4t-neuroprostane isomer. DHA supplementation to LDLR(-/-) mice concomitantly increased total F4-neuroprostanes levels compared to F2-isoprostanes but this effect was more pronounced in the liver than brain.

DHA at nutritional doses restores insulin sensitivity in skeletal muscle by preventing lipotoxicity and inflammation.

Skeletal muscle plays a major role in the control of whole body glucose disposal in response to insulin stimulus. Excessive supply of fatty acids to this tissue triggers cellular and molecular disturbances leading to lipotoxicity, inflammation, mitochondrial dysfunctions, impaired insulin response and decreased glucose uptake. This study was conducted to analyze the preventive effect of docosahexaenoic acid (DHA), a long-chain polyunsaturated n-3 fatty acid, against insulin resistance, lipotoxicity and inflammation in skeletal muscle at doses compatible with nutritional supplementation. DHA (30 µM) prevented insulin resistance in C2C12 myotubes exposed to palmitate (500 µM) by decreasing protein kinase C (PKC)-θ activation and restoring cellular acylcarnitine profile, insulin-dependent AKT phosphorylation and glucose uptake. Furthermore, DHA protected C2C12 myotubes from palmitate- or lipopolysaccharide-induced increase in Ptgs2, interleukin 6 and tumor necrosis factor-α mRNA level, probably through the inhibition of p38 MAP kinase and c-Jun amino-terminal kinase. In LDLR -/- mice fed a high-cholesterol-high-sucrose diet, supplementation with DHA reaching up to 2% of daily energy intake enhanced the insulin-dependent AKT phosphorylation and reduced the PKC-θ activation in skeletal muscle. Therefore, DHA used at physiological doses participates in the regulation of muscle lipid and glucose metabolisms by preventing lipotoxicity and inflammation.

Non-enzymatic cyclic oxygenated metabolites of adrenic, docosahexaenoic, eicosapentaenoic and α-linolenic acids; bioactivities and potential use as biomarkers.

Cyclic oxygenated metabolites are formed in vivo through non-enzymatic free radical reaction of n-6 and n-3 polyunsaturated fatty acids (PUFAs) such as arachidonic (ARA C20:4 n-6), adrenic (AdA 22:4 n-6), α-linolenic (ALA 18:3 n-3), eicosapentaenoic (EPA 20:5 n-3) and docosahexaenoic (DHA 22:6 n-3) acids. These cyclic compounds are known as isoprostanes, neuroprostanes, dihomo-isoprostanes and phytoprostanes. Evidence has emerged for their use as biomarkers of oxidative stress and, more recently, the n-3PUFA-derived compounds have been shown to mediate bioactivities as secondary messengers. Accordingly, this review will focus on the cyclic oxygenated metabolites generated from AdA, ALA, EPA and DHA. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

The omega-3 fatty acid docosahexaenoic acid favorably modulates the inflammatory pathways and macrophage polarization within aorta of LDLR(-/-) mice.

The omega-3 fatty acid docosahexaenoic acid (DHA) has potent anti-atherogenic properties but its mechanisms of action at the vascular level remain poorly explored. Knowing the broad range of molecular targets of omega-3 fatty acids, microarray analysis was used to open-mindedly evaluate the effects of DHA on aorta gene expression in LDLR(-/-) mice and better understand its local anti-atherogenic action. Mice were fed an atherogenic diet and received daily oral gavages with oils rich in oleic acid or DHA. Bioinformatics analysis of microarray data first identified inflammation and innate immunity as processes the most affected by DHA supplementation within aorta. More precisely, several down-regulated genes were associated with the inflammatory functions of macrophages (e.g., CCL5 and CCR7), cell movement (e.g., ICAM-2, SELP, and PECAM-1), and the major histocompatibility complex (e.g., HLA-DQA1 and HLA-DRB1). Interestingly, several genes were identified as specific biomarkers of macrophage polarization, and their changes suggested a preferential orientation toward a M2 reparative phenotype. This observation was supported by the upstream regulator analysis highlighting the involvement of three main regulators of macrophage polarization, namely PPARγ (z-score = 2.367, p = 1.50 × 10(-13)), INFγ (z-score = -2.797, p = 2.81 × 10(-14)), and NFκB (z-score = 2.360, p = 6.32 × 10(-9)). Moreover, immunohistological analysis of aortic root revealed an increased abundance of Arg1 (+111 %, p = 0.01), a specific biomarker of M2 macrophage. The present study showed for the first time that DHA supplementation during atherogenesis is associated with protective modulation of inflammation and innate immunity pathways within aorta putatively through the orientation of plaque macrophages toward a M2 reparative phenotype.