Quantitative profiling of inflammatory and pro-resolving lipid mediators in human adolescents and mouse plasma using UHPLC-MS/MS

Objectives Lipid mediators are bioactive lipids which help regulate inflammation. We aimed to develop an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify 58 pro-inflammatory and pro-resolving lipid mediators in plasma, determine preliminary reference ranges for adolescents, and investigate how total parenteral nutrition (TPN) containing omega-3 polyunsaturated fatty acid (n-3 PUFA) or n-6 PUFA based lipid emulsions influence lipid mediator concentrations in plasma. Methods Lipid mediators were extracted from plasma using SPE and measured using UHPLC-MS/MS. EDTA plasma was collected from healthy adolescents between 13 and 17 years of age to determine preliminary reference ranges and from mice given intravenous TPN for seven days containing either an n-3 PUFA or n-6 PUFA based lipid emulsion. Results We successfully quantified 43 lipid mediators in human plasma with good precision and recovery including several leukotrienes, prostaglandins, resolvins, protectins, maresins, and lipoxins. We found that the addition of methanol to human plasma after blood separation reduces post blood draw increases in 12-hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE), 12S-hydroxyeicosatrienoic acid (12S-HETrE), 14-hydroxydocosahexaenoic acid (14-HDHA) and thromboxane B2 (TXB2). Compared to the n-6 PUFA based TPN, the n-3 PUFA based TPN increased specialized pro-resolving mediators such as maresin 1 (MaR1), MaR2, protectin D1 (PD1), PDX, and resolvin D5 (RvD5), and decreased inflammatory lipid mediators such as leukotriene B4 (LTB4) and prostaglandin D2 (PGD2). Conclusions Our method provides an accurate and sensitive quantification of 58 lipid mediators from plasma samples, which we used to establish a preliminary reference range for lipid mediators in plasma samples of adolescents; and to show that n-3 PUFA, compared to n-6 PUFA rich TPN, leads to a less inflammatory lipid mediator profile in mice

Executive functions and borderline personality features in adolescents with major depressive disorder

BackgroundExecutive functions (EF) consolidate during adolescence and are impaired in various emerging psychiatric disorders, such as pediatric Major Depressive Disorder (pMDD) and Borderline Personality Disorder. Previous studies point to a marked heterogeneity of deficits in EF in pMDD. We examined the hypothesis that deficits in EF in adolescents with pMDD might be related to comorbid Borderline Personality features (BPF).MethodsWe examined a sample of 144 adolescents (15.86 ± 1.32) diagnosed with pMDD. Parents rated their child’s EF in everyday life with the Behavior Rating Inventory of Executive Function (BRIEF) and BPF with the Impulsivity and Emotion Dysregulation Scale (IED-27). The adolescents completed equivalent self-rating measures. Self- and parent-ratings of the BRIEF scores were compared with paired t-Tests. Correlation and parallel mediation analyses, ICC, and multiple regression analyses were used to assess symptom overlap, parent-child agreement, and the influence of depression severity.ResultsOver the whole sample, none of the self- or parent-rated BRIEF scales reached a mean score above T > 65, which would indicate clinically impaired functioning. Adolescents tended to report higher impairment in EF than their parents. Depression severity was the strongest predictor for BPF scores, with Emotional Control predicting parent-rated BPF and Inhibit predicting self-rated BPF. Furthermore, the Behavioral Regulation Index, which includes EF closely related to behavioral control, significantly mediated the relationship between depression severity and IED-27 factors emotional dysregulation and relationship difficulties but not non-suicidal self-injuries.ConclusionOn average, adolescents with depression show only subtle deficits in executive functioning. However, increased EF deficits are associated with the occurrence of comorbid borderline personality features, contributing to a more severe overall psychopathology. Therefore, training of executive functioning might have a positive effect on psychosocial functioning in severely depressed adolescents, as it might also improve comorbid BPF.Clinical trial registrationwww.ClinicalTrials.gov, identifier NCT03167307

Effect of Omega-3 Polyunsaturated Fatty Acid Supplementation on Lipid Mediator Profiles

Lipid mediators are bioactive lipids, produced from polyunsaturated fatty acids (PUFAs), that are important regulators of inflammation. Inflammatory lipid mediators such as leukotriene B4 (LTB4) and 2-series prostanoids aid in the recruitment of leukocytes and are produced from the n-6 PUFA, arachidonic acid (AA). Conversely, the resolution of inflammation is coordinated by a group of lipid mediators termed “specialized pro-resolving mediators” (SPMs) which include lipoxins (LX) produced from AA, as well as n-3 PUFA-derived resolvins (D-series (RvD) or E-series), maresins (MaR), and protectins (PD). Increased intake of n-3 PUFAs is associated with many health benefits, and replacement of the n-6 PUFA rich soybean oil typically used in total parenteral nutrition (TPN) with an n-3 PUFA rich oil is known to prevent TPN-associated liver disease. The benefits of n-3 PUFAs are assumed to come primarily from the increased production of SPMs. Therefore, measurements of lipid mediators provide valuable insight into the inflammatory conditions in a given system

Influence of bacteria on menaquinone concentrations in fermented dairy products

Menaquinones (MK’s) are a form of vitamin K, supplementation with which has been shown to provide health benefits by reducing arterial calcification and improving bone density. Fortifying MK’s in foods may provide the health benefits associated with MK’s to the general population. This study aimed to increase the amount of MK’s in yogurt, which typically contains very little MK’s, by co-fermenting milk with both typical yogurt bacteria and bacteria known to produce MK’s in cheese and natto. A LC-MS/MS method was first developed to measure MK’s as well as all other fat-soluble vitamins. The LC-MS/MS method was able to quantify phylloquinone, MK-4, MK-7, MK-9, cholecalciferol, α-tocopherol, and was able to detect MK-8, but was unable to quantify ergocalciferol or retinol due to ionization suppression from the sample matrix. The LC-MS/MS method was used to measure MK’s in non-fat milk samples fermented with either one bacteria ssp. or a combination of S. thermophilus, L. bulgaricus, and one of L. lactis, L. cremoris, P. shermanii, or B. subtilis. Fermentations were carried out at 30 – 45 °C for 36 h to determine the optimal incubation temperature at which to carry out replicate fermentations. The pH was measured throughout fermentation as an indicator of overall growth and the relative amount of each bacteria ssp. after fermentation was measured using qPCR. Only L. cremoris produced MK-8 and MK-9, while all samples, including negative controls, contained high concentrations of MK-7. The qPCR results showed co-fermentations contained over 99 % S. thermophilus, except in co-fermentations with L. lactis which contained 59 % L. lactis, 41 % S. thermophilus, and < 0.1 % L. bulgaricus. Delaying inoculation of S. thermophilus and L. bulgaricus resulted in almost exclusive growth of the first inoculant. Overall, L. cremoris showed the greatest potential for enhancing the MK content of yogurt as it produced up to 234.4 ng/g MK-9, 174.9 ng/g MK-7, and produced MK-8, but further experiments are required to improve the growth of yogurt and MK producing bacteria.Land and Food Systems, Faculty ofGraduat

Resolvin D1 reduces inflammation in co-cultures of primary human macrophages and adipocytes by triggering macrophages

Obesity leads to chronic inflammation of the adipose tissue which is tightly associated with the metabolic syndrome, type 2 diabetes and cardiovascular disease. Inflammation of the adipose tissue is mainly characterized by the presence of crown-like structures composed of inflammatory macrophages in the neighborhood of adipocytes. Resolvin D1 (RvD1), a potent anti-inflammatory and pro-resolving lipid mediator derived from the omega-3 fatty acid docosahexaenoic acid, has been shown to reduce the inflammatory tone of adipose tissue in animal models but the underlying mechanism is not clear. We investigated the effect of RvD1 on the inflammatory state of a human co-culture system of adipocytes and macrophages. For this, human mesenchymal stem cells were differentiated into mature adipocytes and overlaid with human primary macrophages. In this co-culture, 10-500 nM RvD1 dose-dependently reduced the secretion of the pro-inflammatory cytokine IL-6 (-21%) and its soluble receptor IL-6Rα (-22%), of the chemokine MCP-1 (-13%), and of the adipokine leptin (-22%). Similarly, we observed a reduction in secretion of the soluble receptor IL-6Rα (-20%), and TNF-α (-11%) when macrophages alone were treated with RvD1, while no change of cytokine secretion was observed when adipocytes were treated with RvD1. We conclude that RvD1 polarizes macrophages to an anti-inflammatory phenotype, which in turn modulates inflammation in adipocytes

Quantitative profiling of inflammatory and proresolving lipid mediators in human adolescents and mouse plasma using UHPLC-MS/MS

Objectives Lipid mediators are bioactive lipids which help regulate inflammation. We aimed to develop an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify 58 pro-inflammatory and pro-resolving lipid mediators in plasma, determine preliminary reference ranges for adolescents, and investigate how total parenteral nutrition (TPN) containing omega-3 polyunsaturated fatty acid (n-3 PUFA) or n-6 PUFA based lipid emulsions influence lipid mediator concentrations in plasma. Methods Lipid mediators were extracted from plasma using SPE and measured using UHPLC-MS/MS. EDTA plasma was collected from healthy adolescents between 13 and 17 years of age to determine preliminary reference ranges and from mice given intravenous TPN for seven days containing either an n-3 PUFA or n-6 PUFA based lipid emulsion. Results We successfully quantified 43 lipid mediators in human plasma with good precision and recovery including several leukotrienes, prostaglandins, resolvins, protectins, maresins, and lipoxins. We found that the addition of methanol to human plasma after blood separation reduces post blood draw increases in 12-hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE), 12S-hydroxyeicosatrienoic acid (12S-HETrE), 14-hydroxydocosahexaenoic acid (14-HDHA) and thromboxane B2 (TXB2). Compared to the n-6 PUFA based TPN, the n-3 PUFA based TPN increased specialized pro-resolving mediators such as maresin 1 (MaR1), MaR2, protectin D1 (PD1), PDX, and resolvin D5 (RvD5), and decreased inflammatory lipid mediators such as leukotriene B4 (LTB4) and prostaglandin D2 (PGD2). Conclusions Our method provides an accurate and sensitive quantification of 58 lipid mediators from plasma samples, which we used to establish a preliminary reference range for lipid mediators in plasma samples of adolescents; and to show that n-3 PUFA, compared to n-6 PUFA rich TPN, leads to a less inflammatory lipid mediator profile in mice.ISSN:1434-6621ISSN:1437-433

Lipid emulsion rich in n-3 polyunsaturated fatty acids elicits a pro-resolution lipid mediator profile in mouse tissues and in human immune cells

BACKGROUND Lipid emulsions are a key component of total parenteral nutrition (TPN) and are administered to patients who are unable to ingest their daily required calories orally. Lipid emulsions rich with n-6 (ω-6) PUFAs are known to cause parenteral nutrition-associated liver disease and have inflammatory side effects, whereas n-3 PUFA-rich emulsions have favourable clinical outcomes. OBJECTIVES The present study used targeted lipid mediator analysis to investigate the metabolism of a n-3 PUFA-rich lipid emulsion and a n-6 PUFA-rich lipid emulsion in a mouse model of TPN and in primary human monocyte-derived macrophages (MDMs) and CD4+ T cells. RESULTS Mice given n-3 PUFA-based TPN for 7 d had a less proinflammatory lipid mediator profile compared with those receiving n-6 PUFA-based TPN. This was characterized by higher concentrations of specialized pro-resolving mediators (SPMs) and endocannabinoids, including resolvin D (RvD) 1, maresin (MaR) 1, MaR2, protectin D1 (PD1), protectin DX (PDX), and the endocannabinoids eicosapentaenoyl ethanolamide (EPEA) and docosahexaenoyl ethanolamide (DHEA) in the liver and RvD1, 17R-RvD1, RvD2, RvD3, RvD5, MaR1, MaR2, PD1, PDX, and EPEA and DHEA in the spleen. The spleen was identified as a source of high lipid mediator and SPM formation as lipid mediator concentrations were on average 25-fold higher than in the liver. Additionally, n-3 PUFA-treated primary human MDMs produced RvD5 and the endocannabinoids EPEA and DHEA, which was associated with an increased IL-10 secretion. In contrast, primary human CD4+ T cells showed only an increase in SPM precursors and an increase in the endocannabinoids EPEA and DHEA, which was associated with reduced cytokine expression. CONCLUSIONS This demonstrates that lipid mediators, particularly SPMs and endocannabinoids from spleen, could play a key role in facilitating the favorable clinical outcomes associated with the use of n-3 PUFA-rich lipid emulsions in TPN

Lipid emulsion rich in n-3 polyunsaturated fatty acids elicits a pro-resolution lipid mediator profile in mouse tissues and in human immune cells

Background Lipid emulsions are a key component of total parenteral nutrition (TPN) and are administered to patients who are unable to ingest their daily required calories orally. Lipid emulsions rich with n–6 (ω-6) PUFAs are known to cause parenteral nutrition–associated liver disease and have inflammatory side effects, whereas n–3 PUFA-rich emulsions have favourable clinical outcomes. Objectives The present study used targeted lipid mediator analysis to investigate the metabolism of a n–3 PUFA-rich lipid emulsion and a n–6 PUFA-rich lipid emulsion in a mouse model of TPN and in primary human monocyte-derived macrophages (MDMs) and CD4+ T cells. Results Mice given n–3 PUFA-based TPN for 7 d had a less proinflammatory lipid mediator profile compared with those receiving n–6 PUFA-based TPN. This was characterized by higher concentrations of specialized pro-resolving mediators (SPMs) and endocannabinoids, including resolvin D (RvD) 1, maresin (MaR) 1, MaR2, protectin D1 (PD1), protectin DX (PDX), and the endocannabinoids eicosapentaenoyl ethanolamide (EPEA) and docosahexaenoyl ethanolamide (DHEA) in the liver and RvD1, 17R-RvD1, RvD2, RvD3, RvD5, MaR1, MaR2, PD1, PDX, and EPEA and DHEA in the spleen. The spleen was identified as a source of high lipid mediator and SPM formation as lipid mediator concentrations were on average 25-fold higher than in the liver. Additionally, n–3 PUFA-treated primary human MDMs produced RvD5 and the endocannabinoids EPEA and DHEA, which was associated with an increased IL-10 secretion. In contrast, primary human CD4+ T cells showed only an increase in SPM precursors and an increase in the endocannabinoids EPEA and DHEA, which was associated with reduced cytokine expression. Conclusions This demonstrates that lipid mediators, particularly SPMs and endocannabinoids from spleen, could play a key role in facilitating the favorable clinical outcomes associated with the use of n–3 PUFA-rich lipid emulsions in TPN.ISSN:0002-9165ISSN:1938-320

The effects of farnesoid X receptor activation on arachidonic acid metabolism, NF-kB signaling and hepatic inflammation

Inflammation has a recognized role in non-alcoholic fatty liver disease (NAFLD) progression. The present work studied the effect of high fat diet (HFD) on arachidonic acid metabolism in the liver and investigated the role of the farnesoid X receptor (FXR, NR1H4) in eicosanoid biosynthetic pathways and NF-kB signaling, major modulators of the inflammatory cascade. Mice were fed a HFD to induce NAFLD, then, treated with the FXR ligand obeticholic acid (OCA). Histology and gene expression analysis were performed on liver tissue. Eicosanoid levels were measured from serum and urine samples. The molecular mechanism underlying the effect of FXR activation on arachidonic acid metabolism and NF-kB signaling was studied in Huh7 cells and primary cultured hepatocytes. NAFLD was characterized by higher (~25%) pro-inflammatory (leukotrienes, LTB4) and lower (~3fold) anti-inflammatory (epoxyeicosatrienoic acids, EETs) eicosanoid levels than in chow mice. OCA induced the expression of several hepatic Cyp450 epoxygenases, the enzymes responsible for EET synthesis, and mitigated HFD-induced hepatic injury. In vitro, induction of CYP450 epoxygenases was sufficient to inhibit NF-kB signaling and cell migration. The CYP450 epoxygenase pan-inhibitor gemfibrozil fully abolished the protective effect of OCA indicating that OCA-mediated inhibition of NF-kB signaling was EET-dependent. In summary non-alcoholic fatty liver disease (NAFLD) was characterized by an imbalance in arachidonate metabolism. Farnesoid X receptor (FXR) activation reprogramed arachidonate metabolism by inducing CYP450 epoxygenase expression and EET production. In vitro, FXR-mediated NF-kB inhibition, required active CYP450 epoxygenases