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Prostanoids are potent mediators of many physiological and pathophysiological processes. Of the many analytical methodologies used for their qualitative and quantitative analysis, electrospray tandem mass spectrometry coupled to liquid chromatography (LC/ESI-MS/MS) offers a rapid, sensitive and versatile system applicable to lipidomic analyses. We have developed an LC/ESI-MS/MS assay for twenty-seven mediators including prostaglandins, prostacyclines, thromboxanes, dihydroprostaglandins and isoprostanes. The assay was liner over the concentration range 1-100 pg/microL. The limits of detection and quantitation were 0.5-50 and 2-100 pg, respectively, whilst recoveries were from 83-116% depending on the metabolite. The assay can be applied to the profiling of prostanoids produced by a variety of biological fluids and extracts including brain, liver, plasma and urine, thus facilitating our understanding of the role of these lipid mediators in health and disease, as well as assisting in drug development.

A wide range of biologically active lipids such as eicosanoids, docosanoids, and other lipid mediators are known for their physiological and pathophysiological effects in almost every organ system. It is essential that the full spectrum of these compounds is comprehensively captured, to fully characterize their biological activity. The analytical determination of these lipid mediators is challenging, however, mainly because of their biological diversity and their physicochemical similarity. LC-MS/MS, in particular in combination with high-resolution instruments, is currently the most powerful tool for analysis of lipid mediators. This chapter describes a comprehensive analytical protocol that allows for simultaneous profiling of over 100 polyunsaturated fatty acid (PUFA)-derived metabolites using a single analytical method from a wide range of biological matrices.

Obesity-associated low-grade inflammation of white adipose tissue (WAT) contributes to development of insulin resistance and other disorders. Accumulation of immune cells, especially macrophages, and macrophage polarization from M2 to M1 state, affect intrinsic WAT signaling, namely anti-inflammatory and proinflammatory cytokines, fatty acids (FA), and lipid mediators derived from both n-6 and n-3 long-chain PUFA such as (i) arachidonic acid (AA)-derived eicosanoids and endocannabinoids, and (ii) specialized pro-resolving lipid mediators including resolvins derived from both eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), lipoxins (AA metabolites), protectins and maresins (DHA metabolites). In this respect, potential differences in modulating adipocyte metabolism by various lipid mediators formed by inflammatory M1 macrophages typical of obese state, and non-inflammatory M2 macrophages typical of lean state remain to be established. Studies in mice suggest that (i) transient accumulation of M2 macrophages could be essential for the control of tissue FA levels during activation of lipolysis, (ii) currently unidentified M2 macrophage-borne signaling molecule(s) could inhibit lipolysis and re-esterification of lipolyzed FA back to triacylglycerols (TAG/FA cycle), and (iii) the egress of M2 macrophages from rebuilt WAT and removal of the negative feedback regulation could allow for a full unmasking of metabolic activities of adipocytes. Thus, M2 macrophages could support remodeling of WAT to a tissue containing metabolically flexible adipocytes endowed with a high capacity of both TAG/FA cycling and oxidative phosphorylation. This situation could be exemplified by a combined intervention using mild calorie restriction and dietary supplementation with EPA/DHA, which enhances the formation of "healthy" adipocytes. This article is part of a Special Issue entitled Oxygenated metabolism of PUFA: analysis and biological relevance."

This study shows that prostaglandins in human FM55 melanoma cells and epidermal melanocytes are produced by COX-1. Prostaglandin production in FM55 melanoma cells was unrelated to that of melanin suggesting that the two processes can occur independently. Alpha-melanocyte-stimulating hormone, which had no effect on melanin production in FM55 cells, stimulated PGD(2) production in these cells without affecting PGE(2). While cAMP pathways may be involved in regulating PGD(2) production, our results suggest that alpha-MSH acts independently of cAMP, possibly by regulating the activity of lipocalin-type PGD synthase. This alpha-MSH-mediated effect may be associated with its role as an immune modulator.

A wide range of biologically active lipids such as eicosanoids, docosanoids, and other lipid mediators are known for their physiological and pathophysiological effects in almost every organ system. It is essential that the full spectrum of these compounds is comprehensively captured, to fully characterize their biological activity. The analytical determination of these lipid mediators is challenging, however, mainly because of their biological diversity and their physicochemical similarity. LC-MS/MS, in particular in combination with high-resolution instruments, is currently the most powerful tool for analysis of lipid mediators. This chapter describes a comprehensive analytical protocol that allows for simultaneous profiling of over 100 polyunsaturated fatty acid (PUFA)-derived metabolites using a single analytical method from a wide range of biological matrices.

OBJECTIVES: Ulcerative colitis (UC) is a relapsing inflammatory disorder of unconfirmed aetiology, variable severity and clinical course, characterised by progressive histological inflammation and with elevation of eicosanoids which have a known pathophysiological role in inflammation. Therapeutic interventions targetting eicosanoids (5-aminosalicylates (ASA)) are effective first line and adjunctive treatments in mild-moderate UC for achieving and sustaining clinical remission. However, the variable clinical response to 5-ASA and frequent deterioration in response to cyclo-oxygenase (COX) inhibitors, has prompted an in depth simultaneous evaluation of multiple lipid mediators (including eicosanoids) within the inflammatory milieu in UC. We hypothesised that severity of inflammation is associated with alteration of lipid mediators, in relapsing UC.DESIGN: Study was case-control design. Mucosal lipid mediators were determined by LC-MS/MS lipidomics analysis on mucosal biopsies taken from patients attending outpatients with relapsing UC. Univariate and multivariate statistical analyses were used to investigate the association of mucosal lipid mediators, with the disease state and severity graded histologically.RESULTS: Levels of PGE2, PGD2, TXB2, 5-HETE, 11-HETE, 12-HETE and 15-HETE are significantly elevated in inflamed mucosa and correlate with severity of inflammation, determined using validated histological scoring systems.CONCLUSIONS: Our approach of capturing inflammatory mediator signature at different stages of UC by combining comprehensive lipidomics analysis and computational modelling could be used to classify and predict mild-moderate inflammation; however, predictive index is diminished in severe inflammation. This new technical approach could be developed to tailor drug treatments to patients with active UC, based on the mucosal lipid mediator profile.

Adipocytes store excess energy in the form of triglycerides and signal the levels of stored energy to the brain. Here we show that adipocyte-specific deletion of Arntl (also known as Bmal1), a gene encoding a core molecular clock component, results in obesity in mice with a shift in the diurnal rhythm of food intake, a result that is not seen when the gene is disrupted in hepatocytes or pancreatic islets. Changes in the expression of hypothalamic neuropeptides that regulate appetite are consistent with feedback from the adipocyte to the central nervous system to time feeding behavior. Ablation of the adipocyte clock is associated with a reduced number of polyunsaturated fatty acids in adipocyte triglycerides. This difference between mutant and wild-type mice is reflected in the circulating concentrations of polyunsaturated fatty acids and nonesterified polyunsaturated fatty acids in hypothalamic neurons that regulate food intake. Thus, this study reveals a role for the adipocyte clock in the temporal organization of energy regulation, highlights timing as a modulator of the adipocyte-hypothalamic axis and shows the impact of timing of food intake on body weight.

In this study, we define a new role for lipocalin prostaglandin D synthase (L-PGDS) in the control of metabolic fuel utilization by brown adipose tissue (BAT). We demonstrate that L-PGDS expression in BAT is positively correlated with BAT activity, upregulated by peroxisome proliferator–activated receptor γ coactivator 1α or 1β and repressed by receptor-interacting protein 140. Under cold-acclimated conditions, mice lacking L-PGDS had elevated reliance on carbohydrate to provide fuel for thermogenesis and had increased expression of genes regulating glycolysis and de novo lipogenesis in BAT. These transcriptional differences were associated with increased lipid content in BAT and a BAT lipid composition enriched with de novo synthesized lipids. Consistent with the concept that lack of L-PGDS increases glucose utilization, mice lacking L-PGDS had improved glucose tolerance after high-fat feeding. The improved glucose tolerance appeared to be independent of changes in insulin sensitivity, as insulin levels during the glucose tolerance test and insulin, leptin, and adiponectin levels were unchanged. Moreover, L-PGDS knockout mice exhibited increased expression of genes involved in thermogenesis and increased norepinephrine-stimulated glucose uptake to BAT, suggesting that sympathetically mediated changes in glucose uptake may have improved glucose tolerance. Taken together, these results suggest that L-PGDS plays an important role in the regulation of glucose utilization in vivo.

Nutrition during development affects risk of future cardiovascular disease. Relatively little is known about whether the amount and type of fat in the maternal diet affect vascular function in the offspring. To investigate this, pregnant and lactating rats were fed either 7%(w/w) or 21%(w/w) fat enriched in either18:2n-6, trans fatty acids, saturated fatty acids, or fish oil. Their offspring were fed 4%(w/w) soybean oil from weaning until day 77. Type and amount of maternal dietary fat altered acetylcholine (ACh)-mediated vaso-relaxation in offspring aortae and mesenteric arteries, contingent on sex. Amount, but not type, of maternal dietary fat altered phenylephrine (Pe)-induced vasoconstriction in these arteries. Maternal 21% fat diet decreased 20:4n-6 concentration in offspring aortae. We investigated the role of ?6 and ?5 desaturases, showing that their inhibition in aortae and mesenteric arteries reduced vasoconstriction, but not vaso-relaxation, and the synthesis of specific pro-constriction eicosanoids. Removal of the aortic endothelium did not alter the effect of inhibition of ?6 and ?5 desaturases on Pe-mediated vasoconstriction. Thus arterial smooth muscle 20:4n-6 biosynthesis de novo appears to be important for Pe-mediated vasoconstriction. Next we studied genes encoding these desaturases, finding that maternal 21% fat reduced Fads2 mRNA expression and increased Fads1 in offspring aortae, indicating dysregulation of 20:4n-6 biosynthesis. Methylation at CpG -394 bp 5' to the Fads2 transcription start site predicted its expression. This locus was hypermethylated in offspring of dams fed 21% fat. Pe treatment of aortae for 10 minutes increased Fads2, but not Fads1, mRNA expression (76%; P

High personal UVR doses can be gained during leisure activities, causing intense self-resolving inflammation (sunburn) of unprotected skin. UVR activates release of membrane fatty acids and upregulates their metabolism by cyclooxygenases (COX) and lipoxygenases (LOX) to different eicosanoids. While COX-derived prostaglandin (PG)E2 is a potent mediator of sunburn vasodilatation, LOX-derived 15-hydroxyeicosatetraenoic acid (HETE) and its lipoxin metabolites may contribute to sunburn limitation. We explored the relationships between expression of these lipid mediators and the clinical and histological outcomes, comparing responses of individuals prone and more resistant to sunburn. An acute UVR exposure of 12 SED (standard erythema dose) was applied to buttock skin of 32 white Caucasians (n = 16 phototype I/II, n = 16 phototype III/IV), and over the subsequent 72 h assessments were made of skin erythema, immunohistochemical expression of leukocyte markers, COX-2, 12-LOX, 15-LOX and nitric oxide synthase (NOS), and eicosanoid levels by LC/ESI-MS/MS. Evidence of a significant inflammatory response was seen earlier in phototype I/II with regard to expression of erythema (4h, p < 0.001), neutrophil infiltration (24 h, p = 0.01), epidermal COX-2 (24 h, p < 0.05) and 12-LOX (24 h, p < 0.01), and dermal eNOS (24 h, p < 0.05) proteins, although CD3+ lymphocyte infiltration showed an earlier increase in phototype III/IV (24 h, p < 0.05). Although erythema was equivalent at 72 h in both groups, phototype I/II showed higher PGE2 accompanied by elevated 15-HETE, and a strong positive correlation was seen between these mediators (n = 18, r = 0.805, p = 0.0001). Hence anti-inflammatory eicosanoid 15-HETE may temper the pro-inflammatory milieu in sunburn, having greater influence in those prone to sunburn than those more resistant, given the same high UVR exposure conditions.

A number of recent studies suggest that mitochondrial oxidative damage may be associated with atherosclerosis and the metabolic syndrome. However, much of the evidence linking mitochondrial oxidative damage and excess reactive oxygen species (ROS) with these pathologies is circumstantial. Consequently the importance of mitochondrial ROS in the etiology of these disorders is unclear. Furthermore, the potential of decreasing mitochondrial ROS as a therapy for these indications is not known. We assessed the impact of decreasing mitochondrial oxidative damage and ROS with the mitochondria-targeted antioxidant MitoQ in models of atherosclerosis and the metabolic syndrome (fat-fed ApoE(-/-) mice and ATM(+/-)/ApoE(-/-) mice, which are also haploinsufficient for the protein kinase, ataxia telangiectasia mutated (ATM). MitoQ administered orally for 14weeks prevented the increased adiposity, hypercholesterolemia, and hypertriglyceridemia associated with the metabolic syndrome. MitoQ also corrected hyperglycemia and hepatic steatosis, induced changes in multiple metabolically relevant lipid species, and decreased DNA oxidative damage (8-oxo-G) in multiple organs. Although MitoQ did not affect overall atherosclerotic plaque area in fat-fed ATM(+/+)/ApoE(-/-) and ATM(+/-)/ApoE(-/-) mice, MitoQ reduced the macrophage content and cell proliferation within plaques and 8-oxo-G. MitoQ also significantly reduced mtDNA oxidative damage in the liver. Our data suggest that MitoQ inhibits the development of multiple features of the metabolic syndrome in these mice by affecting redox signaling pathways that depend on mitochondrial ROS such as hydrogen peroxide. These findings strengthen the growing view that elevated mitochondrial ROS contributes to the etiology of the metabolic syndrome and suggest a potential therapeutic role for mitochondria-targeted antioxidants.

In the present work we describe the development of an analytical technique for simultaneous profiling of over 100 biochemically related lipid mediators in biological samples. A multistep procedure was implemented to extract eicosanoids and other bioactive lipids from the biological matrix, chromatographically separate them using fast reversed-phase liquid chromatography, tentatively identify new candidate eicosanoids through a matching process of retention times, isotope distribution patterns, and high-resolution orbitrap MS/MS fragmentation patterns, and subsequently quantify tentative candidates by means of analytical reference standards. Key new aspects of this profiling technique included the classification of bioactive lipids into 12 groups according to their calculated exact masses and the development of optimized liquid chromatographic conditions for these groups to achieve sufficient separation of the numerous isobaric and isomeric species, many of which exhibited virtually identical collision-induced dissociation behavior. Importantly, no analytical standards were required at this screening stage of the assay, and tentative identifications were achieved by matching results to selected reference species from each of the groups. The analytical figures of merit for the orbitrap assay such as linear dynamic range, limit of detection, limit of quantitation, and precision demonstrated that the performance of the assay was very similar to that of a quadrupole linear ion trap assay, which was used for validation purposes. The method allowed us to examine eicosanoid profiles within the signaling cascade in chronic lymphocytic leukemia (CLL) cells under basal conditions and following arachidonic acid stimulation. The preliminary screening based on high-resolution tandem mass spectrometry data along with isotope pattern and retention time matching revealed the presence of 15 bioactive lipids, belonging to a range of prostaglandin, leukotriene, and hydroxy and epoxy fatty acid lipid mediators produced by CLL cells.

Lipidomics aim to generate qualitative and quantitative information on different classes of lipids and their species, and when applied in conjunction with proteomic and genomic assays, facilitate the comprehensive study of lipid metabolism in cellular, organ, or body systems. Advances in mass spectrometry have underpinned the expansion of lipidomic methodologies. Prostanoids are potent autacoids present in a plethora of cellular systems, known best for their intimate role in inflammation. Electrospray ionisation (ESI) allows the efficient ionisation of prostanoids in aqueous systems. ESI can be readily coupled to liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS)-based detection, thus allowing the development of a potent and selective LC/ESI-MS/MS quantitative assays. The protocol we describe in this chapter outlines the steps we follow to (a) extract prostanoids from solid or liquid samples, (b) semi-purify the metabolites using solid phase extraction (c) set-up the HPLC separation using reverse phase chromatography and (d) set-up the MS/MS assay using a triple quadrupole mass spectrometer. The experimental details and notes presented here are based on the detailed protocols followed in our group.

Lipidomics aim to generate qualitative and quantitative information on different classes of lipids and their species, and when applied in conjunction with proteomic and genomic assays, facilitate the comprehensive study of lipid metabolism in cellular, organ, or body systems. Advances in mass spectrometry have underpinned the expansion of lipidomic methodologies. Prostanoids are potent autacoids present in a plethora of cellular systems, known best for their intimate role in inflammation. Electrospray ionisation (ESI) allows the efficient ionisation of prostanoids in aqueous systems. ESI can be readily coupled to liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS)-based detection, thus allowing the development of a potent and selective LC/ESI-MS/MS quantitative assays. The protocol we describe in this chapter outlines the steps we follow to (a) extract prostanoids from solid or liquid samples, (b) semi-purify the metabolites using solid phase extraction (c) set-up the HPLC separation using reverse phase chromatography and (d) set-up the MS/MS assay using a triple quadrupole mass spectrometer. The experimental details and notes presented here are based on the detailed protocols followed in our group.

Introduction Ulcerative colitis (UC) is a relapsing remitting disorder of the colon with a recognised role for certain eicosanoid mediators derived from polyunsaturated lipid substrates. However, a detailed characterisation of the eicosanoids involved in UC is currently lacking. Using a comprehensive lipidomics approach, we profiled eicosanoids that could exhibit both pro- and anti-inflammatory function in inflamed and non-inflamed colonic mucosal biopsies from UC patients. Methods Biopsies were taken from inflamed and nearby non-inflamed colonic mucosa (69 patients, 54 with paired inflamed and non-inflamed mucosa) from patients with symptomatic relapses. Inflammation was scored endoscopically and histologically. Mucosal lipid mediators were determined by LC-MS/MS lipidomics analysis. Univariate and multivariate statistical analyses were used to investigate the association of lipid mediators with the disease state as well as histologically assessed disease severity (using the Gomes scoring system). Results Arachidonic acid (AA), but not eicosapentaenoic acid (EPA), derived eicosanoids (prostaglandin (PG)E2, PGD2, thromboxaneB2, 5-hydroxy-eicosatetraenoic acid (HETE), 11-HETE, 12-HETE and 15-HETE), were significantly (p Conclusion There is an upregulation of AA derived inflammatory mediators in UC. This research suggests new eicosanoid targets for research and therapeutic intervention. Disclosure of Interest None Declared

Introduction Ulcerative colitis (UC) is a colonic inflammatory disorder of unconfirmed aetiology. Eicosanoids, inflammatory mediators involved in UC pathogenesis, are enzymatically converted from dietary polyunsaturated fatty acids (PUFA), arachidonic acid (AA) and eicosapentaenoic acid (EPA), themselves competitive substrates also generated via a fatty acid (FA) biosynthetic cascade. Dietary studies using fish oil-derived EPA have been disappointing in UC; we hypothesised that the PUFA biosynthetic pathway in inflamed tissue is altered. This study evaluated PUFA profile in inflamed and non-inflamed mucosa from UC patients and compared to matched controls. Methods Ethical approval was obtained. Patients were prospectively recruited from outpatients9 clinics. Mucosal biopsies at flexible sigmoidoscopy (FS) were taken from UC patients within inflamed and normal proximal mucosa. Age-sex matched control patients undergoing FS for functional symptoms were compared. Inflammation was scored endoscopically and histologically. Membrane bound FA (MBFA): Biopsies were spiked with deuterated internal standard, followed by liquid-liquid extraction and quantitative gas chromatography mass spectrometry (MS). Free Fatty Acid (FFA): Biopsies were homogenised, followed by solid phase extraction and liquid chromatography orbitrap MS. Data were expressed as percentage abundance. Dietary fatty acid analysis was undertaken. Wilcoxon signed rank pair and Spearman9s correlation analysis were employed. Results 69 active UC patients (54 paired normal/inflamed mucosa) and 69 controls were compared. No biologically significant differences were noted between endoscopically normal mucosa from UC patients and controls other than DPA (p Conclusion Mucosal PUFA bioavailability is altered in active UC, with significant elevation of AA and reduction of LA, αLNA and EPA. This suggests modification of the FA biosynthetic pathway with elevated delivery of AA as a precursor of pro-inflammatory eicosanoids in active UC. These findings may explain the lack of efficacy of supplemental fish oil and suggests new alternative therapeutic targets. Competing interests None declared.

The peroxisome proliferator-activated receptors (PPARs) are a set of three nuclear hormone receptors that together play a key role in regulating metabolism, particularly the switch between the fed and fasted state and the metabolic pathways involving fatty-acid oxidation and lipid metabolism. In addition, they have a number of important developmental and regulatory roles outside metabolism. The PPARs are also potent targets for treating type II diabetes, dyslipidemia and obesity, although a number of individual agonists have also been linked to unwanted side effects, and there is a complex relationship between the PPARs and the development of cancer. This review examines the part that metabolomics, including lipidomics, has played in elucidating the roles PPARs have in regulating systemic metabolism, as well as their role in aspects of drug-induced cancer and xenobiotic metabolism. These studies have defined the role PPARδ plays in regulating fatty-acid oxidation in adipose tissue and the interaction between aging and PPARα in the liver. The potential translational benefits of these approaches include widening the role of PPAR agonists and improved monitoring of drug efficacy.

Glucose is the primary source of energy for the brain but also an important source of building blocks for proteins, lipids, and nucleic acids. Little is known about the use of glucose for biosynthesis in tissues at the cellular level. We demonstrate that local cerebral metabolic activity can be mapped in mouse brain tissue by quantitatively imaging the biosynthetic products deriving from [U-(13)C]glucose metabolism using a combination of in situ electron microscopy and secondary ion mass-spectroscopy (NanoSIMS). Images of the (13)C-label incorporated into cerebral ultrastructure with ca. 100 nm resolution allowed us to determine the timescale on which the metabolic products of glucose are incorporated into different cells, their sub-compartments and organelles. These were mapped in astrocytes and neurons in the different layers of the motor cortex. We see evidence for high metabolic activity in neurons via the nucleus (13)C enrichment. We observe that in all the major cell compartments, such as e.g. nucleus and Golgi apparatus, neurons incorporate substantially higher concentrations of (13)C-label than astrocytes.

Background: Micronutrient supplementation has been extensively explored as a strategy to improve health and reduce risk of chronic diseases. Fat-soluble vitamins like A and E with their antioxidant properties and mechanistic interactions with lipoproteins, have potentially a key impact on lipid metabolism and lipidemia., Objective: The impact of micronutrients on lipid metabolism requires further investigation including characterization of plasma lipidome following supplementation and any cause-effect on circulating lipids., Design: In this study, we elucidate the effect and associations of a multi-micronutrient intervention in Brazilian children and teens with lipoprotein alterations and lipid metabolism., Results: Our analysis suggests a combination of short and long-term impact of supplementation on lipid metabolism, potentially mediated primarily by alpha-tocopherol (vitamin E) and retinol (vitamin A). Among the lipid classes, levels of phospholipids, lysophospholipids, and cholesterol esters were impacted the most along with differential incorporation of stearic, palmitic, oleic and arachidonic acids. Integrated analysis with proteomic data suggested potential links to supplementation-mediated alterations in protein levels of phospholipases and pyruvate dehydrogenase kinase 1 (PDK1)., Conclusions: Associations between the observed differences in lipidemia, total triglyceride, and VLDL-cholesterol levels suggest that micronutrients may play a role in reducing these risk factors for cardiovascular disease in children. This would require further investigation. (C) 2019 The Author(s). Published by Elsevier Ltd.

In mammals, glycogen synthesis and degradation are dynamic processes regulating blood and cerebral glucose-levels within a well-defined physiological range. Despite the essential role of glycogen in hepatic and cerebral metabolism, its spatiotemporal distribution at the molecular and cellular level is unclear. By correlating electron microscopy and ultra-high resolution ion microprobe (NanoSIMS) imaging of tissue from fasted mice injected with (13)C-labeled glucose, we demonstrate that liver glycogenesis initiates in the hepatocyte perinuclear region before spreading toward the cell membrane. In the mouse brain, we observe that (13)C is inhomogeneously incorporated into astrocytic glycogen at a rate ~25 times slower than in the liver, in agreement with prior bulk studies. This experiment, using temporally resolved, nanometer-scale imaging of glycogen synthesis and degradation, provides greater insight into glucose metabolism in mammalian organs and shows how this technique can be used to explore biochemical pathways in healthy and diseased states.