1,721,071 research outputs found
Quantification of esterified oxylipins following HILIC-fractionation of lipid classes
No full textSeveral oxylipins are lipid mediators derived from the oxidation of polyunsaturated fatty acids (PUFAs). The majority of oxylipins in biological samples occurs esterified in neutral lipids (nLs) and phospholipids (PLs). They are commonly quantified indirectly following alkaline hydrolysis providing excellent sensitivity but the information in which lipid classes the oxylipins occurred in is lost. The direct analysis of oxidized lipids is currently not sensitive enough to detect all esterified oxylipins. Here, a new hydrophilic interaction liquid chromatography (HILIC) based lipid class fractionation using solid-phase extraction (SPE) cartridges was developed separating lipids into nLs and 4 PL fractions using a single column. Esterified oxylipins in the fractions were quantified following alkaline hydrolysis to sensitively pinpoint in which lipid classes they are bound in plasma. The fractionation was extensively characterized for different lipid extracts demonstrating high separation efficiency and recovery using labeled standards and untargeted analysis of endogenous lipids. Esterified oxylipins in the fractions were quantitatively detected. Based on the results from two independent human plasma pools including SRM1950 it is shown that: hydroxy-linoleic acid- and hydroxy-α-linolenic acid-derived oxylipins are preferably bound to nLs whereas long chain hydroxy-PUFAs and PUFAs (i.e. ARA EPA and DHA) are predominantly esterified to phospholipid classes. Supplementation of n3-PUFAs for 12 months led to an increase in EPA- and -DHA-derived oxylipins in all lipid fractions with the highest increase of hydroxy-PUFAs in nLs. This demonstrates a precursor PUFA-dependent binding of oxylipins and a direct effect of diet on esterified oxylipins in plasma
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Regulation and functions of 15-lipoxygenases in human macrophages
No full textLipoxygenases (LOXs) catalyze the stereo-specific peroxidation of polyunsaturated fatty acids (PUFAs) to their corresponding hydroperoxy derivatives. Human macrophages express two arachidonic acid (AA) 15-lipoxygenating enzymes classified as ALOX15 and ALOX15B. ALOX15, which was first described in 1975, has been extensively characterized and its biological functions have been investigated in a number of cellular systems and animal models. In macrophages, ALOX15 functions to generate specific phospholipid (PL) oxidation products crucial for orchestrating the nonimmunogenic removal of apoptotic cells (ACs) as well as synthesizing precursor lipids required for production of specialized pro-resolving mediators (SPMs) that facilitate inflammation resolution. The discovery of ALOX15B in 1997 was followed by comprehensive analyses of its structural properties and reaction specificities with PUFA substrates. Although its enzymatic properties are well described, the biological functions of ALOX15B are not fully understood. In contrast to ALOX15 whose expression in human monocyte-derived macrophages is strictly dependent on Th2 cytokines IL-4 and IL-13, ALOX15B is constitutively expressed. This review aims to summarize the current knowledge on the regulation and functions of ALOX15 and ALOX15B in human macrophages
Novel signaling routes and molecular determinants for leukotriene biosynthesis
Full text linkLMs derived from AA and related FAs exert diverse biological activities during inflammation. However, although discovered several decades ago, the knowledge about the regulation of the enzymes involved in LM biosynthesis remains far from completion. This thesis aimed at investigating the molecular mechanism of bacteria-induced LT formation and unveiled exotoxins secreted from pathogenic bacteria as determining factors for in cellulo 5-LOX activation. In case of S. aureus, a human commensal associated to infections of diverse severity, we identified PSMs and their receptor FPR2 as critical factors capable to induce LM biosynthesis in neutrophils. Activation of FPR2, phosphorylation of ERK1/2 as well as an increase of intracellular Ca2+ are clearly involved in PSM-mediated 5-LOX activation. Together, we here present the release of LMs and the stimulation of 5-LOX as novel biological properties of PSMs. Moreover, our findings might encourage ongoing efforts to develop alternative anti- microbial strategies focusing on exotoxin secretion, since the rise of antibiotic resistant bacteria is becoming a relevant threat to humans. In addition, we here addressed structural elements of the integral membrane protein FLAP that acts as unique helper for cellular 5-LOX product formation. Our results uncover the second cytosolic loop of FLAP as crucial domain for sufficient assembly of the LT-synthetic protein complex at the nuclear membrane. Prospective studies and the development of FLAP inhibitors should therefore consider this loop as novel binding site, since previous compounds solely target lipid-exposed domains of FLAP, resulting in highly lipophilic drugs and thus inappropriate pharmacokinetic properties. Taken together, our results significantly contribute to the expansive field of LM research and provide novel insights into the signaling and molecular determinants of LT biosynthesis upon bacterial stimulation as well as its dependency on FLAP
Impact of sex differences and small molecules on proinflammatory lipid mediator biosynthesis
Full text linkIn the first part, the thesis examines the influence of sex on prostanoid formation. Prostaglandins (PG) are generated by cyclooxygenase (COX)-1/2, which convert arachidonic acid (AA) to PGH2 which is further metabolized to distinct PGs by terminal synthases. Recent findings suggest a sex biased regulation in animals. Hence, the regulation of the prostanoid formation in male and female human whole blood and freshly isolated cells was evaluated. Analysis of human blood cells showed higher lymphocyte and neutrophil numbers in females, whereas monocyte numbers were higher in males and long term incubations of whole blood resulted in significantly enhanced levels of PGs in males. The elevated formation in males was not influenced by pre-incubation with sex hormones, independent of COX-1/2 protein and COX-2 mRNA expression and of AA release in leukocytes. In contrast, the involvement of the 15-prostaglandin dehydrogenase and of transcellular interactions of isolated leukocytes could be shown. In the second part, potential 5-lipoxygenase (5-LO) inhibitors were evaluated. Leukotrienes (LT) are bioactive lipid mediators, which are biosynthesized involving 5-LO catalyzing the incorporation of molecular oxygen into AA. Out of three distinct series of potential 5-LO inhibitors, the two most potent compounds F-XII and F-XVI were analyzed in detail. Both compounds showed potent inhibition of 5-LO with IC50 values in the nanomolar range in cell-based and cell-free preparations (F-XII = 70 - 100 nM; F-XVI = 60 - 120 nM). 5-LO inhibition was direct, reversible and not primarily mediated due to radical scavenging and antioxidant properties. Docking simulations proposed binding to the amino acid Asp-166, which is involved in a salt bridge connecting the two domains of 5-LO. Furthermore, inhibition of 5-LO was independent of interference with cellular signals required for the 5-LO activation and were confirmed in human whole blood and in zymosan-induced peritonitis in mice
LC-ESI-HRMS - lipidomics of phospholipids – characterization of extraction, chromatography and detection parameters
No full textLipids are a diverse class of molecules involved in many biological functions including cell signaling or cell membrane assembly. Owing to this relevance, LC-MS/MS based lipidomics emerged as a major field in modern analytical chemistry. Here, we thoroughly characterized the influence of MS and LC settings – of a Q Exactive HF operated in Full MS/data-dependent MS2 TOP N acquisition mode - in order to optimize the semi-quantification of polar lipids. Optimization of MS-source settings improved the signal intensity by factor 3 compared to default settings. Polar lipids were separated on an ACQUITY Premier CSH C18 reversed-phase column (100 x 2.1 mm, 1.7 µm, 130 Å) during an elution window of 28 min, leading to a sufficient number of both data points across the chromatographic peaks, as well as MS2 spectra. Analysis was carried out in positive and negative ionization mode enabling the detection of a broader spectrum of lipids and to support the structural characterization of lipids. Optimal sample preparation of biological samples was achieved by liquid-liquid extraction using MeOH/MTBE resulting in an excellent extraction recovery >85% with an intra-day and inter-day variability <15%. The optimized method was applied on the investigation of changes in the phospholipid pattern in plasma from human subjects supplemented with n3-PUFA (20:5 and 22:6). The strongest increase was observed for lipids bearing 20:5, while 22:4 bearing lipids were lowered. Specifically, LPC 20:5_0:0 and PC 16:0_20:5 were found to be strongest elevated, while PE 18:0_22:4 and PC 18:2_18:2 were decreased by n3-PUFA supplementation. These results were confirmed by targeted LC-MS/MS using commercially available phospholipids as standards
Analytik von CYP-Eicosanoiden und ihre Rolle bei ischämischem Organversagen
Full text linkCytochrom P450 (CYP) Enzyme tragen zur Bioaktivierung von langkettigen mehrfach ungesättigten Fettsäuren bei. Die gebildeten Monoepoxy- und Monohydroxy-Metaboliten werden zusammenfassend als CYP-Eicosanoide bezeichnet und fungieren als Mediatoren bei der Regulation des Gefäßtonus, der Herz- und Nierenfunktion, sowie einer Vielzahl weiterer physiologischer Prozesse, wobei die biologische Aktivität oftmals abhängig von der Positions- und Stereoisomerie der Eicosanoide ist. Prominente Vertreter der CYP-Eicosanoid-Familie sind die aus der Arachidonsäure gebildeten Epoxyeicosatriensäuren (EETs) und 20-Hydroxyeicosatetraensäure (20-HETE). EETs und 20-HETE haben zum Teil gegensätzliche biologische Aktivitäten, die zur Aktivierung bzw. Inhibition antiinflammatorischer und weiterer Zell- und Organ-protektiver Signalwege beitragen. Ziel der vorliegenden Arbeit war es, durch Analyse endogener Metabolitenprofile zum besseren Verständnis der Rolle von CYP-Eicosanoiden bei der Entstehung von Ischämie/Reperfusions (I/R)-bedingten Organschäden beizutragen. Als Hauptergebnisse ergaben sich (i) die Entdeckung und Charakterisierung einer protektiven Rolle von EETs in Tiermodellen der Initiationsphase des akuten Nierenversagens, sowie des therapeutischen Potentials stabiler EET-Analoga; (ii) die Identifizierung von 8,9-EET und 20-HETE als mögliche prädiktive Biomarker für das post-operative Auftreten von akutem Nierenversagen nach offener Herzoperation; und (iii) die Entwicklung und Validierung eines analytischen Verfahrens der chiralen Lipidomik (chiral-LC-ESI-MS/MS), das eine Analyse endogener Enantiomere sowohl von Monoepoxy- als auch Monohydroxy-Eicosanoiden in komplexen biologischen Proben erstmalig ermöglichte und dafür genutzt werden konnte, die stereospezifische Regulation der EETs durch Epoxid-Hydrolasen in vitro wie auch in vivo zu beschreiben.Cytochrome P450 (CYP) enzymes contribute to the bioactivation of long-chain polyunsaturated fatty acids. The monoepoxy- and monohydroxy-metabolites generated by CYP enzymes are collectively termed CYP-eicosanoids. CYP-eicosanoids act as mediators in the regulation of vascular tone, heart- and kidney function and several further physiological processes, mostly in a regio- and stereospecific manner. Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are prominent members of the CYP-eicosanoid family. EETs and 20-HETE show partially opposing biological activities that contribute to the activation or inhibition of anti-inflammatory and other cell- and organ-protective mechanisms. The aim of the present work was to study the role of CYP-eicosanoids in ischemia/reperfusion (I/R) related organ damage by analyses of endogenous metabolite profiles. The main results were (i) discovery and characterization of the EETs protective role in animal models of the initiation phase of acute kidney injury (AKI) and the therapeutic potential of stable EET-analogs, (ii) identification of 8,9-EET and 20-HETE as potential predictive biomarkers for AKI in patients who underwent open heart surgery, (iii) the development and validation of a novel analytical method for chiral lipidomics (chiral LC-ESI-MS/MS) that allows to study endogenous enantiomers of monohydroxy- and monoepoxy-eicosanoids in complex matrices of biological and clinical samples. Furthermore, the approach was applied to describe the stereospecific regulation of EETs by epoxide hydrolases in vitro and in vivo
Plasma oxylipins respond in a linear dose-response manner with increased intake of eicosapentaenoic and docosahexaenoic acids: results from a randomized controlled trial in healthy humans
Full text linkBackgroundThe health effects of long-chain omega-3 polyunsaturated fatty acids (n–3 PUFAs) are partly mediated by their oxidized metabolites, i.e., eicosanoids and other oxylipins. Some intervention studies have demonstrated that eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increase systemic concentrations of n–3 PUFA–derived oxylipins and moderately decrease arachidonic acid–derived oxylipins. There is no information on the dose-response of oxylipin concentrations after n–3 PUFA intake.ObjectiveThe aim of this study was to quantify oxylipins in human plasma samples from an intervention study in which participants were randomly assigned to different daily intakes of EPA and DHA for 12 mo.MethodsHealthy adult men and women with low habitual fish consumption (n = 121) were randomly assigned to receive capsules providing doses of n–3 PUFAs reflecting 3 patterns of consumption of oily fish [1, 2, or 4 portions/wk with 3.27 g EPA + DHA (1:1.2, wt:wt) per portion] or placebo. Oxylipins were quantified in plasma after 3 and 12 mo. Relative and absolute changes of individual oxylipins were calculated and concentrations were correlated with the dose and the content of EPA and DHA in blood lipid pools.ResultsSeventy-three oxylipins, mostly hydroxy-, dihydroxy-, and epoxy-PUFAs, were quantified in the plasma samples. After 3 and 12 mo a linear increase with dose was observed for all EPA- and DHA-derived oxylipins. Cytochrome-P450-derived anti-inflammatory and cardioprotective epoxy-PUFAs increased linearly with n–3 PUFA dose and showed low interindividual variance (r2 > 0.95). Similarly, 5, 12-, and 15-lipoxygenase–derived hydroxy-PUFAs as well as those formed autoxidatively increased linearly. These include the precursors of so-called specialized pro-resolving lipid mediators (SPMs), e.g., 17-hydroxy-DHA and 18-hydroxy-EPA.ConclusionsPlasma concentrations of biologically active oxylipins derived from n–3 PUFAs, including epoxy-PUFAs and SPM-precursors, increase linearly with elevated intake of EPA and DHA. Interindividual differences in resulting plasma concentrations are low. This trial was registered at controlled-trials.com as ISRCTN48398526
Aquat Toxicol
Full text linkThe antimicrobial triclocarban (TCC) is frequently found in personal care products and commonly observed in surface waters and sediments. Due to its long environmental persistence TCC accumulates in sewage sludge. It also shows a high unintended biological activity as a potent inhibitor of the soluble epoxide hydrolase (sEH) and may be an endocrine disruptor. In this study, we investigated bioconcentration, metabolism and elimination of TCC in fish using medaka (Oryzias latipes) as a model. Medaka larvae (7 \ub1 1 days post hatching) were exposed to 63 nM (20 \u3bcg/L) TCC water for 24h. The LC-MS/MS analysis of water and tissues provided bioconcentration of TCC and its metabolites in fish body and rapid excretion into culture water. Results from tissue samples showed a tissue concentration of 34 \u3bcmol/kg and a log bioconcentration factor (BCF) of 2.86. These results are slightly lower than previous findings in snails and algae. A significant portion of the absorbed TCC was oxidatively metabolized by the fish to hydroxylated products. These metabolites underwent extensive phase II metabolism to yield sulfate and glucuronic acid conjugates. The most abundant metabolite in fish tissue was the glucuronide of 2'-OH-TCC. Elimination of TCC after transferring the fish to fresh water was rapid, with a half-life of 1h. This study shows that larval medaka metabolize TCC similarly to mammals. The rapid rate of metabolism results in a lower bioconcentration than calculated from the octanol-water coefficient of TCC.U50 OH007550/OH/NIOSH CDC HHS/United StatesP42 ES004699-26/ES/NIEHS NIH HHS/United StatesR01 ES002710/ES/NIEHS NIH HHS/United StatesP42 ES004699/ES/NIEHS NIH HHS/United StatesR01 ES002710-31/ES/NIEHS NIH HHS/United StatesU54 OH007550/OH/NIOSH CDC HHS/United StatesOH07550/OH/NIOSH CDC HHS/United StatesU54OH007550/ACL/ACL HHS/United States2012-10-01T00:00:00Z21872556PMC32360358873vault:728
Chemosphere
Full text linkThe antibacterial triclocarban (TCC) concentrates in the cellular fraction of blood. Consequently, plasma levels are at least two-fold lower than the TCC amount present in blood. Utilizing whole blood sampling, a low but significant absorption of TCC from soap during showering is demonstrated for a small group of human subjects.P42 ES004699/ES/NIEHS NIH HHS/United StatesPHS OH07550/OH/NIOSH CDC HHS/United StatesR01 ES002710/ES/NIEHS NIH HHS/United State
Environ Sci Technol
Full text linkThe antibacterial soap additive triclocarban (TCC) is widely used in personal care products. TCC has a high environmental persistence. We developed and validated a sensitive online solid-phase extraction-LC-MS/MS method to rapidly analyze TCC and its major metabolites in urine and other biological samples to assess human exposure. We measured human urine concentrations 0-72 h after showering with a commercial bar soap containing 0.6% TCC. The major route of renal elimination was excretion as N-glucuronides. The absorption was estimated at 0.6% of the 70\ub115 mg of TCC in the soap used. The TCC-N-glucuronide urine concentration varied widely among the subjects, and continuous daily use of the soap led to steady state levels of excretion. In order to assess potential biological effects arising from this exposure, we screened TCC for the inhibition of human enzymes in vitro. We demonstrate that TCC is a potent inhibitor of the enzyme soluble epoxide hydrolase (sEH), whereas TCC's major metabolites lack strong inhibitory activity. Topical administration of TCC at similar levels to rats in a preliminary in vivo study, however, failed to alter plasma biomarkers of sEH activity. Overall the analytical strategy described here revealed that use of TCC soap causes exposure levels that warrant further evaluation.U50 OH007550/OH/NIOSH CDC HHSUnited States/PHS OH07550/OH/NIOSH CDC HHSUnited States/R01 ES002710/ES/NIEHS NIH HHSUnited States/P42 ES004699/ES/NIEHS NIH HHSUnited States/R01 ES002710-31/ES/NIEHS NIH HHSUnited States/U54 OH007550/OH/NIOSH CDC HHSUnited States/U54OH007550/ACL/ACL HHSUnited States
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