Adipocyte autophagy limits gut inflammation by controlling oxylipin and IL-10.

Lipids play a major role in inflammatory diseases by altering inflammatory cell functions, either through their function as energy substrates or as lipid mediators such as oxylipins. Autophagy, a lysosomal degradation pathway that limits inflammation, is known to impact on lipid availability, however, whether this controls inflammation remains unexplored. We found that upon intestinal inflammation visceral adipocytes upregulate autophagy and that adipocyte-specific loss of the autophagy gene Atg7 exacerbates inflammation. While autophagy decreased lipolytic release of free fatty acids, loss of the major lipolytic enzyme Pnpla2/Atgl in adipocytes did not alter intestinal inflammation, ruling out free fatty acids as anti-inflammatory energy substrates. Instead, Atg7-deficient adipose tissues exhibited an oxylipin imbalance, driven through an NRF2-mediated upregulation of Ephx1. This shift reduced secretion of IL-10 from adipose tissues, which was dependent on the cytochrome P450-EPHX pathway, and lowered circulating levels of IL-10 to exacerbate intestinal inflammation. These results suggest an underappreciated fat-gut crosstalk through an autophagy-dependent regulation of anti-inflammatory oxylipins via the cytochrome P450-EPHX pathway, indicating a protective effect of adipose tissues for distant inflammation.

Dietary Fatty Acids Directly Impact Central Nervous System Autoimmunity via the Small Intestine.

Growing empirical evidence suggests that nutrition and bacterial metabolites might impact the systemic immune response in the context of disease and autoimmunity. We report that long-chain fatty acids (LCFAs) enhanced differentiation and proliferation of T helper 1 (Th1) and/or Th17 cells and impaired their intestinal sequestration via p38-MAPK pathway. Alternatively, dietary short-chain FAs (SCFAs) expanded gut T regulatory (Treg) cells by suppression of the JNK1 and p38 pathway. We used experimental autoimmune encephalomyelitis (EAE) as a model of T cell-mediated autoimmunity to show that LCFAs consistently decreased SCFAs in the gut and exacerbated disease by expanding pathogenic Th1 and/or Th17 cell populations in the small intestine. Treatment with SCFAs ameliorated EAE and reduced axonal damage via long-lasting imprinting on lamina-propria-derived Treg cells. These data demonstrate a direct dietary impact on intestinal-specific, and subsequently central nervous system-specific, Th cell responses in autoimmunity, and thus might have therapeutic implications for autoimmune diseases such as multiple sclerosis.

LC-ESI-HRMS - lipidomics of phospholipids : Characterization of extraction, chromatography and detection parameters.

Lipids 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 × 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.

Sterol Derivatives Specifically Increase Anti-Inflammatory Oxylipin Formation in M2-like Macrophages by LXR-Mediated Induction of 15-LOX.

The understanding of the role of LXR in the regulation of macrophages during inflammation is emerging. Here, we show that LXR agonist T09 specifically increases 15-LOX abundance in primary human M2 macrophages. In time- and dose-dependent incubations with T09, an increase of 3-fold for ALOX15 and up to 15-fold for 15-LOX-derived oxylipins was observed. In addition, LXR activation has no or moderate effects on the abundance of macrophage marker proteins such as TLR2, TLR4, PPARγ, and IL-1RII, as well as surface markers (CD14, CD86, and CD163). Stimulation of M2-like macrophages with FXR and RXR agonists leads to moderate ALOX15 induction, probably due to side activity on LXR. Finally, desmosterol, 24(S),25-Ep cholesterol and 22(R)-OH cholesterol were identified as potent endogenous LXR ligands leading to an ALOX15 induction. LXR-mediated ALOX15 regulation is a new link between the two lipid mediator classes sterols, and oxylipins, possibly being an important tool in inflammatory regulation through anti-inflammatory oxylipins.

Formation of lipoxins and resolvins in human leukocytes.

Specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins are formed by the consecutive action of 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- or 15-lipoxygenases using arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid as substrate. Lipoxins are trihydroxylated oxylipins which are formed from arachidonic and eicosapentaenoic acid. The latter can also be converted to di- and trihydroxylated resolvins of the E series, whereas docosahexaenoic acid is the substrate for the formation of di- and trihydroxylated resolvins of the D series. Here, we summarize the formation of lipoxins and resolvins in leukocytes. From the data published so far, it becomes evident that FLAP is required for the biosynthesis of most of the lipoxins and resolvins. Even in the presence of FLAP, formation of the trihydroxylated SPMs (lipoxins, RvD1-RvD4, RvE1) in leukocytes is very low or undetectable which is obviously due to the extremely low epoxide formation by 5-LO from oxylipins such as 15-H(p)ETE, 18-H(p)EPE or 17-H(p)DHA. As a result, only the dihydroxylated oxylipins (5 S,15S-diHETE, 5 S,15S-diHEPE) and resolvins (RvD5, RvE2, RvE4) can be consistently detected using leukocytes as SPM source. However, the reported levels of these dihydroxylated lipid mediators are still much lower than those of the typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g. 5-HETE), leukotrienes or cyclooxygenase-derived prostaglandins. Since 5-LO expression is mainly restricted to leukocytes these cells are considered as the main source of SPMs. The low formation of trihydroxylated SPMs in leukocytes, the fact that they are hardly detected in biological samples as well as the lack of functional signaling by their receptors make it highly questionable that trihydroxylated SPMs play a role as endogenous mediators in the resolution of inflammation.

Development of a quantitative proteomics approach for cyclooxygenases and lipoxygenases in parallel to quantitative oxylipin analysis allowing the comprehensive investigation of the arachidonic acid cascade.

Oxylipins derived from the cyclooxygenase (COX) and lipoxygenase (LOX) pathways of the arachidonic acid (ARA) cascade are essential for the regulation of the inflammatory response and many other physiological functions. Comprehensive analytical methods comprised of oxylipin and protein abundance analysis are required to fully understand mechanisms leading to changes within these pathways. Here, we describe the development of a quantitative multi-omics approach combining liquid chromatography tandem mass spectrometry-based targeted oxylipin metabolomics and proteomics. As the first targeted proteomics method to cover these pathways, it enables the quantitative analysis of all human COX (COX-1 and COX-2) and relevant LOX pathway enzymes (5-LOX, 12-LOX, 15-LOX, 15-LOX-2, and FLAP) in parallel to the analysis of 239 oxylipins with our targeted oxylipin metabolomics method from a single sample. The detailed comparison between MRM3 and classical MRM-based detection in proteomics showed increased selectivity for MRM3, while MRM performed better in terms of sensitivity (LLOQ, 16-122 pM vs. 75-840 pM for the same peptides), linear range (up to 1.5-7.4 µM vs. 4-368 nM), and multiplexing capacities. Thus, the MRM mode was more favorable for this pathway analysis. With this sensitive multi-omics approach, we comprehensively characterized oxylipin and protein patterns in the human monocytic cell line THP-1 and differently polarized primary macrophages. Finally, the quantification of changes in protein and oxylipin levels induced by lipopolysaccharide stimulation and pharmaceutical treatment demonstrates its usefulness to study molecular modes of action involved in the modulation of the ARA cascade.

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.

Combined Targeted Proteomics and Oxylipin Metabolomics for Monitoring of the COX-2 Pathway.

The important role of inducible cyclooxygenase-2 (COX-2) in several diseases necessitates analytical tools enabling thorough understanding of its modulation. Analysis of a comprehensive oxylipin pattern provides detailed information about changes in enzyme activities. In order to simultaneously monitor gene expression levels, a targeted proteomics method for human COX-2 is developed. With limits of detection and quantification down to 0.25 and 0.5 fmol (on column) the method enables sensitive quantitative analysis via LC-MS/MS within a linear range up to 2.5 pmol. Three housekeeping proteins are included in the method for data normalization. A tiered approach for method development comprised of in silico and experimental steps is described for choosing unique peptides and selective and sensitive SRM transitions while avoiding isobaric interferences. This method combined with a well-established targeted oxylipin metabolomics method allows to investigate the role of COX-2 in the human colon carcinoma cell lines HCT-116, HT-29, and HCA-7. Moreover, the developed methodology is used to demonstrate the time-dependent prostanoid formation and COX-2 enzyme synthesis in lipopolysaccharide-stimulated human primary macrophages. The described approach is a helpful tool which will be further used as standard operation procedure, ultimately aiming at comprehensive targeted proteomics/oxylipin metabolomics strategies to examine the entire arachidonic acid cascade.

Rapid quantification of fatty acids in plant oils and biological samples by LC-MS.

Analysis of fatty acids (FA) in food and biological samples such as blood is indispensable in modern life sciences. We developed a rapid, sensitive and comprehensive method for the quantification of 41 saturated and unsaturated fatty acids by means of LC-MS. Optimized chromatographic separation of isobaric analytes was carried out on a C8 reversed phase analytical column (100 × 2.1 mm, 2.6 µm core-shell particle) with a total run time of 15 min with back pressure lower than 300 bar. On an old triple quadrupole instrument (3200, AB Sciex), pseudo selected reaction monitoring mode was used for quantification of the poorly fragmenting FA, yielding limits of detection of 5-100 nM. Sample preparation was carried out by removal of phospholipids and triglycerides by solid-phase extraction (non-esterified fatty acids in oils) or saponification in iso-propanol (fatty acyls). This is not only a rapid strategy for quantification of fatty acyls, but allows the direct combination with the LC-MS-based analysis of fatty acid oxidation products (eicosanoids and other oxylipins) from the same sample. The concentrations of fatty acyls determined by means of LC-MS were consistent with those from GC-FID analysis demonstrating the accuracy of the developed method. Moreover, the method shows high precisions with a low intra-day (≤ 10% for almost all fatty acids in plasma and ≤ 15% in oils) and inter-day as well as inter-operator variability (< 20%). The method was successfully applied on human plasma and edible oils. The possibility to quantify non-esterified fatty acids in samples containing an excess of triacylglycerols and phospholipids is a major strength of the described approach allowing to gain new insights in the composition of biological samples.

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.

Early antihypertensive treatment and ischemia-induced acute kidney injury.

Acute kidney injury (AKI) frequently complicates major surgery and can be associated with hypertension and progress to chronic kidney disease, but reports on blood pressure normalization in AKI are conflicting. In the present study, we investigated the effects of an angiotensin-converting enzyme inhibitor, enalapril, and a soluble epoxide hydrolase inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea (TPPU), on renal inflammation, fibrosis, and glomerulosclerosis in a mouse model of ischemia-reperfusion injury (IRI)-induced AKI. Male CD1 mice underwent unilateral IRI for 35 min. Blood pressure was measured by tail cuff, and mesangial matrix expansion was quantified on methenamine silver-stained sections. Renal perfusion was assessed by functional MRI in vehicle- and TPPU-treated mice. Immunohistochemistry was performed to study the severity of AKI and inflammation. Leukocyte subsets were analyzed by flow cytometry, and proinflammatory cytokines were analyzed by quantitative PCR. Plasma and tissue levels of TPPU and lipid mediators were analyzed by liquid chromatography mass spectrometry. IRI resulted in a blood pressure increase of 20 mmHg in the vehicle-treated group. TPPU and enalapril normalized blood pressure and reduced mesangial matrix expansion. However, inflammation and progressive renal fibrosis were severe in all groups. TPPU further reduced renal perfusion on days 1 and 14. In conclusion, early antihypertensive treatment worsened renal outcome after AKI by further reducing renal perfusion despite reduced glomerulosclerosis.

Simple Targeted Assays for Metabolic Pathways and Signaling: A Powerful Tool for Targeted Proteomics.

We introduce STAMPS, a pathway-centric web service for the development of targeted proteomics assays. STAMPS guides the user by providing several intuitive interfaces for a rapid and simplified method design. Applying our curated framework to signaling and metabolic pathways, we reduced the average assay development time by a factor of ∼150 and revealed that the insulin signaling is actively controlled by protein abundance changes in insulin-sensitive and -resistance states. Although at the current state STAMPS primarily contains mouse data, it was designed for easy extension with additional organisms.

Targeting esterified oxylipins by LC-MS - Effect of sample preparation on oxylipin pattern.

A major part of oxygenated metabolites of polyunsaturated fatty acids - i.e. eicosanoids and other oxylipins - in biological samples is found in the esterified form. Yet, their biological role is only poorly understood. For quantification of esterified oxylipins in biological samples current protocols mostly apply alkaline hydrolysis with or without prior lipid extraction to release oxylipins into their free form which can be subsequently quantified via liquid chromatography-mass spectrometry. Herein, a detailed protocol for precise and reproducible quantification of esterified oxylipins in plasma is presented comprising i) extraction of lipids and removal of proteins with iso-propanol, ii) base hydrolysis with potassium hydroxide to saponify lipids and iii) solid phase extraction of the liberated oxylipins on C8/anion exchange mixed mode material. Unequal extraction of internal standards and lipid classes during lipid extraction before hydrolysis led to distorted concentrations, emphasizing that the choice of solvent used in this step is important to minimize discrimination. Regarding the hydrolysis conditions, at least 30 min incubation at 60 °C is required with 0.1 M KOH in sample. Drying of the SPE cartridges is a critical parameter since autoxidation processes of PUFA, which are present in high concentrations after cleavage, lead to artificial formation of epoxy fatty acids. With the developed protocol, inter-day, intra-day and inter-operator variance was <21% for most oxylipins including hydroxy-, dihydroxy-, and epoxy-PUFA. The applicability of the developed methodology is demonstrated by investigating the changes in the oxylipin pattern following omega-3 fatty acid feeding to rats.

Dietary omega-3 PUFA improved tubular function after ischemia induced acute kidney injury in mice but did not attenuate impairment of renal function.

BACKGROUND: Acute kidney injury (AKI) is an important complication after major surgery and solid organ transplantation. Here, we present a dietary omega-3 polyunsaturated fatty acid (n3-PUFA) supplementation study to investigate whether pre-treatment can reduce ischemia induced AKI in mice. METHODS: Male 12-14 week old C57BL/6 J mice received a linoleic acid rich sunflower oil based standard diet containing 10 % fat (STD) or the same diet enriched with n3-PUFA (containing 1 % EPA and 1 % DHA) (STD + n3). After 14 days of feeding bilateral 30 min renal ischemia reperfusion injury (IRI) was conducted to induce AKI and mice were sacrificed at 24 h. Serum creatinine and blood urea nitrogen (BUN) as well as liver enzyme elevation were measured. Kidney damage was analyzed by histology and immunohistochemistry. Furthermore, pro-inflammatory cytokines (IL-6, MCP-1) were determined by qPCR. FA and oxylipin pattern were quantified in blood and kidneys by GC-FID and LC-MS/MS, respectively. RESULTS: n3-PUFA supplementation prior to renal IRI increased systemic and renal levels of n3-PUFA. Consistently, eicosanoids and other oxylipins derived from n3-PUFA including precursors of specialized pro-resolving mediators were elevated while n6-PUFA derived mediators such as pro-inflammatory prostaglandins were decreased. Feeding of n3-PUFA did not attenuate renal function impairment, morphological renal damage and inflammation characterized by IL-6 and MCP-1 elevation or neutrophil infiltration. However, the tubular transport marker alpha-1 microglobulin (A1M) was significantly higher expressed in proximal tubular epithelial cells of STD + n3 compared to STD fed mice. This indicates a better integrity of proximal tubular epithelial cells and thus significant protection of tubular function. In addition, heme oxygenase-1 (HO-1) which protects tubular function was also up-regulated in the treatment group receiving n3-PUFA supplemented chow. DISCUSSION: We showed that n3-PUFA pre-treatment did not affect overall renal function or renal inflammation in a mouse model of moderate ischemia induced AKI, but tubular transport was improved. In conclusion, dietary n3-PUFA supplementation altered the oxylipin levels significantly but did not protect from renal function deterioration or attenuate ischemia induced renal inflammation.

Clinical blood sampling for oxylipin analysis - effect of storage and pneumatic tube transport of blood on free and total oxylipin profile in human plasma and serum.

Quantitative analysis of oxylipins in blood samples is of increasing interest in clinical studies. However, storage after sampling and transport of blood might induce artificial changes in the apparent oxylipin profile due to ex vivo formation/degradation by autoxidation or enzymatic activity. In the present study we investigated the stability of free (i.e. non-esterified) and total oxylipins in EDTA-plasma and serum generated under clinical conditions assessing delays in sample processing and automated transportation: Free cytochrome P450 monooxygenase and 5-lipoxygenase (LOX) formed oxylipins as well as autoxidation products were marginally affected by storage of whole blood up to 4 h at 4 °C, while total (i.e. the sum of free and esterified) levels of these oxylipins were stable up to 24 h and following transport. Cyclooxygenase (COX) products (TxB2, 12-HHT) and 12-LOX derived hydroxy-fatty acids were prone to storage and transport induced changes due to platelet activation. Total oxylipin patterns were generally more stable than the concentration of free oxylipins. In serum, coagulation induced higher levels of COX and 12-LOX products showing a high inter-individual variability. Overall, our results indicate that total EDTA-plasma oxylipins are the most stable blood oxylipin marker for clinical samples. Here, storage of blood before further processing is acceptable for a period up to 24 hours at 4 °C. However, levels of platelet derived oxylipins should be interpreted with caution regarding potential ex vivo formation.

Non-targeted and targeted analysis of oxylipins in combination with charge-switch derivatization by ion mobility high-resolution mass spectrometry.

Eicosanoids and other oxylipins play an important role in mediating inflammation as well as other biological processes. For the investigation of their biological role(s), comprehensive analytical methods are necessary, which are able to provide reliable identification and quantification of these compounds in biological matrices. Using charge-switch derivatization with AMPP (N-(4-aminomethylphenyl)pyridinium chloride) in combination with liquid chromatography ion mobility quadrupole time-of-flight mass spectrometry (LC-IM-QTOF-MS), we developed a non-target approach to analyze oxylipins in plasma, serum, and cells. The developed workflow makes use of an ion mobility resolved fragmentation to pinpoint derivatized molecules based on the cleavage of AMPP, which yields two specific fragment ions. This allows a reliable identification of known and unknown eicosanoids and other oxylipins. We characterized the workflow using 52 different oxylipins and investigated their fragmentation patterns and ion mobilities. Limits of detection ranged between 0.2 and 10.0 nM (1.0-50 pg on column), which is comparable with other state-of-the-art methods using LC triple quadrupole (QqQ) MS. Moreover, we applied this strategy to analyze oxylipins in different biologically relevant matrices, as cultured cells, human plasma, and serum. Graphical abstract.

Polyunsaturated fatty acid metabolites: biosynthesis in Leishmania and role in parasite/host interaction.

Inside the human host, Leishmania infection starts with phagocytosis of infective promastigotes by macrophages. In order to survive, Leishmania has developed several strategies to manipulate macrophage functions. Among these strategies, Leishmania as a source of bioactive lipids has been poorly explored. Herein, we assessed the biosynthesis of polyunsaturated fatty acid metabolites by infective and noninfective stages of Leishmania and further explored the role of these metabolites in macrophage polarization. The concentration of docosahexaenoic acid metabolites, precursors of proresolving lipid mediators, was increased in the infective stage of the parasite compared with the noninfective stage, and cytochrome P450-like proteins were shown to be implicated in the biosynthesis of these metabolites. The treatment of macrophages with lipids extracted from the infective forms of the parasite led to M2 macrophage polarization and blocked the differentiation into the M1 phenotype induced by IFN-γ. In conclusion, Leishmania polyunsaturated fatty acid metabolites, produced by cytochrome P450-like protein activity, are implicated in parasite/host interactions by promoting the polarization of macrophages into a proresolving M2 phenotype.

A strategy for validating concentrations of oxylipin standards for external calibration.

Quantitative analysis of oxylipins by means of chromatography/mass spectrometry is based on (external) calibration with standard compounds. Therefore, the quality of analytical standards is of fundamental importance for accurate results. Recently launched certified standards with an assured concentration within a narrow range are useful tools to verify analytical standards. However, such standards are only available for a few compounds. Based on the exemplary comparison of certified with none certified standards we suggest a tiered approach to validate and control the concentrations when preparing an external calibration based on non-certified oxylipin standards. Concentrations are evaluated by means of liquid chromatography negative electrospray ionization mass spectrometry (LC-ESI(-)-MS) in selected ion monitoring mode and UV spectroscopy. Based on the suggested approach, more than 50% of the standards in our calibration mix could be validated. Though most of the non-certified standards are of good quality, several oxylipin concentrations differ considerably demonstrating that a quality control strategy as suggested here is a mandatory prerequisite for quantitative oxylipin metabolomics.

Formation of trans-epoxy fatty acids correlates with formation of isoprostanes and could serve as biomarker of oxidative stress.

In mammals, epoxy-polyunsaturated fatty acids (epoxy-PUFA) are enzymatically formed from naturally occurring all-cis PUFA by cytochrome P450 monooxygenases leading to the generation of cis-epoxy-PUFA (mixture of R,S- and S,R-enantiomers). In addition, also non-enzymatic chemical peroxidation gives rise to epoxy-PUFA leading to both, cis- and trans-epoxy-PUFA (mixture of R,R- and S,S-enantiomers). Here, we investigated for the first time trans-epoxy-PUFA and the trans/cis-epoxy-PUFA ratio as potential new biomarker of lipid peroxidation. Their formation was analyzed in correlation with the formation of isoprostanes (IsoP), which are commonly used as biomarkers of oxidative stress. Five oxidative stress models were investigated including incubations of three human cell lines as well as the in vivo model Caenorhabditis elegans with tert-butyl hydroperoxide (t-BOOH) and analysis of murine kidney tissue after renal ischemia reperfusion injury (IRI). A comprehensive set of IsoP and epoxy-PUFA derived from biologically relevant PUFA (ARA, EPA and DHA) was simultaneously quantified by LC-ESI(-)-MS/MS. Following renal IRI only a moderate increase in the kidney levels of IsoP and no relevant change in the trans/cis-epoxy-PUFA ratio was observed. In all investigated cell lines (HCT-116, HepG2 and Caki-2) as well as C. elegans a dose dependent increase of both, IsoP and the trans/cis-epoxy-PUFA ratio in response to the applied t-BOOH was observed. The different cell lines showed a distinct time dependent pattern consistent for both classes of autoxidatively formed oxylipins. Clear and highly significant correlations of the trans/cis-epoxy-PUFA ratios with the IsoP levels were found in all investigated cell lines and C. elegans. Based on this, we suggest the trans/cis-epoxy-PUFA ratio as potential new biomarker of oxidative stress, which warrants further investigation.