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.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of methotrexate in human serum and plasma.

OBJECTIVES: To develop an isotope dilution-liquid chromatography-tandem mass spectrometry-(ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for quantification of methotrexate in human serum and plasma. METHODS: Quantitative nuclear magnetic resonance (qNMR) was used to determine absolute methotrexate content in the standard. Separation was achieved on a biphenyl reversed-phase analytical column with mobile phases based on water and acetonitrile, both containing 0.1% formic acid. Sample preparation included protein precipitation in combination with high sample dilution, and method validation according to current guidelines. The following were assessed: selectivity (using analyte-spiked samples, and relevant structural-related compounds and interferences); specificity and matrix effects (via post-column infusion and comparison of human matrix vs. neat samples); precision and accuracy (in a five-day validation analysis). RMP results were compared between two independent laboratories. Measurement uncertainty was evaluated according to current guidelines. RESULTS: The RMP separated methotrexate from potentially interfering compounds and enabled measurement over a calibration range of 7.200-5,700 ng/mL (0.01584-12.54 µmol/L), with no evidence of matrix effects. All pre-defined acceptance criteria were met; intermediate precision was ≤4.3% and repeatability 1.5-2.1% for all analyte concentrations. Bias was -3.0 to 2.1% for samples within the measuring range and 0.8-4.5% for diluted samples, independent of the sample matrix. RMP results equivalence was demonstrated between two independent laboratories (Pearson correlation coefficient 0.997). Expanded measurement uncertainty of target value-assigned samples was ≤3.4%. CONCLUSIONS: This ID-LC-MS/MS-based approach provides a candidate RMP for methotrexate quantification. Traceability of methotrexate standard and the LC-MS/MS platform were assured by qNMR assessment and extensive method validation.

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.

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.

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.

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.

Aspirin alone and combined with a statin suppresses eicosanoid formation in human colon tissue.

Eicosanoids, including prostaglandins (PGs) and thromboxanes, are broadly bioactive lipid mediators and increase colon tumorigenesis possibly through chronic inflammatory mechanisms. Epidemiological and experimental data suggest that acetylsalicylic acid (ASA) helps prevent colorectal cancer (CRC), possibly through cyclooxygenase (COX)-mediated suppression of eicosanoid, particularly PGE2, formation. Recent studies suggest that statins prevent CRC and improve survival after diagnosis. We identified patients on ASA and/or statin treatment undergoing routine colonoscopy and measured eicosanoid levels in colonic mucosa with targeted metabolomics technology (LC-MS/MS). ASA-treated individuals (n = 27) had significantly lower tissue eicosanoid levels of most COX-derived metabolites than untreated individuals (n = 31). In contrast, COX-derived lipid metabolites tended to be higher in patients with statin treatment (n = 7) as compared with those not receiving statins (n = 24). This effect was not discernible in subjects treated with ASA and statins (n = 11): Individuals treated with both drugs showed a pronounced suppression of COX-derived eicosanoids in colon tissue, even compared with subjects treated with ASA alone. Our data from a routine clinical setting support the hypothesis that ASA and statins could inhibit CRC development via lipid mediator modification. Further studies should directly investigate the effect of dual ASA and statin treatment on colon tumorigenesis in humans.

Effect of Omega-3 Fatty Acid Supplementation on Oxylipins in a Routine Clinical Setting.

Omega-6 polyunsaturated fatty acid (n-6 PUFA) is the predominant polyunsaturated fatty acid (PUFA), especially in Western diet. A high omega-6/omega-3 ratio in Western diets is implicated in the development of cardiovascular diseases and inflammatory processes. Studies in animal models and in humans have demonstrated beneficial effects of omega-3 PUFA (n-3 PUFA) in a variety of diseases, including cardiac arrhythmias and inflammatory diseases, as well as breast and colon cancer. The molecular mechanisms underlying the effects of n-3 PUFA are still not well understood. Possible mechanisms include competition between n-3 and n-6 PUFAs at the cyclooxygenase (COX) and lipoxygenase (LOX) and cytochrome P450 levels, and subsequent formation of oxylipins with specific anti-inflammatory or anti-arrhythmic effects. In this study, we report the impact of routine long-term treatment with prescription-grade n-3 PUFA (either 840 mg or 1680 mg per day) on blood cell membrane fatty acid composition, as well as plasma oxylipin patterns, in a patient population with severe hyperlipidemia and cardiovascular disease who are on standard lipid-lowering and cardioprotective medications. Lipidomics analyses were performed by LC/ESI-MS/MS. Supplementation led to a dose-dependent increase in n-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the blood cell fraction. We also observed a dose-dependent increase in EPA- and DHA-derived epoxy metabolites, whereas the effect of n-3 PUFA supplementation on LOX-dependent EPA- and DHA-derived hydroxy metabolites was less pronounced, with a tendency towards lower metabolites in subjects with higher n-3 PUFA levels. These data thus generally confirm effects of n-3 PUFA supplementation observed previously in healthy individuals. Additionally, they indicate a suppressive effect of high n-3 PUFA supplementation on the formation of LOX metabolites in the context of concomitant aspirin medication.

Impact of dextran sulphate sodium-induced colitis on the intestinal transport of the colon carcinogen PhIP.

Colorectal cancer is one of the most frequent cancers in Western countries. Chronic intestinal diseases such as Crohn's disease and ulcerative colitis, in which the intestinal barrier is massively disturbed, significantly raise the risk of developing a colorectal tumour. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a genotoxic heterocyclic aromatic amine that is formed after strongly heating fish and meat. In this study, the hypothesis that PhIP uptake in the gut is increased during chronic colitis was tested. Chronic colitis was induced by oral administration of dextran sulphate sodium (DSS) to Fischer 344 rats. The transport of PhIP in eight different rat intestinal segments was examined in Ussing chambers. The tissues were incubated with 10 µM PhIP for 90 min, and the concentration of PhIP was determined in the mucosal and serosal compartments of the Ussing chambers as well as in the clamped tissues by LC-MS. Although chronic colitis was clearly induced in the rats, no differences in the intestinal transport of PhIP were observed between control and DSS-treated animals. The hypothesis that in the course of chronic colitis more PhIP is taken up by the intestinal epithelium, thereby increasing the risk of developing colorectal cancer, could not be confirmed in the present report.

Effect of acute and chronic DSS induced colitis on plasma eicosanoid and oxylipin levels in the rat.

Eicosanoids and oxylipins are potent lipid mediators involved in the regulation of inflammation. In order to evaluate their role and suitability as biomarkers in colitis, we analyzed their systemic levels in the acute and chronic phase of dextran sulfate sodium (DSS) induced colitis. Male Fischer 344 rats were treated in three cycles with 4% DSS in the drinking water (4 days followed by 10 days recovery) and blood was drawn 3 days prior to the first DSS treatment and on days 4, 11, 32 and 39. Histopathological evaluation of the colon tissue after 42 days showed that the animals developed a mild to severe chronic colitis. Consistently, prostaglandin levels were massively (twofold) elevated in the colonic tissue. LC-MS based targeted metabolomics was used to determine plasma oxylipin levels at the different time points. In the acute phase of inflammation directly after DSS treatment, epoxy-fatty acid (FA), dihydroxy-FA and hydroxy-FA plasma concentrations were uniformly elevated. With each treatment cycle the increase in these oxylipin levels was more pronounced. Our data suggest that in the acute phase of colitis release of polyunsaturated FAs from membranes in the inflamed tissue is reflected by a uniform increase of oylipins formed in different branches of the arachidonic acid cascade. However, during the recovery phases the systemic oxylipin pattern is not or only moderately altered and does not allow to evaluate the onset of chronic inflammation in the colon.

Oral treatment of rodents with soluble epoxide hydrolase inhibitor 1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl]urea (TPPU): Resulting drug levels and modulation of oxylipin pattern.

Epoxides from polyunsaturated fatty acids (PUFAs) are potent lipid mediators. In vivo stabilization of these epoxides by blockade of the soluble epoxide hydrolase (sEH) leads to anti-inflammatory, analgesic and normotensive effects. Therefore, sEH inhibitors (sEHi) are a promising new class of drugs. Herein, we characterized pharmacokinetic (PK) and pharmacodynamic properties of a commercially available potent sEHi 1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl]urea (TPPU). Cell culture studies suggest its high absorption and metabolic stability. Following administration in drinking water to rats (0.2, 1, and 5mg TPPU/L with 0.2% PEG400), TPPU's blood concentration increased dose dependently within the treatment period to reach an almost steady state after 8 days. TPPU was found in all the tissues tested. The linoleic epoxide/diol ratios in most tissues were dose dependently increased, indicating significant sEH inhibition. Overall, administration of TPPU with the drinking water led to systemic distribution as well as high drug levels and thus makes chronic sEH inhibition studies possible.

Comparison of sample preparation methods for the quantitative analysis of eicosanoids and other oxylipins in plasma by means of LC-MS/MS.

Oxylipins are potent lipid mediators. For the evaluation of their biological roles, several LC-MS based methods have been developed. While these methods are similar, the described sample preparation procedures for the extraction of oxylipins differ considerably. In order to deduce the most appropriate method for the analysis of non-esterified oxylipins in human plasma, we evaluated the performance of seven established sample preparation procedures. Six commonly used solid phase extraction (SPE) and one liquid-liquid extraction (LLE) protocol were compared based on the recovery of 13 added internal standards, extraction efficacy of oxylipins from plasma and reduction of ion-suppressing matrix. Dramatic differences in the performance in all three parameters were found. LLE with ethyl acetate was overall not a sufficient sample preparation strategy. The protocols using Oasis- and StrataX-material insufficiently removed interfering matrix compounds. Extraction efficacy of oxylipins on anion-exchanging BondElut cartridges was low, while removal of matrix was nearly perfect. None of the protocols led to a high extraction efficacy of analytes while removing all interfering matrix components. However, SPE on a C18-material with removal of matrix by water and n-hexane prior elution with methyl formate showed the best performance for the analysis of a broad spectrum of oxylipins in plasma.

Targeted metabolomics of the arachidonic acid cascade: current state and challenges of LC-MS analysis of oxylipins.

Quantification of eicosanoids and oxylipins derived from other polyunsaturated fatty acids in biological samples is crucial for a better understanding of the biology of these lipid mediators. Moreover, a robust and reliable quantification is necessary to monitor the effects of pharmaceutical intervention and diet on the arachidonic acid (AA) cascade, one of today's most relevant drug targets. Low (sub-nanomolar) concentrations and a large number of structurally similar analytes, including regioisomers, require high chromatographic resolution and selective and sensitive mass spectrometry analysis. Currently, reversed-phase liquid chromatography in combination with detection on sensitive triple-quadrupole instruments, operating in selected reaction monitoring mode, is the main method of quantitative oxylipin analysis. A lack of standardized sample collection, handling, and preparation procedures, degradation of the analytes during sample preparation, and purity and availability of standards (internal standards) are the major problems of targeted metabolomics approaches for the AA cascade. Major challenges for instrumental analytical methods are the detection of esterified oxylipins, and separation and individual detection of oxylipin isomers. Solving these problems would help to further knowledge of the biology of lipid mediators, and is an important task for bio-analytical research.

Comparison of the effects of long-chain omega-3 fatty acid supplementation on plasma levels of free and esterified oxylipins.

INTRODUCTION: It is believed that many of the beneficial effects of long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) are mediated by their oxidized metabolites, the oxylipins. The formation and biological role of many cytochrome P450 and lipoxygenase derived hydroxy, epoxy and dihydroxy FA, particularly of oxylipins esterified in polar lipids and triglycerides remain unclear. In this study, we compared the impact of twelve weeks of LC n-3 PUFA supplementation on the patterns of free and total (sum of esterified and free) hydroxy, epoxy and dihydroxy FAs. SUBJECTS AND METHODS: Subjects (5 male; 5 female) between 46 and 70 years were supplemented with 1.1g/d of eicosapentaenoic acid (EPA) and 0.74g/d docosahexaenoic acid (DHA) as ethyl esters. Blood samples were drawn before and after twelve weeks of treatment. Oxylipins in plasma were analyzed by LC-MS directly for free oxylipins and after saponification. Relative FA composition in erythrocyte membranes was analyzed by GC. RESULTS: LC n-3 PUFA treatment led to a significant increase in EPA (200%) and DHA (23%) in erythrocyte membranes. Of the oxylipins measured in plasma, total and free EPA-derived metabolites were highly increased (70-150%), while total AA-derived metabolites were decreased on average by 30%. There was no effect on DHA-metabolites. Concentrations of total hydroxy and epoxy FAs in plasma were considerably higher compared to free hydroxy and epoxy FAs (up to 350 times), while levels of most free dihydroxy FAs were in a similar range to total dihydroxy FAs. However, the individual ratios between total and free plasma oxylipins remained unchanged after LC n-3 PUFA treatment. DISCUSSION AND CONCLUSIONS: LC n-3 PUFA supplementation causes a shift in the levels of circulating oxylipins, having the strongest impact on EPA-derived epoxy, dihydroxy and hydroxy FA. The unchanged ratio of free and esterified oxylipins in plasma indicates that both concentrations are valuable biomarkers for assessing the individual status of these lipid mediators.

APOE Genotype Modifies the Plasma Oxylipin Response to Omega-3 Polyunsaturated Fatty Acid Supplementation in Healthy Individuals.

The omega-3 polyunsaturated fatty acids (n-3 PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), mediate inflammation in large part by affecting pro-inflammatory and anti-inflammatory/pro-resolving oxylipin concentrations. Common gene variants are thought to underlie the large inter-individual variation in oxylipin levels in response to n-3 PUFA supplementation, which in turn is likely to contribute to the overall heterogeneity in response to n-3 PUFA intervention. Given its known role in inflammation and as a modulator of the physiological response to EPA and DHA, here we explore, for the first time, the differential response of plasma hydroxy-, epoxy- and dihydroxy-arachidonic acid, EPA and DHA oxylipins according to apolipoprotein E (APOE) genotype using samples from a dose-response parallel design RCT. Healthy participants were given doses of EPA+DHA equivalent to intakes of 1, 2, and 4 portions of oily fish per week for 12 months. There was no difference in the plasma levels of EPA, DHA or ARA between the wildtype APOE3/E3 and APOE4 carrier groups after 3 or 12 months of n-3 PUFA supplementation. At 12 months, hydroxy EPAs (HEPEs) and hydroxy-DHAs (HDHAs) were higher in APOE4 carriers, with the difference most evident at the highest EPA+DHA intake. A significant APOE*n-3 PUFA dose effect was observed for the CYP-ω hydroxylase products 19-HEPE (p = 0.027) and 20-HEPE (p = 0.011). 8-HEPE, which, along with several other plasma oxylipins, is an activator of peroxisome proliferator activated receptors (PPARs), showed the highest fold change in APOE4 carriers (14-fold) compared to APOE3/E3 (4-fold) (p = 0.014). Low basal plasma EPA levels (EPA < 0.85% of total fatty acids) were associated with a greater change in 5-HEPE, 9-HEPE, 11-HEPE, and 20-HEPE compared to high basal EPA levels (EPA > 1.22% of total fatty acids). In conclusion, APOE genotype modulated the plasma oxylipin response to increased EPA+DHA intake, with APOE4 carriers presenting with the greatest increases following high dose n-3 PUFA supplementation for 12 months.

Oxylipin patterns in human colon adenomas.

OBJECTIVE: Cyclooxygenase (COX)-derived prostaglandin E2 (PGE2) is an important lipid mediator in colorectal carcinoma (CRC) pathogenesis. Other lipid mediators derived from lipoxygenases (LOX) have also been implicated in neoplastic processes in the colon. In this study we aimed to characterize lipid mediators, so called oxylipins, in human colon adenomatous polyps. DESIGN: We quantified oxylipins in healthy colon tissue and colorectal adenoma tissue procured during routine colonoscopy examinations. Lipid metabolite profiles were analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: Adenoma tissue showed a distinct prostaglandin profile as compared to normal colon mucosa. Interestingly, PGE2 was not higher in adenoma tissue as compared to normal mucosa. In contrast, we found significantly lower levels of prostaglandin D2, prostaglandin J2, and prostaglandin D1 in adenoma tissue. Furthermore, levels of 5-LOX and 12-LOX pathway products were clearly increased in adenoma biopsy samples. We also investigated the effect of aspirin treatment on prostaglandin profiles in adenoma tissue in a subset of patients and found a trend towards decreased prostaglandin levels in response to aspirin. CONCLUSION: The human data presented here show specific changes of oxylipin profiles in colon adenoma tissue with decreased prostaglandin D2 levels as well as increased 5- and 12-LOX metabolites.

Effects of chronic low-dose aspirin treatment on tumor prevention in three mouse models of intestinal tumorigenesis.

Although early detection and treatment of colorectal cancer (CRC) have improved, it remains a significant health-care problem with high morbidity and mortality. Data indicate that long-term intake of low-dose aspirin reduces the risk of CRC; however, the mechanisms underlying this chemopreventive effect are still unclear. Different mouse models for inflammation-associated, sporadic, and hereditary CRC were applied to assess the efficacy and mechanism of low-dose aspirin on tumor prevention. An initial dosing study performed in healthy mice indicates that aspirin at a dose of 25 mg/kg/d has a similar pharmacodynamic effect as low-dose aspirin treatment in human subjects (100 mg/d). Chronic low-dose aspirin treatment suppresses colitis-associated and to a lesser extent spontaneous tumorigenesis in mice. Aspirin's antitumor effect is most pronounced in a preventive approach when aspirin administration starts before the tumor-initiating genotoxic event and continues for the duration of the experiment. These effects are not associated with alterations in cell proliferation, apoptosis, or activation of signaling pathways involved in CRC. Aspirin-induced reduction in tumor burden is accompanied by inhibition of thromboxane B2 formation, indicating reduced platelet activation. Aspirin treatment also results in decreased colonic prostaglandin E2 formation and tumor angiogenesis. With respect to colitis-triggered tumorigenesis, aspirin administration is associated with a reduction in inflammatory activity in the colon, as indicated by decreased levels of pro-inflammatory mediators, and tumor-associated iNOS-positive macrophages. Our results suggest that low-dose aspirin represents an effective antitumor agent in the context of colon tumorigenesis primarily due to its well-established cyclooxygenase inhibition effects.

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

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

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

Oxylipins, including the well-known eicosanoids, are potent lipid mediators involved in numerous physiological and pathological processes. Therefore, their quantitative profiling has gained a lot of attention during the last years notably in the active field of health biomarker discovery. Oxylipins include hundreds of structurally and stereochemically distinct lipid species which today are most commonly analyzed by (ultra) high performance liquid chromatography-mass spectrometry based ((U)HPLC-MS) methods. To maximize the utility of oxylipin profiling in clinical research, it is crucial to understand and assess the factors contributing to the analytical and biological variability of oxylipin profiles in humans. In this review, these factors and their impacts are summarized and discussed, providing a framework for recommendations expected to enhance the interlaboratory comparability and biological interpretation of oxylipin profiling in clinical research.