Development of an Optimized LC-MS Method for the Detection of Specialized Pro-Resolving Mediators in Biological Samples

The cardioprotective and anti-inflammatory effects of long chain omega-3 polyunsaturated fatty acids (n3 PUFA) are believed to be partly mediated by their oxygenated metabolites (oxylipins). In the last two decades interest in a novel group of autacoids termed specialized pro-resolving mediators (SPMs) increased. These are actively involved in the resolution of inflammation. SPMs are multiple hydroxylated fatty acids including resolvins, maresins, and protectins derived from the n3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as well as lipoxins derived from arachidonic acid (ARA). In the present paper, we developed an LC-MS/MS method for a comprehensive set of 18 SPMs derived from ARA, EPA, and DHA and integrated it into our targeted metabolomics platform. Quantification was based on external calibration utilizing five deuterated internal standards in combination with a second internal standard for quality assessment of sample preparation in each sample. The tandem mass spectrometric parameters were carefully optimized for sensitive and specific detection. The influence of source parameters of the used AB Sciex 6500 QTRAP instrument as well as electronic parameters and the selection of transitions are discussed. The method was validated/characterized based on the criteria listed in the European Medicines Agency (EMA) guideline on bioanalytical method validation and method performance is demonstrated regarding recovery of internal standards (between 78 ± 4% and 87 ± 3% from 500 μL of human serum) as well as extraction efficacy of SPMs in spiked plasma (intra-day accuracy within ±20 and ±15% at 0.1 and 0.3 nM in plasma, respectively). Based on the lower limit of quantification of 0.02–0.2 nM, corresponding to 0.18–2.7 pg on column, SPMs were generally not detectable/quantifiable in plasma and serum supporting that circulating levels of SPMs are very low, i.e., <0.1 nM in healthy subjects. Following septic shock or peritonitis, SPMs could be quantified in the samples of several patients. However, in these studies with a small number of patients no clear correlation with severity of inflammation could be observed

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

IntroductionIt 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 methodsSubjects (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.ResultsLC 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 conclusionsLC 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

Extraction of lipids and oxylipins from plasma for quantification of total oxylipins – Challenges and strategies

Several eicosanoids and other oxylipins are potent lipid mediators which are involved in theregulation of physiological functions such as inflammation. It is believed that they act predominantlyin their free, i.e. non-esterified, form. However, a major portion of oxylipins is found as esters, e.g. inpolar lipids.Only little information is available on the biological activity of these esterified oxylipins. An importantstep towards a better understanding of their biological role is a comprehensive comparison of thechanges in the pattern of free vs. esterified mediators induced by pharmacological intervention ordietary supplementation as well as during onset and progression of diseases.While several LC-MS based methods for the detection of free oxylipins have been developed,esterified oxylipins are commonly quantified as a sum of free and esterified oxylipins following basehydrolysis. However, different approaches have been described for preparation of samples beforehydrolysis and for the solid phase extraction. Here, we present a three-step strategy for thequantification of total oxylipins including extraction of total lipids, saponification to liberate esterifiedoxylipins and solid phase extraction of free oxylipins. We thoroughly investigated different liquidliquidextraction procedures and protein precipitation in terms of extraction efficiency for variouslipid classes from plasma. Moreover, to optimize sample throughout we compared extraction of freeoxylipins via solid phase extraction on cartridges with 96-well plates.In conclusion, our results emphasis the challenges related to the extraction procedures and providedifferent strategies for reliable oxylipin quantification with a focus on efficient and reproducibleextraction

Intra-individual variance of the human plasma oxylipin pattern: Low inter-day variability in fasting blood samples: Versus high variability during the day

Introduction: Several eicosanoids and other oxylipins are important lipid mediators. Reliable quantification in plasma is important to assess the state of disease, action of drugs and the biology of oxylipins. In order to monitor biological changes, low background variability of oxylipin concentrations in biological samples is essential for proper interpretation of oxylipin biology. However, only little is known about the variation in the oxylipin profile in healthy human subjects. Experimental: Inter-day variation in circulating oxylipins after overnight fasting was investigated in healthy young men on either a standardized or non-standardized diet during a (24 to) 48 h time interval. Intra-day variance was investigated during an 8 h time interval (covering breakfast and lunch meals) in men on a standardized diet with blood sampling at 0, 2, 4, 6 and 8 hours. Free oxylipins in plasma were analyzed using a targeted metabolomics platform for the quantification of 160 oxylipins from different precursors. Analytical variation was evaluated based on quality control plasma samples. Results: Free oxylipins in quality control plasma samples showed low variations (<20% for most analytes). Inter-day variations in fasting blood were in the same range, while significant differences were observed within the day (intra-day variance). Conclusion: Based on the low intra-individual inter-day variance in concentrations of free oxylipins, our results demonstrate the suitability of fasting plasma for the investigation of oxylipin biology. In non-fasting plasma samples, the variations were high during the day. Thus, non-fasting plasma samples appear to be unsuitable to evaluate biologically relevant changes, for instance, those caused by disease or drugs. However, it remains to be determined if the same standardized meal results in reproducible modulations of the oxylipin profile allowing evaluation of the oxylipin pattern during the postprandial state

MS-based targeted metabolomics of eicosanoids and other oxylipins: Analytical variability and interlaboratory comparison of esterified oxylipin profile

IntroductionOxylipins are potent lipid mediators involved in numerous physiological and pathological processesand their quantitative profiling has gained a lot of attention [1]. To maximize the utility of theoxylipin profiling in clinical research it is now crucial (i) to assess its analytical variability; (ii) todetermine its comparability between laboratories and (iii) to identify putative critical oxylipins. Thesethree main challenges are addressed within the EU JPI HDHL*-Oxygenate project.Technological and methodological innovationTo address the challenges stated above, a SOP was established by a reference laboratory for the MSbasedtargeted metabolomics of total oxylipins (free + esterified, ~160 oxylipins) in human plasma[2]. The intra- and inter-day variabilities of each oxylipin were assessed. Then, the SOP wastransferred to 4 independent laboratories together with mixtures of internal standards, calibrantsand 7 different pools of plasma to determine the comparability of oxylipin profiles between labs.Results and impactThe cumulated intra-/inter-day variabilities revealed that 68 % of oxylipins (>LLOQ) have a CV<20%.The interlab-variability was low and dependent on the type of plasma analyzed. Overall, our resultsshow that the MS-based profiling of total oxylipins in human plasma is a robust tool for clinicalresearch. Moreover, the comparability of oxylipin profiles will allow generating large-scale databasesallowing a better understanding of the relationships between oxylipins and human health.References[1] Gladine C. et al. 2019. Free Radical Biology and Medicine 144 (2019) 72–89[2] Ostermann et al. 2019. Prostag Oth Lipid M. DOI: 10.1016/j.prostaglandins.2019.106384*Joint Programming Initiative “A healthy diet for a healthy life

Profilage lipidomique des oxylipines pour mieux caractériser le syndrome cardiométabolique et ses liens avec l’alimentation

Session IV: Obésité (Modérateurs : K. Couturier et J.A. Nazare)Le syndrome cardiométabolique est un désordre complexe et progressif qui constitue un facteur de risque significatif de maladies cardiovasculaires et de diabète de type II. Il se caractérise par l’association d’au moins trois anomalies incluant un tour de taille élevé, une pression sanguine haute, une hyperglycémie, une hypertriglycéridémie et un taux faible de HDL-cholestérol. Le diagnostic et la prise en charge du syndrome cardiométabolique reste insatisfaisant car il est souvent trop tardif et pas assez intégratif. Les oxylipines, métabolites issus de l’oxygénation des acides gras polyinsaturés, sont des médiateurs lipidiques impliqués dans la régulation de nombreux processus biologiques en lien avec le développement du syndrome cardiométabolique. Par ailleurs, la synthèse des oxylipines est modulable par l’alimentation. Dans le cadre du projet JPI-HDHL OXYGENATE, nous avons émis l’hypothèse que la signature d’oxylipines pourrait permettre d’identifier des perturbations précoces du statut cardiométabolique et pourrait aider à suivre l’effet d’une intervention nutritionnelle sur la prise en charge du syndrome cardiométabolique. Le projet OXYGENATE a pour but d’identifier et valider les signatures d’oxylipines caractéristiques du statut cardiométabolique et de son évolution

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

BackgroundThe 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 &gt; 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

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

Activation of Lipid Mediator Formation Due to Lipoprotein Apheresis

Lipoprotein apheresis reliably reduces low-density lipoprotein (LDL) cholesterol in patients with atherosclerotic disease and therapy-refractory hypercholesterolemia or elevated lipoprotein (a) (Lp(a)). Besides lowering lipoproteins and triglycerides, apheresis also decreases levels of essential omega-6 and omega-3 polyunsaturated fatty acids (n-6 and n-3 PUFAs) in blood plasma. In contrast, heparin-induced extracorporeal low-density lipoprotein precipitation (HELP) lipid apheresis might increase the formation of potentially pro-inflammatory and pro-thrombotic lipid mediators derived from n-6 and n-3 PUFAs. The study presented here analyzed lipid mediator profiles in the plasma of patients with hyperlipidemia treated by one of three different apheresis methods, either HELP, direct absorption (DA), or membrane filtration (MDF), in a direct pre- and post-apheresis comparison. Using gas chromatography and liquid chromatography tandem mass spectrometry (LC-MS/MS) we were able to analyze fatty acid composition and the formation of lipid mediators called oxylipins. Our data illustrate&#8212;particularly in HELP-treated patients&#8212;significant decreases of essential omega-6 and omega-3 polyunsaturated fatty acids in blood plasma but significant increases of PUFA-derived lipoxygenase-, as well as cyclooxygenase- and cytochrome P450-derived lipid mediators. Given that n-3 PUFAs in particular are presumed to be cardioprotective and n-3 PUFA-derived lipid mediators might limit inflammatory reactions, these data indicate that n-3 PUFA supplementation in the context of lipid apheresis treatment might have additional benefits through apheresis-triggered protective n-3 PUFA-derived lipid mediators