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

Comparaison inter-laboratoires de profils d’oxylipines par lipidomique ciblée dans le cadre du projet OXYGENATE

Actuellement 20 à 25% de la population adulte mondiale présente un syndrome cardiométabolique. Ce syndrome est un exemple de maladie multifactorielle en lien avec l’alimentation et se caractérise par des troubles cardiovasculaires, métaboliques et inflammatoires. Ces troubles amènent au développement du diabète de type II et des maladies cardiovasculaires. Cependant le diagnostic de ce syndrome n’est pas satisfaisant car pas assez intégratif et précoce pour permettre une prise en charge nutritionnelle. Les oxylipines, métabolites issus de l’oxygénation des acides gras polyinsaturés via différentes voies de biosynthèse, sont connues pour être des médiateurs lipidiques impliqués dans la régulation de processus biologiques liés au syndrome cardiométabolique et dont la biosynthèse est modulée par le statut cardiométabolique et aussi par l’alimentation. D’où l’hypothèse qu’un profilage complet des oxylipines pourrait révéler des perturbations précoces du statut cardiométabolique et pourrait être un nouvel outil d’évaluation de l’efficacité des préventions et interventions nutritionnelles. Le projet OXYGENATE a pour but d’identifier et valider des oxylipines discriminant le statut cardiométabolique et la qualité de l’alimentation. Pour cela, il est nécessaire d’avoir une méthode fiable de profilage quantitatif des oxylipines permettant d’obtenir des profils comparables entre différents laboratoires. Une comparaison inter-laboratoires de notre méthode a été réalisée afin d’estimer les variabilités analytiques et d’identifier les oxylipines critiques. Cinq laboratoires ont préparé (en triplicat et sur 2 jours différents) et analysé 7 plasmas présentant des profils d’oxylipines très contrastés. Les variabilités intra- et inter-jour ainsi que la part de variabilité attribuable à la préparation et à l’appareillage sont évalués. Au-delà de l’intérêt pour le projet, cette comparaison inter-laboratoires unique permettra d’harmoniser les profils d’oxylipines réalisés par l’ensemble de la communauté scientifique

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

Rapid assessment of fatty acyls chains of phospholipids and plasmalogens by atmospheric pressure chemical ionization in positive mode and high-resolution mass spectrometry using in-source generated monoacylglycerol like fragments intensities

International audienceWe recently published a new concept using monoacylglycerol-like fragments [MG+H-H2O]+ (ions B) pro-duced in-source by atmospheric pressure photoionization in positive mode and high-resolution massspectrometry for the determination of the fatty acyl (FA) composition of triacylglycerols (TGs) from plantoils. This study extends the concept to the phospholipids (PLs) category and shows that the APCI+ sourcecan also be used. Moreover, the coupling with NP-LC allows to simultaneously analyze different PLsclasses in the same sample. We compared the relative intensities of the ions B produced in-source tothe % composition of FAs determined by GC-FID. In the case of PLs from natural extracts composed ex-clusively of diacyl-PLs, the relative intensities of ions B are close to the % of the FAs obtained by GC-FID.This approach is not directly useable for extracts containing plasmalogens (P-PLs). For these PLs, acidichydrolysis by HCl fumes allows hydrolyzing selectively vinyl ether functions to form lyso-PLs. The analy-sis of hydrolyzed extracts makes it possible to obtain the composition of P-PLs FAs thanks to the lyso-PLsthus formed, while the diacyl-PLs composition remains unchanged. Unlike GC-FID FAs determination, thisapproach allows a distinction between the diacyl-PLs and P-PLs FAs composition. We also found that theion B intensities were consistent among the PL classes (PG, PE, PA, PI, CL, PS and PC) and lyso- forms(LPE and LPC). In the case of the diacyl-PLs extracts analyzed, no statistically significant differences werefound between the PLs FAs compositions calculated from ion B intensities and the corresponding GC-FIDdata. A weighting coefficient was applied to correct ion B intensities issued from polyunsaturated FAswith three or more double bonds. The fatty alkenyls composition of P-PLs could also be calculated fromthe % intensities of specific ions

Harmonized procedures lead to comparable quantification of total oxylipins across laboratories

International audienceOxylipins 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

Improving lipid mapping in Genome Scale Metabolic Networks using ontologies

International audienceIntroduction To interpret metabolomic and lipidomic profiles, it is necessary to identify the metabolic reactions that connect the measured molecules. This can be achieved by putting them in the context of genome-scale metabolic network reconstructions. However, mapping experimentally measured molecules onto metabolic networks is challenging due to differences in identifiers and level of annotation between data and metabolic networks, especially for lipids.Objectives To help linking lipids from lipidomics datasets with lipids in metabolic networks, we developed a new matching method based on the ChEBI ontology. The implementation is freely available as a python library and in MetExplore webserver.Methods Our matching method is more flexible than an exact identifier-based correspondence since it allows establishing a link between molecules even if a different level of precision is provided in the dataset and in the metabolic network. For instance, it can associate a generic class of lipids present in the network with the molecular species detailed in the lipidomics dataset. This mapping is based on the computation of a distance between molecules in ChEBI ontology.Results We applied our method to a chemical library (968 lipids) and an experimental dataset (32 modulated lipids) and showed that using ontology-based mapping improves and facilitates the link with genome scale metabolic networks. Beyond network mapping, the results provide ways for improvements in terms of network curation and lipidomics data annotation.Conclusion This new method being generic, it can be applied to any metabolomics data and therefore improve our comprehension of metabolic modulations

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

International audienceInside 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

Mass spectrometry-based proteomics in clinical practice amyloid typing: state-of-the-art from a French nationwide cohort

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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