Stability of total oxylipins – influence of plasma generation and long-term storage

International audienc

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

Efferocytosis potentiates the expression of arachidonate 15-lipoxygenase (ALOX15) in alternatively activated human macrophages through LXR activation

Macrophages acquire anti-inflammatory and proresolving functions to facilitate resolution of inflammation and promote tissue repair. While alternatively activated macrophages (AAMs), also referred to as M2 macrophages, polarized by type 2 (Th2) cytokines IL-4 or IL-13 contribute to the suppression of inflammatory responses and play a pivotal role in wound healing, contemporaneous exposure to apoptotic cells (ACs) potentiates the expression of anti-inflammatory and tissue repair genes. Given that liver X receptors (LXRs), which coordinate sterol metabolism and immune cell function, play an essential role in the clearance of ACs, we investigated whether LXR activation following engulfment of ACs selectively potentiates the expression of Th2 cytokine-dependent genes in primary human AAMs. We show that AC uptake simultaneously upregulates LXR-dependent, but suppresses SREBP-2-dependent gene expression in macrophages, which are both prevented by inhibiting Niemann-Pick C1 (NPC1)-mediated sterol transport from lysosomes. Concurrently, macrophages accumulate sterol biosynthetic intermediates desmosterol, lathosterol, lanosterol, and dihydrolanosterol but not cholesterol-derived oxysterols. Using global transcriptome analysis, we identify anti-inflammatory and proresolving genes including interleukin-1 receptor antagonist (IL1RN) and arachidonate 15-lipoxygenase (ALOX15) whose expression are selectively potentiated in macrophages upon concomitant exposure to ACs or LXR agonist T0901317 (T09) and Th2 cytokines. We show priming macrophages via LXR activation enhances the cellular capacity to synthesize inflammation-suppressing specialized proresolving mediator (SPM) precursors 15-HETE and 17-HDHA as well as resolvin D5. Silencing LXRα and LXRβ in macrophages attenuates the potentiation of ALOX15 expression by concomitant stimulation of ACs or T09 and IL-13. Collectively, we identify a previously unrecognized mechanism of regulation whereby LXR integrates AC uptake to selectively shape Th2-dependent gene expression in AAMs

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

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

Indirect effect of alpha-1-antitrypsin on endotoxin-induced IL-1β secretion from human PBMCs

Human alpha-1-antitrypsin (AAT) encoded by the SERPINA1 gene, is an acute phase glycoprotein that regulates inflammatory responses via both protease inhibitory and non-inhibitory activities. We previously reported that AAT controls ATP-induced IL-1β release from human mononuclear cells by stimulating the release of small bioactive molecules. In the current study, we aimed to elucidate the identity of these putative effectors released from human PBMCs in response to AAT, which may inhibit the LPS-induced release of IL-1β. We pre-incubated human PBMCs alone or with different preparations of AAT (4 mg/ml) for 30 min at 37°C, 5% CO2, and collected cell supernatants filtered through centrifugal filters (cutoff 3 kDa) to eliminate AAT and other high molecular weight substances. Supernatants passed through the filters were used to culture PBMCs isolated from the autologous or a heterologous donors with or without adding LPS (1 μg/ml) for 6 h. Unexpectedly, supernatants from PBMCs pre-incubated with AAT (Zemaira®), but not with other AAT preparations tested or with oxidized AAT (Zemaira®), lowered the LPS-induced release of IL-1β by about 25%–60% without affecting IL1B mRNA. The reversed-phase liquid chromatography coupled with mass spectrometry did not confirm the hypothesis that small pro-resolving lipid mediators released from PBMCs after exposure to AAT (Zemaira®) are responsible for lowering the LPS-induced IL-1β release. Distinctively from other AAT preparations, AAT (Zemaira®) and supernatants from PBMCs pre-treated with this protein contained high levels of total thiols. In line, mass spectrometry analysis revealed that AAT (Zemaira®) protein contains freer Cys232 than AAT (Prolastin®). Our data show that a free Cys232 in AAT is required for controlling LPS-induced IL-1β release from human PBMCs. Further studies characterizing AAT preparations used to treat patients with inherited AAT deficiency remains of clinical importance

Stability of oxylipins during plasma generation and long-term storage

International audienceOxidized 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 degrees 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 degrees C or 4 h at 20 degrees C before centrifugation of EDTA-whole blood and up to 5 days at - 20 degrees 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 degrees C for 15 months. Overall, we demonstrate that total plasma oxylipins are robust regarding delays during plasma generation and long-term storage at -80 degrees C supporting the application of oxylipin profiling in clinical research

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