Ziel der vorliegenden Promotionsarbeit war die Untersuchung von Sepsis-assoziierten Veränderungen des Arachidonsäure (AA)-Metabolismus und die Identifikation differentiell regulierter AA-Metabolite mit Prüfung ihres diagnostischen Potentials bei Patienten mit Sepsis unter Anwendung eines in-vitro Lipopolysaccharid (LPS) Vollblutaktivierungs-Modells. In Zellüberständen von nicht-aktiviertem und LPS-aktiviertem Heparinblut (25 Sepsis- Patienten, 15 Gesunde) wurden AA-Metabolite mittels Flüssigkeitschromatographie-Tandem- Massenspektrometrie analysiert. In einer unabhängigen Kohorte (10 Sepsis-Patienten, 3 Gesunde) wurden nach RNA-Isolation aus Zellmaterial zusätzlich Target-Gene des AAMetabolismus (Cyclooxygenase (COX)-2 und mikrosomale Prostaglandin-E-Synthase (mPGES)-1 mittels quantitativer Reverse Transkriptase-Polymerase Kettenreaktion (RT-PCR) untersucht. Es konnte eine differentielle Freisetzung von AA, AA-Analoga und der COX-assoziierten Metabolite Prostaglandin (PG) E2, 11-Hydroxyeicosatetraensäure (HETE) und Thromboxan (TX) B2 zwischen Patienten und gesunden Kontrollpersonen gezeigt werden. Sepsis-Patienten wiesen dabei gegenüber Gesunden eine deutlich reduzierte Freisetzung von AA und den COXassoziierten Metaboliten 11-HETE und PGE2 auf. Das Ausmaß der reduzierten Mediatorenfreisetzung bei Sepsis-Patienten war mit der Schwere der Erkrankungssymptomatik und dem klinischen Outcome assoziiert. Auf Genexpressionsebene zeigte sich eine reduzierte Induzierbarkeit der COX-2 mRNA-Expression bei Sepsis-Patienten gegenüber Gesunden, jedoch eine erhaltene Induzierbarkeit auf der Ebene der mPGES-1.:I Bibliographische Beschreibung………………………………………………………2 II Abkürzungen……………………………..……………………………………………4 III Einleitung 1. Epidemiologie und Definition der Sepsis……………………………………………..…6 2. Pathophysiologie der Sepsis……………………………………………………………..7 3. Stellenwert und Limitationen labordiagnostischer Marker bei Sepsis…………………..8 4. Eicosanoide und Sepsis…………………………………………………………………10 5. In-vitro LPS Vollblutaktivierungs-Modell für die Prüfung der Eicosanoidantwort auf Genexpressions- und Mediatorenebene……………..................12 6. Bestimmung von Eicosanoiden mittels LC-MS/MS........................................................16 7. Zielstellung der Arbeit……………………………………………………………….....18 IV Publikation…………………………………………………………….......................19 V Zusammenfassung der Arbeit………………………………………………………29 VI Literatur……………………………………………………………………………...32 VII Erklärung über die eigenständige Abfassung der Arbeit.………..…………….....35 VIII Lebenslauf………………………………………………………………………........36 IX Spezifizierung des wissenschaftlichen Beitrages zur Publikation...........................38 X Danksagung………………………………………………………………………......39

Nano-DESI Mass spectrometry imaging of eicosanoids in mouse kidney tissue using selected ion monitoring. Nano-DESI mass spectrometry imaging (MSI) is a technique for label-free spatial and molecular characterization of surfaces and biological samples. Eicosanoids are lipid mediators derived from eicosapolyenoic acid- products of arachidonic acid oxidation. Eicosanoids have been of interest to the medical field for many years. Major focus on this lipid class came from the development of nonsteroidal anti-inflammatory drugs (NSAIDs), some of these including aspirin, naproxen, ibuprofen, and acetaminophen work by blocking either the formation or the effects of eicosanoids. These lipids also play important roles in various body functions (cardiovascular, renal, gastrointestinal, neuronal) and as mediators of inflammation, asthma, fever, pain, hypertension, and stroke. Typically, eicosanoids occur in subnanomolar concentrations, despite their high level of bioactivity, which makes them significantly more difficult to analyze via direct mass spectrometry. Here, selected ion monitoring (SIM) is used to increase the signal-to-noise of the identified eicosanoids compared to a broadband full scan mode.

Oxidized low-density lipoproteins (OxLDL) mediate many of the pathological events associated with atherosclerosis. Oxysterols, the major cytotoxic component of OxLDL, induce apoptosis in macrophages by a calcium flux mediated activation of cytosolic phospholipase A2 resulting in the release of arachidonic acid (AA). Inhibition of AA metabolism has been shown to protect macrophages from oxysterol-induced apoptosis. The current study explores the steps in the oxysterol-induced apoptosis signaling pathway in murine macrophages subsequent to the liberation of AA. To elucidate this mechanism, two oxysterols, 7-ketocholesterol and 25-hydroxycholesterol (25-OHC), were used to induce apoptosis in murine macrophage cell lines (P388D1, and Raw 264.7) and mouse peritoneal macrophages (MPMs). Pharmacological inhibition of eicosanoid synthesis or genetic knockout of important eicosanoid biosynthetic genes had no significant effect on the induction of apoptosis by oxysterols in macrophages. The induction of apoptosis in macrophage cell lines and MPMs by oxysterols and OxLDL was suppressed by Sandoz 58-035, an inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT). Furthermore, in comparison to wild-type MPMs, ACAT-1 deficient MPMs were found to be resistant to apoptosis induced by oxysterols or OxLDL. Macrophages treated with 7KC accumulated ACAT-derived cholesteryl and 7-ketocholesteryl esters. An inhibitor of cholesterol trafficking, U18666A, specifically prevented the accumulation of cholesteryl esters, but not 7-ketocholesteryl esters nor the induction of apoptosis. An inhibitor of cPLA2 prevented the accumulation of 7-ketocholesteryl esters. This inhibition was reversed by supplementing oleic acid or AA; however, only AA supplementation restored the induction of apoptosis. These results suggest that oxysterols not only initiate the apoptosis pathway by activating cPLA2, but also participate in the downstream signaling pathway when esterified by ACAT to form arachidonyl oxysterols. We also demonstrate that macrophages lacking the cannabinoid type-2 (CB2) receptor have reduced levels of apoptosis when treated with oxysterols or OxLDL in comparison to wild-type macrophages and that a CB2 specific antagonist blocks oxysterol-induced apoptosis in macrophages suggesting that the CB2 receptor is involved in this pathway, perhaps by interacting with the arachidonyl oxysterols.

In clinical diagnostics and research, blood samples are one of the most frequently used materials. Nevertheless, exploring the chemical composition of human plasma and serum is challenging due to the highly dynamic influence of pre-analytical variation. A prominent example is the variability in pre-centrifugation delay (time-to-centrifugation; TTC). Quality indicators (QI) reflecting sample TTC are of utmost importance in assessing sample history and resulting sample quality, which is essential for accurate diagnostics and conclusive, reproducible research. In the present study, we subjected human blood to varying TTCs at room temperature prior to processing for plasma or serum preparation. Potential sample QIs were identified by Ultra high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) based metabolite profiling in samples from healthy volunteers (n = 10). Selected QIs were validated by a targeted MS/MS approach in two independent sets of samples from patients (n = 40 and n = 70). In serum, the hypoxanthine/guanosine (HG) and hypoxanthine/inosine (HI) ratios demonstrated high diagnostic performance (Sensitivity/Specificity > 80%) for the discrimination of samples with a TTC > 1 h. We identified several eicosanoids, such as 12-HETE, 15-(S)-HETE, 8-(S)-HETE, 12-oxo-HETE, (±)13-HODE and 12-(S)-HEPE as QIs for a pre-centrifugation delay > 2 h. 12-HETE, 12-oxo-HETE, 8-(S)-HETE, and 12-(S)-HEPE, and the HI- and HG-ratios could be validated in patient samples.

No / Lipid mediators are produced from the oxidation of polyunsaturated fatty acids through enzymatic and free radical-mediated reactions. When subject to oxygenation via cyclooxygenases, lipoxygenases, and cytochrome P450 monooxygenases, polyunsaturated fatty acids give rise to an array of metabolites including eicosanoids, docosanoids, and octadecanoids. These potent bioactive lipids are involved in many biochemical and signaling pathways, with inflammation being of particular importance. Moreover, because they are produced by more than one pathway and substrate, and are present in a variety of biological milieus, their analysis is not always possible with conventional assays. Liquid chromatography coupled to electrospray mass spectrometry offers a versatile and sensitive approach for the analysis of bioactive lipids, allowing specific and accurate quantitation of multiple species present in the same sample. Here we explain the principles of this approach to mediator lipidomics and present detailed protocols for the assay of enzymatically produced oxygenated metabolites of polyunsaturated fatty acids that can be tailored to answer biological questions or facilitate assessment of nutritional and pharmacological interventions.

Erythema is a hallmark skin response to excessive ultraviolet radiation (UVR) and is associated with cutaneous inflammation. Both are mediated by inflammatory mediators including nitric oxide (NO), prostaglandin E2 (PGE2) and chemoattractants such as 12-hydroxyeicosatetraenoic acid (12-HETE) leading to vasodilation and increased leukocyte infiltration. The erythematous response is more pronounced in individuals with low basal melanin levels or who fail to respond to UVR with a robust up-regulation of melanogenesis. While melanin production is a key function of melanocytes, these cells can also produce NO and PGE2, and are located in close proximity to the dermal vasculature. It has been hypothesized that melanocytes with poor melanogenic capacity may participate in the inflammatory response to UVR. The aim of this project was to investigate the inflammatory response in the skin of individuals with either skin phototype (SPT) 1 or 4 to UVR. Sixteen normal healthy individuals were selected for study (8 SPT-1 & 8 SPT-4). Buttock skin was investigated by immunohistochemistry for leukocyte subtypes, eicosanoid producing enzymes and NO synthases under basal and UVR-stimulated conditions. In addition primary cultures of epidermal melanocytes (EM) were established from 16 individuals (8 SPT-1 & 8 SPT-4) and assessed for the presence of eicosanoid-producing enzymes, melanogenic enzymes and NO synthases, by immunocytochemistry, Polymerase Chain Reaction and Western Blotting and for the production of the main pro-inflammatory eicosanoid PGE2 by ELISA and Mass Spectrometry. Moreover, the fatty acid composition of cultured melanocytes was assessed by Gas Chromatography. Results showed that individuals with SPT-1 had significantly greater neutrophil infiltration into the epidermis than those with SPT-4 at 24 hrs post-UVR. Moreover, CD3+ lymphocyte infiltration into the dermis was significantly greater in individuals with SPT-4 than those with SPT-1 at 24 and 72 hrs post-UVR. NOS-1, NOS-3, 12-LOX and COX-2 expression were significantly increased in SPT-1 skin, while NOS-2 and 15-LOX were significantly increased in SPT-4 skin. As 12-LOX and COX-2 products are chemoattractive (for neutrophils) and pro-inflammatory respectively these data could explain the greater observed neutrophil infiltration in SPT-1. The 15-LOX product (15-HETE) is anti-inflammatory and may suggest that 15-LOX up-regulation in SPT-4 skin may aid resolution of the sunburn response, which in part may be mediated by CD3+ lymphocytes and a class-switch in eicosanoid production from COX to LOX products. Melanocyte primary cultures surprisingly showed that SPT was not correlated with melanin content or melanogenic enzyme expression/activity suggesting that all melanocytes in vitro contained the necessary cellular machinery to produce melanin. This finding may reflect also their equal treatment under these enriched culture conditions, which may or may not be available to these cells in situ. Moreover, all melanocytes expressed the necessary machinery (PLA2, COX-1, cPGES) to produce PGE2. However, only some cultures did so at baseline and in response to UVR, and this was not correlated with SPT. A positive correlation was found however between expression level of dopachrome tautomerase (DCT) and protection against PGE2 production in response to UVR, which may suggest a novel role for DCT unrelated to melanogenesis. In summary this research project has generated data that highlights differences between the skin of individuals with SPT-1 and those with SPT-4, and may provide evidence that the keratinocyte partner contributes significantly to the SPT-associated response. This research may also suggest DCT as a novel therapeutic target to protect EM from participation in the UVR-associated inflammatory response in skin.

Prostanoids are oxygenated derivatives of arachidonic acid with a wide range of physiological effects in vertebrates including modulation of inflammation and innate immune responses. Nonsteroidal anti-inflammatory drugs (NSAIDs) act through inhibition of cyclooxygenase (COX) conversion of arachidonic acid to prostanoids. In order to better understand the potential of environmental NSAIDS for interruption of normal levels COX products in fishes, we developed an LC/MS/MS-based approach for tissue analysis of 7 prostanoids. Initial studies examining muscle, gut and gill demonstrated that prostaglandin E2 (PGE2) was the most abundant of the measured prostanoids in all tissues and that gill tissue had the highest and most consistent concentrations of PGE2. After short-term 48-h laboratory exposures to concentrations of 5, 25, 50 and 100 ppb ibuprofen, 50.0ppb and 100.0 ppb exposure concentrations resulted in significant reduction of gill tissue PGE2 concentration by approximately 30% and 80% respectively. The lower exposures did not result in significant reductions when compared to unexposed controls. Measured tissue concentrations of ibuprofen indicated that this NSAID had little potential for bioaccumulation (BCF 1.3) and the IC50 of ibuprofen for inhibition of PGE2 production in gill tissue was calculated to be 0.4 µM. Short-term laboratory exposure to ibuprofen did not result in significant alteration of concentrations of PGE2 at environmentally relevant concentrations.

Les récepteurs nucléaires LXRα et LXRβ sont activés par la fixation de dérivés oxygénés du cholestérol. Ils régulent l’expression de nombreux gènes appartenant au métabolisme du cholestérol et des acides gras, et jouent un rôle important dans l’inflammation et l’immunité innée. L’activation de LXR inhibe le développement de l’athérosclérose, en augmentant l’efflux de cholestérol des macrophages ainsi que le transport inverse jusqu’au foie et l’excrétion biliaire. De plus, LXR diminue la biosynthèse et la captation du cholestérol dans les tissus périphériques. Enfin, les agonistes synthétiques de LXR administrés à des souris diminuent significativement l’inflammation dans les lésions athérosclérotiques, notamment en inhibant la sécrétion de certaines cytokines inflammatoires. Néanmoins LXR régule également la lipogenèse et la synthèse d’acides gras mono-insaturés, et l’administration d’agonistes de LXR s’accompagne également d’effets indésirables liés à cette régulation, comme une accumulation dérégulée d’acides gras dans le foie et une augmentation du taux de LDLs circulantes. Plusieurs autres mécanismes restent encore à être explorés, comme la synthèse d’acides gras polyinsaturés et les conséquences sur le métabolisme cellulaire. Nos travaux identifient une nouvelle voie régulée entièrement par LXR, le métabolisme des acides gras polyinsaturés. Le récepteur nucléaire LXR régule l’ensemble des enzymes FADS1, FADS2 et ELOVL5, responsables de la synthèse d’acides gras polyinsaturés oméga-6 et oméga-3. Cette régulation s’accompagne d’une incorporation d’acide arachidonique dans les phospholipides, via la régulation de LPCAT3, ce qui prépare les macrophages à une synthèse accrue de dérivés inflammatoires issus de l’acide arachidonique, comme la Prostaglandine E2, suite à une stimulation au lipopolysaccharide. La régulation de cette voie par LXR a également un effet sur le développement de l’athérosclérose, augmentant les taux d’acides gras polyinsaturés oméga-6 et oméga-3 dans les plaques d’athérome. Nos résultats montrent donc que LXR régule la synthèse des acides gras polyinsaturés en plus des acides gras mono-insaturés et de la lipogenèse et que cette régulation a des conséquences sur le profil lipidique des macrophages in vitro et in vivo ainsi que sur leur réponse inflammatoire. / The nuclear receptors LXRα and LXRβ are activated by oxygenated metabolites of cholesterol. They regulate the expression of numerous genes belonging to cholesterol and fatty acids metabolism, and play a central role in inflammation and innate immunity. LXR activation inhibits atherosclerosis development, by increasing cholesterol efflux from macrophages as well as reverse cholesterol transport and biliary excretion. In addition, LXR decreases cholesterol uptake and biosynthesis. Synthetic LXR agonists fed to mice significantly decrease inflammation in atherosclerotic lesions, by inhibiting several inflammatory cytokines. However, LXR also regulate lipogenesis and monounsaturated fatty acids synthesis, and LXR agonists supplementation is accompanied by side effects due to this regulation, such as a deregulated accumulation of fatty acids in the liver and an increase in circulating LDLs. Other mecanisms still need to be characterized, such as polyunsaturated fatty acids synthesis and the consequences on cell metabolism. Our work identify a new pathway regulated by LXR, the metabolism of polyunsaturated fatty acids. The nuclear receptor LXR regulates all enzymes responsible for omega-6 and omega-3 polyunsaturated fatty acids synthesis, FADS1, FADS2 and ELOVL5. This regulation is accompanied by an increase in arachidonic acid incorporation in phospholipids, via LPCAT3 regulation, which subsequently primes human macrophages for an increased inflammatory metabolites secretion derived from arachidonic acid, such as Protaglandin E2, following a LPS stimulation. The regulation of this pathway by LXR has an effect on atherosclerosis, increasing omega-6 and omega-3 ployunsaturated fatty acids in atheroma plaques. Our results show therefore that LXR regulates polyunsaturated fatty acids synthesis in addition to monounsaturated fatty acids and lipogenesis, and that this regulation has direct consequences on lipid profile of macrophages in vitro and in vivo as well as on their inflammatory response.

O Glioblastoma (GBM) é um astrocitoma grau IV, representando o glioma de maior malignidade e o tumor cerebral primário mais frequente em humanos. A terapia indicada para o GBM é a ressecção cirúrgica, quimioterapia e radioterapia, todas com baixíssima eficiência devido a agressividade e as características do GBM. Consequentemente, a sobrevida dos pacientes indicados aos tratamentos convencionais é pouco mais de um ano. A inflamação é, sabidamente, uma das características que participa de modo decisivo do desenvolvimento tumoral, incluindo do GBM, e as relações entre mediadores inflamatórios e câncer são alvos de pesquisa nos últimos anos. Diversos estudos apontam um papel da via dos eicosanoides na modulação de processos patológicos envolvidos na inflamação e no câncer. Os eicosanoides são mediadores lipídicos bioativos, envolvidos em diversos processos fisiológicos e patológicos, em especial os associados à resposta inflamatória. As principais vias de produção de eicosanoides (ciclooxigenases, lipoxigenases, e citocromo P450), assim como seus produtos, são frequentemente alterados em diversos tipos de tumor, associados ao crescimento e a progressão tumoral. Contudo, o perfil dessas vias é consideravelmente pouco compreendido em GBM. O objetivo deste estudo foi analisar in vitro o perfil e o papel das três vias de eicosanoides e seus produtos (eicosanoides ou não) nas linhagens de GBM (U251-MG, U87-MG, A172, T98G e U138-MG), modulando a atividade de enzimas chaves com drogas especificas para, então, analisar parâmetros de proliferação, migração e morte celular. Nossos resultados mostram, em todas as linhagens analisadas, um perfil heterogêneo das enzimas e receptores chaves das três vias. O perfil lipídico evidencia a produção de 13-HODE, produto de 15-lipoxigenase-1 em todas as linhagens, bem como ausência de leucotrienos e 5-HETE do eixo de 5-lipoxigenase. Os inibidores farmacológicos para 15-LOX e 12-LOX/15-LOX foram capazes de reduzir o crescimento, modular o ciclo celular e a migração celular das linhagens U251-MG, U87-MG e A172. O mesmo é visto com a inibição de mPGES-1. A inibição de 5-LOX por outro lado não afetou nenhum parâmetro nas mesmas linhagens. Todos os resultados apontam, portanto, para um papel do eixo de 15-LOX e COX no crescimento e na migração das células de GBM humano. / Glioblastoma (GBM) is a grade IV astrocytoma, the most malignant and the most frequent primary brain tumour in humans. Standard therapies for treating GBM are surgical resection, chemotherapy and radiotherapy, all with low efficiency due to GBM aggressiveness and characteristics. Therefore, patient survival after conventional treatments is about one year. Inflammation is one of the main characteristics that plays a decisive role in tumour development, including in GBM. The relationships between inflammatory mediators and cancer have been the subject of research in recent years. Several studies point to the eicosanoid pathways as modulators of pathological processes between inflammation and cancer. Eicosanoids are bioactive lipid mediators, involved in various physiological and pathological processes, especially those associated with the inflammatory response. The major eicosanoid pathways (cyclooxygenases, lipoxygenases, and cytochrome P450) as well as their products, are frequently altered in several tumours, associated with tumour growth and progression. However, the profile of these pathways is poorly understood in GBM. The objective of this study was to analyse, in vitro, the profile and role of eicosanoid pathways and their products (eicosanoids or not) in GBM cell lines (U251-MG, U87-MG, A172, T98G and U138-MG), modulating the key enzymes with inhibitors, analysing proliferation, migration and cell death. Our results show, in all analysed cell lines, a heterogeneous profile for the key enzymes and receptors of the three pathways. The lipid profile shows the production of 13-HODE, a product of 15-lipoxygenase-1, as well the absence of leukotrienes and 5-HETE of the 5-lipoxygenase axis. The pharmacological inhibitors for 15-LOX and 12-LOX / 15-LOX led to changes in cell cycle, reduced growth, reduced migration of U251-MG, U87-MG and A172 cell lines. The same was seen with the inhibition of mPGES-1. Inhibition of 5-LOX, on the other hand, did not affect any of these parameters in the same cell lines. All results, therefore, point to an important role of 15-LOX and COX pathways in the growth and migration of human GBM cells.

Les acides docosahexaénoïque (DHA) et arachidonique (ArA), qui appartiennent respectivement aux familles n-3 et n-6, sont présents en grande quantité dans les tissus cérébraux, en particulier dans les phospholipides membranaires. Ces deux acides gras polyinsaturés (AGPI) jouent des rôles essentiels dans le fonctionnement cérébral, notamment dans le neurodéveloppement et la neuroinflammation. De plus, ces AGPI sont des précurseurs de plusieurs médiateurs lipidiques oxygénés impliqués dans divers processus physiologiques et pathologiques.Dans ce contexte, l'objectif de cette thèse était de caractériser les métabolites oxygénés dérivés du DHA et de l'ArA dans le cerveau, étape cruciale pour une meilleure compréhension de leurs rôles biologiques dans cet organe. Ainsi, une technique d'analyse à haute performance a été utilisée, la chromatographie liquide couplée à de la spectrométrie de masse en tandem (UHPLC-MS/MS), pour identifier les différents métabolites oxygénés dérivés du DHA et de l'ArA présents dans les cerveaux de rats exsanguinés et non exsanguinés, que ce soit en conditions basales ou inflammatoires. Ainsi, plusieurs produits oxygénés dérivés du DHA et de l'ArA ont été identifiés et quantifiés dans les cerveaux exsanguinés et non-exsanguinés, à la fois à l'état libre dans le cerveau mais également estérifiés dans les phospholipides. Les métabolites mono-hydroxylés sont les principaux dérivés oxygénés du DHA et de l'ArA, cependant, des quantités mesurables de produits di-hydroxylés, tels que le 8,15-diHETE et la protectine DX, ont également été détectées. L'exsanguination permet de différencier les métabolites oxygénés du tissu cérébral de ceux présents dans le sang. De plus, le métabolisme oxygéné du DHA et de l'ArA est impacté lors d'une inflammation cérébrale. En effet, dans ces conditions, la synthèse de certains métabolites est augmentée, notamment celle de la protectine D1, molécule connue pour ces fortes propriétés anti-inflammatoires. Ces résultats méritent de plus amples recherches dans des conditions pathophysiologiques, notamment lors de maladies neurodégénératives, afin d'observer leur impact sur le métabolisme oxygéné de ces AGPI / Docosahexaenoic (DHA) and arachidonic (ArA) acids, two PUFA which belong to n-3 and n-6 families respectively, are both present at high amount in brain tissues, and especially in membrane phospholipids. These two polyunsaturated fatty acids (PUFA) play important roles in brain functioning, notably in neurodevelopment and neuroinflammation. Moreover, these PUFA are precursors of various oxygenated lipid mediators involved in diverse physiological and pathological processes. In this context, the aim of this work was to characterize oxygenated metabolites derived from DHA and ArA in brain, crucial step for better understanding their biological roles in this organ. For this purpose, a high performance analytical approach was usd, the liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), in order to identify the different oxygenated metabolites derived from DHA and ArA in exsanguinated and non-exsanguinated brains, both in basal and inflammatory conditions. Several oxygenated products from DHA and ArA were identified and measured in exsanguinated and non-exsanguinated brain, both in the free form and esterified in phospholipids. Mono-hydroxylated metabolites were the main oxygenated derivatives from DHA and ArA, however, measurable amounts of di-hydroxylated products such as 8,15-diHETE and protectin DX, were also detected. Exsanguination allowed discriminating oxygenated metabolites from brain tissue against those in blood brain. Moreover, DHA and ArA oxygenated metabolism was impacted during brain inflammation. Indeed, in these conditions, the synthesis of some metabolites was increased, including that of protectin D1, molecule known for its strong anti-inflammatory properties. These results deserve further research in pathophysiological conditions, especially in neurodegenerative diseases, in order to observe their impact on the oxygenated metabolism of these PUFA