Human 5-lipoxygenase (5-LOX) oxidizes arachidonic acid to 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HpETE) and leukotriene (LT) A4. In neutrophils, LTA4 is further converted to the potent chemoattractant LTB4. These cells also contain the heme enzyme myeloperoxidase (MPO), which produces several potent oxidants such as hypochlorous acid (HOCl), which are involved in pathogen defense and immune regulation. Here, we addressed the question whether MPO-derived oxidants are able to affect the activity of 5-LOX and the product profile of this enzyme. Human 5-LOX was incubated with increasing amounts of HOCl or HOBr. Afterward, arachidonic acid metabolites of 5-LOX were analyzed by reverse-phase high-performance liquid chromatography as well as by liquid chromatography-electrospray ionization-tandem mass spectrometry. The incubation of 5-LOX with the MPO-derived oxidants significantly changed the product profile of 5-LOX. Thereby, HOCl and HOBr increased the ratio of 5-H(p)ETE to 6-trans-LTB4 in a concentration-dependent manner. At low oxidant concentrations, there was a strong decrease in the yield of 6-trans-LTB4, whereas 5-HpETE did not change or increased. Additionally, the formation of 8-HpETE and 12-HpETE by 5-LOX rose slightly with increasing HOCl and HOBr. Comparable results were obtained with the MPO-H2O2-Cl– system when glucose oxidase and glucose were applied as a source of H2O2. This was necessary because of a strong impairment of 5-LOX activity by H2O2. In summary, MPO-derived oxidants showed a considerable impact on 5-LOX, impairing the epoxidation of 5-HpETE, whereas the hydroperoxidation of arachidonic acid was unaffected. Apparently, this was caused by an oxidative modification of critical amino acid residues of 5-LOX. Further work is necessary to assess the specific type and position of oxidation in the substrate-binding cavity of 5-LOX and to specify whether this interaction between 5-LOX and MPO-derived oxidants also takes place in stimulated neutrophils.

Cytochrom P450 (CYP) Enzyme tragen zur Bioaktivierung von langkettigen mehrfach ungesättigten Fettsäuren bei. Die gebildeten Monoepoxy- und Monohydroxy-Metaboliten werden zusammenfassend als CYP-Eicosanoide bezeichnet und fungieren als Mediatoren bei der Regulation des Gefäßtonus, der Herz- und Nierenfunktion, sowie einer Vielzahl weiterer physiologischer Prozesse, wobei die biologische Aktivität oftmals abhängig von der Positions- und Stereoisomerie der Eicosanoide ist. Prominente Vertreter der CYP-Eicosanoid-Familie sind die aus der Arachidonsäure gebildeten Epoxyeicosatriensäuren (EETs) und 20-Hydroxyeicosatetraensäure (20-HETE). EETs und 20-HETE haben zum Teil gegensätzliche biologische Aktivitäten, die zur Aktivierung bzw. Inhibition antiinflammatorischer und weiterer Zell- und Organ-protektiver Signalwege beitragen. Ziel der vorliegenden Arbeit war es, durch Analyse endogener Metabolitenprofile zum besseren Verständnis der Rolle von CYP-Eicosanoiden bei der Entstehung von Ischämie/Reperfusions (I/R)-bedingten Organschäden beizutragen. Als Hauptergebnisse ergaben sich (i) die Entdeckung und Charakterisierung einer protektiven Rolle von EETs in Tiermodellen der Initiationsphase des akuten Nierenversagens, sowie des therapeutischen Potentials stabiler EET-Analoga; (ii) die Identifizierung von 8,9-EET und 20-HETE als mögliche prädiktive Biomarker für das post-operative Auftreten von akutem Nierenversagen nach offener Herzoperation; und (iii) die Entwicklung und Validierung eines analytischen Verfahrens der chiralen Lipidomik (chiral-LC-ESI-MS/MS), das eine Analyse endogener Enantiomere sowohl von Monoepoxy- als auch Monohydroxy-Eicosanoiden in komplexen biologischen Proben erstmalig ermöglichte und dafür genutzt werden konnte, die stereospezifische Regulation der EETs durch Epoxid-Hydrolasen in vitro wie auch in vivo zu beschreiben. / Cytochrome P450 (CYP) enzymes contribute to the bioactivation of long-chain polyunsaturated fatty acids. The monoepoxy- and monohydroxy-metabolites generated by CYP enzymes are collectively termed CYP-eicosanoids. CYP-eicosanoids act as mediators in the regulation of vascular tone, heart- and kidney function and several further physiological processes, mostly in a regio- and stereospecific manner. Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are prominent members of the CYP-eicosanoid family. EETs and 20-HETE show partially opposing biological activities that contribute to the activation or inhibition of anti-inflammatory and other cell- and organ-protective mechanisms. The aim of the present work was to study the role of CYP-eicosanoids in ischemia/reperfusion (I/R) related organ damage by analyses of endogenous metabolite profiles. The main results were (i) discovery and characterization of the EETs protective role in animal models of the initiation phase of acute kidney injury (AKI) and the therapeutic potential of stable EET-analogs, (ii) identification of 8,9-EET and 20-HETE as potential predictive biomarkers for AKI in patients who underwent open heart surgery, (iii) the development and validation of a novel analytical method for chiral lipidomics (chiral LC-ESI-MS/MS) that allows to study endogenous enantiomers of monohydroxy- and monoepoxy-eicosanoids in complex matrices of biological and clinical samples. Furthermore, the approach was applied to describe the stereospecific regulation of EETs by epoxide hydrolases in vitro and in vivo.

Le syndrome d’apnées obstructives du sommeil (SAOS) est une pathologie caractérisée par des épisodes d’hypoxie intermittente (HI) nocturnes et est un problème de santé publique par sa prévalence dans la population générale (5-20%) et ses nombreuses complications métaboliques et cardiovasculaires. La répétition des épisodes d’HI est considérée comme le facteur principal responsable de cette morbidité cardiovasculaire dont l’athérosclérose fait partie. Le traitement de référence du SAOS par la pression positive continue présente dans certains cas une efficacité limitée, en particulier sur les conséquences cardiovasculaires qui nécessitent d’autres thérapeutiques plus spécifiques. Les mécanismes reliant SAOS et athérosclérose ne sont pas encore totalement connus. Cependant, des perturbations du métabolisme des acides gras (AG) en lien avec le processus athéromateux ont déjà été rapportées au cours du SAOS. Elles concernent en particulier le métabolisme de l’acide arachidonique (AG n-6) avec une augmentation d’eicosanoïdes pro-inflammatoires. Par ailleurs, les AG n-3 peuvent avoir une influence sur le développement et la progression des maladies cardiovasculaires, notamment grâce à une modification de la balance AG n-6 / AG n-3. Ainsi l’objectif de ce travail a donc été dans un premier temps de caractériser expérimentalement l’effet d’une supplémentation en AG n-3 sur le développement de l’athérosclérose dans le contexte d’HIC, et d’évaluer cliniquement la distribution AG n-6 / AG n-3 au niveau érythrocytaire chez des patients atteints d’un SAOS. Nous avons démontré que la supplémentation en AG n-3 permet de prévenir l’accélération de l’athérosclérose dans le contexte de l’HIC et est associée à une modulation de l’expression de certains médiateurs inflammatoires. Ces résultats prometteurs incitent à envisager une étude interventionnelle chez les patients SAOS. Dans un second temps, nous nous sommes intéressés au métabolisme des AG, via la β-oxydation mitochondriale, et aux métabolites intermédiaires produits, les acylcarnitines (ACs). Ces métabolites sont de plus en plus étudiés dans le contexte des pathologies cardiovasculaires. Nous avons étudié l’impact du SAOS sur la β-oxydation et ses conséquences sur la fonction vasculaire. L’étude de ces métabolites semble prometteuse et permettra peut-être l’émergence de marqueurs biologiques en relation avec l’état cardiovasculaire des patients. / Obstructive sleep apnea (OSA) syndrome is a disease characterized by recurrent episodes of nocturnal intermittent hypoxia (IH). OSA is a major public health problem due to its frequency in general population (5 to 20%) and its numerous metabolic and cardiovascular complications. Repetitive apneas lead to IH which is responsible of early atherosclerosis and cardiovascular complications. Gold standard treatment of OSA, that is to say continuous positive airway pressure, has poor effects on OSA cardiovascular consequences in some patients, underlining the need of alternative therapeutic strategies. Underlying mechanisms linking OSA to atherosclerosis are still poorly understood. Nevertheless, a link between polyunsaturated fatty acids (PUFAs) metabolism changes and atheromatous process has already been report during OSA syndrome. Arachidonic acid (n-6 PUFA) metabolism leads to increased biosynthesis of pro-inflammatory eicosanoids during OSA. Moreover, n-3 PUFAs influence cardiovascular complications progression especially by modifying n-6 FA / n-3 FA balance. The aim of this work was first to evaluate the influence of n-3 PUFAs supplementation on a CIH induced atherosclerosis progression model, and to clinically evaluate erythrocyte n-6 PUFA / n-3 PUFA distribution in OSA patients. We have shown that n-3 PUFAs supplementation prevents atherosclerosis acceleration in CIH exposed mice and is associated with a modulation of inflammatory mediators. These promising results encourage us to consider an interventional clinical study in OSA patients. In a second time, we have studied FA mitochondrial β-oxidation metabolism via acylcarnitines (ACs) metabolites. These ACs are increasingly studied especially in cardiovascular diseases context. OSA impact on β-oxidation metabolism and its vascular function consequences have been evaluated. ACs study is promising and will perhaps allow biological markers emergence in relation to cardiovascular pattern.

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. / Wellcome Trust

Ultraviolet radiation (UVR) in solar light is important for skin biology. It is involved in the development acute and chronic skin inflammation, aging and cancer, causing erythema, tanning and local or systemic immunosuppression. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are considered anti- inflammatory and could reduce the damage caused by overexposure to UVR. Although, n-3 PUFA have been considered as photoprotective agents, their exact mechanisms of action is not completely understood. The aim of the work is to determine the effect of UVR and the n-3 PUFA eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on human skin cells (in vitro study), specifically on: cell viability, apoptosis and their metabolism through the cyclooxygenase and lipoxygenase pathways. Also, to study the cellular incorporation and effect of n-3 PUFA on the fatty acid profile of skin cells. A clinical study was undertaken to assess the incorporation of n-3 PUFA supplements in human skin. A clinical study was performed in 40 healthy women (active group) supplemented with 4g/day of EPA (70%) and DHA (10%) and 40 healthy women (placebo group) supplemented with 4g/day of glyceryl tricoprylate coprate (GTCC). After 3 months, both blood samples and skin punch biopsies were collected and analysed for fatty acids by gas chromatography (GC). HaCaT keratinocytes and 46BR.1N fibroblasts were cultured and treated with 10 and 50μM of either EPA, or DHA or oleic acid (OA) for 72h and exposed to 15 and 50 mJ/cm2. Cell viability was measured by the MTT assay and cell apoptosis by a colorimetric method, at 24h post UVR. Cells and culture media were analysed by GC and liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) to assess cellular fatty acids and production of eicosanoids. The clinical a study showed that in RBC saturated fatty acids (SFA) (44.27±7.43%) were the main fatty acid group followed by n-6 PUFA (29.61±5.53%). While in dermal tissue monounsaturated fatty acids (MUFA) (58.90±9.80%) was the main fatty acid group followed by SFA (27.06±6.78%). A significant increase in EPA, DHA and docosapentaenoic acid (DPA) was observed in RBC but only EPA was significantly increased in the dermis post n-3 PUFA supplementation. . The viability of HaCaT keratinocytes and 46BR.1N fibroblasts decreased post UVR and this was further reduced post PUFA treatment. Cell apoptosis increased when cells were exposed to UVR and further increased when cells were treated with EPA and DHA. . In HaCaT keratinocytes MUFA (54.22±8.82%) was the main fatty acid group followed by FAS (37.11±.9.16%), while SFA (51.94±8.68%) was the main group followed by MUFA (27.07±4.79) in 46BR.1N. Treated both cells with EPA and DHA showed significant increased in cellular EPA, DPA and DHA. 46BR.1N fibroblasts produced higher levels of prostaglandins (PG) compared to HaCaT keratinocytes: PGE2 and PGD2 were the main PG in both HaCaT (7.96±3.18 and 1.48±1.19 pg/million cell; respectively) and 46BR.1N with (44.2±23.00 and 17.1±9.71 pg/million cell; respectively). Significant increase in PGE1 and PGE2 occurred when cells were exposed to 15mJ/cm2 UVR. Treatment with n-3 PUFA decreased the level of PGE1 and PGE2, and increase production PGE3 at the baseline and post UVR. Both cell lines produced hydroxy fatty acids and the concentration of these mediators was higher in 46BR.1N than HaCaT. The concentrations of these mediators were significant increased post UVR: treatment with n-3 PUFA decreased the level of HODE and HETE, and increase production of HEPE and HDHA at baseline and post UVR. Overall, n-3PUFA treatment led to increases in the content of EPA and DHA on RBC, dermal tissue and human skin cell lines. EPA and DHA in skin cell lines appear to offer protection by increasing cellular apoptosis, decreasing inflammatory mediators specifically PGE2 and 12-HETE, and increasing anti-inflammatory mediators such as PGE3, 15-HEPE and 17-HDHA.

Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid (PUFA) with neuroprotective and cardioprotective properties. It is thought that some of the actions of EPA may be attributed to its elongated metabolite, the PUFA docosapentaenoic acid (DPA). Docosahexaenoic acid (DHA) and arachidonic acid (AA) are bioactive PUFA ubiquitously expressed in neural tissues. EPA and AA can be converted by cyclooxygenase (COX) to prostanoids and by lipoxygenase (LOX) to hydroxy fatty acids. PUFA can also be converted to ethanolamides in the brain. These mediators are involved in physiological and pathological processes in many bodily systems. The purpose of this study was to examine the production of eicosanoids, hydroxy fatty acids and fatty acid ethanolamides in young and aged rat brain following EPA or DPA enriched diets. The effects of specific PUFA on human platelet eicosanoid production were also investigated as these mediators play a role in adhesion and aggregation. Liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) assays were developed and used to measure lipid mediators in rat brain and human platelets. Ageing in rat brain was accompanied with several changes in the prostanoid and hydroxy fatty acid profiles. Supplementing the diet with EPA or DPA at a daily dose of 200 mg/kg for 8 weeks prevented these changes and decreased levels of PGE2. DPA changed the profile of hydroxy fatty acids synthesised in the brain tissue of young animals. This study has shown that levels of eicosapentaenoylethanolamide (EPA-EA) increase in the brain as a result of ageing and that this is accompanied by an increase in levels of anandamide. Feeding aged animals EPA or DPA further increased the levels of EPA-EA but prevented any change in the level of anandamide. Niacin is used to treat hypercholesterolaemia although it is associated with an unpleasant PGD2 mediated skin flush. This exploratory study has shown that human platelets treated with niacin did not show any changes in their prostanoid and hydroxy fatty acid profiles. Platelets treated with EPA showed increased production of TXB3 and 12-HEPE. Niacin augmented the effects of EPA on human platelet mediator synthesis. Overall, this study has demonstrated that EPA can change brain and platelet lipid mediator synthesis and has provided evidence that could explain some of the neuroprotective and cardioprotective actions of this PUFA.

Dentro da perspectiva da utilização de compostos fenólicos como antioxidantes naturais para minimizar os efeitos in vitro e in vivo do processo oxidativo dos lípides insaturados, foi realizada a monitoração dietética de duas dietas ricas em lípides das séries ω3 e ω6 e a suplementação de um chá de uma mistura de especiarias, em ratos Wistar, com o objetivo de verificar a influência dos compostos fenólicos, presentes nas especiarias, sobre o metabolismo de ácidos graxos das séries ω3 e ω6. Extratos e frações das especiarias mostarda, canela e erva doce foram obtidos e tiveram suas atividades antioxidantes testadas em sistemas aquoso (cooxidação de substratos com o uso de ácido linoléico/β-caroteno) e lipídico (RANCIMAT) e o perfil de compostos fenólicos identificados e quantificados por CGMS. A partir de uma mistura de especiarias, foi elaborado um chá que foi fornecido aos animais de cada grupo dietético (ω3 e ω6). Após 45 dias de tratamento, os animais foram sacrificados e tiveram seus tecidos coletados para análise de TBARs e do perfil lipídico por CGMS. Todos os extratos das especiarias apresentaram atividade antioxidante equivalente ou superior ao BHT. Foram identificados por CGMS os ácidos fenólicos: catecol, salicílico e caféico. Foi obtido nos tecidos dos animais que o somatório do perfil de ácidos graxos saturados e dos insaturados apresentaram diferença entre os grupos testes e controles. Logo, no tecido cerebral, o EPA foi incorporado apenas no grupo ω3 que recebeu o extrato das especiarias. Já para o DHA, do mesmo grupo dietético, os tecidos hepático e renal também apresentaram incorporação aumentada em relação ao controle. No grupo dietético ω6, destaca-se um aumento no percentual de incorporação do ácido linoléico nos tecidos cardíaco e renal no grupo experimental. Enquanto, para o ácido araquidônico, houve diferença em todos os tecidos. Já, para os resultados da lipoperoxidação, observou-se que todos os tecidos dos animais que receberam o extrato das especiarias apresentaram baixos valores em comparação aos seus respectivos controles. Este estudo também avaliou a ação de compostos fenólicos do chá da mistura das especiarias mostarda, canela e erva doce sobre as enzimas lipoxigenase e cicloxigenase. O extrato da mistura das especiarias a 100 e 200 ppm foi adicionado no meio de reação contendo o substrato e a enzima lipoxigenase 1B da Sigma com 112.000 und/mg. A cicloxigenase foi obtida de vesículas seminais de carneiro e seguiu-se o mesmo protocolo de atividade para a lipoxigenase. Indometacina foi o inibidor utilizado como controle positivo da reação. Observou-se que a 200 ppm, como na concentração do chá (0,02%), a lipoxigenase foi inibida em aproximadamente em 90%. Já para a cicloxigenase, o extrato a 200 ppm resultou numa média de 75% de inibição da atividade da enzima, enquanto que a indomentacina apresentou uma média de 77% de inibição. A absorção aparente do chá das especiarias indicou que os fenólicos presentes na mistura foram absorvidos em média de 75%. O estudo histopatológico dos intestinos delgados dos animais não apresentou nenhuma diferença na área de absorção entre o grupo experimental e o controle. Estes dados sugerem, portanto, um efeito antioxidante das substâncias fenólicas identificadas nas especiarias sobre os ácidos graxos das séries ω3 e ω6, podendo agir diretamente: (1) no alimento (óleo), (2) com modificação do perfil lipídico, (3) proteção quanto à oxidação de tecidos e (4) inibição das enzimas da biossíntese dos eicosanóides. / Considering the perspective for the use of phenolic compounds as natural antioxidants to minimize in vitro and in vivo effects of oxidative processes on unsaturated lipids, this work monitored Wistar rats fed with two diets rich in ω3 and ω6 polyunsaturated lipids, supplemented with tea made from a blend of spices. The objective of the work was to study the influence of phenolic compounds present in spices on the metabolism of ω3 and ω6 fatty acids. Extracts and fractions of mustard, cinnamon and anise were obtained and had their antioxidant activity tested in aqueous (co-oxidation of substrates using linoleic acid/β-carotene) and lipidic systems (RANCIMAT). Their phenolic compounds profile was determined and quantified using CGMS. The rats of each diet group (ω3 and ω6) were given tea made from a blend of spices and sacrificed after 45 days. Their tissues were then collected and analyses of TBARS and lipid profile were performed using CGMS. Ali the extracts of spices showed equal or higher antioxidant activity than BHT. The following phenolic acids were identified using CGMS: cathecol, salicilic and cafeic. It was observed that the total amount of the profile of saturated and unsaturated fatty acids in the rat tissues were different in the test group and the control group. In brain tissue, EPA was found only in the ω3 diet group which was given the tea. Concerning DHA, liver and kidney tissues of the same diet group showed higher concentrations than the control group. In the ω6 diet group, an outstanding increase of linoleic acid in cardiac and kidney tissues was found. Concerning the arachidonic acid, a difference in concentration was observed in ali tissues. Ali the tissues from rats given the tea presented a lower level of lipid peroxidation than their respective control groups. The present research also evaluated the action of phenolic compounds found in the tea made from the blend of mustard, cinnamon and anise on the enzymes lipoxygenase and cycloxygenase. The extract of the blend of spices, in the concentrations of 100 and 200 ppm, was added to the substrate and Sigma lipoxygenase 112.000 units/mg. Cycloxygenase was obtained from sheep seminal bladders and underwent the same protocol as lipoxygenase. Indomethacin was the inhibitor used as the positive control of the reaction. It was observed that in the concentration of 200 ppm (that is 0.02%, the same concentration as in the tea), lipoxygenase presented an average 90% inhibition. The extract of cycloxygenase 200 ppm presented 75% inhibition of the enzyme activity, while indomethacin presented an average 77% inhibition. It was found that around 75% of the phenolic compounds present in the tea made from the blend of spices were absorbed, thus leading to the conclusion that apparent absorption of the tea took place. Histopathologic examinations on the small intestines of the rats did not reveal any difference in the absorption area between the experimental and the control groups. Such results suggest therefore an antioxidant effect of the phenolic substances identified in the spices on the ω3 and ω6 fatty acids, this effect being possible directly (1) on the food (oil) , (2) modification of the profile fatty acids, (3) oxidation protection tissues and, (4) inhibition of enzymes of eicosanoids biosinthesys.

Mehrfach ungesättigte omega-3 Fettsäuren (n-3 PUFAs), wie Eicosapentaensäure (EPA) und Docosahexaensäure (DHA), schützen vor kardiovaskulären Erkrankungen, wie tödlichen Arrhythmien. In vitro Untersuchungen belegen, dass rekombinante Cytochrom P450 (CYP) Enzyme nicht nur die n-6 PUFA Arachidonsäure (AA), sondern auch die n-3 PUFAs EPA und DHA als alternative Substrate verwenden. Dabei entstehen bioaktive regio- und stereoisomere Epoxy- und Hydroxymetaboliten, CYP-Eicosanoide, die als sekundäre Botenstoffe bei der Regulation von Gefäß-, Nieren- und Herzfunktionen fungieren. Die genauen molekularen Mechanismen dieser Metabolite sind noch weitgehend unerforscht. In der vorliegenden Arbeit wurde zunächst der ernährungsbedingte Einfluss auf das endogene CYP-Eicosanoidprofil im Menschen untersucht. Die Ergebnisse der klinischen Studie zeigten, dass n-3 PUFAs auch in vivo alternative Substrate von CYP-Enzymen darstellen und wenn verfügbar sogar effektiver zu ihren Metaboliten umgesetzt wurden als AA. Als ein wichtiger Metabolit entsteht nach EPA/DHA-Supplementation 17,18-EEQ, welcher womöglich der eigentliche Vermittler der kardioprotektiven Effekte von n-3 PUFAs ist. Unter Verwendung eines etablierten Zellmodells mit spontan schlagenden neonatalen Rattenkardiomyozyten (NRKMs) wurde der anti-arrhythmische Effekte von 17,18-EEQ genauer untersucht. Der negativ chronotrope Effekt von EPA auf NRKMs wurde tatsächlich durch 17,18-EEQ vermittelt, insbesondere dem R,S-Enantiomer. Mittels Strukturfunktionsanalyse wurden synthetische Analoga mit gleicher Wirksamkeit wie dem 7,18-EEQ gefunden, wobei strikte strukturelle Merkmale für die biologische Funktion identifiziert wurden. Die Suche nach einem molekularen Ziel für CYP-Epoxyeicosanoide führte zu einem möglichen Rezeptorkandidaten, der hinsichtlich seiner Ligandenspezifität untersucht wurde. Dieser oder zukünftige andere Rezeptorkandidaten stellen ein mögliches neues zelluläres Ziel zur Behandlung kardialer Arrhythmien dar. / The n-3 polyunsaturated fatty acids (n-3 PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), protect from cardiovascular disease, especially from fetal arrhythmia. Moreover, in vitro studies proved that recombinant cytochrome P450 (CYP) enzymes not only accept the physiologically most important n-6 PUFA arachidonic acid (AA), but also EPA and DHA as alternative substrates, thereby generating regio- and stereospecific biologically active epoxy- and hydroxymetabolites, CYP-eicosanoids. These metabolites serve as second messengers regulating vascular, renal and cardiac function. The precise underlying molecular mechanisms are only partially understood and need further investigation. The first aim of the thesis was to show that the endogenous CYP-eicosanoid profile depends on the availability of the precursor fatty acids. The results of a clinical trial with 20 volunteers, show that n-3 PUFAs serve also in vivo as alternative CYP-dependent substrates and are even preferentially metabolized compared to AA. After EPA/DHA-supplementation 17,18 EEQ was generated as a major metabolite, potentially an important mediator of cardiovascular effects originally attributed to n-3 PUFAs. To test the anti-arrhythmic effect of EPA and 17,18-EEQ, an established cell model with neonatal rat cardiomyocytes (NRKMs) was used. The negative chronotropic effect of EPA was mimicked by 17,18-EEQ, attributed only to the R,S-enantiomer. A structure activity relationship study revealed synthetic analogs, exerting the same biological effect as 17,18-EEQ. Strict structural requirements were found for agonistic function, hinting at a specific interaction with cellular targets, like GPCRs. The search of a molecular target of CYP-eicosanoids led to a putative receptor, which was tested for ligand binding specificity. If the preliminary results on the ligand binding are confirmed in future experiments this receptor might be a novel target for the treatment of cardiac arrhythmia.

Ultraviolet radiation (UVR) in solar light is important for skin biology. It is involved in the development acute and chronic skin inflammation, aging and cancer, causing erythema, tanning and local or systemic immunosuppression. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are considered anti- inflammatory and could reduce the damage caused by overexposure to UVR. Although, n-3 PUFA have been considered as photoprotective agents, their exact mechanisms of action is not completely understood. The aim of the work is to determine the effect of UVR and the n-3 PUFA eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on human skin cells (in vitro study), specifically on: cell viability, apoptosis and their metabolism through the cyclooxygenase and lipoxygenase pathways. Also, to study the cellular incorporation and effect of n-3 PUFA on the fatty acid profile of skin cells. A clinical study was undertaken to assess the incorporation of n-3 PUFA supplements in human skin. A clinical study was performed in 40 healthy women (active group) supplemented with 4g/day of EPA (70%) and DHA (10%) and 40 healthy women (placebo group) supplemented with 4g/day of glyceryl tricoprylate coprate (GTCC). After 3 months, both blood samples and skin punch biopsies were collected and analysed for fatty acids by gas chromatography (GC). HaCaT keratinocytes and 46BR.1N fibroblasts were cultured and treated with 10 and 50μM of either EPA, or DHA or oleic acid (OA) for 72h and exposed to 15 and 50 mJ/cm2. Cell viability was measured by the MTT assay and cell apoptosis by a colorimetric method, at 24h post UVR. Cells and culture media were analysed by GC and liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) to assess cellular fatty acids and production of eicosanoids. The clinical a study showed that in RBC saturated fatty acids (SFA) (44.27±7.43%) were the main fatty acid group followed by n-6 PUFA (29.61±5.53%). While in dermal tissue monounsaturated fatty acids (MUFA) (58.90±9.80%) was the main fatty acid group followed by SFA (27.06±6.78%). A significant increase in EPA, DHA and docosapentaenoic acid (DPA) was observed in RBC but only EPA was significantly increased in the dermis post n-3 PUFA supplementation. . The viability of HaCaT keratinocytes and 46BR.1N fibroblasts decreased post UVR and this was further reduced post PUFA treatment. Cell apoptosis increased when cells were exposed to UVR and further increased when cells were treated with EPA and DHA. . In HaCaT keratinocytes MUFA (54.22±8.82%) was the main fatty acid group followed by FAS (37.11±.9.16%), while SFA (51.94±8.68%) was the main group followed by MUFA (27.07±4.79) in 46BR.1N. Treated both cells with EPA and DHA showed significant increased in cellular EPA, DPA and DHA. 46BR.1N fibroblasts produced higher levels of prostaglandins (PG) compared to HaCaT keratinocytes: PGE2 and PGD2 were the main PG in both HaCaT (7.96±3.18 and 1.48±1.19 pg/million cell; respectively) and 46BR.1N with (44.2±23.00 and 17.1±9.71 pg/million cell; respectively). Significant increase in PGE1 and PGE2 occurred when cells were exposed to 15mJ/cm2 UVR. Treatment with n-3 PUFA decreased the level of PGE1 and PGE2, and increase production PGE3 at the baseline and post UVR. Both cell lines produced hydroxy fatty acids and the concentration of these mediators was higher in 46BR.1N than HaCaT. The concentrations of these mediators were significant increased post UVR: treatment with n-3 PUFA decreased the level of HODE and HETE, and increase production of HEPE and HDHA at baseline and post UVR. Overall, n-3PUFA treatment led to increases in the content of EPA and DHA on RBC, dermal tissue and human skin cell lines. EPA and DHA in skin cell lines appear to offer protection by increasing cellular apoptosis, decreasing inflammatory mediators specifically PGE2 and 12-HETE, and increasing anti-inflammatory mediators such as PGE3, 15-HEPE and 17-HDHA.

Dentro da perspectiva da utilização de compostos fenólicos como antioxidantes naturais para minimizar os efeitos in vitro e in vivo do processo oxidativo dos lípides insaturados, foi realizada a monitoração dietética de duas dietas ricas em lípides das séries ω3 e ω6 e a suplementação de um chá de uma mistura de especiarias, em ratos Wistar, com o objetivo de verificar a influência dos compostos fenólicos, presentes nas especiarias, sobre o metabolismo de ácidos graxos das séries ω3 e ω6. Extratos e frações das especiarias mostarda, canela e erva doce foram obtidos e tiveram suas atividades antioxidantes testadas em sistemas aquoso (cooxidação de substratos com o uso de ácido linoléico/β-caroteno) e lipídico (RANCIMAT) e o perfil de compostos fenólicos identificados e quantificados por CGMS. A partir de uma mistura de especiarias, foi elaborado um chá que foi fornecido aos animais de cada grupo dietético (ω3 e ω6). Após 45 dias de tratamento, os animais foram sacrificados e tiveram seus tecidos coletados para análise de TBARs e do perfil lipídico por CGMS. Todos os extratos das especiarias apresentaram atividade antioxidante equivalente ou superior ao BHT. Foram identificados por CGMS os ácidos fenólicos: catecol, salicílico e caféico. Foi obtido nos tecidos dos animais que o somatório do perfil de ácidos graxos saturados e dos insaturados apresentaram diferença entre os grupos testes e controles. Logo, no tecido cerebral, o EPA foi incorporado apenas no grupo ω3 que recebeu o extrato das especiarias. Já para o DHA, do mesmo grupo dietético, os tecidos hepático e renal também apresentaram incorporação aumentada em relação ao controle. No grupo dietético ω6, destaca-se um aumento no percentual de incorporação do ácido linoléico nos tecidos cardíaco e renal no grupo experimental. Enquanto, para o ácido araquidônico, houve diferença em todos os tecidos. Já, para os resultados da lipoperoxidação, observou-se que todos os tecidos dos animais que receberam o extrato das especiarias apresentaram baixos valores em comparação aos seus respectivos controles. Este estudo também avaliou a ação de compostos fenólicos do chá da mistura das especiarias mostarda, canela e erva doce sobre as enzimas lipoxigenase e cicloxigenase. O extrato da mistura das especiarias a 100 e 200 ppm foi adicionado no meio de reação contendo o substrato e a enzima lipoxigenase 1B da Sigma com 112.000 und/mg. A cicloxigenase foi obtida de vesículas seminais de carneiro e seguiu-se o mesmo protocolo de atividade para a lipoxigenase. Indometacina foi o inibidor utilizado como controle positivo da reação. Observou-se que a 200 ppm, como na concentração do chá (0,02%), a lipoxigenase foi inibida em aproximadamente em 90%. Já para a cicloxigenase, o extrato a 200 ppm resultou numa média de 75% de inibição da atividade da enzima, enquanto que a indomentacina apresentou uma média de 77% de inibição. A absorção aparente do chá das especiarias indicou que os fenólicos presentes na mistura foram absorvidos em média de 75%. O estudo histopatológico dos intestinos delgados dos animais não apresentou nenhuma diferença na área de absorção entre o grupo experimental e o controle. Estes dados sugerem, portanto, um efeito antioxidante das substâncias fenólicas identificadas nas especiarias sobre os ácidos graxos das séries ω3 e ω6, podendo agir diretamente: (1) no alimento (óleo), (2) com modificação do perfil lipídico, (3) proteção quanto à oxidação de tecidos e (4) inibição das enzimas da biossíntese dos eicosanóides. / Considering the perspective for the use of phenolic compounds as natural antioxidants to minimize in vitro and in vivo effects of oxidative processes on unsaturated lipids, this work monitored Wistar rats fed with two diets rich in ω3 and ω6 polyunsaturated lipids, supplemented with tea made from a blend of spices. The objective of the work was to study the influence of phenolic compounds present in spices on the metabolism of ω3 and ω6 fatty acids. Extracts and fractions of mustard, cinnamon and anise were obtained and had their antioxidant activity tested in aqueous (co-oxidation of substrates using linoleic acid/β-carotene) and lipidic systems (RANCIMAT). Their phenolic compounds profile was determined and quantified using CGMS. The rats of each diet group (ω3 and ω6) were given tea made from a blend of spices and sacrificed after 45 days. Their tissues were then collected and analyses of TBARS and lipid profile were performed using CGMS. Ali the extracts of spices showed equal or higher antioxidant activity than BHT. The following phenolic acids were identified using CGMS: cathecol, salicilic and cafeic. It was observed that the total amount of the profile of saturated and unsaturated fatty acids in the rat tissues were different in the test group and the control group. In brain tissue, EPA was found only in the ω3 diet group which was given the tea. Concerning DHA, liver and kidney tissues of the same diet group showed higher concentrations than the control group. In the ω6 diet group, an outstanding increase of linoleic acid in cardiac and kidney tissues was found. Concerning the arachidonic acid, a difference in concentration was observed in ali tissues. Ali the tissues from rats given the tea presented a lower level of lipid peroxidation than their respective control groups. The present research also evaluated the action of phenolic compounds found in the tea made from the blend of mustard, cinnamon and anise on the enzymes lipoxygenase and cycloxygenase. The extract of the blend of spices, in the concentrations of 100 and 200 ppm, was added to the substrate and Sigma lipoxygenase 112.000 units/mg. Cycloxygenase was obtained from sheep seminal bladders and underwent the same protocol as lipoxygenase. Indomethacin was the inhibitor used as the positive control of the reaction. It was observed that in the concentration of 200 ppm (that is 0.02%, the same concentration as in the tea), lipoxygenase presented an average 90% inhibition. The extract of cycloxygenase 200 ppm presented 75% inhibition of the enzyme activity, while indomethacin presented an average 77% inhibition. It was found that around 75% of the phenolic compounds present in the tea made from the blend of spices were absorbed, thus leading to the conclusion that apparent absorption of the tea took place. Histopathologic examinations on the small intestines of the rats did not reveal any difference in the absorption area between the experimental and the control groups. Such results suggest therefore an antioxidant effect of the phenolic substances identified in the spices on the ω3 and ω6 fatty acids, this effect being possible directly (1) on the food (oil) , (2) modification of the profile fatty acids, (3) oxidation protection tissues and, (4) inhibition of enzymes of eicosanoids biosinthesys.