Caractérisation de métabolites oxygénés issus de l'acide alpha-linolénique (Effets anti-agrégants et anti-inflammatoires)

Les acides gras de la série n-3 et notamment l acide docosahexaénoïque (DHA) jouent un rôle important dans la prévention des maladies cardiovasculaires. Un de ses métabolites, la protectine DX (PDX), qui est un isomère de la protectine D1 (PD1), inhibe l agrégation des plaquettes sanguines. D autres composés similaires appelés "poxytrins", qui possèdent aussi un triène conjugué avec une géométrie E,Z,E, ont également été synthétisés à partir d'autres acides gras polyinsaturés (AGPI) via la lipoxygénase de soja. Ces composés présentent des propriétés anti-agrégantes en inhibant la cyclo-oxygénase plaquettaire et le récepteur du thromboxane A2. Dans cette thèse, nous décrivons de nouveaux composés dihydroxylés synthétisés par la 15-lipoxygénase de soja à partir de l acide alpha-linolénique (18:3n-3), un acide gras polyinsaturé indispensable consommé au niveau du gramme chez l Homme adulte. Il est converti en acides gras monohydroxylés et dihydroxylés. Ces composés ont été séparés par HPLC en phase inverse et caractérisés par GC-MS après dérivation adéquate. Un acide gras monohydroxylé, majoritaire, l acide 13(S)-octadécatriénoïque et quatre acides gras dihydroxylés ont été détectés. Ces derniers présentent tous un spectre UV caractéristique avec une absorption maximale à 270 nm et deux épaulements à 260 et 280 nm. Les spectres UV de deux d'entre eux sont superposables à celui de la PDX, ce qui suggère une géométrie E,Z,E des doubles liaisons de leur triène. La caractérisation complète de ces composés a été réalisée par RMN à haut champ et par GC-MS. Ce sont les acides 9(R),16(S)-dihydroxy-octadéca-10E,12E,14E-triénoïque, 9(S),16(S)-dihydroxy-octadéca-10E,12E,14E-triénoïque, 9(S),16(S)-dihydroxy-octadéca-10E,12Z,14E-triénoïque et 9(R),16(S)-dihydroxy-octadéca-10E,12Z,14E-triénoïque. Ils sont également synthétisés par la 15 lipoxygénase recombinante humaine de type 2. Ces composés dihydroxylés 9,16-diHOTEs ont été testés sur les plaquettes isolées à partir du sang humain. Nous avons observé que seules les molécules ayant la géométrie E,Z,E du triène conjugué inhibent l'agrégation plaquettaire induite par le collagène et inhibent la cyclooxygénase-1 (COX-1) de mouton. Les propriétés anti-inflammatoires de ces produits ont également été étudiés. Tous les isomères 9,16-diHOTEs, possédant un triène conjugué avec une géométrie E,Z,E, inhibent la COX-2 recombinante humaine et seul l acide 9(R),16(S)-dihydroxy-octadéca-10E,12Z,14E-triénoïques inhibe la 5-lipoxygénase des leucocytes, siège de la synthèse des leucotriènes issus de l acide arachidonique. En conclusion, les composés dihydroxlés possédant un triène conjugué E,Z,E, issus du 18:3n-3, ainsi que la PDX, inhibent l activité des COX-1 et 2, et seraient anti-agrégants et anti-inflammatoires. Ces résultats donnent des perspectives pharmacologiques aux recommandations nutritionnelles promouvant la consommation d acide alpha linolénique.N-3 fatty acids, especially docosahexaenoic acid (DHA), play an important role in the prevention of cardiovascular diseases. One metabolite of DHA, protectin DX (PDX), an isomer of protectin D1 (PD1) (Chen P et al., 2009),possesses inhibits blood platelet aggregation. Similar compounds called "poxytrins", which have a conjugated triene with a E,Z,E geometry have also been synthesized from other polyunsaturated fatty acids (PUFA) by soybean lipoxygenase. They have anti-aggregating properties by inhibiting platelet cyclooxygenase and thromboxane A2 receptor (Chen P et al., 2011). In this thesis, we describe new dihydroxy compounds synthesized by the soybean 15-lipoxygenase from alpha-linolenic acid (18:3n-3), an essential PUFA that is consumed in the gram range in human adults . It is converted into monohydroxylated and dihydroxylated derivatives. These compounds were separated by reverse phase high performance liquid chromatography (HPLC) and characterized by gas chromatography-mass spectrometry (GC-MS) after appropriate derivatization. A main monohydroxylated fatty acid, 13(S)-octadecatrienoic acid (13(S)-OH-18:3) and four dihydroxylated fatty acids were detected. The last ones have all a characteristic UV spectrum with a maximum absorbance at 270 nm with two shoulder peaks at 260 and 280 nm. The UV spectra from two of them are superimposable to that of PDX, suggesting a E,Z,E geometry for their conjugated triene. The complete characterization of these compounds was performed by high field nuclear magnetic resonance (NMR) and by GC-MS. These are the 9(R),16(S)-dihydroxy-octadeca-10E,12E,14E-trienoic, 9(S),16(S)-dihydroxy-octadeca-10E,12E,14E-trienoic, 9(S),16(S)-dihydroxy-octadeca-10E,12Z,14E-trienoic and 9(R),16(S)-dihydroxy-octadeca-10E,12Z,14E-trienoic acids. They can also be synthesized by the (type 2) 15 human recombinant lipoxygenase. These dihydroxylated compounds (9,16-diHOTEs)were tested on isolated human blood platelets. We observed that only molecules containing a conjugated triene with a E,Z,E geometry are able to inhibit platelet aggregation induced by collagen, and inhibit sheep cyclooxygenase-1 (COX-1). The anti-inflammatory properties of these products were also studied. All 9,16-diHOTEs isomers having a conjugated triene with a E,Z,E geometry, inhibit human recombinant cyclooxygenase-2 (COX-2) and only 9(R),16(S)-dihydroxy-octadeca-10E,12Z,14E-trienoic acid inhibits polymorphonuclear leukocytes (PMN) 5-lipoxygenase which is involved in the leukotriene synthesis from arachidonic acid. In conclusion, the E,Z,E dihydroxlated compounds from 18:3n-3, as well as PDX, inhibiting the COX-1 and 2 activities appear to be anti-aggregatory and anti-inflammatory agents. These results provide pharmacological perspectives to nutritional recommendations promoting the intake of alpha-linolenic acid.VILLEURBANNE-DOC'INSA-Bib. elec. (692669901) / SudocSudocFranceF

Docosahexaenoic acid-containing choline phospholipid modulates LPS-induced neuroinflammation in vivo and in microglia in vitro

BACKGROUND: Neuroinflammatory processes are considered a double-edged sword, having both protective and detrimental effects in the brain. Microglia, the brain's resident innate immune cells, are a key component of neuroinflammatory response. There is a growing interest in developing drugs to target microglia and control neuroinflammatory processes. In this regard, docosahexaenoic acid (DHA), the brain's n-3 polyunsaturated fatty acid, is a promising molecule to regulate pro-inflammatory microglia and cytokine production. Several works reported that the bioavailability of DHA to the brain is higher when DHA is acylated to phospholipid. In this work, we analyzed the anti-inflammatory activity of DHA-phospholipid, either acetylated at the sn-1 position (AceDoPC, a stable form thought to have superior access to the brain) or acylated with palmitic acid at the sn-1 position (PC-DHA) using a lipopolysaccharide (LPS)-induced neuroinflammation model both in vitro and in vivo. METHODS: In vivo, adult C57Bl6/J mice were injected intravenously (i.v.) with either AceDoPC or PC-DHA 24 h prior to LPS (i.p.). For in vitro studies, immortalized murine microglia cells BV-2 were co-incubated with DHA forms and LPS. AceDoPC and PC-DHA effect on brain or BV-2 PUFA content was assessed by gas chromatography. LPS-induced pro-inflammatory cytokines interleukin IL-1β, IL-6, and tumor necrosis factor (TNF) α production were measured by quantitative PCR (qPCR) or multiplex. IL-6 receptors and associated signaling pathway STAT3 were assessed by FACS analysis and western-blot in vitro. RESULTS: In vivo, a single injection of AceDoPC or PC-DHA decreased LPS-induced IL-6 production in the hippocampus of mice. This effect could be linked to their direct effect on microglia, as revealed in vitro. In addition, AceDoPC or PC-DHA reduced IL-6 receptor while only AceDoPC decreased IL-6-induced STAT3 phosphorylation. CONCLUSIONS: These results highlight the potency of administered DHA-acetylated to phospholipids-to rapidly regulate LPS-induced neuroinflammatory processes through their effect on microglia. In particular, both IL-6 production and signaling are targeted by AceDoPC in microglia.Metabolism in human of a structured phospholipid from marine origin and neural effec

Obesity in a model of haploinsufficiency uncovers a causal role for lipid-derived aldehydes in human metabolic disease and cardiomyopathy

Lipid peroxides and their reactive aldehyde derivatives (LPPs) have been linked to obesity-related pathologies, but whether they have a causal role has remained unclear. Glutathione peroxidase 4 (GPx4) is a selenoenzyme that selectively neutralizes lipid hydroperoxides, and human gpx4 gene variants have been associated with obesity and cardiovascular disease in epidemiological studies. This study tested the hypothesis that LPPs underlie cardio-metabolic derangements in obesity using a high fat, high sucrose (HFHS) diet in gpx4 haploinsufficient mice (GPx4(+/-)) and in samples of human myocardium. METHODS: Wild-type (WT) and GPx4(+/-) mice were fed either a standard chow (CNTL) or HFHS diet for 24 weeks, with metabolic and cardiovascular parameters measured throughout. Biochemical and immuno-histological analysis was performed in heart and liver at termination of study, and mitochondrial function was analyzed in heart. Biochemical analysis was also performed on samples of human atrial myocardium from a cohort of 103 patients undergoing elective heart surgery. RESULTS: Following HFHS diet, WT mice displayed moderate increases in 4-hydroxynonenal (HNE)-adducts and carbonyl stress, and a 1.5-fold increase in GPx4 enzyme in both liver and heart, while gpx4 haploinsufficient (GPx4(+/-)) mice had marked carbonyl stress in these organs accompanied by exacerbated glucose intolerance, dyslipidemia, and liver steatosis. Although normotensive, cardiac hypertrophy was evident with obesity, and cardiac fibrosis more pronounced in obese GPx4(+/-) mice. Mitochondrial dysfunction manifesting as decreased fat oxidation capacity and increased reactive oxygen species was also present in obese GPx4(+/-) but not WT hearts, along with up-regulation of pro-inflammatory and pro-fibrotic genes. Patients with diabetes and hyperglycemia exhibited significantly less GPx4 enzyme and greater HNE-adducts in their hearts, compared with age-matched non-diabetic patients. CONCLUSION: These findings suggest LPPs are key factors underlying cardio-metabolic derangements that occur with obesity and that GPx4 serves a critical role as an adaptive countermeasure

Targeted LC–MS derivatization for aldehydes and carboxylic acids with a new derivatization agent 4-APEBA

Based on the template of a recently introduced derivatization reagent for aldehydes, 4-(2-(trimethylammonio)ethoxy)benzeneaminium dibromide (4-APC), a new derivatization agent was designed with additional features for the analysis and screening of biomarkers of lipid peroxidation. The new derivatization reagent, 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APEBA) contains a bromophenethyl group to incorporate an isotopic signature to the derivatives and to add additional fragmentation identifiers, collectively enhancing the abilities for detection and screening of unknown aldehydes. Derivatization can be achieved under mild conditions (pH 5.7, 10 °C). By changing the secondary reagent (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide instead of sodium cyanoborohydride), 4-APEBA is also applicable to the selective derivatization of carboxylic acids. Synthesis of the new label, exploration of the derivatization conditions, characterization of the fragmentation of the aldehyde and carboxylic acid derivatives in MS/MS, and preliminary applications of the labeling strategy for the analysis of aldehydes in urine and plasma are described

Low-Density Lipoprotein Has an Enormous Capacity To Bind (E)-4-Hydroxynon-2-enal (HNE): Detection and Characterization of Lysyl and Histidyl Adducts Containing Multiple Molecules of HNE

(E)-4-Hydroxynon-2-enal (HNE), an electrophilic bifunctional cytotoxic lipid peroxidation product, forms covalent adducts with nucleophilic side chains of amino acid residues. HNE-derived adducts have been implicated in many pathophysiological processes including atherosclerosis, diabetes, and Alzheimer’s disease. Tritium- and deuterium-labeled HNE (d4-HNE) were used orthogonally to study adduction with proteins and individual nucleophilic groups of histidyl, lysyl, and cysteine residues. Using tritium-labeled HNE, we detected the binding of 486 molecules of HNE per low-density lipoprotein (LDL) particle, significantly more than the total number of all reactive nucleophiles in the LDL particle. This suggests the formation of adducts that incorporate multiple molecules of HNE with some nucleophilic amino acid side chains. We also found that the reaction of a 1:1 mixture of d4-HNE and d0-HNE with N-acetylhistidine, N-acetyl-Gly-Lys-OMe, or N-acetyl cysteine generates 1:1, 2:1, and 3:1 adducts, which exhibit unique mass spectral signatures that aid in structural characterization. A domino-like reaction of initial 1:1 HNE Michael adducts of histidyl or lysyl nucleophiles with multiple additional HNE molecules forms 2:1 and 3:1 adducts that were structurally characterized by tandem mass spectrometry

The “Goldilocks Zoneâ€? from a redox perspectiveâ€â€�Adaptive vs. deleterious responses to oxidative stress in striated muscle

Consequences of oxidative stress may be beneficial or detrimental in physiological systems. An organ system's position on the “hormetic curve� is governed by the source and temporality of reactive oxygen species (ROS) production, proximity of ROS to moieties most susceptible to damage, and the capacity of the endogenous cellular ROS scavenging mechanisms. Most importantly, the resilience of the tissue (the capacity to recover from damage) is a decisive factor, and this is reflected in the disparate response to ROS in cardiac and skeletal muscle. In myocytes, a high oxidative capacity invariably results in a significant ROS burden which in homeostasis, is rapidly neutralized by the robust antioxidant network. The up-regulation of key pathways in the antioxidant network is a central component of the hormetic response to ROS. Despite such adaptations, persistent oxidative stress over an extended time-frame (e.g., months to years) inevitably leads to cumulative damages, maladaptation and ultimately the pathogenesis of chronic diseases. Indeed, persistent oxidative stress in heart and skeletal muscle has been repeatedly demonstrated to have causal roles in the etiology of heart disease and insulin resistance, respectively. Deciphering the mechanisms that underlie the divergence between adaptive and maladaptive responses to oxidative stress remains an active area of research for basic scientists and clinicians alike, as this would undoubtedly lead to novel therapeutic approaches. Here, we provide an overview of major types of ROS in striated muscle and the divergent adaptations that occur in response to them. Emphasis is placed on highlighting newly uncovered areas of research on this topic, with particular focus on the mitochondria, and the diverging roles that ROS play in muscle health (e.g., exercise or preconditioning) and disease (e.g., cardiomyopathy, ischemia, metabolic syndrome)