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Gas chromatographic acquisition of representative 'Total' cellular fatty acid profiles from bacteria or bacteria-containing samples (e.g., environmental or clinical materials) tends to be dependent on the method used to released the fatty acids and convert them to derivatives suitable for analysis. Alkaline saponification or interesterification methods, while preserving acid-sensitive components such as cyclopropane fatty acids, are often insufficient to release amide-linked components, such as hydroxylated fatty acids. Acid-catalyzed hydrolyses or interesterifications, on the other hand, while more efficiently releasing the predominantly amide-linked hydroxylated components, have been shown to cause severe and unpredictable degradation of cyclopropane fatty acids. We report studies of a single-tube method involving sequential alkaline/acid release of fatty acids in which fatty acids released by the alkaline step are partitioned into an organic epiphase during the aqueous acid hydrolysis step. After hydrolysis, the epiphase and the released fatty acids are extracted into an hypophasic solvend and esterified at moderate temperature under relatively low acid concentrations. Under these conditions, cyclopropane as well as hydroxylated fatty acids are recovered in high yield.

La cutine est un polymère d'acides gras oxydés et de glycérol propre aux plantes. Elle forme la matrice structurale de la cuticule qui recouvre l'épiderme des parties aériennes et joue un rôle vital pour les plantes en empêchant la dessiccation. La biosynthèse du polymère de cutine a été étudiée chez la plante modèle Arabidopsis thaliana. Des mutants perte-de-fonction pour la monooxygénase de type cytochrome P450 CYP77A4 ont été isolés. L'analyse de lignées transgéniques exprimant le gène rapporteur GUS sous le contrôle du promoteur de CYP77A4 a montré que le gène était exprimé essentiellement dans les organes floraux et les graines. L'analyse de la cutine dans divers organes a permis de démontrer que le gène était essentiel pour la synthèse d'un acide gras trihydroxylé en C18 présent dans les polyesters des embryons. Une méthode permettant la séparation de l'embryon et des téguments des graines en quantité suffisante pour analyser la cutine a été mise au point. Le profil de monomères des embryons mutants a montré que CYP77A4 est une époxygénase de la voie de biosynthèse des monomères de cutine en C18. L'étude de la physiologie des mutants a par ailleurs permis de démontrer que les acides gras trihydroxylés de la cuticule de l'embryon jouent un rôle important dans la germination de la graine en conditions de stress salin. Dans une deuxième étude, des mutants perte-de-fonction et des suexpresseurs pour le gène d'Arabidopsis BODYGUARD (BDG) codant une protéine de la superfamille des hydrolases à repliement α/β ont été caractérisés. L'analyse des polyesters dans ces lignées a permis de montrer que cette protéine jouait en fait un rôle dans la biosynthèse de la cutine. / Cutin is a polymer of oxidized fatty acids and glycerol specific to plants. It forms the structural matrix of the waxy cuticle covering the epidermis of the aerial parts and plays a vital role in plants by preventing desiccation. Biosynthesis of cutin polymer was studied in the model plant Arabidopsis thaliana. Mutant loss-of-function monooxygenase type cytochrome P450 CYP77A4 were isolated. The analysis of transgenic lines expressing the GUS reporter gene under the control of the promoter of CYP77A4 showed that the gene was expressed mainly in floral organs and seeds. The analysis of various organs in the cutin demonstrated that the gene was essential for the synthesis of a C18 trihydroxy polyesters present in seed polyesters (9,10,18-trihydroxyoctadecenoic acid). A method for the separation of the embryo and seed coat allowing to analyze embryo polyesters was developed. The trihydroxy C18 fatty acid was found to be the major cutin embryo monomer. Profile of cutin monomers in mutant embryos showed that CYP77A4 is an epoxygenase in the biosynthetic pathway of C18 cutin monomers. The study of the physiology of the mutants also showed that the trihydroxy- fatty acids of the embryo cuticle play an important role in the germination of the seed under conditions of salt stress. In a second study, mutant loss-of-function and overexpressors for the Arabidopsis gene BODYGUARD encoding a protein of the α / β hydrolase fold superfamily have been characterized. Analysis of polyesters in these lines showed that this protein, whose role in the formation of the cuticle was not understood, plays in fact a role in cutin biosynthesis.

A ninhydrin-positive, phosphorus-negative lipid from Paracoccus denitrificans ATCC 13543 has been isolated and purified by mild alkaline methanolysis followed by silicic acid column chromatography and preparative thin-layer chromatography. The lipid was identified as an ornithine-containing lipid. The major ester-linked fatty acid was cis vaccenic acid. Major amide-linked fatty acids were 3-OH-20:1 and 3-OH-18:0. Ornithine-containing lipid was a major lipid component of P. denitrificans. Phospholipids made up about 57% and ornithine-containing lipid about 14% of the weight of the total lipid of the organism. The ratios of lipid ornithine: lipid phosphorus were 0.23, 0.65 and 0.58 in cytoplasmic membrane, outer membrane, and an NaCl extract, which is thought to represent chiefly outer membrane, respectively. Thus ornithine-containing lipid appears to be present in larger amounts in outer membrane than cytoplasmic membrane. No substantial variations in lipid ornithine levels were noted in stationary phase versus exposnential phase organisms, organisms grown in complex medium versus organisms grown in minimal medium with and without amino acid supplements, or in organisms grown in low phosphate-containing medium.

no / Chronic wounds often result from prolonged inflammation involving excessive polymorphonuclear leukocyte activity. Studies show that the omega-3 polyunsaturated fatty acids eicosapentaenoic and docosahexaenoic acids found in fish oils generate bioactive lipid mediators that reduce inflammation and polymorphonuclear leukocyte recruitment in numerous inflammatory disease models. The purpose of this study was to test the hypotheses that boosting plasma levels of eicosapentaenoic and docosahexaenoic acids with oral supplementation would alter lipid mediator levels in acute wound microenvironments and reduce polymorphonuclear leukocyte levels. Eighteen individuals were randomized to 28 days of either eicosapentaenoic + docosahexaenoic acid supplementation (Active Group) or placebo. After 28 days the Active Group had significantly higher plasma levels of eicosapentaenoic (p<0.001) and docosahexaenoic acid (p<0.001) than the Placebo Group and significantly lower wound fluid levels of two 15-lipoxygenase products of omega-6 polyunsaturated fatty acids, [9- hydroxyoctadecadienoic (HODE) acid (p = 0.033) and15-hydroxyeicosatrienoic acid (HETrE) (p = 0.006)], at 24 hours post wounding. The Active Group also had lower mean levels of myeloperoxidase, a leukocyte marker, at 12 hours and significantly more re-epithelialization on Day 5 post wounding. We suggest that lipid mediator profiles can be manipulated by altering polyunsaturated fatty acid intake to create a wound microenvironment more conducive to healing.

no / Bioactive lipid mediators derived from polyunsaturated fatty acids (PUFA) and exhibit a range of tissue and cell-specific activities in many physiological and pathological processes. Electrospray tandem mass spectrometry coupled to liquid chromatography (LC/ESI-MS/MS) is a sensitive, versatile analytical methodology for the qualitative and quantitative analysis of lipid mediators. Here we present an LC/ESI-MS/MS assay for the simultaneous analysis of twenty mono- and poly-hydroxy fatty acid derivatives of linoleic, arachidonic, eicosapentaenoic and docosahexaenoic acids. The assay was linear over the concentration range 1-100 pg/¿L, whilst the limits of detection and quantitation were 10-20 and 20-50 pg respectively. The recovery of the extraction methodology varied from 76-122% depending on the metabolite. This system is useful for profiling a range of biochemically-related potent mediators including the newly discovered resolvins and protectins, and their precursor hydroxy-eicosapentaenoic and hydroxy-docosahexaenoic acids, and, consequently, advance our understanding of the role of PUFA in health and disease. / Wellcome Trust, British Heart Foundation