Dissertations / Theses on the topic 'Essential fatty acids Cell Membrane'

Polyunsaturated fatty acids (PUFA) and long chain PUFA (LCPUFA) may play a role in bone health, but evidence is scarce in human males. The objective of this study is to determine if the dietary intake of PUFA and LCPUFA, particularly the omega-3 LCPUFA, and their subsequent levels in red blood cell (RBC) membranes are associated with higher bone mineral density (BMD) of the whole body, spine, hip, and femoral neck in healthy middle aged men. Anthropometric measurements, assessment of dietary and supplement intake, assessment of total and weight-bearing physical activity, quantification of total fatty acid levels in RBC membranes, and assessment of BMD using dual energy x-ray absorptiometry (DXA) was conducted in a cross-sectional sample of healthy middle-aged men. Statistical Analysis using the student t-test for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) intake and status above and below the median was conducted, followed by multiple linear regression analysis to confirm the results of the t-test while accounting for covariates including body mass index (BMI), serum 25-hydroxy vitamin D (25(OH)D), calcium intake, alcohol intake, and physical activity. Higher dietary intake of EPA+DHA was associated with higher whole body and spine BMD and spine z-score. Higher EPA intake was associated with higher spine z-score. Finally, higher EPA status (% in RBC) was associated with higher whole body and spine BMD; and of femoral neck and spine z‐scores. These preliminary results suggest that men with higher intakes of EPA and DHA and higher EPA status have improved bone health. However, it appears the LCPUFA status is a stronger predictor than dietary LCPUFA intake.
Les acides gras polyinsaturés (AGPI) et les acides gras polyinsaturés à longue chaîne (AGPI-LC) peuvent influencer la santé osseuse, mais très peu de preuves existent en ce qui concerne les hommes. L'objectif de cette étude est de déterminer si l'apport alimentaire en AGPI et en AGPI-LC, en particulier en oméga-3 AGPI-LC, et leurs concentrations subséquents dans les membranes des érythrocytes sont associés à une plus grande densité minéral osseuse (DMO) du corps entier, de la colonne vertébrale, des hanches et du col du fémur chez les hommes d'âge moyen en bonne santé. Dans un échantillon transversal d'hommes d'âge moyen en bonne santé, les mesures anthropométriques ont été relevées, l'apport des aliments et des suppléments alimentaires ainsi que les activités physiques totales et celles avec mise en charge évalués, les concentrations totales des acides gras dans les membranes des érythrocytes quantifiées et les DMO mesurées par ostéodensitométrie. L'analyse statistique a été effectuée à la fois pour l'apport et le bilan en acide eicosapentanoique (EPA) et acide docosahexaenoique (DHA) au-dessus et en-dessous de la médiane en utilisant des tests-t de Student, suivi par une analyse de régression linéaire multiple pour confirmer les résultats des tests-t en prenant en compte les covariables. Un apport alimentaire plus élevé en EPA et DHA est associé avec des DMO plus élevées du corps entier et de la colonne vertébrale et un plus grand score-z pour la colonne vertébrale. Un apport plus élevé en EPA est associé avec un score-z réduit de celle-ci. Enfin, bilan plus élevé d'EPA (% présent dans les érythrocytes) est associé avec des DMO plus élevées du corps entier et de la colonne vertébrale et des plus grands score-z pour le col du fémur et la colonne vertébrale. Ces résultats préliminaires suggèrent que les hommes qui consomment plus d'EPA et de DHA et qui ont un bilan en EPA plus élevé ont une meilleure santé osseuse. Cependant, il semblerait que le bilan en AGPI-LC soit un meilleur indicateur que l'apport alimentaire en AGPI-LC.

Red blood cells (RBC) have been shown to mediate plaque development seen in coronary artery disease (CAD). This study determined whether differences in RBC fatty acid (FA) composition were related to CAD risk. FAs were extracted from RBCs of 38 individuals who have undergone cardiac catheterization, 9 of whom had obstructive CAD, and analyzed via gas chromatography. Ferric reducing ability of plasma (FRAP) assay was used to determine oxidative stress. Food frequency questionnaires were used to correlate RBC omega-3 FA to daily intake of omega-3 FA. No correlation was found between RBC content and intake of omega-3 FA. FRAP values and RBC FA composition did not differ between the 2 groups with exception of the saturated FA, palmitic acid (p=0.018). These results suggest that RBC FA composition may differ between individuals with or at risk for CAD. Additional research is needed to validate this biomarker as a predictor of CAD.

Polyunsaturated fatty acids (PUFA) have been used with some success in the treatment of canine atopic dermatitis (CAD). Correspondent in vitro studies revealed that PUFA play a crucial role in the exocytosis of mast cells. n3 PUFA such as α-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), as well as the n6 PUFA linoleic acid (LA) have been shown to arrest the secretion of inflammatory mediators. Contrary, the n-6 PUFA arachidonic acid (AA) has been proven to promote the production of mast cell inflammatory mediators. However, we are still lacking a complete picture of the mode of action. The goal of this work was to further characterize the modulatory effects of PUFA supplementation on the plasma membrane lipid composition of mast cells. Furthermore the consequences of a membrane modulation of mast cells by PUFA on the localization and activity on of the membrane bound enzyme phospholipases D (PLD) were investigated. Canine mastocytoma cells (C2) were supplemented with one of the following PUFA: LNA, EPA, DHA, LA or AA. To investigate the influence of PUFA on the lipid composition of membrane microdomains, lipid rafts were separated from non-raft plasma membranes of mast cells for the first time using a detergent-free isolation technique. Results show that PUFA are significantly increased in rafts as well as in non-rafts microdomains (Publication 1). The incorporation of PUFA into the membrane goes along with an increase of the unsaturation status and the fluidity of the membrane. This rise in membrane fluidity may result in a reorganization of membrane signaling molecules and enzymes such as the PLD. To define the impact of a PUFA supplementation on PLD trafficking, C2 were transfected with green fluorescent protein (GFP) fusion plasmids encoding PLD1 or PLD2. Since the transfection ability of the suspension cell line C2 is limited, a special transfection protocol was established, suitable for non-adherent cell lines. Transfection succeeded using chicken egg white as coating material for the cell culture plates. The transfection efficiency rose to 50% versus 5% in uncoated plates. In addition to the obvious increase in the transfection efficiency, the new technique is simple and economic and might be suitable for a wide range of suspension cell lines (Publication 2). Using this optimized protocol the influence of PUFA on the trafficking of PLD isoforms was studied. LNA, EPA, DHA and LA but not AA prevented the stimulation-induced translocation of PLD1 to the plasma membrane. Since the translocation of PLD1 is important for mast cell exocytosis, LNA, EPA, DHA and LA do have an inhibiting effect on the stimulation-induced release of pro-inflammatory mediators. All PUFA tested boosted the total PLD activity. In order to rule out, which PLD isoform was affected by the PUFA, the mast cells were supplemented with DHA or AA in the presence of specific PLD isoform inhibitors. DHA completely abolished the inhibitiory effect of the PLD1 inhibitor but had no effect on the inhibitory effect of PLD2 inhibitor. On the other hand, AA suppressed the inhibitory effect of both PLD1 and PLD2 inhibitor (Publication 3). Taking together, the studies provide a mechanistic base for the role of PUFA in the exocytosis processes of mast cells. PUFA of the n3 and the n6 families impact the lipid composition of membrane microdomains, which in turn lead to a modulation of the physiochemical properties of the membrane. LNA, EPA, DHA and LA suppress the release of inflammatory mediators through their inhibitory action on the stimulation-induced translocation of the PLD1. Contrariwise, AA permits the stimulation-induced migration of PLD1 to the plasma membrane and increases the activity of both PLD isoforms. Therefore, LNA, EPA, DHA and LA but not AA inhibit the release of mast cell inflammatory mediators upon stimulation
Mehrfach ungesättigte Fettsäuren (PUFA) können mit einigem Erfolg zur Behandlung der caninen atopischen Dermatitis (CAD) eingesetzt werden. In vitro-Studien zeigten, dass PUFA eine entscheidende Rolle in der Exozytose von Mastzellen spielen. N-3-PUFA wie α-Linolensäure (LNA), Eicosapentaensäure (EPA), Docosahexaensäure (DHA) sowie die n-6-PUFA Linolsäure (LA) können die Sekretion von Entzündungsmediatoren vermindern. Arachidonsäure (AA) als n-6 mehrfach ungesättigte Fettsäure hingegen fördert die Entzündungsmediatoren-Freisetzung aus den Mastzellen. Eine vollständige Aufklärung der Wirkungsweise fehlt aber weiterhin. Das Ziel dieser Arbeit war eine weitergehende Charakterisierung der modulierenden Effekte einer PUFA-Supplementierung auf die Lipidzusammensetzung der Plasmamembran von Mastzellen. Darüber hinaus wurden die Auswirkungen von PUFA auf die Lokalisation und Aktivität des Membran-gebundenen Enzyms Phospholipase D (PLD) untersucht. Canine Mastozytom-Zellen (C2) wurden mit einer der folgenden PUFA kultiviert: LNA, EPA, DHA, LA oder AA. Um den Einfluss von PUFA auf die Lipidzusammensetzung der Membran-Mikrodomänen zu untersuchen, konnten sowohl Lipid Raft als auch Nicht-Raft Plasmamembran-Anteile von Mastzellen zum ersten Mal mittels einer Detergenzien-freien Isolationsmethode getrennt werden. Hervorzuheben ist, dass PUFA signifikant vermehrt in Raft- sowie in Nicht-Raft Membranmikrodomänen eingelagert werden (Publikation 1). Die Integration von PUFA in die Membran geht mit einer Steigerung der Doppelbindungsanzahl und der Fluidität der Membran einher. Diese Erhöhung der Membranfluidität kann zu einer Reorganisation von membranären Signalmolekülen und Enzymen wie der PLD führen. Um die Auswirkungen einer PUFA-Supplementierung auf den intrazellulären Transport der PLD in C2 zu bestimmen, wurden die Zellen mit PLD1- oder PLD2-codierenden grün fluoreszierenden Protein-(GFP-)Fusionsplasmiden transfiziert. Da die Transfektionsfähigkeit der Suspensions-Zelllinie C2 begrenzt ist, wurde ein für nicht-adhärente Zelllinien geeignetes Transfektionsprotokoll etabliert. Mit Hühnereiweiß als Beschichtungsmaterial für die Zellkultur-Platten stieg die Transfektionseffizienz auf 50% im Vergleich zu 5% bei unbeschichteten Platten. Neben der deutlichen Erhöhung der Transfektionseffizienz ist die neu etablierte Technik einfach durchzuführen sowie wirtschaftlich und kann für eine Vielzahl von Suspension-Zelllinien geeignet sein (Publikation 2). Unter Verwendung dieses optimierten Protokolls wurde der Einfluss von PUFA auf die Translokation der PLD-Isoformen untersucht. LNA, EPA, DHA und LA, nicht aber AA verhindern die stimulationsinduzierte Translokation der PLD1 an die Plasmamembran. Die Translokation der PLD1 ist wichtig für die Mastzell-Exozytose. LNA, EPA, DHA und LA haben hier eine hemmende Wirkung auf die stimulationsinduzierte Freisetzung von proinflammatorischen Mediatoren. Alle getesteten PUFA verstärken die Gesamt-PLD-Aktivität. Um zu unterscheiden, welche PLD-Isoform durch PUFA beeinflusst ist, wurden die Mastzellen mit DHA oder AA in Gegenwart von PLD-Isoform-Inhibitoren supplementiert. DHA hebt die inhibitorische Wirkung des PLD1-Inhibitors vollständig auf, zeigte aber keinen Einfluss auf die hemmende Wirkung des PLD2-Inhibitors. Andererseits unterdrückt AA die hemmende Wirkung des PLD1- als auch des PLD2-Inhibitors (Publikation 3). Zusammenfassend bietet die Studie eine mechanistische Basis für die Rolle von PUFA bei Exozytose-Prozessen von Mastzellen. PUFA der n-3- und n-6-Familie beeinflussen die Lipidzusammensetzung von membranären Mikrodomänen, was wiederum zu einer Modulation der physikalisch-chemischen Eigenschaften der Membran führt. LNA, EPA, DHA und LA verhindern die Freisetzung von Entzündungsmediatoren durch ihre hemmende Wirkung auf die stimulationsinduzierte Translokation der PLD1. Umgekehrt erlaubt AA eine stimulationsinduzierte Migration der PLD1 zur Plasmamembran und steigert die Aktivität der beiden Isoformen der PLD. Somit hemmen LNA, EPA, DHA und LA, aber nicht AA die Freisetzung von Mastzell-Entzündungsmediatoren nach Stimulation

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Este ensaio teve como objetivo avaliar a suplementação alimentar com dois óleos vegetais (soja e linhaça) e parede celular de Saccharomyces cerevisiae em tilápias do Nilo sobre o desempenho produtivo, inflamação aguda induzida por Streptococcus agalactiae inativado, inflamação crônica por corpo estranho por meio do implante de lamínulas de vidro no tecido subcutâneo e a taxa de sobrevivência frente ao desafio com S. agalactiae. Foram utilizadas 840 tilápias, invertidas sexualmente, distribuídas em 24 caixas de 500L (n=35). O delineamento utilizado foi o inteiramente casualizado com oito tratamentos e três repetições, no qual foram testadas duas fontes de ácidos graxos essenciais (óleo de soja (OS) e óleo de linhaça (OL)) e dois níveis de parede celular de S. cerevisiae (PC) na dieta (0,0 e 0,3%) constituindo os seguintes tratamentos: OS; OL; OS+OL; OS+PC; OL+PC; OS+OL+PC; PC e controle. Os peixes receberam as rações teste durante três meses, duas vezes ao dia, ad libitum. Um lote de peixes foi avaliado quanto ao acúmulo de células na inflamação induzida por S. agalactiae inativado, na bexiga natatória após 12, 24 e 48 horas. O segundo lote de peixes foi submetido ao implante de lamínulas de vidro no tecido subcutâneo por dois, quatro, seis e oito dias, quando foram colhidas e avaliadas quanto ao acúmulo de macrófagos e formação de gigantócitos. Dentre os parâmetros hematológicos foi realizado eritrograma completo (contagem de células vermelhas, percentual de hematócrito e taxa de hemoglobina). Além disso, foi avaliada a atividade respiratória de leucócitos sanguíneos. No terceiro lote de peixes, foram avaliados os parâmetros de desempenho produtivo e a sobrevivência frente ao desafio bacteriano com S. agalactiae. No estudo da inflamação aguda constatou-se o efeito da suplementação alimentar com os óleos vegetais (soja e/ou linhaça)...
This test was designed to evaluate dietary supplementation with two vegetable oils (soybean and linseed) and cell wall of Saccharomyces cerevisiae in Nile tilapia growth performance, acute inflammation induced by Streptococcus agalactiae inactivated, chronic inflammation of foreign body through implantation of glass coverslips into the subcutaneous tissue and survival upon challenge with S. agalactiae. 840 tilapia, sexually inverted, were distributed into 24 aquarium of 500L (n = 35). The design was completely randomized with eight treatments and three replicates which two sources of essential fatty acids (soybean oil (SO) and linseed oil (LO)) and two levels of the cell wall of S. cerevisiae (PC) in diet (0.0 and 0.3%), and tested to the following treatments: OS, OL, OL + OS, OS + PC, PC + OL; OS + OL + PC, PC and control. The fish were fed with diets for three months, twice daily, ad libitum. In one group of fish the accumulation of cells in the inflammation induced by S. agalactiae inactivated in bladder after 12, 24 and 48 hours were evaluated. The second group of fish was subjected to implantation of glass coverslips into the subcutaneous tissue by two, four, six and eight days when they were harvested and evaluated for macrophage accumulation and formation of giant cell. Among the hematological parameters the complete erythrocyte (red blood cell count, percentage of hematocrit and hemoglobin) was performed. Furthermore, the respiratory activity of blood leukocytes was evaluated. The third group of fish, the productive performance parameters and survival to the bacterial challenge with S. agalactiae was evaluated. In the study of acute inflammation was found the effect of dietary supplementation with vegetable oils (soybean and / or linseed), as well as the yeast cell wall increased accumulation of total cells in the exudate of the swim bladder... (Complete abstract click electronic access below)

Resumo: Este ensaio teve como objetivo avaliar a suplementação alimentar com dois óleos vegetais (soja e linhaça) e parede celular de Saccharomyces cerevisiae em tilápias do Nilo sobre o desempenho produtivo, inflamação aguda induzida por Streptococcus agalactiae inativado, inflamação crônica por corpo estranho por meio do implante de lamínulas de vidro no tecido subcutâneo e a taxa de sobrevivência frente ao desafio com S. agalactiae. Foram utilizadas 840 tilápias, invertidas sexualmente, distribuídas em 24 caixas de 500L (n=35). O delineamento utilizado foi o inteiramente casualizado com oito tratamentos e três repetições, no qual foram testadas duas fontes de ácidos graxos essenciais (óleo de soja (OS) e óleo de linhaça (OL)) e dois níveis de parede celular de S. cerevisiae (PC) na dieta (0,0 e 0,3%) constituindo os seguintes tratamentos: OS; OL; OS+OL; OS+PC; OL+PC; OS+OL+PC; PC e controle. Os peixes receberam as rações teste durante três meses, duas vezes ao dia, ad libitum. Um lote de peixes foi avaliado quanto ao acúmulo de células na inflamação induzida por S. agalactiae inativado, na bexiga natatória após 12, 24 e 48 horas. O segundo lote de peixes foi submetido ao implante de lamínulas de vidro no tecido subcutâneo por dois, quatro, seis e oito dias, quando foram colhidas e avaliadas quanto ao acúmulo de macrófagos e formação de gigantócitos. Dentre os parâmetros hematológicos foi realizado eritrograma completo (contagem de células vermelhas, percentual de hematócrito e taxa de hemoglobina). Além disso, foi avaliada a atividade respiratória de leucócitos sanguíneos. No terceiro lote de peixes, foram avaliados os parâmetros de desempenho produtivo e a sobrevivência frente ao desafio bacteriano com S. agalactiae. No estudo da inflamação aguda constatou-se o efeito da suplementação alimentar com os óleos vegetais (soja e/ou linhaça) ...(Resumo completo, clicar acesso eletrônico abaixo)
Abstract: This test was designed to evaluate dietary supplementation with two vegetable oils (soybean and linseed) and cell wall of Saccharomyces cerevisiae in Nile tilapia growth performance, acute inflammation induced by Streptococcus agalactiae inactivated, chronic inflammation of foreign body through implantation of glass coverslips into the subcutaneous tissue and survival upon challenge with S. agalactiae. 840 tilapia, sexually inverted, were distributed into 24 aquarium of 500L (n = 35). The design was completely randomized with eight treatments and three replicates which two sources of essential fatty acids (soybean oil (SO) and linseed oil (LO)) and two levels of the cell wall of S. cerevisiae (PC) in diet (0.0 and 0.3%), and tested to the following treatments: OS, OL, OL + OS, OS + PC, PC + OL; OS + OL + PC, PC and control. The fish were fed with diets for three months, twice daily, ad libitum. In one group of fish the accumulation of cells in the inflammation induced by S. agalactiae inactivated in bladder after 12, 24 and 48 hours were evaluated. The second group of fish was subjected to implantation of glass coverslips into the subcutaneous tissue by two, four, six and eight days when they were harvested and evaluated for macrophage accumulation and formation of giant cell. Among the hematological parameters the complete erythrocyte (red blood cell count, percentage of hematocrit and hemoglobin) was performed. Furthermore, the respiratory activity of blood leukocytes was evaluated. The third group of fish, the productive performance parameters and survival to the bacterial challenge with S. agalactiae was evaluated. In the study of acute inflammation was found the effect of dietary supplementation with vegetable oils (soybean and / or linseed), as well as the yeast cell wall increased accumulation of total cells in the exudate of the swim bladder... (Complete abstract click electronic access below)
Orientador: Flávio Ruas de Moraes
Coorientador: Fabiana Pilarski
Banca: Ana Lúcia Salaro
Banca: Áureo Evangelista Santana
Banca: Marco Antônio de Andrade Belo
Banca: Margarida Maria Barros
Doutor

Les acides gras à très longues chaine (VLCFA) sont essentiels dans le développement, particulièrement dans les mécanismes de trafic vésiculaires, de différenciation et division cellulaire. Cependant, le rôle de ces VLCFA dans ces différents processus chez les plantes n’est pas encore bien compris. Afin d’identifier de nouveaux acteurs associés à la biosynthèse ou la fonction des VLCFA, un crible suppresseur multicopies a été réalisé dans un mutant d’élongation des VLCFA de levure. La perte de l’activité déshydratase PHS1 chez la levure et de PASTICCINO2 chez les plantes perturbe la croissance et induit des défauts de cytokinèse. La PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) historiquement caractérisée comme une déshydratase inactive est capable de restaurer les défauts de croissance et d’élongation de phs1 mais non de pas2. PTPLA interagit avec plusieurs membres du complexe élongase dans le RE et son absence conduit à l’accumulation 3-hydroxyacyl-CoA, signature des déshydratases impliquées dans l’élongation des acides gras. Cependant, la perte de PTPLA conduit à une augmentation des VLCFA, probablement dépendante de PAS2 montrant que PTPLA serait un répresseur potentiel de l’élongation. Les deux déshydratases ont des profils d’expression divergents dans la racine. PAS2 est majoritairement exprimé dans l’endoderme tandis que PTPLA s’exprime uniquement dans les tissus vasculaires et le péricycle. La comparaison de l’expression ectopique de PAS2 et PTPLA dans leur tissus respectif confirme l’existence de deux complexe élongase indépendant associé à PAS2 ou PTPLA et interagissant de manière non cellule autonome. Les cytokinines pourraient constituer le signal entre les deux complexes élongase du fait que la biosynthèse de ces hormones est réprimée par les VLCFA. Les VLCFA répriment ainsi l'expression d'IPT3 dans les racines comme observées pour la partie apicale. Les cytokinines semblent aussi réguler la teneur en VLCFA dans la racine suggérant la présence de boucles de rétrocontrôles entre ces hormones et les VLCFA
Very long chain fatty acids (VLCFA) are involved in plant development and particularly in several cellular processes such as membrane trafficking, cell division and cell differentiation. However, the precise role of VLCFA in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFA, a yeast multicopy suppressor screen was carried out in a yeast mutant strain defective for fatty acid elongation. Loss of function of the elongase dehydratase PHS1 in yeast and PASTICCINO2 in plants prevents growth and induces cytokinesis defects. PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) previously characterized as an inactive dehydratase was able to restore yeast phs1 growth and VLCFA elongation but not the plant pas2 defects. PTPLA interacted with elongase members in the ER and its absence induced the accumulation of 3-hydroxyacyl-CoA as expected from a dehydratase involved in fatty acid (FA) elongation. However, loss of PTPLA function led to increased VLCFA levels, effect that was dependent of the presence of PAS2 indicating that PTPLA activity repressed FA elongation. The two dehydratases have specific expression profiles in the root with PAS2, mostly restricted in the endodermis, while PTPLA was confined in the vascular tissue and pericycle cells. Comparative ectopic expression of PTPLA and PAS2 in their respective domains confirmed the existence of two independent elongase complexes comprising PAS2 or PTPLA that were functionally interacting in a non-cell autonomous manner. A putative regulating signal could involve cytokinins that were described to be regulated by VLCFA. VLCFA were indeed found to repress IPT3 expression in roots like in leaves. Cytokinins were also found to regulate VLCFA levels suggesting the existence of regulatory feedback loops between cytokinins and VLCFA

Lors de lesions nerveuses par retrait de co::(2), l'etude du metabolisme des lipides a montre qu'un traitement a la cdp-choline ne modifie pas la distribution et la synthese des phospholipides et des acides gras mais provoque par contre une diminution de la liberation d'acides gras. La cdp-choline diminue l'activite phospholiposique a1 au niveau des membranes et protege donc celles-ci de la degradation

Indiana University-Purdue University Indianapolis (IUPUI)
Each biological cell is surrounded by a membrane that consists of many different kinds of lipids. The lipids are mainly composed of phospholipids, which form a fluid bilayer that serves as the platform for the function of membrane bound proteins regulating cellular activity. In the research described in this thesis we employed solid state 2H NMR, complemented by DSC (differential scanning calorimetry) and MD (molecular dynamics) simulations, to study the effect of PUFA (polyunsaturated fatty acids) and TFA (trans fatty acids) on molecular organization in protein-free model membranes of controlled composition. These two classes of unsaturated fatty acid incorporate into membrane lipids and have, respectively, a beneficial and harmful impact on health. The aim is to gain insight into the molecular origin of this behavior. DHA (docosahexaenoic acid), which with 6 "natural" cis double bonds is the most highly unsaturated PUFA found in fish oils, and EA (elaidic acid), which with only a single "unnatural" trans double bond is the simplest manmade TFA often found in commercially produced food, were the focus. 2H NMR spectra for [2H31]-N-palmitoylsphingomyelin ([2H31]16:0SM) in SM/16:0-22:6PE (1-palmitoyl-2-docosahexaenoylphosphatidylethanolamine)/cholesterol (1:1:1 mol) mixed membranes were recorded. This system served as our PUFA-containing model. The spectra are consistent with lateral separation into nano-sized (< 20 nm) domains that are SM-rich/cholesterol-rich (raft), characterized by higher chain order, and DHA-rich/cholesterol-poor (non-raft), characterized by lower chain order. The aversion cholesterol has for DHA, as opposed to the affinity cholesterol has for predominantly saturated SM, excludes the sterol from DHA-containing PE-rich domains and DHA from SM-rich/cholesterol-rich domains. It is the formation of highly disordered membrane domains that we hypothesize is responsible, in part, for the diverse health benefits associated with dietary consumption of DHA. 2H NMR spectra for 1-elaidoyl-2-[2H35]stearoylphosphatidylcholine (t18:1-[2H35]18:0PC) and 1-oleoyl-2-[2H35]stearoylphosphatidylcholine (c18:1-[2H35]18:0PC) were recorded to compare membranes with respect to a trans vs. cis ("natural") double bond. The spectra indicate that while a trans double bond produces a smaller deviation from linear conformation than a cis double bond, membrane order is decreased by a comparable amount because the energy barrier to rotation about the C-C single bonds either side of a trans or cis double bond is reduced. Although EA adopts a conformation somewhat resembling a saturated fatty acid, the TFA is almost as disordered as its cis counterpart oleic acid (OA). We speculate that EA could be mistaken for a saturated fatty acid and infiltrate lipid rafts to disrupt the high order therein that is necessary for the function of signaling proteins.

This study explored the effects of dietary unsaturated fatty acids on feline lipid metabolism. It was hypothesized that high dietary linoleic acid (18:2n-6, LA) would enhance conversion to arachidonic acid (20:4n-6), enrichment of dietary long chain n-3 FA (LCn-3FA) would affect lipid parameters, and n-3 FAs incorporation may blunt n-6 FA incorporation. Twenty-nine cats were randomized into groups (n = 9, 10, 10), and fed for 28 days with blood collections on days 0, 14, and 28. Experimental diets consisted of a commercial diet, supplemented with 8g oil/100g kibble. Oil supplements and subsequent diets were: high-oleic sunflower (H diet) with 82% oleic acid (18:1n-9), Menhaden fish (M diet) with LCn-3FA, and safflower (S diet) with 75% 18:2n-6. Dietary 20:4n-6 content was: 0.03 for H and S, and 0.09 for M (g FA/kg diet). Nonesterified fatty acid (NEFA), triacylglycerol (TG), total cholesterol (TC), lipoproteincholesterol (LP-C), plasma phospholipid (PL) FAs, red blood cell membrane (RBC) FAs, and ∆5 and ∆6 desaturase indices were measured. Statistical analyses were performed with SAS PROC MIXED with p < 0.05 determining significance. Neither TC nor NEFA showed significant effects. Diet M resulted in significant TG lowering, despite typically low feline TGs. Similarly, pre-β LP-C (i.e. TG-rich VLDL) was decreased in diet M. Plasma PL FAs revealed significant accumulations of the following: 18:1n-9 in diet H, 18:2n-6 in diet S, and LCn-3FA in diet M. Despite high dietary 18:2n-6, plasma PL 20:4n-6 was not increased in diet S over diets H or M. Increased docosadienoic acid (20:2n-6) in diet S demonstrated that 18:2n-6 chain elongation occurred in deference to its ∆6 desaturation further substantiating low feline ∆6 desaturase activity. Interestingly, no diet M blunting of 20:4n-6 incorporation occurred because fish oil supplementation provided additional 20:4n-6. Tissue 20:4n-6 content appears to be diet-dependent. Accumulation of eicosapentaenoic acid (20:5n-3), but low affinity for docosahexaenoic acid (22:6n-3) occurred in diet M RBC membranes. After 28 days, plasma PLs reflect dietary intake more readily than RBC membranes. Fish oil supplementation resulted in plasma PL LCn-3FA enrichment and lowered plasma TG concentrations, both of which may have physiological significance in cats.

Polyunsaturated fatty acids (PUFA) have been used with some success in the treatment of canine atopic dermatitis (CAD). Correspondent in vitro studies revealed that PUFA play a crucial role in the exocytosis of mast cells. n3 PUFA such as α-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), as well as the n6 PUFA linoleic acid (LA) have been shown to arrest the secretion of inflammatory mediators. Contrary, the n-6 PUFA arachidonic acid (AA) has been proven to promote the production of mast cell inflammatory mediators. However, we are still lacking a complete picture of the mode of action. The goal of this work was to further characterize the modulatory effects of PUFA supplementation on the plasma membrane lipid composition of mast cells. Furthermore the consequences of a membrane modulation of mast cells by PUFA on the localization and activity on of the membrane bound enzyme phospholipases D (PLD) were investigated. Canine mastocytoma cells (C2) were supplemented with one of the following PUFA: LNA, EPA, DHA, LA or AA. To investigate the influence of PUFA on the lipid composition of membrane microdomains, lipid rafts were separated from non-raft plasma membranes of mast cells for the first time using a detergent-free isolation technique. Results show that PUFA are significantly increased in rafts as well as in non-rafts microdomains (Publication 1). The incorporation of PUFA into the membrane goes along with an increase of the unsaturation status and the fluidity of the membrane. This rise in membrane fluidity may result in a reorganization of membrane signaling molecules and enzymes such as the PLD. To define the impact of a PUFA supplementation on PLD trafficking, C2 were transfected with green fluorescent protein (GFP) fusion plasmids encoding PLD1 or PLD2. Since the transfection ability of the suspension cell line C2 is limited, a special transfection protocol was established, suitable for non-adherent cell lines. Transfection succeeded using chicken egg white as coating material for the cell culture plates. The transfection efficiency rose to 50% versus 5% in uncoated plates. In addition to the obvious increase in the transfection efficiency, the new technique is simple and economic and might be suitable for a wide range of suspension cell lines (Publication 2). Using this optimized protocol the influence of PUFA on the trafficking of PLD isoforms was studied. LNA, EPA, DHA and LA but not AA prevented the stimulation-induced translocation of PLD1 to the plasma membrane. Since the translocation of PLD1 is important for mast cell exocytosis, LNA, EPA, DHA and LA do have an inhibiting effect on the stimulation-induced release of pro-inflammatory mediators. All PUFA tested boosted the total PLD activity. In order to rule out, which PLD isoform was affected by the PUFA, the mast cells were supplemented with DHA or AA in the presence of specific PLD isoform inhibitors. DHA completely abolished the inhibitiory effect of the PLD1 inhibitor but had no effect on the inhibitory effect of PLD2 inhibitor. On the other hand, AA suppressed the inhibitory effect of both PLD1 and PLD2 inhibitor (Publication 3). Taking together, the studies provide a mechanistic base for the role of PUFA in the exocytosis processes of mast cells. PUFA of the n3 and the n6 families impact the lipid composition of membrane microdomains, which in turn lead to a modulation of the physiochemical properties of the membrane. LNA, EPA, DHA and LA suppress the release of inflammatory mediators through their inhibitory action on the stimulation-induced translocation of the PLD1. Contrariwise, AA permits the stimulation-induced migration of PLD1 to the plasma membrane and increases the activity of both PLD isoforms. Therefore, LNA, EPA, DHA and LA but not AA inhibit the release of mast cell inflammatory mediators upon stimulation.
Mehrfach ungesättigte Fettsäuren (PUFA) können mit einigem Erfolg zur Behandlung der caninen atopischen Dermatitis (CAD) eingesetzt werden. In vitro-Studien zeigten, dass PUFA eine entscheidende Rolle in der Exozytose von Mastzellen spielen. N-3-PUFA wie α-Linolensäure (LNA), Eicosapentaensäure (EPA), Docosahexaensäure (DHA) sowie die n-6-PUFA Linolsäure (LA) können die Sekretion von Entzündungsmediatoren vermindern. Arachidonsäure (AA) als n-6 mehrfach ungesättigte Fettsäure hingegen fördert die Entzündungsmediatoren-Freisetzung aus den Mastzellen. Eine vollständige Aufklärung der Wirkungsweise fehlt aber weiterhin. Das Ziel dieser Arbeit war eine weitergehende Charakterisierung der modulierenden Effekte einer PUFA-Supplementierung auf die Lipidzusammensetzung der Plasmamembran von Mastzellen. Darüber hinaus wurden die Auswirkungen von PUFA auf die Lokalisation und Aktivität des Membran-gebundenen Enzyms Phospholipase D (PLD) untersucht. Canine Mastozytom-Zellen (C2) wurden mit einer der folgenden PUFA kultiviert: LNA, EPA, DHA, LA oder AA. Um den Einfluss von PUFA auf die Lipidzusammensetzung der Membran-Mikrodomänen zu untersuchen, konnten sowohl Lipid Raft als auch Nicht-Raft Plasmamembran-Anteile von Mastzellen zum ersten Mal mittels einer Detergenzien-freien Isolationsmethode getrennt werden. Hervorzuheben ist, dass PUFA signifikant vermehrt in Raft- sowie in Nicht-Raft Membranmikrodomänen eingelagert werden (Publikation 1). Die Integration von PUFA in die Membran geht mit einer Steigerung der Doppelbindungsanzahl und der Fluidität der Membran einher. Diese Erhöhung der Membranfluidität kann zu einer Reorganisation von membranären Signalmolekülen und Enzymen wie der PLD führen. Um die Auswirkungen einer PUFA-Supplementierung auf den intrazellulären Transport der PLD in C2 zu bestimmen, wurden die Zellen mit PLD1- oder PLD2-codierenden grün fluoreszierenden Protein-(GFP-)Fusionsplasmiden transfiziert. Da die Transfektionsfähigkeit der Suspensions-Zelllinie C2 begrenzt ist, wurde ein für nicht-adhärente Zelllinien geeignetes Transfektionsprotokoll etabliert. Mit Hühnereiweiß als Beschichtungsmaterial für die Zellkultur-Platten stieg die Transfektionseffizienz auf 50% im Vergleich zu 5% bei unbeschichteten Platten. Neben der deutlichen Erhöhung der Transfektionseffizienz ist die neu etablierte Technik einfach durchzuführen sowie wirtschaftlich und kann für eine Vielzahl von Suspension-Zelllinien geeignet sein (Publikation 2). Unter Verwendung dieses optimierten Protokolls wurde der Einfluss von PUFA auf die Translokation der PLD-Isoformen untersucht. LNA, EPA, DHA und LA, nicht aber AA verhindern die stimulationsinduzierte Translokation der PLD1 an die Plasmamembran. Die Translokation der PLD1 ist wichtig für die Mastzell-Exozytose. LNA, EPA, DHA und LA haben hier eine hemmende Wirkung auf die stimulationsinduzierte Freisetzung von proinflammatorischen Mediatoren. Alle getesteten PUFA verstärken die Gesamt-PLD-Aktivität. Um zu unterscheiden, welche PLD-Isoform durch PUFA beeinflusst ist, wurden die Mastzellen mit DHA oder AA in Gegenwart von PLD-Isoform-Inhibitoren supplementiert. DHA hebt die inhibitorische Wirkung des PLD1-Inhibitors vollständig auf, zeigte aber keinen Einfluss auf die hemmende Wirkung des PLD2-Inhibitors. Andererseits unterdrückt AA die hemmende Wirkung des PLD1- als auch des PLD2-Inhibitors (Publikation 3). Zusammenfassend bietet die Studie eine mechanistische Basis für die Rolle von PUFA bei Exozytose-Prozessen von Mastzellen. PUFA der n-3- und n-6-Familie beeinflussen die Lipidzusammensetzung von membranären Mikrodomänen, was wiederum zu einer Modulation der physikalisch-chemischen Eigenschaften der Membran führt. LNA, EPA, DHA und LA verhindern die Freisetzung von Entzündungsmediatoren durch ihre hemmende Wirkung auf die stimulationsinduzierte Translokation der PLD1. Umgekehrt erlaubt AA eine stimulationsinduzierte Migration der PLD1 zur Plasmamembran und steigert die Aktivität der beiden Isoformen der PLD. Somit hemmen LNA, EPA, DHA und LA, aber nicht AA die Freisetzung von Mastzell-Entzündungsmediatoren nach Stimulation.

Indiana University-Purdue University Indianapolis (IUPUI)
Cellular membranes contain a staggering diversity of lipids. The lipids are heterogeneously distr ibuted to create regions, or domains, whose physical properties differ from the bulk membrane and play an essential role in modulating the function of resident proteins. Many basic questions pertaining to the formation of these lateral assemblies remain. T his research employs model membranes of well - defined composition to focus on the potential role of polyunsaturated fatty acids (PUFAs) and their interaction with cholesterol (chol) in restructuring the membrane environment. Omega - 3 (n - 3) PUFAs are the main bioactive components of fish oil, whose consumption alleviates a variety of health problems by a molecular mechanism that is unclear. We hypothesize that the incorporation of PUFAs into membrane lipids and the effect they have on molecular organization may be, in part, responsible. Chol is a major constituent in the plasma membrane of mammals. It determines the arrangement and collective properties of neighboring lipids, driving the formation of domains via differential affinity for different lipids . T he m olecular organization of 1 -[ 2 H 31 ]palmitoyl -2- eicosapentaenoylphosphatidylcholine (PEPC - d 31 ) and 1 -[ 2 H 31 ]palmitoyl -2- docosahexaenoylphosphatidylcholine (PDPC -d 31 ) in membran es with sphingomyelin (SM) and chol (1:1:1 mol) was compared by solid - state 2 H NMR spectroscopy. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the two major n - 3 PUFAs found in fish oil, while PEPC -d 31 and PDPC -d 31 are phospholipids containing the respective PUFAs at the sn - 2 position and a perdeuterated palmitic acid a t the sn - 1 position . Analysis of s pectra recorded as a function of temperature indicate s that in both cases, formation of PUFA - rich (less ordered) and SM - rich (more ordered) domains occurred. A surprisingly substantial proportion of PUFA was found to infil trate the more ordered domain. There was almost twice as much DHA (65%) as EPA (30%) . The implication is that n - 3 PUFA s can incorporate into lipid rafts, which are domains enriched in SM and chol in the plasma membrane, and potentially disrupt the activity of signaling proteins that reside therein. DHA, furthermore, may be the more potent component of fish oil. PUFA - chol interactions were also examined through affinity measurements. A novel method utilizing electron paramagnetic resonance (EPR) was develope d, to monitor the partitioning of a spin - labeled analog of chol , 3β - doxyl - 5α - cholestane (chlstn), between large unilamellar vesicles (LUVs) and met hyl - β - cyclodextrin (mβCD). The EPR spectra for chlstn in the two environments are distinguishable due to the substantial differences in tumbling rates , allowing the population distribution ratio to be determined by spectral simulation. Advantages of this approach include speed of implementation and a vo idance of potential artifact s associated with physical separation of LUV and mβCD . Additionally, in a check of the method, t he relative partition coefficients between lipids measured for the spin label analog agree with values obtained for chol by isothermal titration calorimetry (ITC). Results from LUV with different composition confirmed a hierarchy of decreased sterol affinity for phospholipids with increasing acyl chain unsaturation , PDPC possessing half the affinity of the corresponding monounsaturated phospholipid. Taken together, the results of these studies on model membranes demonstrate the potential for PUFA - driven alteration of the architecture of biomembranes, a mechanism through which human health may be impacted.