Dietary fatty acid composition and the homeostatic regulation of mitochondrial phospholipid classes in red muscle of rainbow trout (Oncorhynchus mykiss).

Although dietary lipid quality markedly affects fatty acid (FA) composition of mitochondrial membranes from rainbow trout red muscle (Oncorhynchus mykiss), mitochondrial processes are relatively unchanged. As certain classes of phospholipids interact more intimately with membrane proteins than others, we examined whether specific phospholipid classes from these muscle mitochondria were more affected by dietary FA composition than others. To test this hypothesis, we fed trout with two diets differing only in their FA composition: Diet 1 had higher levels of 18:1n-9 and 18:2n-6 than Diet 2, while 22:6n-3 and 22:5n-6 were virtually absent from Diet 1 and high in Diet 2. After 5 months, trout fed Diet 2 had higher proportions of phosphatidylcholine (PC) and less phosphatidylethanolamine (PE) in mitochondrial membranes than those fed Diet 1. The FA composition of PC, PE and cardiolipin (CL) showed clear evidence of regulated incorporation of dietary FA. For trout fed Diet 2, 22:6n-3 was the most abundant FA in PC, PE and CL. The n-6 FA were consistently higher in all phospholipid classes of trout fed Diet 1, with shorter n-6 FA being favoured in CL than in PC and PE. Despite these marked changes in individual FA levels with diet, general characteristics such as total polyunsaturated FA, total monounsaturated FA and total saturated FA were conserved in PE and CL, confirming differential regulation of the FA composition of PC, PE and CL. The regulated changes of phospholipid classes presumably maintain critical membrane characteristics despite varying nutritional quality. We postulate that these changes aim to protect mitochondrial function.

Disseminated neoplasia in the soft-shell clam Mya arenaria: membrane lipid composition and functional parameters of circulating cells.

In a previous study we compared lipid composition and functional parameters of circulating cells from Cerastoderma edule affected or not by disseminated neoplasia (neoplastic cells vs hemocytes) (Le Grand et al. Chem Phys Lipids 167:9-20 2013). Neoplastic cells presented morpho-functional modifications concomitant to striking membrane lipid alterations: the proportion of particular plasmalogen molecular species was drastically decreased. We wanted to test whether this pattern was representative of bivalve neoplastic cells. For the purpose, a similar study was conducted on another bivalve species affected by disseminated neoplasia, the soft-shell clam (Mya arenaria). Although total reactive oxygen species production was unaffected, M. arenaria neoplastic cells presented some functional alterations: phagocytosis activity was reduced by 33 %. However, lipid compositions were not drastically altered. Particularly, sterol and plasmalogen levels did not differ between both cell types (about 43 % of membrane lipids and 35 % of phospholipids, respectively in hemocytes and neoplastic cells). This could be related to the fact that disseminated neoplasia was not related to hemolymph cell proliferation in M. arenaria (0.9 ± 0.2 10(6)cell mL(-1), considering both healthy and neoplastic clams, n = 6). Nevertheless this study highlighted minor but specific alterations of membrane lipid composition in M. arenaria neoplastic cells. The only phospholipid subclass in which the fatty acid profile strongly differed between both cell types was serine plasmalogen (PlsSer), with neoplastic cells presenting lower specific enrichment of 20:1n-11 in PlsSer. Such specific alteration of membrane lipid composition strengthened the assumption of an implication of key plasmalogen molecular species in this leukemia-like disease in bivalves.

Altered membrane lipid composition and functional parameters of circulating cells in cockles (Cerastoderma edule) affected by disseminated neoplasia.

Membrane lipid composition and morpho-functional parameters were investigated in circulating cells of the edible cockle (Cerastoderma edule) affected by disseminated neoplasia (neoplastic cells) and compared to those from healthy cockles (hemocytes). Membrane sterol levels, phospholipid (PL) class and subclass proportions and their respective fatty acid (FA) compositions were determined. Morpho-functional parameters were evaluated through total hemocyte count (THC), mortality rate, phagocytosis ability and reactive oxygen species (ROS) production. Both morpho-functional parameters and lipid composition were profoundly affected in neoplastic cells. These dedifferentiated cells displayed higher THC (5×), mortality rate (3×) and ROS production with addition of carbonyl cyanide m-chloro phenylhydrazone (1.7×) but lower phagocytosis ability (½×), than unaffected hemocytes. Total PL amounts were higher in neoplastic cells than in hemocytes (12.3 and 5.1 nmol×10(-6) cells, respectively). However, sterols and a particular subclass of PL (plasmalogens; 1-alkenyl-2-acyl PL) were present in similar amounts in both cell type membranes. This led to a two times lower proportion of these membrane lipid constituents in neoplastic cells when compared to hemocytes (20.5% vs. 42.1% of sterols in total membrane lipids and 21.7% vs. 44.2% of plasmalogens among total PL, respectively). Proportions of non-methylene interrupted FA- and 20:1n-11-plasmalogen molecular species were the most impacted in neoplastic cells when compared to hemocytes (â…“× and »×, respectively). These changes in response to this leukemia-like disease in bivalves highlight the specific imbalance of plasmalogens and sterols in neoplastic cells, in comparison to the greater stability of other membrane lipid components.

Electrochemistry of cytochrome c immobilized on cardiolipin-modified electrodes: a probe for protein-lipid interactions.

Electrochemistry of cytochrome c (cyt c) immobilized on a cardiolipin (CL)/phosphatidylcholine (PC) film supported on a glassy carbon electrode was investigated using variable-frequency AC voltammetry. At low ionic strength, we observed two redox-active subpopulations characterized by distinct values of potential (E1/2) and electron transfer rate constant (k(ET)). At high ionic strength, only one subpopulation was detected, consistent with the existence of very stable cyt c-CL adducts, most probably formed by hydrophobic interactions between the protein and the fatty acid (FA) chains carried by CL. This subpopulation exhibits a comparatively high k(ET) value (> 300 s(-1)) apparently changing with the structure of the FA chains of CL, i.e. 18:2(n - 6) or 14:0. Our study suggests that electrochemistry can be a useful technique for probing protein-lipid interactions, and more particularly the role played by the specific structure of the FA chains of CL on cyt c binding.

Membrane phospholipid composition of hemocytes in the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum.

The detailed sterol (free sterol proportions and compositions) and phospholipid (PL) compositions (relative proportions of PL classes and subclasses and their respective fatty acid (FA) compositions) of hemocyte membranes were investigated in two bivalve mollusks: the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum. Hemocyte membrane lipids of both species revealed similar general composition: i) their free sterol/PL ratio was above 0.4 and ii) their PL were predominated by the diacyl+alkyl forms of glycerophosphatidylcholine (PC), the plasmalogen form of glycerophosphatidylethanolamine (PE) and ceramide aminoethylphosphonate (CAEP). Free sterols were predominated by cholesterol in both species. Plasmalogen forms of PE and glycerophosphatidylserine (PS) represented 82-83% and 46-55% of total PE and PS, respectively. When compared to their respective diacyl+alkyl forms, plasmalogen forms of PE and PS were specifically enriched in non-methylene-interrupted (NMI) FA and 20:1n-11, suggesting a functional significance of these PL molecular species in bivalve hemocytes. Lysoglycerophosphatidylcholine (LysoPC) levels were found to be fairly high in hemocytes, accounting for about 8% of the PL. Some species-specific features were also found. LysoPC and glycerophosphatidylinositol (PI) FA compositions differed between Ruditapes philippinarum and Crassostrea gigas. CAEP proportion was higher in R. philippinarum than in C. gigas (14.5% and 27.9% of the PL, respectively). Hemolymph cell monolayer observations and flow-cytometric analyses revealed species-specific hemocyte morphology and sub-populations which could account for some of the observed species-specific membrane lipid compositions.

High levels of icosapentaenoic acid in the lipids of oyster Crassostrea gigas ranging over both Japan and France.

The lipid and fatty acids of the Pacific oyster, Crassostrea gigas, collected both in Japan and France, were analyzed. Triacylglycerols and sterols were the major classes in the neutral lipids of both populations of C. gigas between the two countries, and they had significant levels of phospholipids (phosphatidylethanolamine and phosphatidylcholine) with ceramideaminoethylphosphonate in their polar tissue lipids. The differences of 18:3n-3 and 22:6n-3 (docosahexaenoic acid; DHA) levels in the depot triacylglycerols suggest the influence of dietary phytoplankton in different environments of the two countries. Although the levels of the fatty acids slightly differed, that of 20:5n-3 (icosapentaenoic acid; EPA, 20.5-24.8%) in the triacylglycerols was specifically the highest fatty acid in all the samples. The major polyunsaturated fatty acids in the polar lipids of all samples were EPA (13.2-17.8% for phosphatidylethanolamine and 13.4-22.7% for phosphatidylcholine) and DHA (18.9-26.8% for phosphatidylethanolamine and 13.1-22.5% for phosphatidylcholine). The fluctuation of total PUFA levels in the polar lipids between samples of both countries suggests the influence of diet. In addition, the consistently high EPA levels in the phospholipids may compensate for the variation in DHA levels.

Occurrence of the cis-4,7,10, trans-13-22:4 fatty acid in the family Pectinidae (Mollusca: Bivalvia).

The present study aimed to elucidate the effective phylogenetic specificity of distribution of a cis-4,7,10, trans-13-22:4 (22:4(n-9)Delta13trans) among pectinids. For this purpose, we extended the analysis of membrane glycerophospholipids FA composition to 13 species of scallops, covering 11 genera and 7 tribes representatives of the three subfamilies Chlamydinae, Palliolinae and Pectininae and the subgroup Aequipecten. In species belonging to the subfamily Pectininae and the Aequipecten subgroup, 22:4(n-9)Delta13trans was found in substantial amounts, but it was absent in other species belonging to the subfamilies Chlamydinae and Palliolinae. Homologous non-methylene-interrupted (NMI) FA, also hypothesized to differ along phylogenetic lines in bivalves, were totally absent or present only in trace amounts in representatives of the Aequipecten subgroup but ranged from 0.3 to 4.5% of the total FA in Pectinidae, Chlamydinae, and Palliolinae subfamilies. The species-specific occurrence of NMI and 22:4(n-9)Delta13trans FA in membrane lipids of pectinids agrees with the most recent phylogenies based on shell morphology and molecular characteristics. We examined the potential timing of the appearance of 22:4(n-9)Delta13trans in pectinids on a geologic time scale.

Prevalence, intensity, and aneuploidy patterns of disseminated neoplasia in cockles (Cerastoderma edule) from Arcachon Bay: seasonal variation and position in sediment.

The present report presents the first evidence of disseminated neoplasia (DN) in cockles Cerastoderma edule from Arcachon Bay (France). Aneuploidy of neoplastic cells allowed the use of flow cytometry (FCM) to diagnose and stage DN. A 1year survey (2007) of the prevalence and intensity (% of aneuploid circulating cells in neoplastic cockles) was conducted. Prevalences ranged from 2.2% (June) to 13.6% (May), and disease intensity ranged from 18.7% (June) to 95.5% (September). These percentages were not correlated with seawater temperature, but rather showed unexplained oscillations over the year. Prevalence and intensity of DN were higher in cockles found at the surface of sediment compared to those buried normally (11.8% vs. 6.7% and 53.0% vs. 40.6%, respectively, p<0.05). DN could thus be one mechanism leading to unexplained presence of cockles at the surface of the sediment in Arcachon Bay. Ploidy characteristics of neoplastic cells were also investigated using FCM, revealing an unusual, broad continuum of ploidy distribution from 1.6 to 9.6n. Ploidy values were not in whole numbers in contrast to the rounded values reported in other studies. Ploidy varied according to DN intensity, with the ploidy distribution of neoplastic cells from lightly-diseased cockles being unimodal (3.7n median). In contrast, highly-diseased cockles showed a bimodal ploidy distribution (3.0n and 4.7n medians). This suggests that, in cockles from Arcachon Bay, mechanisms leading to aneuploidy are complex, developing during disease progression.

Fish oil attenuates adrenergic overactivity without altering glucose metabolism during an oral glucose load in haemodialysis patients.

Haemodialysis patients display an increased cardiac mortality, which may be partly related to increased sympathoadrenal activity and insulin resistance. Fish oil decreases adrenal activation induced by mental stress and has an insulin sensitizing effect in healthy subjects. Whole-body glucose metabolism after oral glucose was studied in eight haemodialysis patients before and after a 3-week oral fish oil supplementation (i.e. EPA + DHA at 1.8 g/d). Plasma glucose fluxes were traced by using [6,6- (2)H2]glucose infusion. Substrate oxidation was determined by using indirect calorimetry. Each patient was studied in the basal state and over the 6 h following absorption of a 1 g/kg glucose load. Energy expenditure in response to glucose re-increased over the last 2 h of the experiment (P < 0.05), which coincided with an increase in plasma catecholamines, especially epinephrine (P < 0.05), strongly suggesting a sympathoadrenal overactivity. Fish oil supplementation blunted both re-increase in thermogenic response and concomitant increase in plasma epinephrine, but not in plasma norepinephrine, over the last 2 h of the experiment. Fish oil did not alter either whole-body glucose metabolism or substrate oxidation. These data show that in haemodialysis patients, fish oil attenuates adrenal overactivity induced by oral glucose but does not modulate whole-body glucose metabolism and insulin sensitivity.

Changes in mitochondrial oxidative capacities during thermal acclimation of rainbow trout Oncorhynchus mykiss: roles of membrane proteins, phospholipids and their fatty acid compositions.

Changes in the properties of mitochondria from oxidative muscle of rainbow trout Oncorhynchus mykiss were examined during warm (5 degrees C to 15 degrees C) acclimation. Trout were studied shortly after the initial thermal change and after 8 weeks acclimation to 15 degrees C. To identify potential mechanisms by which oxidative capacities change, the modifications of phospholipid composition, membrane proteins and functional capacities of red muscle mitochondria were examined. Marked functional changes of isolated muscle mitochondria during warm acclimation of rainbow trout were reflected by a host of modifications in phospholipid composition, but by few shifts in protein components. Shortly after transfer of trout from 5 degrees C to 15 degrees C, the maximal oxidative capacity of mitochondria measured at 15 degrees C increased slightly, but rates at both assay temperatures (5 degrees C and 15 degrees C) decreased markedly after warm acclimation. The increase in capacity in short-term warm exposed trout was most pronounced when rates at 15 degrees C were expressed relative to cytochrome a and c(1) levels. Non-phosphorylating (State 4) rates of oxygen uptake increased with short-term warm exposure before returning to initial levels after warm acclimation. Cytochrome c oxidase (CCO) activity in the mitochondrial preparations decreased with warm acclimation. The thermal sensitivity of the ADP affinity was markedly modified during short-term warm exposure, when the ADP/O ratio increased, but warm acclimation returned these values to those observed initially. ADP affinity increased after warm acclimation. Changes in the mitochondrial content of cytochromes and adenine nucleotide translocase (ANT) could not explain these patterns. On the other hand, changes in the proportions of the lipid classes and in the acyl chain composition of certain phospholipid classes mirror the modifications in functional properties. Short-term exposure to 15 degrees C decreased the ratio of diacylphosphatidylethanolamine/diacylphosphatidylcholine (diacylPE/diacylPC), whereas warm acclimation led to restructuring of fatty acids (FA) and to increases of plasmalogen forms of PE and PC. Modification of overall membrane unsaturation did not appear to be the primary aim of restructuring membrane FA during warm acclimation, as total mitochondrial phospholipids and the major phospholipid classes only showed slight shifts of their acyl composition with warm acclimation. On the other hand, natural lysophosphatidylcholine (LysoPC) showed dramatic changes in FA content, as 16:0 and 18:1n-9 doubled whereas 22:6n-3 decreased from around 50% to 32% in warm acclimated trout. Similarly, in cardiolipin (CL), the levels of 16:0 and 18:1n-7 halved while 18:2n-6 increased to over 20% of the FA with warm acclimation. Given the central role of CL in modulating the activity of CCO, F(0)F(1)-ATPase and ANT, these changes suggest that specific compositional changes in CL are important modulators of mitochondrial capacities. The many structural changes in membrane lipids contrast with the limited modifications of the membrane protein components examined and support the concept of lipid structure modulating mitochondrial capacities.

cis-4,7,10,trans-1 3-22:4 fatty acid distribution in phospholipids of pectinid species Aequipecten opercularis and Pecten maximus.

The distribution of cis-4,7,10,trans-13-docosatetraenoic (c4,7,10,t13-22:4), a peculiar FA previously isolated in the glycerophospholipids of some pectinid bivalves, was investigated in glycerophospholipid classes and subclasses of separated organs (gills, mantle, gonads, and muscle) of the queen scallop Aequipecten opercularis and the king scallop Pecten maximus. Plasmalogen (Pls) and diacyl + alkyl (Ptd) forms of serine, ethanolamine, and choline glycerophospholipids were isolated by HPLC and their FA compositions analyzed by GC-FID. PIs and Ptd forms of serine glycerophospholipids (PlsSer and PtdSer), and to a lesser extend the Pls form of ethanolamine glycerophospholipids (PlsEtn), were found to be specifically enriched with c4,7,10,t13-22:4. This specificity was found to decrease in the tested organs in the following order: gills, mantle, gonad, and muscle. In gills, c4,7,10,t13-22:4 was shown to be the main unsaturated FA of serine glycerophospholipids in both Pls and Ptd forms (23.8 and 19.4 mol%, respectively, for A. opercularis, and 21.0 and 26.2 mol% for P. maximus). These results represent the first comprehensive report on the FA composition of plasmalogen serine subclass isolated from pectinid bivalves. The specific association of the PlsSer with the c4,7,10,t13-22:4 for the two pectinid species can be paralleled to the specific association of the PlsSer with the non-methylene interrupted (NMI) FA and 20:1 (n-11) observed in mussels, clams, and oysters (Kraffe, E., Soudant, P., and Marty, Y. (2004) Fatty Acids of Serine, Ethanolamine and Choline Plasmalogens in Some Marine Bivalves, Lipids 39, 59-66.) This, led us to hypothesize a similar functional significance for c4,7,10,t13-22:4, NMI FA, and 20:1 (n-11) associated with PlsSer subclass of bivalves.

Docosahexaenoic acid- and eicosapentaenoic acid-enriched cardiolipin in the Manila clam Ruditapes philippinarum.

The FA composition of cardiolipin (CL) from the Manila clam Ruditapes philippinarum was investigated in whole body and individual organs. CL was isolated by HPLC and its chemical structure characterized using NMR spectroscopy. Two prominent FA, EPA and DHA, were found in approximately equal proportions, contributing together up to 73 mol% of the total FA. The FA composition of CL is presumed to reflect a specific synthesis pathway independent of diet and of total glycerophospholipid FA composition. To the best of our knowledge, this is the first time that a CL dominated by the two PUFA 22:6n-3 and 20:5n-3 has been characterized and described. This EPA + DHA specificity of the CL in the Manila clam is thought to reflect a functional and structural modification of mitochondrial membranes of this bivalve species compared with scallops, oysters, and mussels that possess a CL dominated by DHA. The FA composition and levels of CL differed little between separated organs, and the large pool of DHA and EPA was found fairly equally distributed in gills, mantle, foot, siphon, and muscle. However, whereas DHA and PUFA levels were most stable between organs, EPA and arachidonic acid were significantly more variable and seemed to be interrelated.

Incorporation and modification of dietary fatty acids in gill polar lipids by two bivalve species Crassostrea gigas and Ruditapes philippinarum.

Two bivalve species Crassostrea gigas and Ruditapes philippinarum were fed eight weeks with three mono-specific algae diets: T-Isochrysis galbana, Tetraselmis suecica, Chaetoceros calcitrans, selected on the basis of their polyunsaturated fatty acid (PUFA) composition. The incorporation and the modification of dietary fatty acids in C. gigas and R. philippinarum gill lipids were analysed and compared. Essential PUFA (20:4n-6, 20:5n-3 and 22:6n-3) and non-methylene interrupted PUFAs (known to be synthesised from monounsaturated precursors) contents of gill polar lipid of both species were greatly influenced by the dietary conditioning. Interestingly, oysters and clams responded differentially to the mono-specific diets. Oysters maintained higher 20:5n-3 level and higher 22:2j/22:i and n-7/n-9 ratio in gill polar lipids than clams. To better discriminate dietary and species influences on the fatty acid composition, a Principal Component Analysis followed by a MANOVA on the two most explicative components was performed. These statistical analyses showed that difference in fatty acid compositions attributable to species were just as significant as the diet inputs. The differences of gill fatty acid compositions between oysters and clams are speculated to result of an intrinsic species characteristic and perhaps of a group characteristic: Fillibranch vs. Eulamellibranch.

Fatty acids of serine, ethanolamine, and choline plasmalogens in some marine bivalves.

The FA composition of glycerophospholipid (GPL) classes and subclasses was investigated in whole animals of three marine bivalve mollusks: the Japanese oyster Crassostrea gigas, the blue mussel Mytilus edulis, and the Manila clam Ruditapes philippinarum. Individual organs (gills, mantle, foot, siphon, and muscle) of the Manila clam also were examined. The PS plasmalogen (PSplsm), PE plasmalogen (PEplsm), and PC plasmalogen (PCplsm) subclasses were isolated by HPLC, and their individual FA compositions were examined using GC. Plasmalogen forms of PS and PE, when compared to their respective diacyl forms, were found to be specifically enriched with non-methylene-interrupted (NMI) FA (7,15-22:2, 7,13-22:2, and their precursors) and 20:1 n-11 FA. Such a clear specific association was not found for PCplsm. Interestingly, this trend was most apparent in PSplsm, and the above FA were found to be, respectively, the predominant PUFA and monounsaturated FA in the PSplsm isolated from the three species. This specificity was maintained in all the analyzed organs of the Manila clam but varied in proportions: The highest level of plasmalogens, NMI FA, and 20:1 n-11 was measured in gills and the lowest was in muscle. These results represent the first comprehensive report on a FA composition of the PSplsm subclass isolated from mollusks. The fact that NMI FA and 20:1 n-11, which are thought to be biosynthesized FA, were mainly associated with aminophospholipid plasmalogens (PE and PS) is likely to have a functional significance in bivalve membranes.

Evidence of a tetradocosahexaenoic cardiolipin in some marine bivalves.

Separation of phospholipid classes in lipid extracts from the scallop Pecten maximus, the Pacific oyster Crassostrea gigas, and the blue mussel Mytilus edulis was conducted using HPLC. An isolated polar lipid fraction was found to contain a very high level of DHA, up to 80 mol% of the total FA. MS with electrospray ionization in the positive-ion mode, tandem MS (MS-MS) and multidimensional NMR spectroscopy were used to analyze the detailed chemical structure of this polar lipid fraction. The isolated fraction contained exclusively cardiolipin (CL) molecules, predominantly in a form with four docosahexaenoyl chains (Do4CL). To the best of our knowledge, this is the first time that such a CL form has been analytically characterized and described in these three bivalve species. This tetradocosahexaenoic CL is presumed to reflect a specific adaptation in bivalves that enhances the structural and functional mechanisms of biomembranes in response to variations in environmental conditions (temperature, salinity, emersion).