Synthesis of phosphatidylcholine through phosphatidylethanolamine N-methylation in tissues of the mussel Mytilus galloprovincialis.

We previously demonstrated the importance of upregulation of phosphatidylethanolamine N-methylation pathway in euryhaline fish and crustaceans facing hyperosmotic conditions. In marine molluscs phosphatidylcholine synthesis through N-methylation of phosphatidylethanolamine has not been described until now. In vivo labeling of the mussel Mytilus galloprovincialis with [1-(3)H]-ethanolamine showed that the digestive gland is the tissue expressing the highest incorporation into lipids. A sustained increase in lipid labeling was observed up to 72 h following label injection with 79-92% of radioactivity concentrated into phosphatidylethanolamine and phosphatidylcholine. A direct correlation (r = 0.47, p < 0.01) between the specific radioactivities of phosphatidylcholine in plasma and the digestive gland was observed. Moreover, the phosphatidylcholine fatty acid compositions of plasma and the digestive gland were similar but differed from those of phosphatidylcholine purified from other tissues. In vitro incubation of tissues with [1-(3)H]-ethanolamine or L-[3-(3)H]-serine showed that a significant labeling of the choline moiety of phosphatidylcholine was observed in the digestive gland and hemocytes. Pulse-chase experiments with [1-(3)H]-ethanolamine also demonstrated that hemocytes are exchanging the newly formed phospholipids with plasma. Finally, phosphatidylethanolamine N-methyltransferase assays demonstrated salinity-dependent activities in the digestive gland and hemocytes. We conclude that in M. galloprovincialis an active phosphatidylcholine synthesis through N-methylation of phosphatidylethanolamine occurs in the digestive gland and hemocytes and that this newly formed phosphatidylcholine is partly exchanged with plasma.

Salinity regulates N-methylation of phosphatidylethanolamine in euryhaline crustaceans hepatopancreas and exchange of newly-formed phosphatidylcholine with hemolymph.

Phosphatidylcholine (PC), the main phospholipid in eukaryotes, is synthesized via two different routes, the phosphatidylethanolamine N-methyl transferase (PEMT) and the CDP-choline pathways. We previously showed in euryhaline fish that salinity impacts the relative contribution of the two pathways for PC biosynthesis, with PEMT pathway being activated in the liver of sea water (SW)-adapted animals. To address the occurrence of such phenomenon in other animals we performed in vivo metabolic studies in two crustacean species: the Chinese crab (Eriocheir sinensis) and the green crab (Carcinus maenas). In both species, the levels of PC and phosphatidylethanolamine in hepatopancreas and hemolymph were not modified by SW-adaptation. In E. sinensis, SW-adaptation activated PC labeling from L-(U-(14)C)-serine in the hepatopancreas and resulted in an increased ratio of PC specific activities between hemolymph and hepatopancreas. In C. maenas, incorporation of L-(3-(3)H)-serine and L-(2-(14)C)-ethanolamine into PC of hepatopancreas was strongly inhibited after acclimation to fresh water (FW). The results show that PC synthesis via the PEMT pathway and its subsequent release into hemolymph are both activated in SW- compared to FW-adapted animals. SW-adaptation also resulted in increased tissue concentrations of betaine and labeling from L-(U-(14)C)-serine, suggesting that the PEMT-derived PC is used for the synthesis of organic osmolytes. The physiological relevance of these observations is discussed.

Interactions of free copper (II) ions alone or in complex with iron (III) ions with erythrocytes of marine fish Dicentrarchus labrax.

As a consequence of human activity, various toxicants - especially metal ions - enter aquatic ecosystems and many fish are exposed to considerable levels. As the free ion and in some complexes, there is no doubt that copper promotes damage to cellular molecules and structures through radical formation. Therefore, we have investigated the influence of copper uptake by the red blood of the sea bass (Dicentrarchus labrax), and its oxidative action and effects on cells in the presence of complexed and uncomplexed Fe3+ ions. Erythrocytes were exposed to various concentrations of CuSO4, Fe(NO3)3, and K3Fe(CN)6 for up to 5h, and the effects of copper ions alone and in the combination with iron determined. The results show that inside the cells cupric ion interacts with hemoglobin, causing methemoglobin formation by direct electron transfer from heme Fe2+ to Cu2+. Potassium ferricyanide as a source of complexed iron decreases Met-Hb formation induced by copper ions unlike Fe(NO3)3. We also found that incubation of fish erythrocytes with copper increased hemolysis of cells. But complexed and uncomplexed iron protected the effect of copper. CuSO4 increased the level of lipid peroxidation and a protective effect on complexed iron was observed. Incubation of erythrocytes with copper ions resulted in the loss of a considerable part of thiol content at 10 and 20 microM. This effect was decreased by potassium ferricyanide and Fe(NO3)3 only after 1 and 3h of incubation. The level of nuclear DNA damage assayed by comet assay showed that 20 microM CuSO4 as well as 20 microM Fe(NO3)3 and 10 mM K3Fe(CN)6 induce single- and double-strand breaks. The lower changes were observed after the exposure of cells to K3Fe(CN)6. The data suggest that complexed iron can act protectively against copper ions in contrast to Fe(NO3)3.

Correlation between changes in blood fatty acid composition and visual sustained attention performance in children with inattention: effect of dietary n-3 fatty acids containing phospholipids.

BACKGROUND: Increasing evidence supports n-3 fatty acid (FA) supplementation for patients with psychiatric disorders, such as attention deficit hyperactivity disorder. However, the exact metabolic fate of dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on different glyceride carriers remains unclear. OBJECTIVE: We investigated whether conjugation of EPA and DHA to phospholipid (PL-n-3) or to triacylglycerol (fish oil; FO) affects their incorporation in blood compartments and influences executive functioning. DESIGN: Children aged 8-13 y with impaired visual sustained attention performance received placebo, 250 mg/d EPA + DHA esterified to PL-n-3 (300 mg/d phosphatidylserine), or FO for 3 mo in a randomized double-blind manner. Main outcome measures included plasma and erythrocyte FA profile and continuous performance test results (Test of Variables of Attention; TOVA). RESULTS: Sixty of the 83 children enrolled completed the interventions (n = 18-21 per group). There was an enrichment of EPA (1.5-2.2-fold), docosapentaenoic acid (DPA; 1.2-fold), and DHA (1.3-fold) in the PL fraction in the plasma of FO- and PL-n-3-fed children. In erythrocytes, only PL-n-3 resulted in a significant reduction (approximately 30%) of very-long-chain saturated FAs (C20-24) and in an increase (1.2- and 2.2-fold, respectively) in linoleic acid and DPA. Total TOVA scores increased in the PL-n-3 (mean +/- SD: 3.35 +/- 1.86) and FO (1.72 +/- 1.67) groups but not in the placebo group (-0.42 +/- 2.51) (PL-n-3 > FO > placebo; P < 0.001). A significant correlation between the alterations in FAs and increased TOVA scores mainly occurred in the PL-n-3 group. CONCLUSION: Consumption of EPA+DHA esterified to different carriers had different effects on the incorporation of these FAs in blood fractions and on the visual sustained attention performance in children. This trial was registered at clinicaltrials.gov as NCT00382616.

Changes in the fatty acid composition of phospholipids in tissues of farmed sea bass (Dicentrarchus labrax) during an annual cycle. Roles of environmental temperature and salinity.

We quantified seasonal effects on fatty acid composition of tissue phospholipids in farmed sea bass. Major changes in percent phosphatidylethanolamine and phosphatidylcholine were observed in all tissues between February and March, and the phosphatidylcholine/phosphatidylethanolamine ratio was drastically reduced at this time. Different changes in the fatty acid composition of total phospholipids were observed in all tissues examined. Fish fed all year on the same commercial diet showed a significant correlation between water salinity and percentage of 22:6n-3 in muscle, liver and gill phospholipids, but no correlation was found between percent 22:6n-3 of phospholipids and water temperature. In each tissue, we observed annual variation in the 20:5n-3/20:4n-6 ratio in phospholipids, but maximum and minimum values occurred at different times in each organ. From these results, we conclude that salinity can play a significant role in modulating the activities of enzymes acting on lipid metabolism during their natural circannual cycles.

Palmitate and oleate metabolism of rainbow trout in vivo.

The turnover rates of palmitate and oleate were measured in vivo by continuous infusion of 1-[14C]palmitate and 9,10-[3H]oleate in rainbow trout. Our goals were: (1) to quantify the incorporation of a saturated and of a monounsaturated fatty acid into other classes of plasma lipids (neutral lipids, NL, and phospholipids, PL); and (2) to determine whether they could both be used as tracers to quantify fluxes of total non-esterified fatty acids (NEFA). We found that both acids play very different physiological roles because palmitate is preferentially channeled towards plasma PL, whereas oleate is mainly incorporated in circulating NL. Consequently, palmitate is predominantly involved in membrane PL turnover and oleate in the metabolism of circulating NL that may be used to shuttle oxidative fuel in teleosts. Despite this striking difference in their metabolism, palmitate and oleate have flux rates that are proportional to their relative abundance in plasma NEFA (i.e. they have the same fractional turnover rate). They can therefore both be used as reliable tracers to quantify the kinetics of total NEFA.