Fatty acid metabolism in marine fish: Low activity of fatty acyl Δ5 desaturation in gilthead sea bream ( Sparus aurata ) cells

Marine fish are known to have an absolute dietary requirement for C20 and C22 highly unsaturated fatty acids. Previous studies using cultured cell lines indicated that underlying this requirement in marine fish was either a deficiency in fatty acyl Δ5 desaturase or C18-20 elongase activity. Recently, Ghioni et al. (Biochim. Biophys. Acta, 1437, 170-181, 1999) presented evidence that in turbot cells there was low activity of C18-20 elongase whereas Δ5 desaturase had high activity. In the present study, the fatty acid desaturase/elongase pathway was investigated in a cell line (SAF-1) from another carnivorous marine fish, sea bream. The metabolic conversions of a range of radiolabelled polyunsaturated fatty acids that comprised the direct substrates for Δ6 desaturase ([1-14C]18:2n-6 and [1-14C]18:3n-3), C18-20 elongase ([U-14C]18:4n-3), Δ5 desaturase ([1-14C]20:3n-6 and [U-14C]20:4n-3) and C20-22 elongase ([1-14C]20:4n-6 and [1-14C]20:5n-3) were utilized. The results showed that fatty acyl Δ6 desaturase in SAF-1 cells was highly active and there was substantial C18-20 elongase and C20-22 elongase activities. A deficiency in the desaturation/elongation pathway was clearly identified at the level of the fatty acyl Δ5 desaturase which was very low, particularly with 20:4n-3 as substrate. In comparison, the apparent activities of Δ6 desaturase, C18-20 elongase and C20-22 elongase were approximately 94-fold, 27-fold and 16-fold greater than that for Δ5 desaturase towards their respective n-3 polyunsaturated fatty acid substrates. The evidence obtained in the SAF-1 cell line is consistent with the dietary requirement for C20 and C22 highly unsaturated fatty acids in the marine fish, the sea bream, being primarily due to a deficiency in fatty acid Δ5 desaturase activity

Effects of dietary vegetable oil on atlantic salmon hepatocyte fatty acid desaturation and liver fatty acid compositions

Fatty acyl desaturase activities, involved in the conversion of the C18 EFA, 18:2n-6 and 18:3n-3, to the highly unsaturated fatty acids (HUFA) 20:4n-6, 20:5n-3 and 22:6n-3, are known to be under nutritional regulation. Specifically, the activity of the desaturation/elongation pathway is depressed when animals, including fish, are fed fish oils rich in n-3HUFA compared to animals fed vegetable oils rich in C18 EFA. The primary aims of the present study were a) to establish the relative importance of product inhibition (n-3HUFA) versus increased substrate concentration (C18 EFA) and, b) to determine whether 18:2n-6 and 18:3n-3 differ in their effects, on the hepatic fatty acyl desaturation/elongation pathway in Atlantic salmon (Salmo salar). Smolts were fed ten experimental diets containing blends of two vegetable oils, linseed (LO) and rapeseed oil (RO), and fish oil (FO) in a triangular mixture design for 50 weeks. Fish were sampled after 32 and 50 weeks, lipid and fatty acid composition of liver determined, fatty acyl desaturation/elongation activity estimated in hepatocytes using [1-14C]18:3n-3 as substrate, and the data subjected to regression analyses. Dietary 18:2n-6 was positively correlated, and n-3HUFA negatively correlated, with lipid content of liver. Dietary 20:5n-3 and 22:6n-3 were positively correlated with liver fatty acids with a slope greater than unity suggesting relative retention and deposition of these HUFA. In contrast, dietary 18:2n-6 and 18:3n-3 were positively correlated with liver fatty acids with a slope of less than unity suggesting metabolism via β-oxidation and/or desaturation/elongation. Consistent with this, fatty acyl desaturation/elongation in hepatocytes was significantly increased by feeding diets containing vegetable oils. Dietary 20:5n-3 and 22:6n-3 levels were negatively correlated with hepatocyte fatty acyl desaturation. At 32 weeks, 18:2n-6 but not 18:3n-3, was positively correlated with hepatocyte fatty acyl desaturation activity whereas the reverse was true at 50 weeks. The data indicate that both feedback inhibition through increased n-3HUFA and decreased C18 fatty acyl substrate concentration are probably important in determining hepatocyte fatty acyl desaturation activities, and that 18:2n-6 and 18:3n-3 may differ in their effects on this pathway

Egg quality determinants in cod (Gadus morhua L.): egg performance and lipids in eggs from farmed and wild broodstock

Lipids and essential fatty acids, particularly the highly unsaturated fatty acids, 20:5n-3 (eicosapentaenoic acid; EPA), 22:6n-3 (docosahexaenoic acid; DHA) and 20:4n-6 (arachidonic acid, AA) have been shown to be crucial determinants of marine fish reproduction directly affecting fecundity, egg quality, hatching success, larval malformation and pigmentation. In Atlantic cod (Gadus morhua L.) culture, eggs from farmed broodstock can have much lower fertilisation and hatching rates than eggs from wild broodstock. The present study aimed to test the hypothesis that potential quality and performance differences between eggs from different cod broodstock would be reflected in differences in lipid and fatty acid composition. Thus eggs were obtained from three broodstock, farmed, wild/fed and wild/unfed, and lipid content, lipid class composition, fatty acid composition and pigment content were determined and related to performance parameters including fertilisation rate, symmetry of cell division and survival to hatching. Eggs from farmed broodstock showed significantly lower fertilisation rates, cell symmetry and survival to hatching rates than eggs from wild broodstock. There were no differences in total lipid content or the proportions of the major lipid classes between eggs from the different broodstock. However, eggs from farmed broodstock were characterised by having significantly lower levels of some quantitatively minor phospholipid classes, particularly phosphatidylinositol. There were no differences between eggs from farmed and wild broodstock in the proportions of saturated, monounsaturated and total polyunsaturated fatty acids. The DHA content was also similar. However, eggs from farmed broodstock had significantly lower levels of AA, and consequently significantly higher EPA/AA ratios than eggs from wild broodstock. Total pigment and astaxanthin levels were significantly higher in eggs from wild broodstock. Therefore, the levels of AA and phosphatidylinositol, the predominant AA-containing lipid class, and egg pigment content were positively related to egg quality or performance parameters such as fertilisation and hatching success rates, and cell symmetry

Highly unsaturated fatty acid synthesis in marine fish: Cloning, functional characterization, and nutritional regulation of fatty acyl delta6 desaturase of Atlantic cod (Gadus morhua L.)

Fish contain high levels of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. Biosynthesis of HUFA requires enzyme-mediated desaturation of fatty acids. Here we report cloning and functional characterisation of a ∆6 fatty acyl desaturase of Atlantic cod (Gadus morhua), and describe its tissue expression and nutritional regulation. PCR primers were designed based on the sequences of conserved motifs in available fish desaturases and used to isolate a cDNA fragment from liver of cod. The full-length cDNA was obtained by Rapid Amplification of cDNA Ends (RACE). The cDNA for the putative fatty acyl desaturase was shown to comprise 1980bp which included a 5’-UTR of 261bp and a 3’-UTR of 375bp. Sequencing revealed that the cDNA included an ORF of 1344 bp that specified a protein of 447 amino acids. The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the haem-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. The cDNA displayed Δ6 desaturase activity in a heterologous yeast expression system. Quantitative real time PCR assay of gene expression in cod showed that the ∆6 desaturase gene, was highly expressed in brain, relatively highly expressed in liver, kidney, intestine, red muscle and gill, and expressed at much lower levels in white muscle, spleen and heart. In contrast, the abundance of a cod fatty acyl elongase transcript was high in brain and gill, with intermediate levels in kidney, spleen, intestine and heart, and relatively low expression in liver. The expression of the Δ6 desaturase gene and the PUFA elongase gene may be under a degree of nutritional regulation, with levels being marginally increased in livers and intestine of fish fed a vegetable oil blend by comparison with levels in fish fed fish oil. However, this was not reflected in increased Δ6 desaturase activity in hepatocytes or enterocytes, which showed very little highly unsaturated fatty acid biosynthesis activity irrespective of diet. The study described has demonstrated that Atlantic cod express a fatty acid desaturase gene with functional Δ6 activity in a yeast expression system. This is consistent with an established hypothesis that the poor ability of marine fish to synthesise HUFA is not due to lack of a Δ6 desaturase, but rather to deficiencies in other parts of the biosynthetic pathway. However, further studies are required to determine why the Δ6 desaturase appears to be barely functional in cod under the conditions tested

Cultured fish cells metabolize octadecapentaenoic acid (all-cis delta3,6,9,12,15–18∶5) to octadecatetraenoic acid (all-cis delta6,9,12,15–18∶4) via its 2-trans intermediate (trans delta2, all-cis delta6,9,12,15–18∶5)

Octadecapentaenoic acid (all-cis Δ3,6,9,12,15-18:5; 18:5n-3) is an unusual fatty acid found in marine dinophytes, haptophytes and prasinophytes. It is not present at higher trophic levels in the marine food web but its metabolism by animals ingesting algae is unknown. Here we studied the metabolism of 18:5n-3 in cell lines derived from turbot (Scophthalmus maximus), gilthead sea bream (Sparus aurata) and Atlantic salmon (Salmo salar). Cells were incubated in the presence of approximately 1 μM [U-14C] 18:5n-3 methyl ester or [U-14C] 18:4n-3 (octadecatetraenoic acid; all-cis Δ6,9,12,15-18:4) methyl ester, both derived from the alga Isochrysis galbana grown in H14CO3, and also with 25 μM unlabelled 18:5n-3 or 18:4n-3. Cells were also incubated with 25 μM trans Δ2, all-cis Δ6,9,12,15-18:5 (2-trans 18:5n-3) produced by alkaline isomerization of 18:5n-3 chemically synthesized from docosahexaenoic acid (all-cis Δ4,7,10,13,16,19-22:6; 22:6n-3). Radio- and mass analyses of total fatty acids extracted from cells incubated with 18:5n-3 were consistent with this fatty acid being rapidly metabolized to 18:4n-3 which was then elongated and further desaturated to eicosatetraenoic acid (all-cis Δ8,11,14,17,19-20:4; 20:4n-3) and eicosapentaenoic acid (all-cis Δ5,8,11,14,17-20:5; 20:5n-3). Similar mass increases of 18:4n-3 and its elongation and further desaturation products occurred in cells incubated with 18:5n-3 or 2-trans 18:5n-3. We conclude that 18:5n-3 is readily converted biochemically to 18:4n-3 via a 2-trans 18:5n-3 intermediate generated by a Δ3,Δ2-enoyl-CoA-isomerase acting on 18:5n-3. Thus, 2-trans 18:5n-3 is implicated as a common intermediate in the β-oxidation of both 18:5n-3 and 18:4n-3

Influence of Dietary Oil Content and Conjugated Linoleic Acid (CLA) on Lipid Metabolism Enzyme Activities and Gene Expression in Tissues of Atlantic Salmon (Salmo salar L.)

The overall objective is to test the hypothesis that conjugated linoleic acid (CLA) has beneficial effects in Atlantic salmon through affecting lipid and fatty acid metabolism. The specific aims of the present study were to determine the effects of CLA on some key pathways of fatty acid metabolism including fatty acid oxidation and highly unsaturated fatty acid (HUFA) synthesis. Salmon smolts were fed diets containing two levels of fish oil (low, ~18% and high, ~34%) containing three levels of CLA (a 1:1 mixture of 9-cis,trans-11 and trans-10,cis-12 at 0, 1 and 2% of diet) for 3 months. The effects of dietary CLA on HUFA synthesis and β-oxidation were measured and the expression of key genes in the fatty acid oxidation and HUFA synthesis pathways, and potentially important transcription factors, peroxisome proliferators activated receptors (PPARs), determined in selected tissues. Liver HUFA synthesis and desaturase gene expression was increased by dietary CLA and decreased by high dietary oil content. Carnitine palmitoyltransferase-I (CPT-I) activity and gene expression were generally increased by CLA in muscle tissues although dietary oil content had relatively little effect. In general CPT-I activity or gene expression was not correlated with β-oxidation. Dietary CLA tended to increase PPARα and β gene expression in both liver and muscle tissues, and PPARγ in liver. In summary, gene expression and activity of the fatty acid pathways were altered in response to dietary CLA and/or oil content, with data suggesting that PPARs are also regulated in response to CLA. Correlations were observed between dietary CLA, liver HUFA synthesis and desaturase gene expression, and liver PPARα expression, and also between dietary CLA, CPT-I expression and activity, and PPARα expression in muscle tissues. In conclusion, this study suggests that dietary CLA has effects on fatty acid metabolism in Atlantic salmon and on PPAR transcription factors. However, further work is required to assess the potential of CLA as a dietary supplement, and the role of PPARs in the regulation of lipid metabolism in fish

A Vibrational Study by Raman Spectroscopy of Some Dinuclear Gold Ylide Complexes

A vibrational study of the dinuclear gold ylide complexes [Au(CH2)2PPh2]2 and [Au(CH2)2PPh2]2X2 (X = Cl, Br or I) has been undertaken by Raman spectroscopy. The non-bonding AuAu interaction in the AuI dimer, [Au(CH2)2PPh2)2, at 64 cm−1 shifts to higher wavenumber in the single-bonded AuII halogen complexes, with bands at 162, 132 and 103 cm−1 for X = Cl, Br and I, respectively, being assigned to ν(AuAu). The Au-X vibration was also identified. The general trends in AuAu and Au-X stretching vibrations with changing halogen are compared with those for other dinuclear metal-metal bonded complexes, with a metal-metal bond order of one, and with those for mononuclear gold-halogen complexes