A vertebrate fatty acid desaturase with Delta 5 and Delta 6 activities.

Delta5 and Delta6 fatty acid desaturases are critical enzymes in the pathways for the biosynthesis of the polyunsaturated fatty acids arachidonic, eicosapentaenoic, and docosahexaenoic acids. They are encoded by distinct genes in mammals and Caenorhabditis elegans. This paper describes a cDNA isolated from zebrafish (Danio rerio) with high similarity to mammalian Delta6 desaturase genes. The 1,590-bp sequence specifies a protein that, in common with other fatty acid desaturases, contains an N-terminal cytochrome b(5) domain and three histidine boxes, believed to be involved in catalysis. When the zebrafish cDNA was expressed in Saccharomyces cerevisiae it conferred the ability to convert linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) to their corresponding Delta6 desaturated products, 18:3n-6 and 18:4n-3. However, in addition it conferred on the yeast the ability to convert di-homo-gamma-linoleic acid (20:3n-6) and eicosatetraenoic acid (20:4n-3) to arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3), respectively, indicating that the zebrafish gene encodes an enzyme having both Delta5 and Delta6 desaturase activity. The zebrafish Delta5/Delta6 desaturase may represent a component of a prototypic vertebrate polyunsaturated fatty acids biosynthesis pathway.

Replacement of fish oil with rapeseed oil in diets of Atlantic salmon (Salmo salar) affects tissue lipid compositions and hepatocyte fatty acid metabolism.

Duplicate groups of Atlantic salmon post-smolts were fed five practical-type diets in which the added lipid was 100% fish oil [FO; 0% rapeseed oil (0% RO)], 90% FO + 10% RO (10% RO), 75% FO + 25% RO (25% RO), 50% FO + 50% RO (50% RO) or 100% RO, for a period of 17 wk. There were no effects of diet on growth rate or feed conversion nor were any histopathological lesions found in liver, heart, muscle or kidney. The greatest accumulation of muscle lipid was in fish fed 0% RO, which corresponded to significantly lower muscle protein in this group. The highest lipid levels in liver were found in fish fed 100% RO. Fatty acid compositions of muscle lipid correlated with RO inclusion in that the proportions of 18:1(n-9), 18:2(n-6) and 18:3(n-3) all increased with increasing dietary RO (r = 0.98-1.00, P < 0.013). The concentrations of eicosapentaenoic acid [20:5(n-3)] and docosahexaenoic acid [22:6(n-3)] in muscle lipid were significantly reduced (P < 0.05), along with total saturated fatty acids, with increasing dietary RO. Diet-induced changes in liver fatty acid compositions were broadly similar to those in muscle. Hepatic fatty acid desaturation and elongation activities, measured using [1-(14)C] 18:3(n-3), were increased with increasing dietary RO. Limited supplies of marine fish oils require that substitutes be found if growth in aquaculture is to be maintained such that fish health and product quality are not compromised. Thus, RO can be used successfully as a substitute for fish oil in the culture of Atlantic salmon in sea water although at levels of RO >50% of dietary lipid, substantial reductions occur in muscle 20:5(n-3), 22:6(n-3) and the (n-3)/(n-6) polyunsaturated fatty acid (PUFA) ratio, which will result in reduced availability of the (n-3) highly unsaturated fatty acids that are beneficial for human health.

Red blood cell fatty acid compositions in a patient with autistic spectrum disorder: a characteristic abnormality in neurodevelopmental disorders?

The fatty acid compositions of red blood cell (RBC) phospholipids from a patient with autistic spectrum disorder (ASD) had reduced percentages of highly unsaturated fatty acids (HUFA) compared to control samples. The percentage of HUFA in the RBC from the autistic patient was dramatically reduced (up to 70%) when the sample was stored for 6 weeks at -20 degrees C. However, only minor HUFA reductions were recorded in control samples stored similarly, or when the autistic sample was stored at -80 degrees C. A similar instability in RBC HUFA compositions upon storage at -20 degrees C has been recorded in schizophrenic patients. In a number of other neurodevelopmental conditions, including attention deficit hyperactivity disorder (ADHD) and dyslexia, reduced concentrations of RBC HUFA have been recorded. The extent and nature of these aberrations require further assessment to determine a possible common biochemical origin of neurodevelopmental disorders in general. To facilitate this, a large scale assessment of RBC fatty acid compositions in patients with ASD, and related disorders, should be performed as a matter of urgency. Supplementing cells in culture with the tryptophan metabolite indole acrylic acid (IAA) affected the levels of cellular HUFA and prostaglandin production. Indole acroyl glycine (IAG), a metabolite of IAA excreted in urine, is found in high concentrations in patients with neurodevelopmental disorders including ASD, ADHD, dyslexia, Asperger's syndrome and obsessive compulsive disorder.

Depletion of alpha-tocopherol and astaxanthin in Atlantic salmon (Salmo salar) affects autoxidative defense and fatty acid metabolism.

Duplicate groups of Atlantic salmon post-smolts were fed four purified diets supplemented with both vitamin E and the carotenoid astaxanthin (Ax) (+E, +Ax), or supplemented with either vitamin E or Ax (-E, +Ax and +E, -Ax) or deficient in both vitamin E and Ax (-E, -Ax) for 22 wk. There were no effects of diet on growth rate, but an extensive lipoid liver degenerative lesion was observed in 15% of fish fed diets deficient in vitamin E. Tissue vitamin E concentrations varied in accordance with dietary vitamin E in liver, muscle, heart, plasma, brain and eye; levels were reduced to approximately 3% in liver but only to 40% in eye of fish fed diets deficient in vitamin E compared with those fed diets supplemented with vitamin E. An interactive sparing of Ax supplementation on tissue vitamin E concentration was observed, but only in brain. Dietary deficiency of both vitamin E and Ax significantly increased the recovery of desaturated and elongated products of both [1-(14)C] 18:3(n-3) and [1-(14)C] 20:5(n-3) in isolated hepatocytes, suggesting that conversion of fatty acids to their long-chain highly unsaturated products can be stimulated by a deficiency of lipid-soluble antioxidants. The antioxidant synergism of vitamin E and Ax was supported by their ability to reduce malondialdehyde formation in an in vitro stimulation of microsomal lipid peroxidation and to reduce plasma levels of 8-isoprostane. The results of this study suggest that both vitamin E and the carotenoid Ax have antioxidant functions in Atlantic salmon.

Low C18 to C20 fatty acid elongase activity and limited conversion of stearidonic acid, 18:4(n-3), to eicosapentaenoic acid, 20:5(n-3), in a cell line from the turbot, Scophthalmus maximus.

The TF cell line, derived from a top predatory, carnivorous marine teleost, the turbot (Scophthalmus maximus), is known to have a limited conversion of C18 to C20 polyunsaturated fatty acids (PUFA). To illuminate the underlying processes, we studied the conversions of stearidonic acid, 18:4(n-3), and its elongation product, 20:4(n-3), in TF cells and also in a cell line, AS, derived from Atlantic salmon (Salmo salar), by adding unlabelled (25 microM), U-14C (1 microM) or deuterated (d5; 25 microM) fatty acids. Stearidonic acid, 18:4(n-3), was metabolised to 20:5(n-3) in both cells lines, but more so in AS than in TF cells. Delta5 desaturation was more active in TF cells than in AS cells, whereas C18 to C20 elongation was much reduced in TF as compared to AS cells. Only small amounts of docosahexaenoic acid (22:6(n-3)) were produced by both cell lines, although there was significant production of 22:5(n-3) in both cultures, especially when 20:4(n-3) was supplemented. We conclude that limited elongation of C18 to C20 fatty acids rather than limited fatty acyl Delta5 desaturation accounts for the limited rate of conversion of 18:3(n-3) to 20:5(n-3) in the turbot cell line, as compared to the Atlantic salmon cell line. The results can account for the known differences in conversions of C18 to C20 PUFA by the turbot and the Atlantic salmon in vivo.

Growth, mortality, tissue histopathology and fatty acid compositions, eicosanoid production and response to stress, in juvenile turbot fed diets rich in gamma-linolenic acid in combination with eicosapentaenoic acid or docosahexaenoic acid.

Three diets containing either borage oil (BO) and southern hemisphere fish oil Marinol (MO), or BO and tuna orbital oil (TO), or a northern hemisphere fish oil (FO) were fed to duplicate groups of turbot (Scophthalmus maximus) of initial mean weight 1.2 g for a period of 12 weeks. The BO/MO and BO/TO diets were enriched in gamma-linolenic (18:3n-6, GLA) and eicosapentaenoic (20:5n-3, EPA) acids, and GLA and docosahexaenoic acid (22:6n-3, DHA), respectively. No differences were observed in final weights or growth rates, either between duplicate tanks or between dietary treatments. Half of the FO-fed fish sampled showed a histopathological lesion indicative of lipoid liver degeneration while the other treatments only showed a slight incidence of the same pathology. The fatty acid compositions of carcass and tissues broadly reflected the dietary input. In general, fish fed the BO/MO diet had increased levels of 18:2n-6, 18:3n-6, 20:3n-6 and 20:5n-3, but a lower level of 22:6n-3, compared to fish fed FO. In fish fed the BO/TO diet, levels of 18:2n-6, 18:3n-6, 20:3n-6 and 20:4n-6 were increased while levels of 20:5n-3 and 22:5n-3 were reduced, compared to fish fed FO. Concentrations of thromboxanes B (TXB) and leukotrienes B (LTB), derived from 20:4n-6 and 20:5n-3, were measured in plasma and stimulated blood cells. Levels of TXB2 were greatest in fish fed the BO/TO diet compared to both other treatments, while LTB4 was decreased in fish fed the BO/MO diet compared to both other treatments. In a stress test which involved anaesthesia followed by measurement of recovery times, fish fed the BO/MO diet had significantly lower recovery times compared to fish fed the FO diet.

Effects of dietary gamma-linolenic acid-rich borage oil combined with marine fish oils on tissue phospholipid fatty acid composition and production of prostaglandins E and F of the 1-, 2- and 3-series in a marine fish deficient in delta5 fatty acyl desaturase.

The effects of gamma-linolenic acid-rich borage oil (BO), in combination with different marine oils, namely an eicosapentaenoic acid (EPA) rich oil (MO) or a DHA-rich oil (TO), on tissue fatty acid composition and prostaglandin production were investigated in turbot, a species which lacks appreciable delta5 fatty acyl desaturase activity. The juvenile turbot grew well on the experimental diets and there were no significant differences in final weights between dietary treatments. Irrespective of the marine oil component, both the BO-containing diets increased tissue phospholipid levels of 18:2n-6 and 18:3n-6, and their respective elongation products, 20:2n-6 and 20:3n-6, compared to fish fed a control diet containing a standard Northern hemisphere fish oil. Both the BO-containing diets increased the production of 1-series prostaglandins (PG), this being observed across all tissues investigated with PGF and especially PGE. The BO/MO diet also reduced 20:4n-6 in tissue phospholipids without affecting 20:5n-3, whereas the BO/TO combination decreased 20:5n-3 but increased 20:4n-6. The production of 2-series and 3-series PGs was also altered by the dietary treatments but the changes were less dependent upon the tissue levels of their respective precursor fatty acids, 20:4n-6 and 20:5n-3. The BO-containing diets had very significant effects on gross fatty acid compositions of the phospholipids including increased proportions of saturated fatty acids and n-6 polyunsaturated fatty acids (PUFA) and decreased proportions of monounsaturated fatty acids and n-3 PUFA. Overall, this study shows that eicosanoid production in turbot tissues can be influenced by dietary fatty acids, not only by changes in the absolute and relative levels of specific eicosanoid precursor PUFA in tissue phospholipids, but also by general effects on membrane composition, structure and function induced by gross fatty acid compositional changes.

Fish oils and human diet.

Trends in global fish catches are described together with fish landings and fish consumption in the UK. The importance of n-6 and n-3 polyunsaturated fatty acids as essential constituents of human diets is considered and the role of oily fish as a dietary source of the long-chain n-3 polyunsaturates, docosahexaenoic acid and eicosapentaenoic acid, is emphasized. The origin of n-3 polyunsaturates in, the marine phytoplankton and their transmission via zooplankton to fish is described as a means of understanding the composition of different fish body oils. The ease with which the fatty acid composition of fish body oils can be manipulated by altering the fatty acid composition of their feeds is emphasized and the dietary requirements of marine and freshwater fish for n-3 and n-6 polyunsaturates considered. Farming fish on diets containing principally fish meal and fish oil, as used in salmon production in Scotland, generates a high quality product with levels of long-chain n-3 polyunsaturates equalling or exceeding those of wild fish. Farming fish on high quality marine oils rich in docosahexaenoic and eicosapentaenoic acids is an efficient means of delivering these essential nutrients in human diets and also efficiently exploiting a strictly limited marine bioresource.

Fatty acid composition, eicosanoid production and permeability in skin tissues of rainbow trout (Oncorhynchus mykiss) fed a control or an essential fatty acid deficient diet.

Rainbow trout (Oncorhynchus mykiss) were fed either a control diet containing fish oil or an essential fatty acid (EFA) deficient diet containing only hydrogenated coconut oil and palmitic acid as lipid source (93.4% saturated fatty acids) for 14 weeks and the fatty acid compositions of individual phospholipid classes from skin and opercular membrane (OM) determined. The permeability of skin and OM to water and the production of eicosanoids in skin and gills challenged with the Ca2+ ionophore A23187 were also measured. Phospholipid (PL) fatty acid compositions were substantially modified in EFA-deficient fish, with increased saturated fatty acids and decreased polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) and eicosapentaenoic acid (EPA), while docosahexaenoic acid (DHA) was largely retained. The onset of EFA deficiency was shown by the appearance of n-9 PUFA, particularly 20:3n-9. The main effects of EFA deficiency on phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were to increase saturated fatty acids and monoenes, especially 16:1 and 18:1, and to decrease EPA and DHA. The content of DHA in phosphatidylserine (PS) was high in control animals (40% in skin and 35% in opercular membrane) and was mostly retained in EFA deficient animals. Arachidonic acid (AA) was the most abundant PUFA esterified to phosphatidylinositol (PI) and was significantly reduced in EFA deficient animals (from 31% to 13% in skin), where a large amount of 20:3n-9 (9% in skin) was also present. Influxes and effluxes of water through skin and opercular membrane were measured in vitro. No differences were detected between rainbow trout fed the control or the EFA deficient diet. 12-Hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHE) could not be detected in skin from control or EFA deficient fish. There was no difference between control and EFA deficient trout in the levels of leukotriene C4 (LTC4) and leukotriene C5 (LTC5) in skin cells challenged with the calcium ionophore A23187, and of prostaglandin F2alpha (PGF2alpha), 12-HETE and 12-HEPE in gill cells challenged similarly. Prostaglandin F3alpha (PGF3alpha) production by ionophore stimulated gill cells was significantly reduced in fish fed the EFA-deficient diet. 14-HDHE produced by gill cells was 3.3 fold higher in EFA deficient fish compared to controls.

The effect of dietary lipid on polyunsaturated fatty acid metabolism in Atlantic salmon (Salmo salar) undergoing parr-smolt transformation.

The aim of this study was to measure the changes in lipid metabolism which occur during smoltification and seawater transfer in Atlantic salmon (Salmo salar). Duplicate groups of Atlantic salmon parr were fed diets containing either fish oil (FO) or a blend of linseed and rapeseed oils, vegetable oil (VO), from October (week 0) to seawater transfer in May (week 26). From May to August (weeks 26-43), all fish were fed a fish oil-containing diet. Fatty acyl desaturation and elongation activity were followed in isolated hepatocytes incubated with radioactive 18:3n-3 and 18:2n-6. Metabolism of 18:3n-3 was consistently around 5-fold greater than metabolism of 18:2n-6, and total metabolism of both substrate polyunsaturated fatty acids (PUFA) was increased in fish fed both VO and FO up to seawater transfer after which desaturation activities were reduced. Desaturation activities with both 18:3n-3 and 18:2n-6 were significantly greater in fish fed VO, compared to fish fed FO, at 22 and 26 wk. Arachidonic acid (20:4n-6; AA) in liver polar lipids (PL) of fish fed VO increased consistently from weeks 0-22 but varied after seawater transfer. In fish fed FO, AA in liver PL remained constant up to week 17 before increasing at seawater transfer and leveling off thereafter. Eicosapentaenoic acid (20:5n-3; EPA) in liver PL of fish fed VO decreased significantly from week 0-22 before rising at seawater transfer and increasing rapidly posttransfer. EPA in liver PL of fish fed FO showed a similar trend except EPA was always greater in the freshwater phase compared to fish fed VO. Docosahexaenoic acid (DHA) levels in liver PL of fish fed VO remained constant in the seawater phase before increasing following seawater transfer. In fish fed FO, DHA in liver PL increased from weeks 0-17 reducing and leveling off postseawater transfer. The levels of PGF(2 alpha) and PGF(3 alpha) were measured in isolated gill cells stimulated with calcium ionophore A23187. PGF(2 alpha) production in fish fed VO increased significantly between 0-7 wk before decreasing toward seawater transfer. After transfer, PGF(2 alpha), production increased to a peak at 35 wk. PGF(2 alpha) production in fish fed FO was not significantly altered during the trial period. The changes in PGF(3 alpha) production were broadly similar to those occurring with PGF(2 alpha), but the latter was always in excess of the former (2- to 4-fold). Plasma chloride concentrations in fish subjected to seawater challenge at 20 wk were significantly lower in fish fed VO compared to those fed FO. This study has provided new information on the changes in lipid metabolism which accompany parr-smolt transformation and suggests that diets which have a fatty acid composition more similar to that in aquatic invertebrates may be beneficial in effecting successful seawater adaptation.

Fatty acyl desaturation in isolated hepatocytes from Atlantic salmon (Salmo salar): stimulation by dietary borage oil containing gamma-linolenic acid.

The effects of different dietary oils on the fatty acid compositions of liver phospholipids and the desaturation and elongation or [1-14C]18:3n-3 and [1-14C]18:2n-6 were investigated in isolated hepatocytes from Atlantic salmon. Atlantic salmon smolts were fed diets containing either a standard fish oil (FO) as a control diet, a 1:1 blend of Southern Hemisphere marine oil and tuna orbital oil (MO/TO), sunflower oil (SO), borage oil (BO), or olive oil (OO) for 12 wk. The SO and BO diets significantly increased the percentages of 18:2n-6, 18:3n-6, 20:2n-6, 20:3n-6, and total n-6 polyunsaturated fatty acids (PUFA) in salmon liver lipids in comparison with the FO diet. The BO diet also increased the percentage of 20:4n-6. Both the SO and BO diets significantly reduced the percentages of all n-3 PUFA in comparison with the FO diet. The OO diet significantly increased the percentages of 18:1n-3, 18:2n-6, total monoenes, and total n-6 PUFA in liver lipids compared to the FO diet, and the percentages of all n-3 PUFA were significantly reduced. With [1-14C]18:3n-3, the recovery of radioactivity in the products of delta 6 desaturation was significantly greater in the hepatocytes from salmon fed SO, BO, and OO in comparison with the FO diet. The BO diet also increased the recovery of radioactivity in the products of delta 5 desaturation. Only the BO diet significantly affected the desaturation of [1-14C]18:2n-6, increasing recovery of radioactivity in both delta 6- and delta 5-desaturation products. In conclusion, dietary BO, enriched in gamma-linolenic acid (18:3n-6), significantly increased the proportions of both 20:3n-6 and 20:4n-6 in salmon liver phospholipids and also significantly increased the desaturation of both 18:2n-6 and 18:3n-3 in salmon hepatocytes. The possible relationships between dietary fatty acid composition, tissue phospholipid fatty acid composition, and desaturation/elongation activities are discussed.

Stimulation of proliferation of an essential fatty acid-deficient fish cell line by C20 and C22 polyunsaturated fatty acids and effects on fatty acid composition.

Recently we reported the development of a fish cell line, EPC-EFAD, derived from the carp (Cyprinus carpio) epithelial papilloma line, EPC, that could survive and proliferate in essential fatty acid-deficient (EFAD) medium. The EPC-EFAD cell line may be a useful model system in which to study the cellular biochemical effects of EFA deficiency and has advantages in studies of polyunsaturated fatty acid (PUFA) and eicosanoid metabolism in fish in that the complications introduced by culture in relatively n-6 PUFA-rich mammalian sera are removed. In the present study, the effects on cell proliferation rate of supplementing EPC-EFAD cells with various n-3 and n-6 PUFA were investigated to determine the possible role(s) of PUFA in cell growth and division. The selectivity of incorporation of specific PUFA into individual glycerophospholipid classes and the feasibility of reproducing in vivo fatty acid compositions in vitro were also investigated. Proliferation of the EPC-EFAD cell line was stimulated by arachidonic (20:4 n-6), eicosapentaenoic (20:5 n-3) and docosahexaenoic (22:6 n-3) fatty acids but not by 18:2 n-6 or 18:3 n-3. The differential effects of PUFA on cellular proliferation may be related to the lack of significant delta 5 desaturase activity in the cells at 22 degrees C and may implicate a role for eicosanoids in the mechanism of stimulation of proliferation. PUFA supplementation increased the cytotoxic effects of longer term culture, an effect that was partly alleviated by inclusion of vitamin E in the culture medium. The cells could generally be supplemented with PUFA to produce cellular fatty acid compositions in vitro that were similar to in vivo compositions.

Modification of membrane fatty acid composition, eicosanoid production, and phospholipase A activity in Atlantic salmon (Salmo salar) gill and kidney by dietary lipid.

Atlantic salmon post-smolts were fed diets containing either fish oils (Fosol, FO and Marinol, MO) rich in long-chain n-3 polyunsaturated fatty acids (PUFA), or plant oils rich in 18:2n-6 (sunflower oil, SO) or 18:3n-3 (linseed oil, LO) for 12 wk. The major PUFA in individual phospholipids from gill and kidney were related to the dietary lipid intake. Levels of n-6 PUFA were highest while levels of n-3 PUFA were lowest in fish fed SO. Fish fed LO generally had lower levels of 20:4n-6 compared to the other treatments while fish fed SO generally had the highest levels of 20:4n-6. In all phospholipid classes except phosphatidylinositol (PI) 20:5n-3 was greatest in fish fed MO followed by FO, LO, and SO. In PI, 20:5n-3 was also highest in fish fed MO but those fed LO contained more 20:5n-3 than those fed FO. This resulted in the ratio of the eicosanoid precursors, 20:4n-6/20:5n-3, being significantly greater in fish fed SO, for all phospholipid classes, compared to fish fed the other three dietary oils. The activity of gill phospholipase A was greatest in fish fed FO and was lowest in fish fed SO. The concentration of PGF3 alpha was significantly increased in gill homogenates from fish fed MO compared to the other three treatments while PGF2 alpha was significantly increased in fish fed SO compared to those fed LO. The concentration of PGE3 was significantly reduced in kidney homogenates from fish fed SO compared to the other three treatments while PGE2 was significantly increased in fish fed SO compared to those fed either FO or LO.

Production of eicosanoids derived from 20:4n-6 and 20:5n-3 in primary cultures of turbot (Scophthalmus maximus) brain astrocytes in response to platelet activating factor, substance P and interleukin-1 beta.

Primary cultures of turbot (Scophthalmus maximus) brain astroglial cells established in medium containing fetal bovine serum contain increased proportions of 18:1(n-9), total (n-9) and (n-6) polyunsaturated fatty acids (PUFA) and greatly reduced (n-3) PUFA in comparison with turbot brain. Supplementation with a mixture of 5 microM eicosapentaenoic [20:5(n-3)] and 25 microM docosahexaenoic [22:6(n-3)] acids for 4 days significantly increased the percentages of these acids in total cellular lipid of turbot astrocytes and restored the (n-3) PUFA composition of the cells to that found in turbot brain. The production of prostaglandins (PG) E and F of the 2- and 3-series and leukotrienes (LT) C4 and C5 in response to various agonists was determined in PUFA-supplemented astrocytes. Calcium ionophore A23187, platelet activating factor and substance P stimulated the production of both PGF and PGE. Interleukin-1 beta significantly stimulated the production of PGF only. There were differences between the agonists in their effects on the relative levels of 2- and 3-series PGs produced. Only very low amounts of LTC were produced by the turbot astrocytes, with only substance P showing a minor stimulatory effect.

Dietary lipid affects phospholipid fatty acid compositions, eicosanoid production and immune function in Atlantic salmon (Salmo salar).

Atlantic salmon (Salmo salar) post-smolts were fed diets containing either Fosol (FO), a North Sea fish oil, sunflower oil (SO), linseed oil (LO) or Marinol K (MO), a southern hemisphere fish oil rich in 20:5(n-3) for 12 weeks. A macrophage-enriched leucocyte preparation was obtained from head kidney and the fatty acid compositions of the individual membrane phospholipids measured. In general phospholipids from SO- and LO-fed fish had increased 18:2(n-6), 20:2(n-6) and 20:3(n-6) compared to the fish oil treatments while LO-fed fish had lower 20:4(n-6) than any other dietary treatment. Fish fed LO also had increased 18:3(n-3), 18:4(n-3), 20:3(n-3) and 20:4(n-3). The 20:5(n-3) content of kidney macrophage-enriched leucocyte phospholipids was highest in MO-fed fish followed by FO- and LO-fed fish with the lowest level in fish fed SO. The overall effect on the ratio of eicosanoid precursors, 20:4/20:5, showed the highest value in SO-fed fish and the lowest in fish fed LO. Production of LTB5 by kidney macrophage-enriched leucocytes stimulated with A23187 was highest in MO-fed fish and lowest in those fed SO. Production of LTB4 was greatest in SO-fed fish and lowest in fish fed LO. Serum Ig levels were significantly affected by dietary treatment with highest values in fish fed FO and SO and lowest in fish fed MO and LO.

The desaturation and elongation of linolenic acid and eicosapentaenoic acid by hepatocytes and liver microsomes from rainbow trout (Oncorhynchus mykiss) fed diets containing fish oil or olive oil.

The products of desaturation and elongation of [1-14C]18:3(n - 3) and [1-14C]20:5(n - 3) were studied using hepatocytes and microsomes prepared from livers of trout maintained on diets containing either olive oil or fish oil, to establish the extent to which the formation of 22:6(n - 3) was enhanced in the absence of dietary 22:6(n - 3) and to investigate the pathway(s) of conversion of 18:3(n - 3) and 20:5(n - 3) to 22:6(n - 3). Levels of 20:5(n - 3) and 22:6(n - 3) in the total lipid of hepatocytes from trout fed olive oil were 20-fold and 10-fold, respectively, lower than in cells from trout fed fish oil. For both dietary groups, [1-14C]18:3(n - 3) was incorporated into hepatocyte lipid to a greater extent than [1-14C] 20:5(n - 3). Almost 70% of the total radioactivity from [1-14C]18:3(n - 3) was recovered in hepatocyte triacylglycerols, whereas radioactivity from [1-14C]20:5(n - 3) was recovered almost equally in neutral lipids (52%) and polar lipids (48%). The products of desaturation and elongation from both labelled substrates were esterified mainly into hepatocyte polar lipids, whereas elongation products of [1-14C]18:3(n - 3) were preferentially incorporated into neutral lipids. Radioactivity recovered in the 22:6(n - 3) of polar lipids of hepatocytes from trout fed olive oil, from both 14C substrates, was approximately double that in hepatocytes from trout fed fish oil. No radioactivity from either [1-14C]18:3(n - 3) or [1-14C]20:5(n - 3) was incorporated into 22:6(n - 3) by microsomes isolated from livers from either group of fish and incubated in the presence of acetyl-CoA, malonyl-CoA, NADH, NADPH, ATP and coenzyme A. However, significant radioactivity was recovered in 24:5(n - 3) and 24:6(n - 3) from [1-14C]20:5(n - 3) and more radioactive 24:6(n - 3) accumulated in microsomes from trout fed olive oil than from trout fed fish oil. The results establish that the formation of 22:6(n - 3) from both 18:3(n - 3) and 20:5(n - 3) in hepatocytes of rainbow trout is stimulated by omitting 22:6(n - 3) from the diet and are consistent with the biosynthesis of 22:6(n - 3) in trout liver cells proceeding via 24:5(n - 3) and 24:6(n - 3) intermediates.

Development of an in vitro model of essential fatty acid deficiency in fish cells.

Levels of eicosapentaenoic acid (EPA; 20:5, n-3) greatly exceed those of arachidonic acid (AA; 20:4, n-6) in the tissue phospholipids of most fish species. Despite this, it is 20:4, n-6-derived eicosanoids that are produced predominantly in fish cells. The development of an essential fatty acid (EFA)-deficient fish cell line would greatly assist the study of this selectivity and so several fish cell lines were cultured in EFA-deficient (EFAD) media. All n-3 and n-6 polyunsaturated fatty acids (PUFA) and total PUFA were considerably reduced in all lines, except turbot fin (TF) in which total n-9 PUFA doubled from 13.8% to 27.5% of total fatty acids. In the topminnow hepatocarcinoma cell line (PLHC-1), there was almost complete depletion of both n-3 and n-6 PUFA and in TF cells, no n-3 PUFA were detected. In the carp epithelial papilloma cell line (EPC), both n-6 and n-3 PUFA were reduced by approximately 70%. The reduced PUFA in cells cultured in EFAD media was compensated to a large extent in most cell lines by significantly increased percentages of monounsaturated fatty acids, particularly 18:1, n-9. Total n-9 PUFA were significantly increased in all cell lines by culture in EFAD media, with 20:2, n-9 significantly increased in all cell lines. There were relatively small increases, but often significant, in 20:3, n-9 in all cell lines. Of the cell lines investigated, only EPC and PLHC-1 showed proliferation after four passages in EFAD medium, although the growth rates were reduced in comparison with media supplemented with serum, but EPC was the only cell line able to survive and proliferate in long-term culture on EFAD medium. The EFAD-EPC line is a potentially useful model system for the study of the effects of EFA deficiency on cell structure and function and eicosanoid metabolism in fish.

Diets rich in eicosapentaenoic acid and gamma-linolenic acid affect phospholipid fatty acid composition and production of prostaglandins E1, E2 and E3 in turbot (Scophthalmus maximus), a species deficient in delta 5 fatty acid desaturase.

Duplicate groups of juvenile turbot, (Scophthalmus maximus), were fed diets containing either Marinol K (MO), a marine fish oil rich in eicosapentaenoic acid (EPA; 20:5, n-3) or borage oil (BO), rich in gamma-linolenic acid (GLA; 18:3, n-6), for a period of 12 weeks. Individual phospholipid fatty acid compositions from hearts of fish fed BO had significantly more 18:2, n-6, GLA, 20:2, n-6, dihomo-gamma-linolenic acid (DHGLA; 20:3, n-6) and total n-6 polyunsaturated fatty acids (PUFA), but significantly less arachidonic acid (AA; 20:4, n-6), compared to fish fed MO. In both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from heart, the DHGLA was increased by over 50-fold in fish fed BO while AA was reduced by over two-thirds, compared to fish fed MO. In brain, EPA was the major C20 PUFA, i.e. potential eicosanoid precursor in all phospholipids from fish fed MO, with the EPA level being twice that of AA in brain phosphatidylinositol (PI). DHGLA was the major C20 PUFA in all phospholipid classes from fish fed BO. In kidney and gill, EPA was the predominant C20 PUFA in all phospholipid classes, except PI, in fish fed MO. In kidney of fish fed BO, DHGLA was the major C20 PUFA in all phospholipid classes, except PE. In gill of fish fed BO, DHGLA was the major C20 PUFA in all phospholipid classes, including PI, where DHGLA was over 2.5-fold greater than AA. In homogenates of heart, kidney and gill from BO-fed fish the prostaglandin E1 (PGE1) concentration was significantly increased compared to MO-fed fish. In heart and kidney homogenates from fish fed MO the PGE3 concentration was significantly increased compared to fish fed BO. The ratio of PGE2/PGE1 was significantly reduced in brain, heart, kidney and gill homogenates from fish fed BO compared to those fed MO.

Dietary deficiency of docosahexaenoic acid impairs vision at low light intensities in juvenile herring (Clupea harengus L.).

In the retina of herring (Clupea harengus L.), rods are recruited from about 8 wk after hatching, and from this time there is a linear relationship between the number of rods in the photoreceptor cell population and the content of di22:6n-3 molecular species of phospholipids. Juvenile herring were reared from four weeks' post-hatching for 15 wk on either Artemia nauplii deficient in 22:6n-3 or on enriched Artemia nauplii containing 4.3% 22:6n-3. The visual performance of the fish was then determined at three light intensities (0.01, 0.1, and 1.0 lux) by observing their frequency of striking at live Artemia nauplii using infrared video recording. Herring reared on the diet containing no 22:6n-3 were less active predators, especially at the lowest light intensity where very few strikes were observed. The eyes of these fish contained greatly reduced levels of di22:6n-3 molecular species of total phospholipid, 2.1% vs. 12.0% in fish supplemented with 22:6n-3. The contribution of saturated and monounsaturated fatty acids in the molecular species of phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) was virtually unchanged, while 20:5n-3 and 22:5n-3 largely replaced 22:6n-3. There was an almost complete disappearance of di22:6n-3 PC, while the amounts of di22:6n-3 PE and PS fell by 18.1 and 20.6% to 2.7 and 7.6%, respectively. The dipolyunsaturated molecular species di20:5n-3, 20:5n-3/22:5n-3, and di22:5n-3 made up a substantial part of the deficit. We conclude that a dietary deficiency of 22:6n-3 during the period early in rod development impairs visual performance such that the fish can no longer feed at low light intensities.

Effect of supplementation with 20:3(n-6), 20:4(n-6) and 20:5(n-3) on the production of prostaglandins E and F of the 1-, 2- and 3-series in turbot (Scophthalmus maximus) brain astroglial cells in primary culture.

The production of prostaglandins E and F of the 1-, 2- and 3-series was determined in primary cultures of turbot (Scophthalmus maximus) brain astroglial cells after supplementation with 25 microM dihomo-gamma-linolenic (20:3(n-6)), arachidonic (20:4(n-6)) and eicosapentaenoic (20:5(n-3)) acids. Supplementation by 20:3(n-6), 20:4(n-6) and 20:5(n-3) for 4 days significantly increased the percentages of the respective acids in the total cellular lipid of the turbot astrocytes. The predominant prostaglandins formed by turbot astrocytes in response to stimulation with calcium ionophore A23187 were prostaglandin E2 and prostaglandin F2 alpha under all experimental conditions. The production of prostaglandin E2 was stimulated 2.6-fold, but prostaglandin F2 alpha production was unaffected after supplementation of cultures with 20:4(n-6). However, prostaglandin E2 production in astrocytes was significantly inhibited 3- and 4-fold, and prostaglandin F2 alpha was inhibited 1.6- and 14.6-fold by supplementation with 20:3(n-6) and 20:5(n-3), respectively. Supplementation with 20:3(n-6) also significantly increased the production of prostaglandin E1 (almost 4-fold) and prostaglandin F1 alpha (2.2-fold) whereas supplementation with 20:5(n-3) did not significantly increase the production of prostaglandin E3. Prostaglandin F3 alpha but not prostaglandin E1 were significantly reduced in 20:5(n-3)-supplemented cultures.