Incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids in phospholipid classes in cultured rainbow trout (Salmo gairdneri) cells

The incorporation and metabolism of various (n-3) and (n-6) polyunsaturated fatty acids supplemented to the culture medium was investigated in the rainbow trout cell line, RTG-2. The distribution, and the occurrence and relative extent of further desaturation and elongation of the incorporated acids was determined in individual phospholipid classes by analysis of the fatty acid compositions. RTG-2 cells exhibited Δ6 and Δ5 desaturase activities whereas Δ4 desaturase activity was almost totally absent. The percentage of precursor acids was greatest in the phosphatidic acid/cardiolipin fraction (PA/CL), suggesting a role for possibly PA in the initial incorporation of these acids into the phospholipid pool. The compositional data indicated that individual intermediates and products of the desaturation pathways were associated with specific phospholipid classes probably via mechanisms depending upon the specificities of the acylating enzymes. The composition of phosphatidylinositol (PI) and the tightly controlled mechanisms for generating/maintaining it are consistent with a role for this phospholipid in providing precursor fatty acid for eicosanoid synthesis.     

Polyunsaturated fatty acid metabolism in cultured fish cells: Incorporation and metabolism of (n-3) and (n-6) series acids by Atlantic salmon (Salmo salar) cells

The incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids (PUFA) supplemented to growing cultures were studied in Atlantic salmon (AS) cells. A fatty acid concentration of 25 μM considerably altered the fatty acid composition of AS cells without increasing the neutral lipid content of the cells or inducing the production of cytoplasmic lipid droplets. Whereas Δ6 and Δ5 desaturase activities were significantly expressed in AS cells, Δ4 desaturase activity was very low. Both the Δ6 desaturase activity and the Δ5 desaturase activity showed some preference for (n-3) PUFA.     

The incorporation and metabolism of polyunsaturated fatty acids in phospholipids of cultured cells from chum salmon (Oncorhynchus keta)

The incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids were studied in a cell line derived from chum salmon heart (CHH-1). Supplementing media with 25 M fatty acid considerably altered the cellular fatty acid composition but did not affect the lipid class composition or cause the appearance of cytoplasmic lipid droplets. CHH-1 cells exhibited considerable -6-desaturase activity but showed no preference between (n-3) and (n-6)PUFA substrates. CHH-1 cells also possess -5-desaturase activity which showed preference towards (n-3)PUFA, but -4-desaturase activity was totally absent. Elongation of 20-carbon PUFA was especially active in CHH-1 cells with 22-carbon PUFA being specifically incorporated into PE and PS lipid classes. The fatty acid composition of PI indicated specific incorporation of 20-carbon PUFA into this lipid class. Supplementation with 22:6(n-3) generated fatty acid compositions more closely resembling those of intact salmonid hearts. Substantial chain shortening of 22:6(n-3) to 20:5(n-3) occurred.Abbreviations BHT butylated hydroxytoluene - BSA bovine serum albumin - CL cardiolipin - FCS fetal calf serum - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - PUFA polyunsaturated fatty acid - SM sphingomyelin     

The effect of culture on morphology, lipid and fatty acid composition, and polyunsaturated fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells

Rainbow trout (Oncorhynchus mykiss) skin cell cultures were obtained by trypsinization of the tissue and grown in Leibovitz L-15 medium. Lipid class compositions, and fatty acid profiles of total lipids and individual phospholipid classes were determined at different times of culture. The metabolism of polyunsaturated fatty acids (PUFA) was investigated by incubating primary cultures after 7 and 14 days with [1-¹⁴C]18:2n-6 and [1-¹⁴C-]18:3n-3. The change in morphology between epithelial-like primary cultures and fibroblastic-like secondary subcultures was accompanied by alterations in the lipid composition. Polar lipids became predominant by 14 days in culture. The relative proportions of phosphatidylcholine (PC), the most abundant phospholipid, phosphatidylinositol and cholesterol increased significantly, while sphingomyelin decreased. Saturated fatty acids, 18:1n-9, n-6 and n-9PUFA were more abundant in total lipid in cultures at 14 days and 4 months than in cells initially isolated which contained higher percentages of longer chain monoenes and n-3PUFA. The changes in fatty acid composition with time in culture were observed in all the major phospholipid classes. Rainbow trout skin cells in culture desaturated and elongated both 18:2n-6 and 18:3n-3, with 20:4n-6 and 20:5n-3 being the most abundant products, respectively. PC presented the highest incorporation of radioactivity, especially following incubation with 18:3n-3. Lipid metabolism in general increased with the age of primary cultures, with both the amount of C18 PUFA incorporated and metabolized by desaturation/elongation significantly increased in 14 day cultures compared to 7 day cultures. Product/precursor ratios calculated for both n-6 and n-3 fatty acids showed that, while 6 desaturase activity was increased significantly with cell age, 5 desaturase activity was more affected by the fatty acid series, with 18:3n-3 being more readily transformed to 20:5n-3 than 18:2n-6 to 20:4n-6. Further desaturation of 20:5n-3 to hexaenes was low. Overall, the data suggested that the trout skin cell cultures were more similar to mammalian skin fibroblasts than mammalian epidermal/keratinocyte cultures.     

Metabolism of 18:4n-3 (stearidonic acid) and 20:4n-3 in salmonid cells in culture and inhibition of the production of prostaglandin F2α (PGF2α) from 20:4n-6 (arachidonic acid)

Arachidonic acid (AA; 20:4n-6) is the precursor of a range of highly biologically active derivatives, collectively termed eicosanoids, including prostaglandins, thromboxanes, leukotrienes and lipoxins, that act as autocrine hormones regulating many physiological processes including haemostasis, reproduction, immune and inflammatory responses. Eicosapentaenoic (EPA; 20:5n-3) and dihomo-γ-linolenic (20:3n-6) acids modulate eicosanoid metabolism by both inhibiting the conversion of AA to eicosanoids whilst simultaneously being converted to eicosanoids with different, often attenuated, properties compared to their AA homologues. Eicosatetraenoic acid (20:4n-3) is a naturally occurring C₂₀ polyunsaturated fatty acid (PUFA), present in fish oil at levels of around 1–2%, that has been suggested to be the active metabolite responsible for the anti-inflammatory effects of plant oils containing stearidonic acid (18:4n-3). However, the biochemical properties of 20:4n-3 in terms of cellular biology have rarely been investigated, partly due to difficulties in obtaining the fatty acid in high purity. In this paper, we describe methods for the medium scale laboratory preparation of high purity 20:4n-3, and investigate its metabolism in fish cell culture systems which normally contain significant amounts of n-3 PUFA. Thus the incorporation and metabolism of 18:4n-3 and 20:4n-3, and their distribution in phospholipid classes was studied in an established cell line from Atlantic salmon (Salmo salar) (AS), and the effects of 20:4n-3 on eicosanoid production studied in freshly isolated macrophages from rainbow trout (Oncorhynchus mykiss). Both 18:4n-3 and 20:4n-3 were preferentially esterified into phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in contrast with the accumulation of AA in phosphatidylinositol. Incorporated 18:4n-3 was readily converted to 20:4n-3, and both fatty acids were further desaturated and elongated to EPA and 22:5n-3 but not 22:6n-3. Supplementation with 20:4n-3 decreased the conversion of AA into prostaglandins, as demonstrated by the decreased levels of PGF_2α produced in trout macrophages supplemented with 20:4n-3 and AA compared to cells supplemented with AA alone. In addition, 20:4n-3 was converted into eicosanoids in fish cells as indicated by the presence of Δ^17,18 12-HETE, Δ^17,18 PGE1 and Δ^17,18 PGF_1α in extracts from rainbow trout macrophages incubated with 20:4n-3.     

Effects of temperature and feed composition on essential fatty acid (n-3 and n-6) retention in Atlantic salmon (Salmo salar L.) parr

Retentions of total n-3 and n-6 essential fatty acids (EFAs) were assessed in Atlantic salmon (Salmo salar L.) parr held at 8 °C and 2 °C until they increased in weight from ca. 19 g to 38 g. Feeds contained sandeel oil or a rapeseed:linseed oil blend at 21 and 34% dietary fat. EFA retention efficiencies [(g EFA gained g EFA ingested-1) × 100] were estimated by the 'mass balance method' from measurements of feed intake, changes in biomass for each tank of fish, and fatty acid compositions of the feeds and fish. The n-3 EFA retentions were higher (overall mean 71%) across feed treatments and temperatures than the n-6 EFA retentions (overall mean 63%). Retentions of the n-3 fatty acids were higher in the fish given the feeds with the lower fat content (77% vs. 65%), implying improved retention with reduced n-3 EFA availability. n-3 EFA retention tended to be higher at 2 °C than at 8 °C, although this was not consistent across feeds. At low temperature there was very high retention of the n-3 EFAs in feeds containing sandeel oil (80%). Such high retention may represent an adaptation response to low temperature. Lower n-6 EFA retentions imply that more n-6 fatty acids were metabolized than n-3 EFAs. Feed oil influenced retention of the n-6 fatty acids, retention being lower for the salmon parr given the feeds containing sandeel oil (56% vs. 71%). This could indicate a higher tissue deposition of n-6 fatty acids when they are freely available via the diet. Abbreviations: AA – arachidonic acid (C20:4 n-6); DHA – docosahexaenoic acid (C22:6 n-3); EFA – essential fatty acid; EPA – eicosapentaenoic acid (C20:5 n-3); HUFA – highly-unsaturated fatty acid (\ge4 double bonds); MUFA – monounsaturated fatty acid (1 double bond); PL – phospholipid; PUFA – poly-unsaturated fatty acids (\ge2 double bonds); SFA – saturated fatty acid (no double bond); TAG – triacylglycerol. This revised version was published online in July 2006 with corrections to the Cover Date.     

Polyunsaturated fatty acid metabolism in a cell culture model of essential fatty acid deficiency in a freshwater fish, carp (Cyprinus carpio)

Proliferation of an essential fatty acid deficient cell line from carp (EPC-EFAD; epithelioma papillosum carp-essential fatty acid deficient) is stimulated by supplementing the cells with C₂₀, but not C₁₈ polyunsaturated fatty acids (PUFA). It is hypothesized that the differential ability of the PUFA to stimulate proliferation of the EPC-EFAD cells may be related to the extent of the cells' ability to desaturate and elongate C₁₈ PUFA. In the present study, the metabolism of ¹⁴C-labeled C₁₈ and C₂₀ PUFA was investigated in EPC-EFAD cells in comparison with normal EPC cells. The incorporation of all the PUFA was significantly greater in EPC-EFAD cells but the rank order, 20:5n-3 > 18:3n-3 = 18:2n-6 >20:4n-6 was the same in both cell lines. The proportion of radioactivity from all labeled PUFA recovered in phosphatidylethanolamine and total polar lipids was significantly lower in EPC-EFAD cells compared to EPC cells, whereas the proportion of radioactivity recovered in all the other phospholipid classes and total neutral lipid was greater in EPC-EFAD cells. Both cell lines desaturated[1-¹⁴C]18:3n-3 and [1-¹⁴C]20:5n-3 to a greater extent than the corresponding (n-6) substrates but the desaturation of all the ¹⁴ C-labeled PUFA was significantly greater in EPC-EFAD cells compared to EPC cells. The results showed that, although essential fatty acid deficiency had several significant effects on PUFA metabolism in EPC cells, the fatty acid desaturation/elongation pathway was not impaired in EPC-EFAD cells and so they can desaturate 18:3n-3 to 20:5n-3 and 22:6n-3, and 18:2n-6 to 20:4n-6. However, 20:4n-3 and 20:3n-6, and not 20:4n-6 and 20:5n-3, were the predominant C₂₀ PUFA produced by the elongation and desaturation of [1-¹⁴C]18:3n-3 and [1-¹⁴C]18:2n-6, respectively. Therefore, the previously reported inability of 18:3n-3 and 18:2n-6, compared to 20:5n-3 and 20:4n-6, to stimulate proliferation of the cells is apparently not due to a general deficiency in the fatty acid desaturation pathway in EPC-EFAD cells but may be related to potential differences in eicosanoid profiles in cells supplemented with C₁₈ PUFA compared to C₂₀ PUFA.     

Fatty Acid Porfiles of Farm-Raised Catfish Fillet:

Fatty acid profiles of the total lipids and the phospholipid classes of farm-raised channel catfish (Ictalurus punctatus) fillets were analyzed. Monoenes represented 49.22 mol% of the total lipid fatty acids while polyunstaturated fatty acids (PUFA) accounted for 19.37 mol%. The total n-3 PUFA content was low (4.22 mol%). The total saturated fatty acids as 31.44 mol %. Catfish contained phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), lysophosphatidylcholine (LPC), and sphingomyelin (SPH). Analysis for the individual phospholipid class fatty acid profiles indicated that PI had a high concentration of 18:0 (33.20 mol%) and 20:4 n-6 (13.08 mol%). Phosphatidylethanolamine had the highest concentrations of combined eicosapentaenoic acid, and docoshahexaenoic acid, of 17.37 mol%. Higher ratios of n-3/n-6 PUFA were found in the phospholipid classes than the total lipids. LPC and SPH did not contain measurable PUFA of the n-3 series. The level of 18:1 n-9 in PC, PE, and LPC was approximately 31 mol% whereas in PI, PS, and SPH, the level was approximately 16 mol%. The understanding of the basic distribution of fatty acids in the individual phospholipid classes of channel catfish may be an essential first step in explaining the protective role of 18:1 n-9 and n-3 PUFA against thrombotic and cardiovascular disorder in humans.     

Relationship between dietary phospholipid classes and neutral lipid absorption in newly-weaned turbot, shape Scophthalmus maximus

A 28-day feeding trial was conducted for comparing the effect of different dietary phospholipid (PL) classes on the growth of post-larval turbot and on the incorporation of dietary neutral lipid fatty acids into their body lipids. Prior to the experiment the turbot were weaned for one week on a PL-free diet. The nine experimental diets were isolipidic and contained an equal amount of highly unsaturated fatty acids in the form of ethyl esters. They differed by their PL content (0, 1 or 2%) and by the PL class composition of the added soybean PL fractions.Compared to the PL-free diet, diets enriched with phosphatidylcholine (PC) resulted in a better growth, a higher triglyceride content (% body dry matter) and increased levels of docosahexaenoic acid (% total fatty acids) in each of the examined body lipid classes (neutral lipid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol). The effects of the other soybean PL fractions were less explicit than those noted for soybean PC.The results support the idea that dietary PC plays a role in the intestinal absorption of neutral lipid fatty acids. This might, at least partially, explain the superiority of PC for enhancing growth. Abbreviations: DHA – docosahexaenoic acid (22:6n-3); EPA – eicosapentaenoic acid (20:5n-3); HUFA – highly unsaturated fatty acid; PA – phosphatidic acid; PC – phosphatidylcholine; PE – phosphatidylethanolamine; PI – phosphatidylinositol; PL – phospholipid; PS – phosphatidylserine; PUFA – polyunsaturated fatty acid.     

Effects of salinity on the fatty acid compositions of total lipid and individual glycerophospholipid classes of Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus) cells in culture

Cells from a relatively stenohaline marine species, turbot (Scophthalmus maximus) (TF) and an anadromous species, Atlantic salmon (AS) were cultured in media supplemented with NaCl to produce OPs varying from 300 to 500 mOsm kg^–1 and the direct effects of OP (salinity) on the fatty acid compositions of the main glycerophospholipid classes were determined. The most dramatic effects of salinity on total lipid fatty acids were observed in polyunsaturated fatty acids (PUFA) in TF cells. There was a graded decrease in the percentage of 18:2n-9, and consequently total n-9 PUFA, and concomitantly increased percentages of both total n-3 and n-6 PUFA with increasing salinity. The increased n-3 and n-6 PUFA was due to significantly increased percentages of the major fatty acids in each of these groups, namely 22:6n-3 and 20:4n-6, respectively. The reciprocal changes in n-9 PUFA and n-3/n-6 PUFA in TF cell total lipid resulted in the percentage of total PUFA not being significantly affected by changes in salinity. The graded decrease in 18:2n-9 with increasing salinity in TF cells was observed in all the major glycerophospholipids but especially PE, PI and PS. Increasing salinity resulted in graded increases in the percentages of 22:6n-3 in PE and PS in TF cells. The quantitatively greatest increase in the percentage of n-6 PUFA in TF cells occurred with 20:4n-6 in PC, PE and PL. There were less significant changes in the fatty acid compositions of glycerophospholipids in AS cells. However, the proportion of total n-3 + n-6 PUFA in PE varied reciprocally with the proportion of dimethylacetals in response to salinity. Similar reciprocal changes between fatty acids in response to salinity were also evident in the quantitatively more minor glycerophospholipids PS and Pl. In PS, the percentage of 22:6n-3 was significantly lower at 400 mOsm kg^–1 whereas the proportion of total monoenes was significantly higher at that salinity. A similar inverse relationship between total monoenes and 20:4n-6 (and, to a lesser extent total saturates) in response to salinity was noted in PI. The results show that environmental salinity, without whole-body physiological stimuli, has direct effects on the fatty acid composition of major glycerophospholipid classes in fish cells and that these effects differ in cells from different fish speciesAbbreviations ANOVA analysis of variance - BHT butylated hydroxytoluene - BSA bovine serum albumin - DMA dimethylacetals - EMEM Eagle's minimal essential medium - FCS fetal calf serum - GC gas chromatography - HBSS Hank's balanced salt solution (without Ca²⁺ and Mg²⁺) - OP osmotic pressure - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - PUFA polyunsaturated fatty acid - TLC thin-layer chromatography     

The fatty acid compositions of established fish cell lines after long-term culture in mammalian sera

The effect of long-term culture of fish cells in mammalian serum on the phospholipid fatty acid composition was investigated. All the cell lines studied had much lower levels of polyunsaturated fatty acids (PUFA) than those found in intact fish tissues. In particular (n-3) PUFA were considerably depleted in the cultured cell lines, leading to very low (n-3)/(n-6) ratios in all the phospholipid classes. In general the cells were rich in 18:1, 16:0, 18:0 and 16:1 with 20:4(n-6) and 22:6(n-3) as the major PUFA. The fatty acid composition reflected the composition of the fetal calf serum added to the media rather than their fish tissue origins. The results were discussed in relation to the roles of PUFA in general cell metabolism and more specifically the role of (n-3) PUFA in fish cells.     

Administration of two oils rich in n-3 long-chain polyunsaturated fatty acids to rat pups of dams fed a diet high in fat and low in n-3 polyunsaturated fatty acids

Long-chain polyunsaturated fatty acids (LCPUFA) with 20 or 22 carbons are considered important to the development of infants and sometimes added to infant formulae. In this study, two characteristic sources of n-3 LCPUFA (fish oil and microalgal oil) were orally administrated to rat pups of mildly n-3 PUFA — deficient dams to compare the consequences of the administration. The milk from the dams fed a n-3 PUFA — restricted diet contained less n-3 LCPUFA than that of the dams fed a control diet. Pups were administered 1 mg/g weight of the test oil at the age of 5–7 days. At the age of 7 days, they were sacrificed before or after the administration and fatty acid compositions of the stomach and serum lipid were studied. The administration changed docosahexaenoic acid (DHA; 22:6n−3) levels in the stomach contents and serum lipids with time. Eicosapentaenoic acid (EPA; 20:5n−3) levels increased immediately after the administration of fish oil. The administration of microalgal oil also affected the serum lipid EPA level, in spite of a lack of EPA. In this study, both oils effectively supplemented DHA. Fish oil returned the serum EPA level close to the control value while microalgae oil had little effect.     

Fatty acid metabolism in European sea bass (Dicentrarchus labrax): effects of n-6 PUFA and MUFA in fish oil replaced diets

Monounsaturated fatty acids (MUFA)-rich and n-6 polyunsaturated fatty acid (n-6 PUFA)-rich vegetable oils are increasingly used as fish oil replacers for aquafeed formulation. The present study investigated the fatty acid metabolism in juvenile European sea bass (Dicentrarchus labrax, 38.4 g) fed diets containing fish oil (FO, as the control treatment) or two different vegetable oils (the MUFA-rich canola/rapeseed oil, CO; and the n-6 PUFA-rich cottonseed oil, CSO) tested individually or as a 50/50 blend (CO/CSO). The whole-body fatty acid balance method was used to deduce the apparent in vivo fatty acid metabolism. No effect on growth performance and feed utilization was recorded. However, it should be noted that the fish meal content of the experimental diets was relatively high, and thus the requirement for n-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA) may have likely been fulfilled even if dietary fish oil was fully replaced by vegetable oils. Overall, relatively little apparent in vivo fatty acid bioconversion was recorded, whilst the apparent in vivo β-oxidation of dietary fatty acid was largely affected by the dietary lipid source, with higher rate of β-oxidation for those fatty acids which were provided in dietary surplus. The deposition of 20:5n-3 and 22:6n-3, as % of the dietary intake, was greatest for the fish fed on the CSO diet. It has been shown that European sea bass seems to be able to efficiently use n-6 PUFA for energy substrate, and this may help in minimizing the β-oxidation of the health benefiting n-3 LC-PUFA and thus increase their deposition into fish tissues.     

Lipids of arctic charr,Salvelinus alpinus (L.) II. Influence of dietary fatty acids on the elongation and desaturation of linoleic and linolenic acid

Arctic charr,Salvelinus alpinus L. were fed five test diets containing 0% or 1% of different polyunsaturated fatty acids (PUFA) for 93 days. The fish were injected intraperitoneally with (1–¹⁴C)–18:2(n–6) or (1–¹⁴C)–18:3(n–3), and the bioconversion to longer chain PUFA studied. The conversion rate in neutral lipids was slow, with most label found as the fatty acid injected, while extensive modification took place prior to or during incorporation into polar lipids. Linolenic acid was preferred over linoleic acid as substrate for elongation and desaturation regardless of diet. In polar lipids, the predominant products of (1–¹⁴C)–18:2(n–6) metabolism were generally 20:3(n–6) and 20:4(n–6), while 18:4(n–3), 20:5(n–3) and 22:6(n–3) were the major products of (1–¹⁴C)–18:3(n–3) metabolism. The lack of radioactivity in 22:5(n–6) suggests that 4 desaturation is specific for (n–3) PUFA. Feeding the PUFA deficient diet reduced the 5 desaturation compared to fish maintained on PUFA supplemented diets. The 6 desaturation was only reduced in fish fed C18 PUFA and injected with (1–¹⁴C)–18:3(n–3). Longer chain C20 and C22 PUFA, particularly those of the (n–3) family, exerted some inhibition on the elongation and desaturation of injected fatty acids compared to those fed C18 PUFA. The incorporation of radiolabelled fatty acids into polar lipids of fish fed a commercial diet was very low, and the desaturation neglectible in both polar and neutral lipids, showing that Arctic charr under culture conditions do not convert short chain PUFA to longer chain metabolites.     

Incorporation and metabolism of14C-labelled polyunsaturated fatty acids in wild-caught juveniles of golden grey mullet,Liza aurata,in vivo

The incorporation, and the capacity for desaturation and elongation in vivo, of intraperitoneally-injected,¹⁴C-labelled n-3 and n-6 C₁₈ and C₂₀ polyunsaturated fatty acids (PUFA) were investigated in juvenile golden grey mullet,Liza aurata. The results indicate that juvenile mullet have only limited ability to convert C₁₈ polyunsaturated fatty acids to C₂₀ and C₂₂ highly unsaturated fatty acids (HUFA)in vivo. This suggests that juvenile golden grey mullet require the provision of preformed C_20/22 HUFA, such as eicosapentaenoic and docosahexaenoic acids, in the diet. The impairment in the desaturase/elongase pathway was similar to that found in turbot,Scophthalmus maximus, and gilthead sea bream,Sparus aurata, being primarily at the level of Δ5-desaturase. The data from the largely herbivorous golden grey mullet juveniles are consistent with the hypothesis that marine fish in general, irrespective of dietary habits, have limited capacity for the desaturation and elongation of C₁₈ PUFA. The defect in Δ5-desaturase activity combined with the consistent finding that arachidonic acid is selectively incorporated and retained in membrane phosphatidylinositol suggests that, like turbot and gilthead sea bream, golden grey mullet may also have a requirement for preformed arachidonic acid in the diet.     

Essential fatty acids in Atlantic salmon: time course of changes in fatty acid composition of liver, blood and carcass induced by a diet deficient in n-3 and n-6 fatty acids

Atlantic salmon fry (4 g) were fed a semi-synthetic diet devoid of n-3 and n-6 polyunsaturated fatty acids (PUFA) for 4 months. External signs of nutritional imbalance during the period were slow growth rate and increased mortality. Some symptoms of essential fatty acid deficiency appeared in the fatty acid composition of the blood and liver during the fourth month. At that time, the percentages of n-3 and n-6 PUFA of triacylglycerols (TAG) were nearly exhausted, and the percentages of 22:6 n-3 and of 20:5 n-3 in phospholipids (PL) showed a marked decrease. This decrease in the PUFA level of the PL was paralleled by the appearance of 20:3 n-9, whereas in the TAG an increase in the percentage of 18:1 n-9 was observed. After 4 months the monounsaturated fatty acid 18:1 n-9 constituted nearly 50% of the fatty acids in the TAG fraction of the liver. The time course of the changes in fatty acid composition of liver and blood lipids was quite similar, whereas the carcass lipid composition appeared to respond slowly to a diet devoid of essential fatty acids.     

Effect of broodstock diets on total lipids and fatty acid composition of larvae of gilthead sea bream (Sparus aurata L.) during yolksac stage

Total lipid content and total lipid fatty acid compositions were studied in larvae at the yolksac stage of gilthead sea bream from two different broodstocks. The two broodstock diets had the same total lipid content but were different with respect to their fatty acid levels. There were differences in the fatty acid composition of total lipids in the two groups of larvae, reflecting the influence of fatty acid levels in the diets fed to the broodstock. The main fatty acids in both larvae lots during yolksac stage were 16:0, 18:1n-9 and 22:6n-3. The desaturation index Δx = [P + Σ (n − x) − Σ (n − x) diet]/P allowed the determination of the relative importance of the Δ9, Δ6, Δ5 and Δ4 desaturations during the egg and larval yolksac stages. The levels of total lipid fatty acids in eggs and larvae during yolksac period were almost constant and very low desaturation and elongation activities were apparent. A competitive inhibition was observed between the fatty acids of the n-6 series with respect to the ones of the n-3 series in those eggs and larvae from broodstock whose diet contained a high proportion of linoleic acid and its derivatives.     

Effects of essential fatty acid-deficient diets on growth, mortality, tissue histopathology and fatty acid compositions in juvenile turbot (Scophthalmus maximus)

Three diets in which the lipid component was supplied either as fish oil (FO), linseed oil (LO) or olive oil (OO) were fed to duplicate groups of juvenile turbot (Scophthalmus maximus) of initial weight 1.2 g for a period of up to 12 weeks. The latter two diets resulted in a significant reduction in specific growth rate and an increased mortality compared to the FO (control) fed fish. A liver histopathology was evident in around half of the fish fed the LO and OO diets but was absent in fish fed FO. The lesion showed indications of cellular alterations consisting of foci of densely basophilic cells but without evidence of inflammatory activity. The total lipid fatty acid composition of the carcass from fish fed LO had increased percentages of 18:2n-6 and 18:3n-3, but decreased percentages of all other polyunsaturated fatty acids (PUFA) including the physiologically important 20:4n-6, 20:5n-3 and 22:6n-3, compared to fish fed FO. Almost 2/3 of the total fatty acids in the carcass of OO-fed fish were monounsaturated while the percentages of total saturated fatty acids and all other PUFA, except 18:2n-6, were significantly reduced compared to fish fed FO. Broadly similar effects on total lipid fatty acid composition were observed in liver. In the liver glycerophospholipid classes of fish fed LO, percentages of 18:2n-6, 18:3n-3 and 20:3n-3 were significantly increased whereas all C20 and C22 PUFA, with the exception of 20:5n-3 in PI, were significantly reduced compared to fish fed FO. The liver glycerophospholipids of fish fed OO all showed significantly increased total monounsaturates, 18:2n-6, 20:2n-6, 18:2n-9 and 20:2n-9 as well as reduced percentages of 20:4n-6 and 22:6n-3, compared to fish fed FO. The brain glycerophospholipids showed broadly similar changes in response to dietary treatment although the magnitude of fatty acid alterations was less than those observed in liver. The greater mortalities in the OO-fed fish compared to the LO-fed fish suggests that incorporation of 18:3n-3 into tissue phospholipids can offset losses of long-chain PUFA more effectively than incorporation of 18:1n-9. However, levels of dietary long-chain PUFA must be optimised to allow normal growth and development. We conclude that the very low flux through the fatty acid desaturase/elongase pathways in turbot is not up-regulated by diets deficient in 20:5n-3 and 22:6n-3.     

Effects of dichlorvos and formalin on fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells in primary culture

The effects of sub-lethal doses of dichlorvos and formalin, antimicrobial/parasitic agents used in aquaculture, on lipid composition and metabolism of rainbow trout skin cells in primary culture were investigated. [1-¹⁴C]Stearic (18:0), [1-¹⁴C]lin 18:2n-6) and [1-¹⁴C]linolenic (18:3n-3) acids were used as tracers to determine effects on fatty acid incorporation and metabolism. Formalin increased cell numbers and reduced the lipid content of the cells and the incorporation of radioactive fatty acids. The effects of dichlorvos were qualitatively similar but quantitatively less. Formalin induced relatively small but significant changes in lipid class composition including a decreased proportion of phosphatidycholine with increased proportions of sphatidylethanolamine and phosphatidylserine. Dichlorvos had no significant effect on lipid class compositions. The trout primary skin cells expressed substantial 9, 6 and 5 fatty acyl desaturase activities. Although, as expected, the cells were m active towards [1-¹⁴C]18:3n-3, the cells were unusually active towards [1-¹⁴C]18:2n-6. Both dichlorvos and, especially, formalin appeared to significantly inhibit 9 and 6 desaturation. Changes in the distribution of radioactivity between individual spholipid classes was also influenced by formalin and dichlorvos, and this may be related to changes in desaturase activity. This study has shown that topically active agents used in aquaculture, formalin and dichlorvos, had a range of effects on the rainbow trout skin cell cultures that may affect cell proliferation and lipid and fatty acid metabolism. Both agents significantly inhibited desaturation of fatty acids, particularly of 18:2n-6 to 20:4n-6 and, as 20:4n-6 is a major eicosanoid precursor ish and considering the importance of eicosanoids in the biochemistry of skin, it is suggested that these agents may have direct effects on fish skin that could have important consequences for fish health in general.     

Effects of diets rich in linoleic (18:2n - 6) and α-linolenic (18:3n - 3) acids on the growth,lipid class and fatty acid compositions and eicosanoid production in juvenile turbot (Scophthalmus maximus L.)

Three practical-type diets utilizing fishmeal and casein as the protein sources and containing fish oil (FO), safflower oil (SO) or linseed oil (LO) were fed to duplicate groups of juvenile turbot (Scophthalmus maximus) of initial weight 1.2 g for a period of 12 weeks. No differences in final weight, mortality or development of pathological lesions were evident either between duplicate tanks or between dietary treatments over this period. Fish fed diets containing SO and LO contained significantly greater amounts of liver triacylglycerol compared to fish fed FO. The major C₁₈ polyunsaturated fatty acids (PUFA) in SO and LO diets, 18:2(n-6) and 18:3(n-3) respectively, were readily incorporated into both total lipid and individual phospholipids of turbot tissues. There was no accumulation of the Δ6-desaturation products of these fatty acids, namely 18:3(n-6) and 18:4(n-3), in any of the tissues examined. The products of elongation of 18:2(n-6) and and 18:3(n-3), 20:2(n-6) and 20:3(n-3) respectively, accumulated in both total lipid and phospholipids with the highest levels of 20:2(n-6) in liver PC and 20:3(n-3) in liver PE. Eicosapentaenoic acid [EPA, 20:5(n-3)] levels exceeded those of arachidonic acid [AA, 20:4(n-6)] in phosphatidylinositol (PI) from liver and gill of fish fed LO. EPA levels in liver PI from fish fed LO were 3-fold and 2-fold greater than SO-fed and FO-fed fish, respectively. Fish fed diets containing SO and LO had significantly reduced levels of AA in liver and muscle total lipid and lower AA in individual phospholipid classes of liver and gill compared to FO-fed fish. The concentration of thromboxane B₂ was significantly reduced in plasma and isolated gill cells stimulated with calcium ionophore A23187 of fish fed SO and LO compared to those fed FO. Prostaglandin E produced by isolated gill cells stimulated with A23187 was significantly reduced in fish fed both SO and LO compared to fish fed FO.