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 μM), U-¹⁴C (1 μM) or deuterated (d5; 25 μM) 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. Δ5 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 Δ5 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.     

Polyunsaturated fatty acid metabolism in fish cells: differential metabolism of (n-3) and (n-6) series acids by cultured cells originating from a freshwater teleost fish and from a marine teleost fish

1. The incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids (PUFA) supplemented to growing cultures were studied in rainbow trout (RTG-2) and turbot (TF) cell lines. 2. A fatty acid concentration of 20 microM considerably altered the fatty acid composition of the cells without affecting lipid class composition or the appearance of cytoplasmic lipid droplets. 3. Both cell lines exhibited considerable delta 6 desaturase activities. 4. Whereas delta 5 desaturase activity was expressed in RTG-2 cells, delta 4 desaturase activity was absent and, conversely, delta 4 desaturase activity was expressed in TF cells, but there was an apparent deficiency in the C18 to C20 elongase multi-enzyme complex. 5. The delta 6 desaturase activity in both cell lines showed little preference between 18:2(n-6) and 18:3(n-3) but the delta 5 desaturase activity of RTG-2 cells and the delta 4 desaturase activity of TF cells showed a preference for (n-3)PUFA. 6. Two fish oil concentrates were assessed for their ability to generate fatty acid compositions in the cell lines more closely resembling those of intact fish tissues.     

Effect of polyunsaturated fatty acids on the growth of fish cells in culture

• 1.1. The effects on growth of supplementing the medium with (n-3) and (n-6) polyunsaturated fatty acids (PUFA) were investigated in Atlantic salmon (AS) and turbot (TF) cell lines.
• 2.2. Neither cell line grew in the absence of serum, and addition of increasing percentages of serum resulted in graded increases in cell growth in both cell lines.
• 3.3. The growth of AS cells was stimulated by supplementing the medium with both (n-6)PUFA and (n-3)PUFA at 5–25 μM, especially 18:3(n-3) and 20:5(n-3).
• 4.4. Intermediate concentrations (15–20 μM) of 18:2(n-6) and 18:3(n-3) increased cell growth in TF cells, although only after 8 days in culture.
• 5.5. In contrast, both (n-3) and (n-6)PUFA at 25 μM tended to inhibit the growth of TF cells, and in longer incubations caused cell death.
• 6.6. The inhibition of TF cell growth rate and, in particular, the cell death induced by 25 μM PUFA could be abolished by the addition of vitamin E to the medium.

     

Alternate pathways in the desaturation and chain elongation of linolenic acid, 18:3(n-3), in cultured glioma cells.

Cultured C6 glioma cells rapidly incorporate and metabolize the essential fatty acids, 18:2(n-6) and 18:3(n-3), to 20- and 22-carbon polyunsaturated fatty acids. Using several deuterated fatty acid substrates we have obtained data that suggest alternate pathways, one possibly involving delta 8-desaturation, may exist in glioma cells for formation of 20:5(n-3) and 22:6(n-3) from 18:3(n-3). With 18:3(n-3)-6,6,7,7-d4 practically no 18:4(n-3)-6,7-d2 or 20:4(n-3)-8,9-d2 was detected whereas 20:3(n-3)-8,8,9,9-d4 accounted for 3.4% and delta 5,11,14,17-20:4-8,8,9,9-d4 for 21.1% of the total deuterated fatty acids recovered in phospholipids after a 16 h incubation; 20:5(n-3)-8,9-d2, 22:5(n-3)-10,11-d2, and 22:6(n-3)-10,11-d2 accounted for 42.4%, 13.2%, and 2.8% of deuterated acyl chains, respectively. When added exogneously, 20:3-8,8,9,9,-d4 was extensively converted to delta 5,11,14,17-20:4(n-3)-8,8,9,9-d4 (45%) and 20:5(n-3)-8,9-d2 (24%); a small amount (4%) of 18:3(n-3)-d4 also was detected. Both 20:4(n-3)-8,9-d2 and 18:4(n-3)-12,13,15,16-d4 were also converted to 20:5(n-3) and 22:6(n-3) with 8 and 0% of the respective original deuterated substrate remaining after 16 h. A possible pathway for 18:3(n-3) metabolism in glioma cells is described whereby an initial chain elongation step is followed by successive delta 5 and delta 8 desaturation reactions resulting in 20:5(n-3) formation and accounting for the ordered removal of deuterium atoms. Alternatively, extremely effective retroconversion may occur to chain shorten 20:3(n-3)-d4 to 18:3(n-3)-d4 followed by rapid conversion through the classical desaturation and chain elongation sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

The esterification and modification of n-3 and n-6 polyunsaturated fatty acids by hepatocytes and liver microsomes of turbot (Scophthalmus maximus)

The present study was undertaken to establish whether the formation of 22:6n-3 from 18:3n-3 and/or 20:5n-3 can occur in turbot liver and if this conversion is consistent with the operation of a Delta4 desaturase-independent pathway. At the same, time the effects of feeding a diet devoid of long chain polyunsaturated fatty acids (PUFA) on the patterns of esterification and modification of 18:3n-3, 20:5n-3 and 18:2n-6 by turbot hepatocytes and liver microsomes were examined. For this purpose, two groups of fish (25-30 g) were employed: one was fed a commercial diet containing fish oil (FO) and thus rich in long chain n-3 PUFA and the other was fed an experimental diet based on olive oil (OO). After 5 months of feeding, hepatocytes and liver microsomes isolated from individuals in the two groups of fish were incubated with [1-(14)C]-PUFA [either 18:3n-3, 20:5n-3 or 18:2n-6]. After 3 h of incubation, most radioactivity from all three radiolabelled substrates incorporated into lipids by hepatocytes and microsomes was recovered in the original substrate. The formation of desaturation products of n-3 radiolabelled substrates was higher in hepatocytes isolated from OO-fed than FO-fed fish. Small amounts of radiolabelled 22:6n-3 were formed from [1-(14)C]18:3n-3 and [1-(14)C]20:5n-3, but only by hepatocytes from fish fed OO, which also synthesised a small amount of radiolabelled 24:6n-3 from 14C-20:5n-3. Elongation products predominated over desaturation products in hepatic microsomes from both groups of fish studied, particularly in microsomes from fish fed FO. The results confirm that regardless of the long chain PUFA content of the diet, the production of 22:6n-3 in turbot liver from 18:3n-3 and/or 20:5n-3, and of 20:4n-6 from 18:2n-6, is very limited. The presence of radiolabelled 24:6n-3 in microsomes coupled with the absence of radiolabelled 22:6n-3 suggests that the formation of 22:6n-3 that does occur in turbot liver cells, may involve C24 intermediates and peroxisomal beta-oxidation.     

Effects of essential fatty acid deficiency and supplementation with docosahexaenoic acid (DHA; 22:6n-3) on cellular fatty acid compositions and fatty acyl desaturation in a cell culture model

The desaturation of [1-(14)C] 18:3n-3 to docosahexaenoic acid (DHA; 22:6n-3) is enhanced in an essential fatty acid deficient cell line (EPC-EFAD) in comparison with the parent cell line (EPC) from carp. In the present study, the effects of DHA on lipid and fatty acid compositions, and the metabolism of [1-(14)C]18:3n-3 were investigated in EPC-EFAD cells in comparison with EPC cells. DHA supplementation had only relatively minor effects on lipid content and lipid class compositions in both EPC and EPC-EFAD cells, but significantly increased the amount of DHA, 22:5n-3, eicosapentaenoic acid (EPA; 20:5n-3), total n-3 polyunsaturated fatty acids (PUFA), total PUFA and saturated fatty acids in total lipid and total polar lipid in both cell lines. Retroconversion of supplemental DHA to EPA was significantly greater in EPC cells. Monounsaturated fatty acids, n-9 and n-6PUFA were all decreased in total lipid and total polar lipid in both cell lines by DHA supplementation. The incorporation of [1-(14)C]18:3n-3 was greater into EPC-EFAD compared to EPC cells but DHA had no effect on the incorporation of [1-(14)C]18:3n-3 in either cell line. In contrast, the conversion of [1-(14)C]18:3n-3 to tetraenes, pentaenes and total desaturation products was similar in the two cell lines and was significantly reduced by DHA supplementation in both cell lines. However, the production of DHA from [1-(14)C]18:3n-3 was significantly greater in EPC-EFAD cells compared to EPC cells and, whereas DHA supplementation had no effect on the production of DHA from [1-(14)C]18:3n-3 in EPC cells, DHA supplementation significantly reduced the production of DHA from [1-(14)C] 18:3n-3 in EPC-EFAD cells. Greater production of DHA in EPC-EFAD cells could be a direct result of significantly lower levels of end-product DHA in these cells' lipids compared to EPC cells. Consistent with this, the suppression of DHA production upon DHA supplementation was associated with increased cellular and membrane DHA concentrations in EPC-EFAD cells. However, an increase in cellular DHA content to similar levels failed to suppress DHA production in DHA-supplemented EPC cells. A possible explanation is that greatly increased levels of EPA, derived from retroconversion of the added DHA, acts to offset the suppression of the pathway by DHA by stimulating conversion of EPA to DHA in DHA-supplemented EPC cells.     

NGF blocks polyunsaturated fatty acids biosynthesis in n-3 fatty acid-supplemented PC12 cells

Regulation of polyunsaturated fatty acid (PUFA) biosynthesis in proliferating and NGF-differentiated PC12 pheochromocytoma cells deficient in n-3 docosahexaenoic acid (DHA 22:6n-3) was studied. A dose- and time-dependent increase in eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3) and DHA in phosphatidylethanolamine (PtdEtn) and phosphatidylserine (PtdSer) glycerophospholipids (GPL) via the elongation/desaturation pathway following alpha-linolenic acid (ALA, 18:3n-3) supplements was observed. That was accompanied by a marked reduction of eicosatrienoic acid (Mead acid 20:3n-9), an index of PUFA deficiency. EPA supplements were equally effective converted to 22:5n-3 and 22:6n-3. On the other hand, supplements of linoleic acid (LNA, 18:2n-6) were not effectively converted into higher n-6 PUFA intermediates nor did they impair elongation/desaturation of ALA. Co-supplements of DHA along with ALA did not interfere with 20:5n-3 biosynthesis but reduced further elongation to 22-hydrocarbon PUFA intermediates. A marked decrease in the newly synthesized 22:5n-3 and 22:6n-3 following ALA or EPA supplements was observed after nerve growth factor (NGF)-induced differentiation. NGF also inhibited the last step in 22:5n-6 formation from LNA. These results emphasize the importance of overcoming n-3 PUFA deficiency and raise the possibility that growth factor regulation of the last step in PUFA biosynthesis may constitute an important feature of neuronal phenotype acquisition.     

The pathway from arachidonic to docosapentaenoic acid (20:4n-6 to 22:5n-6) and from eicosapentaenoic to docosahexaenoic acid (20:5n-3 to 22:6n-3) studied in testicular cells from immature rats

The concentration-dependent metabolism of 1-(14)C-labelled precursors of 22:5n-6 and 22:6n-3 was compared in rat testis cells. The amounts of [(14)C]22- and 24-carbon metabolites were measured by HPLC. The conversion of [1-(14)C]20:5n-3 to [3-(14)C]22:6n-3 was more efficient than that of [1-(14)C]20:4n-6 to [3-(14)C]22:5n-6. At low substrate concentration (4 microM) it was 3.4 times more efficient, reduced to 2.3 times at high substrate concentration (40 microM). The conversion of [1-(14)C]22:5n-3 to [1-(14)C]22:6n-3 was 1.7 times more efficient than that of [1-(14)C]22:4n-6 to [1-(14)C]22:5n-6 using a low, but almost equally efficient using a high substrate concentration. When unlabelled 20:5n-3 was added to a cell suspension incubated with [1-(14)C]20:4n-6 or unlabelled 22:5n-3 to a cell suspension incubated with [1-(14)C]22:4n-6, the unlabelled n-3 fatty acids strongly inhibited the conversion of [1-(14)C]20:4n-6 or [1-(14)C]22:4n-6 to [(14)C]22:5n-6. In the reciprocal experiment, unlabelled 20:4n-6 and 22:4n-6 only weakly inhibited the conversion of [1-(14)C]20:5n-3 and [1-(14)C]22:5n-3 to [(14)C]22:6n-3. The results indicate that if both n-6 and n-3 fatty acids are present, the n-3 fatty acids are preferred over the n-6 fatty acids in the elongation from 20- to 22- and from 22- to 24-carbon atom fatty acids. In vivo the demand for 22-carbon fatty acids for spermatogenesis in the rat may exceed the supply of n-3 precursors and thus facilitate the formation of 22:5n-6 from the more abundant n-6 precursors.     

An alternative n-3 fatty acid elongation pathway utilising 18:3n-3 in barramundi (Lates calcarifer)

Desaturase and elongase are two key enzyme categories in the long-chain polyunsaturated fatty acid (LCPUFA) pathway that convert dietary α-linolenic acid (18:3n-3) to docosahexaenoic acid (22:6n-3). The Δ6 desaturase is considered as rate limiting in the conversion. In a previous study in barramundi we demonstrated that the desaturase had a low Δ6 activity but noted that the enzyme also possessed Δ8 ability that utilised 20-carbon fatty acids. This observation suggests that an alternative pathway may exist in the barramundi via elongases to form 20-carbon metabolites from 18:3n-3 to 20:3n-3 and then Δ6/8 desaturase to 20:4n-3. Cloning of the barramundi elongation of very long-chain fatty acid gene (ELOVL) and heterologous expression of the corresponding elongase were performed to examine activity with regard to time course, substrate concentration and substrate preference. Results revealed that the barramundi elongase showed a broad range of substrate specificity including 18-carbon PUFA (including 18:3n-3 and 18:2n-6), 20- and 22-carbon LCPUFA, with greater activity towards omega-3 (n-3) than n-6 fatty acids. The findings from this study provide molecular evidence for an alternative n-3 fatty acid elongation pathway utilising 18:3n-3 in barramundi.     

Fatty acid metabolism in Atlantic salmon (Salmo salar L.) hepatocytes and influence of dietary vegetable oil

Isolated hepatocytes from Atlantic salmon (Salmo salar), fed diets containing either 100% fish oil or a vegetable oil blend replacing 75% of the fish oil, were incubated with a range of seven (14)C-labelled fatty acids. The fatty acids were [1-(14)C]16:0, [1-(14)C]18:1n-9, 91-(14)C]18:2n-6, [1-(14)C]18:3n-3, [1-(14)C]20:4n-6, [1-(14)C]20:5n-3, and [1-(14)C]22:6n-3. After 2 h of incubation, the hepatocytes and medium were analysed for acid soluble products, incorporation into lipid classes, and hepatocytes for desaturation and elongation. Uptake into hepatocytes was highest with [1-(14)C]18:2n-6 and [1-(14)C]20:5n-3 and lowest with [1-(14)C]16:0. The highest recovery of radioactivity in the cells was found in triacylglycerols. Of the phospholipids, the highest recovery was found in phosphatidylcholine, with [1-(14)C]16:0 and [1-(14)C]22:6n-3 being the most prominent fatty acids. The rates of beta-oxidation were as follows: 20:4n-6>18:2n-6=16:0>18:1n-9>22:6n-3=18:3n-3=20:5n-3. Of the fatty acids taken up by the hepatocytes, [1-(14)C]16:0 and [1-(14)C]18:1n-9 were subsequently exported the most, with the majority of radioactivity recovered in phospholipids and triacylglycerols, respectively. The major products from desaturation and elongation were generally one cycle of elongation of the fatty acids. Diet had a clear effect on the overall lipid metabolism, with replacing 75% of the fish oil with vegetable oil resulting in decreased uptake of all fatty acids and reduced incorporation of fatty acids into cellular lipids, but increased beta-oxidation activity and higher recovery in products of desaturation and elongation of [1-(14)C]18:2n-6 and [1-(14)C]18:3n-3.     

The biosynthesis of docosahexaenoic acid [22:6(n-3)] from linolenic acid in primary hepatocytes isolated from wild northern pike

Primary hepatocytes from wild northern pike Esox lucius were incubated with radiolabelled linolenic acid ([l-¹⁴C]-18:3(n-3)) to assess their ability to synthesize docosahexaenoic acid [22:6(n-3)]. The distribution of radioactivity in lipid classes and hepatocyte polyunsaturated fatty acids (PUFA) was measured over the time-course of 24h. The majority of radioactivity from [l-¹⁴C]-18:3(n-3) was recovered in hepatocyte triacylglycerols (TAG) and phosphatidylcholine (PC). The levels of radioactivity in TAG and in most of phospholipids, including PC, increased significantly over the incubation period. Radioactivity from [1-¹⁴C]-18:3(n-3) was recovered in several hepatocyte PUFA, including 22:6(n-3), and the Δ6 and Δ5-desaturation products 18:4(n-3) and 20:5(n-3). The presence of radioactivity in C₂₄ (n-3) PUFA may be evidence that the biosynthesis of 22:6(n-3) in pike proceeds via a pathway independent of Δ4-desaturation. Analysis by radio gas chromatography revealed that radiolabelled 24:6(n-3) was present among the desaturation and elongation products of [l-¹⁴C]-18:3(n-3). The results establish that, under the in vitro conditions employed, pike hepatocytes are able to convert linolenic acid to 20:5(n-3) and 22:6(n-3).     

Docosahexaenoic acid synthesis from n-3 polyunsaturated fatty acids in differentiated rat brain astrocytes

DHA, the main n-3 PUFA in the brain, is synthesized from n-3 PUFA precursors by astrocytes. To assess the potential of this process to supply DHA for the brain, we investigated whether the synthesis in astrocytes is dependent on DHA availability. Rat brain astrocytes differentiated with dibutyryl cAMP and incubated in media containing 10% fetal bovine serum synthesized DHA from alpha-linolenic acid ([1-(14)C]18:3n-3), docosapentaenoic acid ([3-(14)C]22:5n-3), tetracosapentaenoic acid ([3-(14)C]24:5n-3), and tetracosahexaenoic acid ([3-(14)C]24:6n-3). When DHA was added to media containing a 5 microM concentration of these (14)C-labeled n-3 PUFA, radiolabeled DHA synthesis was reduced but not completely suppressed even when the DHA concentration was increased to 15 microM. Radiolabeled DHA synthesis also was reduced but not completely suppressed when the astrocytes were treated with 30 microM DHA for 24 h before incubation with 5 microM [1-(14)C]18:3n-3.These findings indicate that although the DHA synthesis in astrocytes is dependent on DHA availability, some synthesis continues even when the cells have access to substantial amounts of DHA. This suggests that DHA synthesis from n-3 PUFA precursors is a constitutive process in the brain and, therefore, is likely to have an essential function.     

Incorporation of 14C-labelled polyunsaturated fatty acids by juvenile turbot, Scophthalmus maximus (L.) in vivo

The ability of juvenile turbot, Scophthalmus maximus (L.), to elongate and desaturate various polyunsaturated fatty acids (PUFA) was examined in relation to their lipid composition. Triacylglycerols were the most abundant lipid class present in the fish and phosphatidylcholine was the predominant phospholipid. In all lipid classes examined the levels of (n-3) PUFA exceeded that of (n-6) PUFA. 18C PUFA were minor components in comparison with 20:5(n-3) and 22:6(n-3). 20:4(n-6) was present in highest concentration in phosphatidylinositol in which it accounted for 16.9% of the fatty acids. When the fish were injected with either ¹⁴C-labelled 18:2(n-6), 18:3(n-3), 20:4(n-6), 20:5(n-3) or 22:6(n-3) the highest percentage recovery of radioactivity (69%) in body lipid was observed with 22:6(n-3). With all labelled substrates free fatty acids contained only a small proportion of the total recovered radioactivity whereas triacylglycerols were highly labelled. Phosphatidylcholine/sphingomyelin was the most highly labelled polar lipid fraction. With ¹⁴C-20:4(n-6) as injected substrate, 23.2% of the radioactivity recovered in total lipid was present in phosphatidylinositol in comparison with less than 6% with the other substrates. Only small proportions of radioactivity from ¹⁴C-18:2(n-6) and ¹⁴C-18:3(n-3) were recovered in the 20 and 22C fatty acids of triacylglycerols and total polar lipid. With ¹⁴C-20:5(n-3) as substrate, 27 and 33% of the total radioactivity recovered in the fatty acids of triacylglycerols and polar lipids respectively was present in 22C fatty acids. The corresponding values for ^l4C-20:4(n-6) as substrate were 19 and 18%. The results confirm the limited capacity of turbot to convert 18C PUFA to longer chain PUFA but demonstrate their ability to synthesize 22C PUFA from 20C PUFA. They also suggest a small but specific requirement for 20:4(n-6).     

Cryptophyceae and rhodophyceae; chemotaxonomy, phylogeny, and application

The biochemical compositions of seven strains of marine cryptomonad and a rhodophyte were determined in logarithmic phase batch (1.4 L flask) and semi-continuous (10 L carboy) culture. Lipid ranged from 13% to 28%, protein ranged from 53% to 68%, and carbohydrate ranged from 9% to 24% of the organic weight. The major lipid classes in the species examined were polar lipids (78-88% of total lipid). The major sterol in the Cryptophyceae and the Rhodophyceae was 24-methylcholesta-5,22E-dien-3beta-ol (62-99% of total sterols); which is also the major sterol in some diatoms and haptophytes. Smaller proportions of cholest-5-en-3beta-ol (1-17.7%) were also found in the Cryptophyceae. Most cryptomonads contained high proportions of the n-3 polyunsaturated fatty acids (PUFA), 18:3n-3 (20.7-29.9% of the total fatty acids), 18:4n-3 (12.5-30.2%), 20:5n-3 (7.6-13.2%) and 22:6n-3 (6.4-10.8%). However, the blue-green cryptomonad Chroomonas placoidea was characterized by a low proportion of 22:6n-3 (0.2% of total fatty acids), and a significant proportion of 22:5n-6 (4.5%), and the presence of 24-ethylcholesta-5,22E-dien-3beta-ol (35.5% of total sterols). The fatty acid composition of the rhodophyte Rhodosorus sp. was similar to those of the Cryptophyceae except for lower proportions of 18:4n-3 and lack of C21 and C22 PUFA. It is postulated that the primary endosymbiosis of a photosynthetic n-3 C18 PUFA-producing prokaryote and a eukaryotic host capable of chain elongation and desaturation of exogenous PUFA, resulted in the Rhodophyceae capable of producing n-3 C20 PUFA. The secondary endosymbiosis of a photosynthetic n-3 C20 PUFA-producing eukaryote (such as a Rhodosorus sp. like-rhodophyte) and a eukaryotic host capable of further chain elongation and desaturation, resulted in the Cryptophyceae being capable of producing n-3 C20 and C22 PUFA de novo. Selected isolates were examined further in feeding trials with juvenile Pacific oysters (Crassostrea gigas). Rhodomonas salina CS-24(containing elevated 22:6n-3) produced high growth rates in oysters; equivalent to the microalga commonly used in aquaculture, Isochrysis sp. (T.ISO).     

Incorporation into phospholipid classes and metabolism via desaturation and elongation of various 14C-labelled (n-3) and (n-6) polyunsaturated fatty acids in trout astrocytes in primary culture

The incorporation and metabolism of [1-14C]18:3(n-3), [1-14C]20:5(n-3), [1-14C]18:2(n-6), and [1-14C]20:4(n-6) were studied in primary cultures of trout brain astrocytes. There were no significant differences between the amounts of individual fatty acids incorporated into total lipid at 22 degrees C, with greater than 90% of all the fatty acids being incorporated into polar lipid classes. The distributions of 18:2(n-6), 18:3(n-3), and 20:5(n-3) in individual phospholipid classes at 22 degrees C were very similar, with 57-63 and 18-24% being incorporated into phosphatidylcholine and phosphatidylethanolamine, respectively. Approximately equal amounts of 20:4(n-6), approximately 30% of the total, were incorporated into each of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. The metabolism of the (n-3) fatty acids to longer-chain and more unsaturated species was significantly greater than that of (n-6) acids, but delta 4-desaturase activity was very low. A culture temperature of 10 degrees C increased the incorporation of all the fatty acids into total lipid and that of C20 fatty acids into polar lipid. At 10 degrees C, the incorporation of C20 fatty acids into phosphatidylethanolamine and phosphatidylinositol was increased, and the incorporation into phosphatidylcholine and phosphatidylserine was decreased. The distribution of C18 fatty acids was unchanged at the lower temperature, as was the desaturation and elongation of all the polyunsaturated fatty acids incorporated.     

Metabolism of hexacosatetraenoic acid (C26:4,n-6) in immature rat brain.

Rat brain was recently found to contain polyenoic very-long-chain fatty acids (VLCFA) belonging to the n-3 and n-6 series with four, five and six double bonds and even-carbon chain lengths from 24 to 38 [Robinson, Johnson & Poulos (1990) Biochem. J. 265, 763-767]. In the present paper, the metabolism in vivo of hexacosatetraenoic acid (C26:4,n-6) was studied in neonatal rat brain. Rats were injected intracerebrally with [1-14C]C26:4,n-6 and the labelled metabolites were examined after 4 h. Radioactivity was detected mainly in non-esterified fatty acids, with smaller amounts in other neutral lipids and phospholipids. Radiolabelled fatty acid products included C28-36 tetraenoic and C26-28 pentaenoic VLCFA formed by elongation and desaturation of the substrate, and C14-24 saturated, C16-24 monoenoic, C18-24 dienoic, C18-22 trienoic and C20-24 tetraenoic fatty acids formed from released [1-14C]acetate either by synthesis de novo or by elongation of endogenous fatty acids. The data suggest that polyenoic VLCFA are synthesized in brain from shorter-chain precursor fatty acids and undergo beta-oxidation.     

C19 odd-chain polyunsaturated fatty acids (PUfas) are metabolized to C21-PUfas in a rat liver cell line, and curcumin, gallic acid, and their related compounds inhibit their desaturation

It was demonstrated that the rat liver cell line BRL-3A converted exogenous C19 odd chain-polyunsaturated fatty acids (PUFAs) into the corresponding C21- and C23-PUFAs as follows: 21:3n-8, 21:4n-8, 23:3n-8, and 23:4n-8 (from 19:3n-8); 21:4n-5, 21:5n-5, 23:4n-5, and 23:5n-5 (from 19:4n-5); 21:5n-2, 21:6n-2, 23:5n-2, and 23:6n-2 (from 19:5n-2). It presumed that these C19 PUFAs were converted through the mimic route to docosahexaenoic acid (22:6n-3) from eicosapentaenoic acid (20:5n-3). In addition, the characterization of the change of fatty acid composition of cellular lipids in rat liver cells were examined, using 19:4n-5 and several fatty acid desaturation inhibitors. Curcumin related compounds, curcumin, capsaicin, isoeugenol, 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, and gallic acid esters with near five carbon numbered alcohol had great changes of fatty acid composition of cellular lipids based on inhibition of the A6 desaturation of C24-PUFAs in rat liver cells.     

Fatty acid utilisation and metabolism in caecal enterocytes of rainbow trout (Oncorhynchus mykiss) fed dietary fish or copepod oil

A combined fatty acid metabolism assay was employed to determine fatty acid uptake and relative utilisation in enterocytes isolated from the pyloric caeca of rainbow trout. In addition, the effect of a diet high in long-chain monoenoic fatty alcohols present as wax esters in oil derived from Calanus finmarchicus, compared to a standard fish oil diet, on caecal enterocyte fatty acid metabolism was investigated. The diets were fed for 8 weeks before caecal enterocytes from each dietary group were isolated and incubated with [1-14C]fatty acids: 16:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:1n-9, 20:4n-6, 20:5n-3, and 22:6n-3. Uptake was measured over 2 h with relative utilisation of different [1-14C]fatty acids calculated as a percentage of uptake. Differences in uptake were observed, with 18:1n-9 and 18:2n-6 showing the highest rates. Esterification into cellular lipids was highest with 16:0 and C18 fatty acids, accounting for over one-third of total uptake, through predominant incorporation in triacylglycerol (TAG). The overall utilisation of fatty acids in phospholipid synthesis was low, but highest with 16:0, the most prevalent fatty acid recovered in intracellular phosphatidylcholine (PC) and phosphatidylinositol (PI), although exported PC exhibited higher proportions of C20/C22 polyunsaturated fatty acids (PUFA). Other than 16:0, incorporation into PC and PI was highest with C20/C22 PUFA and 20:4n-6 respectively. Recovery of labelled 18:1n-9 in exported TAG was 3-fold greater than any other fatty acid which could be due to multiple esterification on the glycerol 'backbone' and/or increased export. Approximately 20-40% of fatty acids taken up were beta-oxidised, and was highest with 20:4n-6. Oxidation of 20:5n-3 and 22:6n-3 was also surprisingly high, although 22:6n-3 oxidation was mainly attributed to retroconversion to 20:5n-3. Metabolic modification of fatty acids by elongation-desaturation was generally low at <10% of [1-14C]fatty acid uptake. Dietary copepod oil had generally little effect on fatty acid metabolism in enterocytes, although it stimulated the elongation and desaturation of 16:0 and elongation of 18:1n-9, with radioactivity recovered in longer n-9 monoenes. The monoenoic fatty acid, 20:1n-9, abundant in copepod oil as the homologous alcohol, was poorly utilised with 80% of uptake remaining unesterified in the enterocyte. However, the fatty acid composition of pyloric caeca was not influenced by dietary copepod oil.     

Polyunsaturated fatty acid biosynthesis from [1-14C]20:3 n-6 acid in rat cultured Sertoli cells. Linoleic acid effect

Primary culture is a suitable system to study lipid metabolism and polyunsaturated fatty acid biosynthesis. Sertoli cell-enriched preparations were used to determine the fatty acid composition after 5 and 7 days in culture (serum free) as well as the uptake and metabolism of [1-14C]eicosa-8,11,14-trienoic acid. The addition of unlabeled linoleic acid (0.2 and 2.0 microg/ml) was also evaluated. Fatty acid methyl esters derived from cellular lipids were analyzed by gas liquid chromatography and radiochromatography. After 5 days in culture, cells had significantly less 18:2, 20:4, 22:5 and 24:5 and more 18:3, 20:3, 22:4 and 24:4 n-6 fatty acids than non-cultured cells. On day 7, an additional increment in 22:4 n-6 and a decrease in linoleic, gamma-linoleic and 24:4 n-6 fatty acids were observed. The presence of linoleic acid (low dose) produced a significant decrease in saturated and monounsaturated acids and an increase in 18:2, 20:4 and 22:5 n-6 fatty acids. At a high concentration almost all fatty acids belonging to 18:2 n-6 increased significantly. The drop in 20:4 n-6/20:3 n-6 ratio was considered as an indirect evidence of a Delta 5 desaturase activity depression. This assumption was corroborated by studying the transformation of [1-14C]eicosa-8,11,14-trienoic acid into 20:4, 22:4, 22:5, 24:4 and 24:5 n-6 fatty acids. We conclude that Sertoli cells after 7 days in culture evidenced changes in the fatty acid profile similar to those described under fat deprivation. The addition of linoleic acid reverted this pattern and indicated that the Delta 5 desaturase activity is a limiting step in the polyunsaturated fatty acid biosynthesis.     

Changes in the fatty acid composition of phospholipids from turbot (Scophthalmus maximus) in relation to dietary polyunsaturated fatty acid deficiencies

Young turbot (1-20 g) were maintained for not less than 14 weeks on three diets: (1) a control diet containing normal amounts of polyunsaturated fatty acids (PUFA); (2) a diet totally deficient in PUFA; (3) a diet deficient in the (n-6) series of PUFA but containing (n-3) PUFA. At 14 weeks the fatty acid compositions of the phospholipids from liver, gut, gills and muscle were analysed. Large changes in the amounts of PUFA in the phospholipids were found. Fish maintained on the totally PUFA deficient diet 2 had retained arachidonic acid, 20:4(n-6), and docosahexaenoic acid, 22:6(n-3), at the expense of eicosapentaenoic acid, 20:5(n-3). Fish maintained on the (n-6) PUFA-deficient diet (3) contained decreased amounts of 20:4(n-6) and 22:6(n-3) while retaining 20:5(n-3). In all cases phosphatidylinositol had the lowest n-3/n-6 ratios. These results are discussed in terms of PUFA function.