Incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids in phospholipid classes in cultured turbot (Scophthalmus maximus) cells

The incorporation and metabolism of various (n-3) and (n-6) polyunsaturated fatty acids (PUFA) supplemented to the culture medium was investigated in a turbot cell line (TF). The distribution, and the occurrence and extent of further metabolism of incorporated PUFA via desaturation/elongation mechanisms in specific phospholipid classes was determined from the different fatty acid compositions. The cells contained Δ6 and Δ4 desaturase activities but were generally deficient in C18–20 elongase activity. Δ5 Desaturase activity was generally masked by this deficiency but was present. The compositional data indicated that there was a high degree of specificity between individual phospholipid classes and particular fatty acids probably driven by the specificities of the acylating enzymes. The highest percentages of the supplemented acids were generally observed in the phosphatidic acid/cardiolipin fraction (PA/CL), suggesting a role for PA in the incorporation of the supplemented acids into the phospholipid pool. PI had a characteristic composition consistent with a putative role as a pool of precursor fatty acid for eicosanoid synthesis. Mechanisms were evident for generating and/or maintaining this composition.     

Effects of dietary lipids on the fatty acid composition of triglycerides and phospholipids in tissues of white sturgeon

Eight purified diets were fed to juvenile white sturgeon, Acipenser transmontanus Rick, for 9 weeks to investigate the effect of dietary lipids on the fatty acid composition of phospholipids and triglycerides from muscle, liver and brain. The diets contained 150 g kg^?1 of oils from canola, corn, cod liver, lard, linseed, soybean, safflower, or a control mixture (corn oil/cod liver oil/lard, 1:1:1, by wt). Dietary lipids significantly (P≤ 05) affected the composition of tissue triglycerides and phospholipids. Tissue triglyceride fatty acid composition ranged widely, in parallel with the dietary lipids, while phospholipids changes were more conservative. Brain phospholipid fatty acid composition was less responsive to diet compared with that in muscle and liver. Considerable amounts of n-6 and n-3 long chain polyun-saturated fatty acids (> C₂₀) were found in triglycerides and phospholipids with all diets, demonstrating that white sturgeon can desaturate and elongate linoleic acid (18:2n–6) and linolenic acid (18:3n–3). Further, the products of the Δ6 desaturase, i.e. 18:3 n–6 and 18:4n–3, were relatively abundant in triglyceride, suggesting that the Δ6 desaturase might not be a limiting step in the process in white sturgeon. Nevertheless, accumulation of both EPA and DHA was greater in the sturgeon fed fish oil than those fed linseed oil, indicating that muscle triglyceride EPA and DHA levels are best enhanced by diets rich in preformed EPA and DHA.     

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     

两种复海鞘油脂中的脂肪酸成分分析

不饱和脂肪酸尤其是ω-3多不饱和脂肪酸是对人类健康具有重要意义的一类营养成分。对海鞘(Aplidium constellatum)和大洋纵列海鞘(Symplegma oceania)两种复海鞘的油脂类成分进行相对含量分析,发现海鞘(A.constellatum)油脂中含有EPA、DHA两种ω-3 PUFAs,含量分别为19.5%和12.7%,大洋纵列海鞘油脂中EPA、DHA的含量分别为18.2%和7.73%。结果表明,两种海鞘油脂均具有较好的开发前景。     

重组抗菌肽的制备及其对水产养殖中常见病原菌的抑菌效果

采用YEPD培养基诱导发酵生产重组抗菌肽。经 72h培养 ,由重组酵母表达并分泌到培养基中的抗菌肽活性可高达 5 0 0 0U/ml。用纸片扩散法和试管稀释法测定重组抗菌肽对水产养殖常见病原菌的抑菌效果。结果表明 ,抗菌肽对水产养殖常见病原菌嗜水气单胞菌 (Aeromonashydrophila)、梅氏弧菌 (Vibriometschnikovi)、迟钝爱德华氏菌 (Edwardsiellatarda)及温和气单胞菌 (A .sobria)有较好的抑菌效果 ,最小抑菌质量浓度为 4 μg/ml、杀菌质量浓度为 8μg/ml。对抗菌肽抑菌过程的超微结构观察表明 ,抗菌肽的抑菌方式是通过作用于细菌的细胞膜而导致菌体裂解死亡。本研究目的在于探讨基因工程抗菌肽在水产养殖中的应用。     

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.     

Essential fatty acids in Atlantic salmon: effects of increasing dietary doses of n-6 and n-3 fatty acids on growth, survival and fatty acid composition of liver, blood and carcass

Atlantic salmon fry (4 g) were fed for 4 months on semi-synthetic diets containing fatty acid methyl esters of either 18:2 n-6, 18:3 n-3 or a mixture of equal amounts of 20:5 n-3 and 22:6 n-3. The different amounts of polyunsaturated fatty acids added were 0, 0.1, 0.2, 0.5, 1 and 2% (by dry weight). Increasing levels of dietary n-3 fatty acids up to 1% gave faster growth rates in salmon fry, and fish fed the mixture of 20:5 n-3 and 22:6 n-3 seemed to grow faster than fish fed only 18:3 n-3. No significant effect on growth rate was seen when the dietary level of 18:2 n-6 was increased. Dietary inclusions of n-3 fatty acids reduced the mortality of salmon, while dietary 18:2 n-6 had no such beneficial effects.
The dietary treatments caused substantial changes in the fatty acid composition of blood and liver phospholipids (PL), whereas the total lipid fraction of the carcass was less affected. Increasing doses of 18:2 n-6 in the diet resulted in an increased percentage of 20:4 n-6 in liver and blood PLs, while the percentage of 20:3 n-9 decreased. The percentage of 18:2 n-6 also increased in liver, blood and carcass. Dietary 18:3 n-3 resulted in increased percentages of 18:3 n-3 and 20:5 n-3 in liver PLs, while the percentage of 20:3 n-9 decreased. There was, however, no significant increase in the percentage of 22:6 n-3. Dietary 18:3 n-3 produced no significant changes in the composition of blood fatty acids, but increased the percentage of 18:3 n-3 in the carcass. The dietary combination of the n-3 fatty acids 20:5 and 22:6 resulted in an increased percentage of 22:6 n-3 in blood and liver lipids and a decreased percentage of 20:3 n-9, but there were no changes in the percentage of 20:5 n-3.     

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.     

The desaturation and elongation of 14C-labelled polyunsaturated fatty acids by pike (Esox lucius L.) in vivo

To examine the ability of pike (Esox lucius L.) to modify exogenous PUFA by desaturation and elongation, ¹⁴C-labelled 18:2(n-6), 18:3(n-3), 20:4(n-6) and 20:5(n-3) were injected intraperitoneally and the distribution of radioactivity in tissue lipid classes and liver PUFA measured. In all tissues examined, radioactivity from all ¹⁴C-PUFA was recovered in many classes of acyl lipids and the level of recovery generally reflected the relative abundance of the lipid classes. Triacylglycerols, CGP and EGP usually contained high levels of all incorporated ¹⁴C-PUFA. PI contained higher levels of radioactivity from ¹⁴C-20:4(n-6) than from other injected substrates. In liver lipid, the 6 desaturation products of ¹⁴C-18:2(n-6) and ¹⁴C-18:3(n-3) contained no measurable radioactivity although the elongation products of the 6 desaturation products were labelled, as were the direct elongation products of these injected substrates. No radioactivity from ¹⁴C-18:2(n-6) or ¹⁴C-18:3(n-3) was detected in C₂₀ or C₂₂ products of 5 and 4 desaturation. Almost all radioactivity from injected ¹⁴C-20:4(n-6) was recovered in this PUFA. Of the total radioactivity from ¹⁴C-20:5(n-3) incorporated into liver lipid, 7% was present as 24:5 and 16.4% was recovered in hexaenoic fatty acids. In liver, 24:5(n-3) and 24:6(n-3) each accounted for 1% of the mass of total fatty acids and were located almost exclusively in triacylglycerols. The presence of radioactivity in these C₂₄ PUFA suggests that in pike the synthesis of 22:6(n-3) from 20:5(n-3) may proceed without 4 desaturase via the pathway which involves chain shortening of 24:6(n-3). It is concluded that under the circumstances employed in this study pike, do not exhibit 5 desaturase activity and are unable to synthesize 20:4(n-6) and 20:5(n-3) from 18:2(n-6) and 18:3(n-3), respectively. This suggests that pike may require 20:4(n-6) and 20:5(n-3) preformed in the diet.Abbreviations CGP choline glycerophospholipids - CL cardiolipin - EGP ethanolamine glycerophospholipids - PG phosphatidylglycerol - PI phosphatidylinositol - PS phosphatidylserine - PUFA polyunsaturated fatty acids - SM sphingomyelin - TLC thin-layer chromatography     

Development of intimal thickening of coronary arteries over the lifetime of Atlantic salmon, Salmo salar L., fed different lipid sources

The objective of the present study was to investigate the development of intimal changes of coronary arteries over the lifetime of farmed Atlantic salmon, Salmo salar L., fed either a 100% fish oil or a 100% vegetable oil blend. The study was performed as a randomized observer blinded controlled trial with parallel group design. At the start of the project, the fish were divided in two groups and sampled at five different time points throughout their life span. The total study sample consisted of 259 healthy fish. Serial sections were taken from the coronary artery lying on the bulbus arteriosus for histopathological evaluation and for area measurements using semi-quantitative and quantitative methods. The earliest onset of vascular changes was detected in fish from both groups in the freshwater stage prior to smoltification. The mean range lesion (MRL), used to describe the severity of the lesions observed, increased significantly for both groups from sea transfer throughout the study period. Comparison of the two groups based on the overall material corrected for time of sampling did not show any difference (P = 0.20) between the two groups with regard to MRL. The percentage lumen loss (PLL) measured by a quantitative method and used as a measure to indicate lesion severity showed an incremental, non-significant increase from week 72 to week 92 and further to week 115 in both diet groups during the seawater phase. Comparison of the groups corrected for time of sampling indicated a difference of PLL in favour of VO (P = 0.02). Heart weight, body weight and body length were all positively and significantly correlated to Log MRL. The partial correlation analysis indicated that heart weight was the most dominant variable in the set. Early vascular changes were found in the major bifurcation of the coronary artery at the apex and beyond the flow divider into the daughter branches. The latter represented the dominant changes and were found throughout the entire lifecycle of the fish. Increasing in size over time they formed pads or cushions that were regularly located close to the outer walls of the bifurcation. The origin of the cells forming the intimal thickening has not been conclusively determined, but immunohistochemical findings indicate a smooth muscle cell origin, possibly of a myointimal type. Our findings suggest there is no correlation between diet and intimal changes. The severity of the changes, MRL and PLL, of the coronary vessels correlate with heart weight and fish weight growth and growth rate and mechanical factors are implicated in intimal development, but rather than being induced by external injury due to the location of the coronaries, haemodynamic factors and low shear stress are proposed as the main mechanism behind these changes.     

Essential fatty acid deficiency in freshwater fish: the effects of linoleic, α-linolenic, γ-linolenic and stearidonic acids on the metabolism of [1-14C]18:3n-3 in a carp cell culture model

The desaturation of [1-¹⁴C]18:3n-3 to 20:5n-3 and 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 competing, unlabeled C₁₈ polyunsaturated fatty acids (PUFA), linoleic (18:2n-6), -linolenic (18:3n-3), -linolenic (18:3n-6) and stearidonic (18:4n-3) acids, on the metabolism of [1-¹⁴C]18:3n-3 were investigated in EPC-EFAD cells in comparison with EPC cells. The incorporation of [1-¹⁴C]18:3n-3 in both cell lines was significantly reduced by competing C₁₈ PUFA, with the rank order being 18:4n-3>18:3n-3 = 18:2n-6>18:3n-6. In the absence of competing PUFA, radioactivity from [1-¹⁴C]18:3n-3 in EPC cells was predominantly recovered in phosphatidylethanolamine followed by phosphatidylcholine. This pattern was unaffected by competing n-6PUFA, but n-3PUFA reversed this pattern as did essential fatty acid deficiency in the presence of all competing PUFA. The altered lipid class distribution was most pronounced in cells supplemented with 18:4n-3. Competing C₁₈ PUFA significantly decreased the proportions of radioactivity recovered in 22:6n-3, pentaene and tetraene products, with the proportions of radioactivity recovered in 18:3n-3 and 20:3n-3 increased, in both cell lines. However, the inhibitory effect of competing C₁₈ PUFA on the desaturation of [1-¹⁴C]18:3n-3 was significantly greater in EPC-EFAD cells. The magnitude of the inhibitory effects of C₁₈ PUFA on [1-¹⁴C]18:3n-3 desaturation was dependent upon the specific fatty acid with the rank order being 18:4n-3>18:3n-3>18:2n-6, with 18:3n-6 having little inhibitory effect on the metabolism of [1-¹⁴C]18:3n-3 in EPC cells. The differential effects of the C₁₈ PUFA on [1-¹⁴C]18:3n-3 metabolism were consistent with mass competition in combination with increased desaturation activity in EPC-EFAD cells and the known substrate fatty acid specificities of desaturase enzymes. However, the mechanism underpinning the greater efficacy with which the unlabeled C₁₈PUFA competed with [1-¹⁴C]18:3n-3 in the desaturation pathway in EPC-EFAD cells was unclear.