Dietary Docosahexaenoic Acid (22:6) Incorporates into Cardiolipin at the Expense of Linoleic Acid (18:2): Analysis and Potential Implications

Cardiolipin is a signature phospholipid of major functional significance in mitochondria. In heart mitochondria the fatty acid composition of cardiolipin is commonly viewed as highly regulated due to its high levels of linoleic acid (18:2n − 6) and the dominant presence of a 4×18:2 molecular species. However, analysis of data from a comprehensive compilation of studies reporting changes in fatty acid composition of cardiolipin in heart and liver mitochondria in response to dietary fat shows that, in heart the accrual of 18:2 into cardiolipin conforms strongly to its dietary availability at up to 20% of total dietary fatty acid and thereafter is regulated. In liver, no dietary conformer trend is apparent for 18:2 with regulated lower levels across the dietary range for 18:2. When 18:2 and docosahexaenoic acid (22:6n − 3) are present in the same diet, 22:6 is incorporated into cardiolipin of heart and liver at the expense of 18:2 when 22:6 is up to ~20% and 10% of total dietary fatty acid respectively. Changes in fatty acid composition in response to dietary fat are also compared for the two other main mitochondrial phospholipids, phosphatidylcholine and phosphatidylethanolamine, and the potential consequences of replacement of 18:2 with 22:6 in cardiolipin are discussed.     

Dietary fatty acid composition induces comparable changes in cardiolipin fatty acid profile of heart and brain mitochondria

The fatty acid profile of cardiolipin (CL) from brain and cardiac mitochondria was measured to determine whether CL isolated from these two tissue sources responded similarly to alterations in dietary fat composition. Male Wistar rats were fed 20% (w/w) diets containing 2 to 12% (w/w) 18∶2n-6 for four weeks. Despite higher baseline levels of CL 18∶2n-6 in cardiac (54±1% of total fatty acids) compared to brain (13±1%) mitochondria, CL 18∶2n-6 levels increased in proportion to dietary 18∶2 levels. The degree of change in 18∶2n-6 was comparable with both tissues showing an approximate 1.5- to 2-fold increase. The time course of changes in CL fatty acid profile was examined in a subsequent experiment in which animals were fed 20% (w/w) fat diets containing either 3 or 15% α-linoleate. Changes in cardiac CL 18∶1, 18∶2n-6, and 22∶6n-3 levels were observed within one week of feeding. While statistically significant differences were not observed in brain CL until the second week of feeding, the time course did not differ substantively from that observed in heart. The results from this study suggest that while baseline fatty acid profile of cardiac and neural CL differ, mitochondria from both tissues show comparable sensitivity to changes in dietary fat composition. Furthermore, it would appear that the turnover rate of fatty acids in CL is similar in both tissues.     

Dietary long-chain polyunsaturated fatty acids modify heart,kidney, and lung fatty acid composition in weanling rats

The fatty acid composition of heart, kidney, and lung was studied in weanling rats fed three diets differing in their polyunsaturated fatty acid content for 0, 2, and 4 wk. The first group had a 10% w/w fat semipurified diet which consisted of a mixture of olive oil (62.5%), soybean oil (11.1%), and refined coconut oil (26.4%) and provided 18:1n-9, 18:2n-6, and 18:3n-3 in similar amounts to a maternal human milk (diet HO). The second group received 7% of HO fat and 3% fish oil (0.4% 20:4n-6 and 5% 22:6n-3 of total fatty acids) (diet FO), and the third group was fed 7% HO fat, 1.5% of the same fish oil, and 1.5% of a purified pig brain phospholipid concentrate (0.6% 20:4n-6 and 3.5% 22:6n-3 of total fatty acids) (diet FO+BPL). The experimental diets increased tissue monounsaturated fatty acids in comparison with rats at weaning. Tissue lipid content of 20:4n-6 was increased and 22:6n-3 decreased in Group HO compared with weanling rats, whereas opposite changes were observed in Group FO. Feeding diet FO+BPL increased 22:6n:3 in tissue lipids compared with diet HO, and increased 20:4n-6 content in relation to diet FO. Our results indicate that rat heart, kidney, and lung are highly responsive to dietary n-3 and n-6 long-chain polyunsaturated fatty acids during postnatal life.     

Dietary arachidonic acid and hepatic desaturation of fatty acids in obese zucker rats

The effect of low levels of dietary arachidonic acid (20:4n-6) on Δ6 desaturation of linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3), and on Δ5 desaturation of dihomo-γ-linolenic acid (20:3n-6) were studied in liver microsomes of obese Zucker rats, in comparison with their lean littermates. Fatty acid composition of serum total lipids and of phospholipids from liver microsomes and from total heart and kidney was determined to see whether modifications of desaturation rate, if any, were reflected in the tissue fatty acid profiles. Animals fed for 12 wk on a balanced diet, containing 20:4n-6 and 18:2n-6, were compared to those fed 18:2n-6 only. The low amount of dietary 20:4n-6 greatly inhibited Δ6 desaturation of 18:2n-6 and Δ5 desaturation of 20:3n-6, whereas Δ6 desaturation of 18:3n-3 was slightly increased in obese rats. Inhibition of the biosynthesis of long-chain n-6 fatty acids by dietary arachidonic acid was only slightly reflected in the 20:4n-6 content of liver microsome phospholipids. On the contrary, the enrichment of serum total lipids and heart and kidney phospholipids in this fatty acid was pronounced, more in obese than in lean animals. Our results show that, although the desaturation rate of the n-6 fatty acids in liver microsomes was greatly decreased by the presence of arachidonic acid in the diet, the tissue phospholipid content in arachidonic acid was not depressed. The potentiality of synthesis of eicosanoids of the 2 family from this fatty acid is consequently not lower, especially in obese rats, in which certain tissues are deficient in arachidonic acid, in comparison with their lean littermates.     

Trans-Membrane Uptake and Intracellular Metabolism of Fatty Acids in Atlantic Salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) Hepatocytes

To elucidate if the trans-membrane uptake of fatty acids is protein-mediated, the uptake of oleic acid (18:1n-9), linoleic acid (18:2n-6), alpha-linolenic acid (18:3n-3), eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) was investigated in vitro in Atlantic salmon (Salmo salar L.) primary hepatocytes. Firstly, optimal fatty acid incubation time and concentration were established for trans-membrane 18:n-9 uptake. Based on saturation kinetics, a 2-h incubation time and 37.5 μM were used for the following experiments. Secondly, in order to identify whether trans-membrane fatty acid uptake in hepatocytes was mainly passive or protein mediated, hepatocytes were pre-incubated with membrane protein inhibitors followed by 2 h of incubation with [1-¹⁴C] labelled 18:1n-9, 18:2n-6, 18:3n-3, 20:5n-3 and 22:6n-3. Fatty acid uptake into hepatocytes was highest with 20:5n-3 and 22:6n-3 and lowest with 18:1n-9. Phloretin was the most potent fatty acid uptake inhibitor, inhibiting uptake in the following order: 20:5n-3 > 18:3n-3 = 22:6n-3 > 18:2n-6 > 18:1n-9. The uptake of FA in Atlantic salmon hepatocytes seem to be due to both saturable and inhibitable protein mediated uptake, as well as passive uptake processes with more unsaturated and long fatty acids (20:n-3 > 22:6n-3 = 18:3n-3 > 18:2n-6) being more dependent on membrane protein mediated uptake compared to 18:1n-9.     

Dietary Source of Stearidonic Acid Promotes Higher Muscle DHA Concentrations than Linolenic Acid in Hybrid Striped Bass

Rapid expansion of aquacultural production is placing increasing demand on fish oil supplies and intensified the search for alternative lipid sources. Many of the potential alternative sources contain low concentrations of long chain n-3 fatty acids and the conversion of dietary linolenic acid to longer chain highly unsaturated fatty acids is a relatively inefficient process in some species. A 6-week study was conducted to compare tissue fatty acid (FA) concentrations in hybrid striped bass fed either 18:3n-3 (α-linolenic acid; ALA) or 18:4n-3 (stearidonic acid; SDA). Hybrid striped bass were fed either a control diet containing fish oil, or diets containing ALA or SDA at three different levels (0.5, 1 and 2% of the diet). There were no significant differences in whole animal responses between fish fed ALA or SDA. Liver and muscle concentrations of ALA and SDA were responsive to dosages fed. However, only 22:6n-3 concentrations in muscle were significantly affected by dietary source of 18 carbon precursors. Muscle 22:6n-3 concentrations were significantly higher in fish fed SDA compared to fish fed ALA. Based on these data, it appears that feeding SDA can increase long chain n-3 fatty acid concentrations in fish muscle.     

Muscle Lipids and Fatty Acid Profiles of the Sea Catfish (Arius madagascariensis) in Madagascar Inland Waters

Muscle lipids and fatty acids (FA) of catfish Arius madagascariensis were determined in catfish caught in the Betsiboka River, Madagascar, during a 5-month sampling period. Total lipids from muscle were extracted and quantified. Fatty acids were identified by means of gas chromatography–mass spectrometry of FA methyl esters and FA pyrrolidides, leading to the identification of 42 FA. Lipid content was relatively high in our fish sample and ranged from 4.3 to 6.6% of wet muscle. Three FA dominated the FA composition: palmitic acid (C16:0, 22.9–32.6%), oleic acid (C18:1n-9, 11.3–13.4%) and stearic acid (C18:0, 10.8–12.0%). A number of polyunsaturated FA (PUFA) were present in appreciable amounts, including arachidonic acid (C20:4n-6, 4.7–7.6%), docosahexaenoic acid (C22:4n-6, 3.0–8.1%), eicosapentaenoic acid (C20:5n-3, 0.6–1.0%), n-3 docosapentaenoic acid (C22:5n-3, 1.1–1.6%), n-6 docosatetraenoic acid (C22:4n-6, 0.7–1.2%) and n-6 docosapentaenoic acid (22:5n-6, 0.9–1.8%). The sum of the n-6 PUFA and n-3 PUFA was 11.3–18.8 and 7.5–13.4%, respectively. These results indicate that A. madagascariensis, an abundant freshwater fish in Madagascar rivers, may be good source of dietary PUFA.     

Nonessential fatty acids in formula fat blends influence essential fatty acid metabolism and composition in plasma and organ lipid classes in piglets

The n-6 and n-3 fatty acid status of developing organs is the cumulative result of the diet lipid composition and many complex events of lipid metabolism. Little information is available, however, on the potential effects of the saturated fatty acid chain length (8:0–16:0) or oleic acid (18:1) content of the diet on the subsequent metabolism of the essential fatty acids 18:2n-6 and 18:3n-3 and their elongated/desaturated products. The effects of feeding piglets formulas with fat blends containing either coconut oil (12:0±14:0) or medium chain triglycerides (MCT, 8:0±10:0) but similar levels of 18:1, 18:2n-6 and 18:3n-3, or MCT with high or low 18:1 but constant 18:2n-6 and 18:3n-3 on the fatty acid composition of plasma, liver and kidney triglycerides, phospholipids and cholesteryl esters, and of brain total lipid, were studied. Diet-induced changes in the fatty acid composition of lipid classes were generally similar for plasma, liver and kidney. Dietary 18:1 content was reflected in tissue lipids and was inversely associated with levels of 18:2n-6. Lower percentage of 18:2n-6, however, was not associated with lower levels of its elongated/desaturated product 20:4n-6 but was associated with higher levels of 22:6n-3. Feeding coconut oilvs. MCT resulted in lower 18:1 levels in all lipids, and higher percentages of 20:4n-6 in tissue phospholipid. Increasing the dietary n-6/n-3 ratio from 5 to 8 significantly increased tissue percentage of 18:2n-6 and decreased phospholipid 22:6n-3. In contrast to plasma, liver and kidney, brain lipid fatty acid composition was not influenced by the formula saturated fatty acid chain length, content of 18:1, or n-6/n-3 ratio. In summary, the studies show that the dietary requirement for n-6 and n-3 fatty acids may be influenced by the nonessential saturated and monounsaturated fatty acids fed concurrently.     

Relationship between dietary supply of long-chain fatty acids and membrane composition of long- and very long chain essential fatty acids in developing rat photoreceptors

The present study was designed to determine if dietary supply of long-chain fatty acid (LCFA, C20∶4n-6, and/or C22∶6n-3), reflecting levels that might be incorporated into infant formulas, influences the fatty acid composition of the visual cell membrane. The rod outer segment (ROS) of the retina was analyzed from rats fed diets varying in the ratio of 18∶2n-6 to 18∶3n-3 with or without 20∶4n-6 [arachidonic acid (AA)] and 22∶6n-3 (docosahexaenoic acid) from birth to six weeks of age. The level of very long chain fatty acids (VLCFA, C₂₄−C₃₆) was identified using gas chromatography and gas chromatography-mass spectrometry. In the ROS, the highest relative percent of AA was attained in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of animals fed 1% AA diet, whereas feeding 0.7% docosahexaenoic acid (DHA) diet significantly increased the DHA level in PC, phosphatidylserine, and phosphatidylinositol compared to feeding diets containing AA. VLCFA of n-6 and n-3 up to C₃₆ were found in PC, with the most abundant fatty acids being C₃₂ and C₃₄. In PC, phosphatidylserine and PE, the n-6 tetraenoic VLCFA level was highly increased in animals fed 1% AA compared to other dietary groups. This study suggests that dietary fat containing small amounts of AA or DHA is an important factor influencing membrane fatty acid composition of the visual cell during development. Based on a presentation at the AOCS Annual Meeting & Expo in San Antonio, Texas, May 7–11, 1995.     

Unique phospholipid metabolism in mouse heart in response to dietary docosahexaenoic or α-linolenic acids

Diet and fatty acid metabolism interact in yet unknown ways to modulate membrane fatty acid composition and certain cellular functions. For example, dietary precursors or metabolic products of n-3 fatty acid metabolism differ in their ability to modify specific membrane components. In the present study, the effect of dietary 22∶6n−3 or its metabolic precursor, 18∶3n−3, on the selective accumulation of 22∶6n−3 by heart was investigated. The mass and fatty acid compositions of individual phospholipids (PL) in heart and liver were quantified in mice fed either 22∶6n−3 (from crocodile oil) or 18∶3n−3 (from soybean oil) for 13 wk. This study was conducted to determine if the selective accumulation of 22∶6n−3 in heart was due to the incorporation of 22∶6n−3 into cardiolipin (CL), a PL most prevalent in heart and known to accumulate 22∶6n−3. Although heart was significantly enriched with 22∶6n−3 relative to liver, the accumulation of 22∶6n−3 by CL in heart could not quantitatively account for this difference. CL from heart did accumulate 22∶6n−3, but only in mice fed preformed 22∶6n−3. Diets rich in non-22∶6n−3 fatty acids result in a fatty acid composition of phosphatidylcholine (PC) in heart that is unusually enriched with 22∶6n−3. In this study, the mass of PC in heart was positively correlated with the enrichment of 22∶6n−3 into PC. The increased mass of PC was coincident with a decrease in the mass of phosphatidylethanolamine, suggesting that 22∶6n−3 induced PC synthesis by increasing phosphatidylethanolamine-N-methyltransferase activity in the heart.     

Inter-Tissue Differences in Fatty Acid Incorporation as a Result of Dietary Oil Manipulation in Port Jackson Sharks (Heterodontus portusjacksoni)

Fatty acid profile analysis is a tool for dietary investigation that may complement traditional stomach contents analysis. While recent studies have shown that the liver of sharks fed different diets have differing fatty acid profiles, the degree to which diet is reflected in shark blood serum and muscle tissue is still poorly understood. An 18-week controlled feeding experiment was undertaken using captive Port Jackson sharks (Heterodontus portusjacksoni). Sharks were fed exclusive diets of artificial pellets treated with fish or poultry oil and sampled every 6 weeks. The fatty acid profiles from liver, blood serum, and muscle were affected differently, with the period from which significant differences were observed varying by tissue and diet type. The total fatty acid profiles of fish oil and poultry oil fed sharks were significantly different from week 12 onwards in the liver and blood serum, but significant differences were only observed by week 18 in the muscle tissue of sharks fed different diets. The drivers of dissimilarity which aligned with dietary input were 14:0, 18:2n-6, 20:5n-3, 18:1n-9 and 22:6n-3 in the liver and blood serum. Dietary fatty acids accumulated more consistently in the liver than in the blood plasma or muscle, likely due to its role as the central organ for fat processing and storage. Blood serum and muscle fatty acid profiles were influenced by diet, but fluctuated over-time. The low level of correlation between diet and muscle FA profiles is likely a result of low levels of fat (<1 %) in the muscle and the domination of structural, cell-membrane phospholipids in shark muscle tissues. Our findings describe inter-tissue differences in the incorporation of fatty acids from the diet to consumer, which should be taken into account when interpreting dietary patterns from fatty acid profiles.     

The effects of partially hydrogenated marine oils on the mitochondrial function and membrane phospholipid fatty acids in rat heart

The influence of dietary partially hydrogenated marine oils containing docosenoic acid on rat heart mitochondrial membrane phospholipid fatty acid composition was studied with particular reference to cardiolipin and oxidative phosphorylation. Five groups of male weanling rats were fed diets containing 20% (w/w) peanut oil (PO), partially hydrogenated peanut oil (HPO), partially hydrogenated Norwegian capelin oil (HCO), partially hydrogenated herring oil (HHO), and rapeseed oil (RSO) for 10 weeks. All the cardiac phospholipids investigated were influenced by the experimental diets. An increased amount of arachidonic acid observed in phosphatidylethanolamine (PE) after feeding partially hydrogenated oils suggests a changed regulation of the arachidonic acid metabolism in comparison with PO treatment. 22∶1 originating from the dietary oils was incorporated only to a small extent into phosphatidylcholine (PC) and PE. A selective incorporation of 18∶1 isomers into the 1- and 2-positions of PC and PE with respect to geometry and position of the double bond was observed. Large amounts of 18∶1trans were incorporated into the 1-position of PC and PE, irrespective of the amount of 18∶2 supplemented to the diets, replacing a considerable proportion of stearic acid in this position. After feeding HHO and RSO, the content of 22∶1 in mitochondrial cardiolipin of rat heart was found to be 3% (mainly cetoleic acid) and 10% (mainly erucic acid), respectively, indicating a high affinity forcis isomers of 22∶1, but also a considerable resistance against incorporation oftrans isomers was observed. The ability of rat cardiac mitochondria to oxidize palmitoylcarnitine and to synthesize ATP was depressed after feeding HHO and RSO. Dietarycis isomers of 22∶1 seem to have a specific ability to interfere with cardiac ATP synthesis and also to alter the fatty acid composition of cardiolipin of rat heart.     

Erucic Acid is Differentially Taken up and Metabolized in Rat Liver and Heart

Because X-linked adrenoleukodystrophy is treated using erucic acid (22:1n-9), we assessed its metabolism in rat liver and heart following infusion of [14-¹⁴C]22:1n-9 (170 Ci/kg) under steady-state-like conditions. In liver, 2.3-fold more tracer was taken up as compared to heart, accounted entirely by increased incorporation into the organic fraction (4.2-fold). The amount of tracer entering the aqueous fraction, which represents β-oxidation, was not different between groups; however a significantly elevated proportion of tracer was in the heart aqueous fraction. In both tissues, 76% of the radioactivity found in the organic fraction was esterified in neutral lipids, while only about 10% was found esterified into phospholipids. In liver, 56% of lipid radioactivity was found in cholesteryl esters, whereas in heart 64% was found in triacylglycerols. Because 22:1n-9 can be chain shortened, we assessed tracer metabolism using phenacyl fatty acid derivatives esterified from saponified esterified neutral lipid (triacylglycerol/cholesteryl ester) and phospholipid fractions. In heart esterified neutral lipids, 75% of tracer was recovered as 22:1n-9 and only 10% as oleic acid (18:1n-9), while in liver only 25% of the tracer was recovered as 22:1n-9, while 50% was found as stearic acid (18:0) and 10% as 18:1n-9. In liver and heart phospholipids, the tracer was distributed amongst the n-9 fatty acid family. Thus, 22:1n-9 under went tissue selective metabolism, with conversion to 18:0 the dominant pathway in the liver presumably for export in the neutral lipids, while in heart it was found primarily as 22:1n-9 in neutral lipids and used for β-oxidation.     

n−3 and n−6 fatty acid enrichment by dietary fish oil and phospholipid sources in brain cortical areas and nonneural tissues of formula-fed piglets

Sufficient availability of both n-3 and n-6 long-chain polyunsaturated fatty acids (LCPUFA) is required for optimal structural and functional development in infancy. The question has been raised as to whether infant formulae would benefit from enrichment with 20 and 22 carbon fatty acids. To address this issue, we determined the effect of fish oil and phospholipid (LCPUFA) sources on the fatty acid composition of brain cortical areas and nonneural tissues of newborn piglets fed artificially for 2 wk. They were fed sow milk, a control formula, or the formula enriched with n-3 fatty acids from a low-20:5n-3 fish oil added at a high or a low concentration, or the formula enriched with n-3 and n-6 fatty acids from either egg yolk- or pig brain-phospholipids. Both the fish oil- and the phospholipid-enriched formula produced significantly higher plasma phospholipid 22:6n-3 concentrations than did the control formula. The 22:6n-3 levels in the brain, hepatic, and intestinal phospholipids were significantly correlated with plasma values, whereas cardiac 22:6n-3 content appeared to follow a saturable dose-response. Feeding sow milk resulted in a much higher 20:4n-6 content in nonneural tissues than did feeding formula. Supplementation with egg phospholipid increased the 20:4n-6 content in the heart, red blood cells, plasma, and intestine in comparison to the control formula, while pig brain phospholipids exerted this effect in the heart only. The addition of 4.5% fish oil in the formula was associated with a decline in 20:4n-6 in the cortex, cerebellum, heart, liver, and plasma phospholipids, whereas using this source at 1.5% limited the decline to the cerebellum, liver, and plasma. Whatever the dietary treatment, the phosphatidylethanolamine 20:4n-6 level was 10–20% higher in the brain temporal lobe than in the parietal, frontal, and occipital lobes in the temporal lobe by administering the formula enriched with egg or brain phospholipids. In conclusion, feeding egg phospholipids to neonatal pigs increased both the 22:6n-3 content in the brain and the 20:4n-6 content in the temporal lobe cortex. This source also increased the 22:6n-3 levels in nonneural tissues with only minor alterations of 20:4n-6. These data support the notion that infant formulae should be supplemented with both 22:6n-3 and 20:4n-6 rather than with 22:6n-3 alone.     

Influence of triacylglycerol structure and fatty acid profile of dietary fats on milk triacylglycerols in the rat. A two-generation study

We investigated the influence of dietary fatty acid profile and triacylglycerol structure on the fatty acid profile and triacylglycerol structure of milk lipids in two generations of rats. Three groups of rats received diets containing 20% fat of which approximately 20% was n-3 fatty acids located in different positions of the triacylglycerol: a fish oil-based diet [docosahexaenoic acid (22:6n-3) predominantly in thesn-2 position], a seal oil-based diet (22:6n-3) predominantly in thesn-1/sn-3 position or a plant oil-based diet [α-linolenic acid (18:3n-3) distributed evenly between the three positions]. This design allowed us to investigate (i) the effect of the triacylglycerol structure of the dietary fat; (ii) the effect of receiving the n-3 fatty acids as long-chain derivatives or as the precursor, 18:3n-3; and (iii) the long-term effects over two generations. The fatty acid profiles of the milk lipids largely reflected the diets, but in the second generation, the level of medium-chain fatty acids was higher (P<0.05) in the milk from rats fed the fish oil diet (24%) compared with the other dietary groups (15 and 18%). This suggests an increased endogenous synthesis of fatty acids in the mammary glands of the fish oil-fed rats. The levels of long-chain n-3 fatty acids in milk were higher (P<0.05) in rats fed maire n-3 fatty acids in milk were higher (P<0.05) in rats fed marie oils (8–12%) compared with rats fed vegetable oil (1%) in both generations. The level of long-chain n-3 fatty acids was significantly higher in the milk from the fish oil-fed rats (12.3%) compared to the seal-oil fed rats (8.0%) in the first generation, but not in the second generation (8.9 vs. 9.1%). The general structure of milk triacylglycerols was maintained in the three experimental groups with 16:0 acylated in thesn-2 position and 18:1 in thesn-1/sn-3 positions. The triacylglycerol structure of mammalian milk appears to be conserved even during extreme dietary manipulation over two generations and an extensive enrichment with long-chain n-3 polyunsaturated fatty acids requires their presence in the diet.     

Dietary Fish Oil Alters DNA Methylation of Genes Involved in Polyunsaturated Fatty Acid Biosynthesis in Muscle and Liver of Atlantic Salmon (Salmo salar)

Adequate dietary supply of eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) is required to maintain health and growth of Atlantic salmon (Salmo salar). However, salmon can also convert α-linolenic acid (18:3n-3) into eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) by sequential desaturation and elongation reactions, which can be modified by 20:5n-3 and 22:6n-3 intake. In mammals, dietary 20:5n-3 + 22:6n-3 intake can modify Fads2 expression (Δ6 desaturase) via altered DNA methylation of its promoter. Decreasing dietary fish oil (FO) has been shown to increase Δ5fad expression in salmon liver. However, it is not known whether this is associated with changes in the DNA methylation of genes involved in polyunsaturated fatty acid synthesis. To address this, we investigated whether changing the proportions of dietary FO and vegetable oil altered the DNA methylation of Δ6fad_b, Δ5fad, Elovl2, and Elovl5_b promoters in liver and muscle from Atlantic salmon and whether any changes were associated with mRNA expression. Higher dietary FO content increased the proportions of 20:5n-3 and 22:6n-3 and decreased Δ6fad_b mRNA expression in liver, but there was no effect on Δ5fad, Elovl2, and Elovl5_b expression. There were significant differences between liver and skeletal muscle in the methylation of individual CpG loci in all four genes studied. Methylation of individual Δ6fad_b CpG loci was negatively related to its expression and to proportions of 20:5n-3 and 22:6n-3 in the liver. These findings suggest variations in dietary FO can induce gene-, CpG locus-, and tissue-related changes in DNA methylation in salmon.     

LC-PUFA Biosynthesis in Rainbow Trout is Substrate Limited: Use of the Whole Body Fatty Acid Balance Method and Different 18:3n-3/18:2n-6 Ratios

Five experimental diets with constant total C₁₈ PUFA and varying 18:3n-3/18:2n-6 ratios were fed to rainbow trout over an entire production cycle. The whole-body fatty acid balance method demonstrated a clear trend of progressively reduced fatty acid bioconversion activity along the n-3 and n-6 pathways, up to the production of 20:5n-3 and 20:4n-6, respectively. This suggests that the pathway exhibits a “funnel like” progression of activity rather than the existence of a single rate limiting step. The production of 22:5n-3 and 22:6n-3 was more active than that of 20:5n-3. However, despite this trend in reduced apparent in vivo net enzyme activity, the efficiency of the various bioconversion steps (measured as % of bioconverted substrate) confirmed an opposing trend. A 3.2-fold higher Δ-6 desaturase affinity towards 18:3n-3 over 18:2n-6 and an 8-fold greater Δ-5 desaturase affinity towards 20:4n-3 over 20:3n-6 were recorded. The main results of the study were that (1) rainbow trout are quite efficient at bioconverting 18:3n-3 to 22:6n-3, and (2) the LC-PUFA biosynthetic pathway is substrate limited. Fillet n-3 LC-PUFA concentrations increased with the increasing dietary supply of 18:3n-3. Despite an almost identical dietary supply of n-3 LC-PUFA, originating from the fish meal fraction of the diets, the fillets of trout fed the diet richest in 18:3n-3 were 2-fold higher in n-3 LC-PUFA than fish fed low 18:3n-3 diets. Nevertheless, fillets of trout fed a fish oil control diet contained more than double the amount of n-3 LC-PUFA compared to fish fed the diets richest in 18:3n-3.     

Unusually High Levels of n-6 Polyunsaturated Fatty Acids in Whale Sharks and Reef Manta Rays

Fatty acid (FA) signature analysis has been increasingly used to assess dietary preferences and trophodynamics in marine animals. We investigated FA signatures of connective tissue of the whale shark Rhincodon typus and muscle tissue of the reef manta ray Manta alfredi. We found high levels of n-6 polyunsaturated fatty acids (PUFA), dominated by arachidonic acid (20:4n-6; 12–17 % of total FA), and comparatively lower levels of the essential n-3 PUFA—eicosapentaenoic acid (20:5n-3; ~1 %) and docosahexaenoic acid (22:6n-3; 3–10 %). Whale sharks and reef manta rays are regularly observed feeding on surface aggregations of coastal crustacean zooplankton during the day, which generally have FA profiles dominated by n-3 PUFA. The high levels of n-6 PUFA in both giant elasmobranchs raise new questions about the origin of their main food source.     

Cell Proliferation and Long Chain Polyunsaturated Fatty Acid Metabolism in a Cell Line From Southern Bluefin Tuna (Thunnus maccoyii)

Southern bluefin tuna (SBT, Thunnus maccoyii) aquaculture is a highly valuable industry, but research on these fish is hampered by strict catch quotas and the limited success of captive breeding. To address these limitations, we have developed a SBT cell line (SBT-E1) and here we report on fatty acid metabolism in this cell line. The SBT-E1 cells proliferated well in standard Leibovitz’s L-15 cell culture medium containing fetal bovine serum (FBS) as the source of fatty acids. Decreasing the FBS concentration decreased the cell proliferation. Addition of the C₁₈ polyunsaturated fatty acids (PUFA) α-linolenic acid (ALA, 18:3n-3) or linoleic acid (LNA, 18:2n-6) to the cell culture medium had little effect on the proliferation of the cells, whereas addition of the long-chain PUFA (LC-PUFA) arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) or docosahexaenoic acid (DHA, 22:6n-3) significantly reduced the proliferation of the cells, especially at higher concentrations and especially for DHA. Addition of vitamin E to the culture medium overcame this effect, suggesting that it was due to oxidative stress. The fatty acid profiles of the total lipid from the cells reflected those of the respective culture media with little evidence for desaturation or elongation of any of the fatty acids. The only exceptions were EPA and ARA, which showed substantial elongation to 22:5n-3 and 22:4n-6, respectively, and DHA, which was significantly enriched in the cells compared with the culture medium. The results are discussed in light of the dietary PUFA requirements of SBT in the wild and in aquaculture.     

Replacement of Dietary Fish Oils by Alpha-Linolenic Acid-Rich Oils Lowers Omega 3 Content in Tilapia Flesh

A 20-week feeding trial was conducted to determine whether increasing linolenic acid (18:3n-3) in vegetable oil (VO) based diets would lead to increased tissue deposition of 22:6n-3 in Nile tilapia (Oreochromis niloticus). Five isonitrogenous and isoenergetic diets were supplemented with 3% of either linseed oil (LO), a mixture of linseed oil with refined palm olein oil (PO) (LO–PO 2:1) and a mixture of refined palm olein oil with linseed oil (PO–LO 3:2) or with fish oil (FO) or corn oil (CO) as controls. The PO–LO, LO–PO and LO diets supplied a similar amount of 18:2n-6 (0.5% of diet by dry weight) and 0.5, 0.7 and 1.1% of 18:3n-3, respectively. Increased dietary 18:3n-3 caused commensurate increases in longer-chain n-3 PUFA and decreases in longer-chain n-6 PUFA in the muscle lipids of tilapia. However, the biosynthetic activities of fish fed the LO-based diets were not sufficient to raise the tissue concentrations of 20:5n-3, 22:5n-3 and 22:6n-3 to those of fish fed FO. The study suggests that tilapia (O. niloticus) has a limited capacity to synthesise 20:5n-3 and 22:6n-3 from dietary 18:3n-3. The replacement of FO in the diet of farmed tilapia with vegetable oils could therefore lower tissue concentrations of 20:5n-3 and 22:6n-3, and consequently produce an aquaculture product of lower lipid nutritional value for the consumer.