Toxicological evaluation of a fish oil concentrate containing Very Long Chain Fatty Acids.

Very long chain fatty acids (VLCFA) have a chain length ≥24 carbons. Fish contain low levels of these fatty acids. A commercial oil called EPAX® Evolve 05 with an up-concentration of VLCFAs of approximately 10 times, has been developed as a dietary supplement by Epax Norway AS. A series of toxicological studies were performed using mice and rats to determine the safety and toxicity of repeat dosing with a gavage administered VLCFA formulation. The results suggest transient lipid accumulation in kidneys and liver. Lipid accumulation was seen with the test item and with the soya control but was not dose related. Liver and kidney lipid accumulation, whilst present in 14- day repeat dose study, was absent in a 90-day rat study. No treatment-effect was seen in urine analysis in any of the studies. No treatment-related effects were seen with a functional observation battery, ophthalmological examination, haematology, urine analysis, oestrus cycle, thyroid hormones, organ weight, or histopathology. In the 90-day study the liver enzymes ALP, AST and ALT were statistically significantly increased with test item but within control values. There were no associated histological findings in the liver suggesting there was no toxic effect and the normalisation of values for all liver enzymes in the recovery groups suggests an adaptive response rather than a prevailing toxic response. The no-observed-adverse-effect level (NOAEL) was determined as 1200 mg VLCFA/kg b.w./day.

Deposition and metabolism of dietary n-3 very-long-chain PUFA in different organs of rat, mouse and Atlantic salmon.

There is limited knowledge about the metabolism and function of n-3 very-long-chain PUFA (n-3 VLC-PUFA) with chain lengths ≥ 24. They are known to be produced endogenously in certain tissues from EPA and DHA and not considered to originate directly from dietary sources. The aim of this study was to investigate whether n-3 VLC-PUFA from dietary sources are bio-available and deposited in tissues of rat, fish and mouse. Rats were fed diets supplemented with a natural fish oil (FO) as a source of low dietary levels of n-3 VLC-PUFA, while Atlantic salmon and mice were fed higher dietary levels of n-3 VLC-PUFA from a FO concentrate. In all experiments, n-3 VLC-PUFA incorporation in organs was investigated. We found that natural FO, due to its high EPA content, to a limited extent increased endogenous production of n-3 VLC-PUFA in brain and eye of mice with neglectable amounts of n-3 VLC-PUFA originating from diet. When higher dietary levels were given in the form of concentrate, these fatty acids were bio-available and deposited in both phospholipids and TAG fractions of all tissues studied, including skin, eye, brain, testis, liver and heart, and their distribution appeared to be tissue-dependent, but not species-specific. When dietary EPA and DHA were balanced and n-3 VLC-PUFA increased, the major n-3 VLC-PUFA from the concentrate increased significantly in the organs studied, showing that these fatty acids can be provided through diet and thereby provide a tool for functional studies of these VLC-PUFA.

Lipid Deposition and Mobilisation in Atlantic Salmon Adipocytes.

The present study aimed to elucidate how Atlantic salmon adipocytes pre-enriched with palmitic (16:0, PA), oleic (18:1n-9, OA), or eicosapentaenoic (20:5n-3, EPA) acid respond to a fasting condition mimicked by nutrient deprivation and glucagon. All experimental groups were supplemented with radiolabeled PA to trace secreted lipids and distribution of radioactivity in different lipid classes. There was a higher content of intracellular lipid droplets in adipocytes pre-enriched with OA than in adipocytes pre-enriched with PA or EPA. In the EPA group, the radiolabeled PA was mainly esterified in phospholipids and triacylglycerols, whereas in the OA and PA groups, the radioactivity was mainly recovered in phospholipids and cholesterol-ester. By subjecting the experimental groups to nutrient-deprived media supplemented with glucagon, lipolysis occurred in all groups, although to a lower extent in the OA group. The lipids were mainly secreted as esterified lipids in triacylglycerols and phospholipids, indicating mobilization in lipoproteins. A significant proportion was secreted as free fatty acids and glycerol. Leptin secretion was reduced in all experimental groups in response to fasting, while the mitochondria area responded to changes in the energy supply and demand by increasing after 3 h of fasting. Overall, different lipid classes in adipocytes influenced their mobilization during fasting.

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.

n-3 Canola oil effectively replaces fish oil as a new safe dietary source of DHA in feed for juvenile Atlantic salmon.

Limited availability of fish oils (FO), rich in n-3 long-chain (≥C20) PUFA, is a major constraint for further growth of the aquaculture industry. Long-chain n-3 rich oils from crops GM with algal genes are promising new sources for the industry. This project studied the use of a newly developed n-3 canola oil (DHA-CA) in diets of Atlantic salmon fingerlings in freshwater. The DHA-CA oil has high proportions of the n-3 fatty acids (FA) 18 : 3n-3 and DHA and lower proportions of n-6 FA than conventional plant oils. Levels of phytosterols, vitamin E and minerals in the DHA-CA were within the natural variation of commercial canola oils. Pesticides, mycotoxins, polyaromatic hydrocarbons and heavy metals were below lowest qualifiable concentration. Two feeding trials were conducted to evaluate effects of two dietary levels of DHA-CA compared with two dietary levels of FO at two water temperatures. Fish increased their weight approximately 20-fold at 16°C and 12-fold at 12°C during the experimental periods, with equal growth in salmon fed the FO diets compared with DHA-CA diets. Salmon fed DHA-CA diets had approximately the same EPA+DHA content in whole body as salmon fed FO diets. Gene expression, lipid composition and oxidative stress-related enzyme activities showed only minor differences between the dietary groups, and the effects were mostly a result of dietary oil level, rather than the oil source. The results demonstrated that DHA-CA is a safe and effective replacement for FO in diets of Atlantic salmon during the sensitive fingerling life-stage.

The long-chain monounsaturated cetoleic acid improves the efficiency of the n-3 fatty acid metabolic pathway in Atlantic salmon and human HepG2 cells.

The present study aimed to determine if the long-chain MUFA cetoleic acid (22 : 1n-11) can improve the capacity to synthesise the health-promoting n-3 fatty acids EPA and DHA in human and fish models. Human hepatocytes (HepG2) and salmon primary hepatocytes were first enriched with cetoleic acid, and thereafter their capacities to convert radio-labelled 18 : 3n-3 (α-linolenic acid, ALA) to EPA and DHA were measured. Increased endogenous levels of cetoleic acid led to increased production of radio-labelled EPA + DHA in HepG2 by 40 % and EPA in salmon hepatocytes by 12 %. In order to verify if dietary intake of a fish oil rich in cetoleic acid would have the same beneficial effects on the n-3 fatty acid metabolic pathway in vivo as found in vitro, Atlantic salmon were fed four diets supplemented with either sardine oil low in cetoleic acid or herring oil high in cetoleic acid at two inclusion levels (Low or High). The diets were balanced for EPA + DHA content within the Low and within the High groups. The salmon were fed these diets from 110 to 242 g. The level of EPA + DHA in liver and whole-body retention of docosapentaenoic acid and EPA + DHA relative to what was eaten, increased with increased dietary cetoleic acid levels. Thus, it is concluded that cetoleic acid stimulated the synthesis of EPA and DHA from ALA in human HepG2 and of EPA in salmon hepatocytes in vitro and increased whole-body retention of EPA + DHA in salmon by 15 % points after dietary intake of cetoleic acid.

Effects of dietary n-3 fatty acids on Toll-like receptor activation in primary leucocytes from Atlantic salmon (Salmo salar).

The shortage of the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the international markets has led to increasing substitution of fish oil by plant oils in Atlantic salmon (Salmo salar) feed and thereby reducing the EPA and DHA content in salmon. However, the minimum required levels of these fatty acids in fish diets for securing fish health are unknown. Fish were fed with 0, 1 or 2% EPA or DHA alone or in combination of both over a period, growing from 50 to 400 g. Primary head kidney leucocytes were isolated and stimulated with Toll-like receptor (TLR) ligands to determine if EPA and DHA deficiency can affect expression of important immune genes and eicosanoid production. Several genes related to viral immune response did not vary between groups. However, there was a tendency that the high-level EPA and DHA groups expressed lower levels of IL-1ß in non-stimulated leucocytes. These leucocytes were also more responsive to the TLR ligands, inducing higher expression levels of IL-1ß and Mx1 after stimulation. The levels of prostaglandin E2 and leukotriene B4 in serum and media from stimulated leucocytes were lower in both low and high EPA and DHA groups. In conclusion, leucocytes from low EPA and DHA groups seemed to be less responsive towards immunostimulants, like TLR ligands, indicating that low levels or absence of dietary EPA and DHA may have immunosuppressive effects.

Requirements of n-3 very long-chain PUFA in Atlantic salmon (Salmo salar L): effects of different dietary levels of EPA and DHA on fish performance and tissue composition and integrity.

Farmed salmon feeds have changed from purely marine-based diets with high levels of EPA and DHA in the 1990s to the current 70 % plant-based diets with low levels of these fatty acids (FA). The aim of this study was to establish the impacts of low dietary EPA and DHA levels on performance and tissue integrity of Atlantic salmon (Salmo salar). Atlantic salmon (50 g) in seawater were fed fourteen experimental diets, containing five levels (0, 0·5, 1·0, 1·5 and 2·0 %) of EPA, DHA or a 1:1 EPA+DHA plus control close to a commercial diet, to a final weight of 400 g. Lack of EPA and DHA did not influence mortality, but the n-3-deficient group exhibited moderately slower growth than those fed levels above 0·5 %. The heart and brain conserved EPA and DHA levels better than skeletal muscle, liver, skin and intestine. Decreased EPA and DHA favoured deposition of pro-inflammatory 20 : 4n-6 and 20 : 3n-6 FA in membrane phospholipids in all tissues. When DHA was excluded from diets, 18 : 3n-3 and EPA were to a large extent converted to DHA. Liver, skeletal and cardiac muscle morphology was normal in all groups, with the exception of cytoplasm packed with large or foamy vacuoles and sometimes swollen enterocytes of intestine in both deficient and EPA groups. DHA supplementation supported normal intestinal structure, and 2·0 % EPA+DHA alleviated deficiency symptoms. Thus, EPA and DHA dietary requirements cannot be based exclusively on growth; tissue integrity and fish health also need to be considered.

Minor lipid metabolic perturbations in the liver of Atlantic salmon (Salmo salar L.) caused by suboptimal dietary content of nutrients from fish oil.

The present study was conducted to evaluate the effects on Atlantic salmon hepatic lipid metabolism when fed diets with increasing substitution of fish oil (FO) with a vegetable oil (VO) blend. Four diets with VOs replacing 100, 90, 79 and 65 % of the FO were fed for 5 months. The levels of eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) in the experimental diets ranged from 1.3 to 7.4 % of fatty acids (FAs), while cholesterol levels ranged from 0.6 to 1.2 g kg(-1). In hepatocytes added [1-(14)C] α-linolenic acid (ALA, 18:3n-3), more ALA was desaturated and elongated to EPA and DHA in cells from fish fed 100 % VO, while in fish fed 65 % VO, ALA was elongated to eicosatrienoic acid (ETE; 20:3n-3), indicating reduced Δ6 desaturation activity. Despite increased desaturation activity and activation of the transcription factor Sp1 in fish fed 100 % VO, liver phospholipids contained less EPA and DHA compared with the 65 % VO group. The cholesterol levels in the liver of the 100 % VO group exceeded the levels in fish fed the 65 % VO diet, showing an inverse relationship between cholesterol intake and liver cholesterol content. For the phytosterols, levels in liver were generally low. The area as a proxy of volume of lipid droplets was significantly higher in salmon fed 100 % VO compared with salmon fed 65 % VO. In conclusion, the current study suggests that suboptimal dietary levels of cholesterol in combination with low levels of EPA and DHA (1.3 % of FAs) can result in minor metabolic perturbations in the liver of Atlantic salmon.

Fatty acids and gene expression responses to bioactive compounds in Atlantic salmon (Salmo salar L.) hepatocytes.

OBJECTIVES: Effects of bioactive compounds on expression of genes involved in lipid metabolism and fatty acid composition were investigated in Atlantic salmon (Salmo salar L.) primary hepatocytes. METHODS: Five treatments were investigated: I) genistein 0.005 mM, II) genistein 0.025 mM, III) lipoic acid 0.2 mM, IV) sesamin/episesamin 0.05 mM, V) sesamin 0.05 mM and compared to controls. The relative expression of genes involved in lipid homeostasis was analysed after 12h and 48h. RESULTS: Incubation with lipoic acid, sesamin and episesamin/sesamin for 48h had significant effect on all analysed genes involved in lipid uptake, ß-oxidation, elongation and desaturation, some effects were detected on the expression of peroxisome prolifertor-activated receptor (PPARs). Also effects on the fatty acid composition were found. CONCLUSION: The strongest effect of bioactive copounds on hepatocyte gene expression was detected after 48 hours.

Medium-chain and long-chain fatty acids have different postabsorptive fates in Atlantic salmon.

An increasingly larger proportion of the oils used in diets for farmed fish are plant derived and rapeseed oil is most commonly used. Despite high dietary lipid levels and a marked change in lipid composition, the transport and metabolic fate of absorbed fatty acids is not fully understood in teleost fish. The main purpose of this study was to trace the postabsorptive metabolic fate of 2 fatty acids of different chain length: oleic acid [(3)H-18:1(n-9)], constituting 70% of fatty acids in rapeseed oil, and the medium-chain decanoic acid [(14)C-10:0], which does not require carrier molecules for membrane passage. The fatty acids and their metabolites were traced in portal and peripheral blood, liver, heart, skeletal muscle, and visceral adipose tissue at time intervals from 3 to 48 h after feeding. The portal vein was the primary transport route for both 10:0 and 18:1(n-9) from the intestine to the liver the first 6 h after feed intake. From 12 to 48 h, the peripheral route became increasingly more important. The study also indicates a possible direct transport route of fatty acids from the intestine to the surrounding viscera. Our data demonstrate that whereas 18:1(n-9) is primarily deposited as TG in skeletal muscle and visceral adipose tissue, 10:0 is used by the heart and skeletal muscle as a source for rapid energy production.

Alterations in oxidative stress status modulate terminal differentiation in Atlantic salmon adipocytes cultivated in media rich in n-3 fatty acids.

Regulation of oxidative stress (OS) in adipocytes is an important mediator of their development and dysfunction. Highly unsaturated fatty acids (HUFAs) play essential roles in marine fish, where they have anti-lipogenic effects, but they are prone to peroxidation. The aim of this study was to investigate how the effects of HUFAs in fish adipocytes are modulated by changes in their intracellular redox status. Adipocytes from Atlantic salmon were cultivated on HUFA-rich media and treated with buthionine sulfoximine (BSO), which is known to deplete stores of the antioxidant glutathione (GSH) thus increasing OS, and alpha-tocopherol (alpha-TOC), which protects from OS. Gene expression was assessed by qPCR. In addition, phospholipid composition, total fatty acid (FA) composition, TBARS, the activities of pro-apoptotic caspase 3 (CASP3) and antioxidant superoxide dismutase (SOD) were determined. BSO treatment decreased the expression of genes encoding GSH-based antioxidant enzymes glutathione peroxidase (GPX) 2 and GPX3. Consequently, depletion of GSH resulted in the highest level of peroxidation products TBARS despite the increased activity of SOD in this group. Significant reduction of TBARS was achieved by alpha-TOC. Further, in comparison to two alpha-TOC supplemented groups, GSH-depleted cells accumulated less fat and their gene expression profile of adipogenic markers was lower. The formation of large intracellular vesicles was prominent in the control and BSO groups while reduction of OS by alpha-TOC coincided with the increased gene expression of the activating transcription factor 6 (ATF6), a transducer of the endoplasmic reticulum (ER)-stress response. CASP3 assay showed no difference between groups; however, depletion of GSH resulted in the increased gene expression of several apoptotic markers. Up-regulation of the apoptosis inducible factor (AIF) implied higher probability of CASP3-independent apoptosis in cells under increased OS. In conclusion, the study provides several lines of evidence in favour of anti-adipogenic effects of OS in a cold blooded vertebrate.

Atlantic salmon (Salmo salar) muscle precursor cells differentiate into osteoblasts in vitro: polyunsaturated fatty acids and hyperthermia influence gene expression and differentiation.

The formation and mineralisation of bone are two critical processes in fast-growing Atlantic salmon (Salmo salar). The mechanisms of these processes, however, have not been described in detail. Thus, in vitro systems that allow the study of factors that influence bone formation in farmed Atlantic salmon are highly warranted. We describe here a method by which unspecialized primary cells from salmon white muscle can differentiate to osteoblasts in vitro. We have subsequently used the differentiated cells as a model system to study the effects of two factors that influence bone formation in Atlantic salmon under commercial farming conditions, namely polyunsaturated fatty acids, PUFAs, and temperature. Muscle precursor cells changed their morphology from triangular or spindle-shaped cells to polygonal or cubical cells after 3 weeks in osteogenic medium. In addition, gene expression studies showed that marker genes for osteoblastogenesis; alp, col1a1, osteocalcin, bmp2 and bmp4 increased after 3 weeks of incubation in osteogenic media showing that these cells have differentiated to osteoblasts at this stage. Adding CLA or DHA to the osteoblast media resulted in a reduced PGE(2) production and increased expression of osteocalcin. Further, temperature studies showed that differentiating osteoblasts are highly sensitive to increased incubation temperature at early stages of differentiation. Our studies show that unspecialized precursor cells isolated from salmon muscle tissue can be caused to differentiate to osteoblasts in vitro. Furthermore, this model system appears to be suitable for the study of osteoblast biology in vitro.

N-3 HUFAs affect fat deposition, susceptibility to oxidative stress, and apoptosis in Atlantic salmon visceral adipose tissue.

We have investigated how n-3 highly unsaturated fatty acids (HUFAs) in the diet affect fatty acid (FA) utilization, fat storage and oxidative stress (OS) in Atlantic salmon (Salmo salar) white adipose tissue (WAT). Four groups of Atlantic salmon were fed for 21 weeks on one of the four diets supplemented with 23% (of dry matter) lipid. Docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3) levels increased from 10% of total FAs in the rapeseed oil (RO) diet, to 20% in the fish oil (FO) diet, and to 50% and 55% in the DHA-enriched and EPA-enriched diets, respectively. Increased dietary levels of n-3 HUFAs resulted in lower fat percentage in WAT. Furthermore, mitochondrial FA beta-oxidation activity was higher in the FO group than it was in the RO group. The relative levels of DHA and EPA in phospholipids (PLs) from WAT and mitochondrial membranes increased with the increasing dietary levels of these HUFAs. In general, the mitochondrial membrane PLs were characterised by lower relative levels of n-3 HUFAs and higher relative levels of linoleic acid (LA; 18:2 n-6) than WAT membrane PLs. The predominance of LA relative to n-3 HUFAs in mitochondrial membrane PLs may help to protect these PLs from peroxidation. Cytochrome c oxidase measurements revealed higher incidence of disrupted mitochondrial membranes in the DHA and EPA dietary groups than in the FO and RO dietary groups. This disruption further affected the mitochondrial function, resulting in a marked reduction in FA beta-oxidation capacities. The reduction in mitochondrial function and the increase in the activity of superoxide dismutase (SOD) in the DHA and EPA groups showed that high dietary dose of DHA and EPA resulted in oxidative stress (OS). The increased activity of caspase 3 in the high n-3 HUFA groups suggested the induction of apoptosis and increased incidence of cell death in WAT, which may be one of the factors explaining the lower fat percentage found in these groups.

Effects of 3-thia fatty acids on expression of some lipid related genes in Atlantic salmon (Salmo salar L.).

In this study, the effects of in vivo administration of 3-thia fatty acids (FAs) on lipid metabolism in muscle and liver of Atlantic salmon were investigated. Prior to analysis, the fish were kept in tanks supplied with 5 degrees C seawater for 20 weeks. The fish were fed fish meal and fish oil (FO)-based diets supplemented with either nothing (FO), or 0.3% and 0.6% of the 3-thia FAs dodecylthioacetic acid (DTA) and tetradecylthioacetic acid (TTA) respectively. The fish grew from an initial weight of 110 g to 220 g in the FO group and to approximately 160 g in the 3-thia FA groups. There was a significant higher mortality (66%) in fish fed 0.6% TTA than in fish fed the 0.3% DTA (15%) and FO diets (15%). None of the 3-thia FA diets affected the lipid content of the salmon muscle. The liver index, however, was significantly higher and the total liver fat content lower in the TTA group than in the FO group. Both DTA and TTA were incorporated into the lipid fraction of muscle and liver (0.4% to 0.9%). There were no major differences in the total FA composition of liver and muscle between the dietary groups; except for a small increase of n-3 polyunsaturated FAs (PUFAs) in liver of the DTA group. The mRNA expression of peroxisome proliferator-activated receptor (PPAR) alpha, apolipoprotein AI (ApoAI), apolipoprotein CII (ApoCII) and low-density lipoprotein receptor (LDL-R) was down-regulated in liver of the salmon fed 0.3% DTA. PPARalpha and ApoAI transcripts were also reduced in liver of salmon fed 0.6% TTA. Additionally, the hepatic lipoprotein lipase (LPL) mRNA level was 3.8 fold increased in TTA fish relative to the FO group. In muscle there were no significant changes in gene expression pattern of any of the genes investigated. This is the first report on the effects of 3-thia FAs on gene expression in Atlantic salmon.

Effects of 3-thia fatty acids on feed intake, growth, tissue fatty acid composition, beta-oxidation and Na+,K+-ATPase activity in Atlantic salmon.

Atlantic salmon (Salmo salar) with an initial mass of 86 g were reared in 12 degrees C seawater for 8 weeks to a final average mass of 250 g. The fish were fed fish meal and fish oil-based diet supplemented with either 0%, 0.3% or 0.6% of tetradecylthioacetic acid (TTA), a 3-thia fatty acid. The specific growth rate (SGR) decreased with increasing dietary dose of TTA. The SGR of the group fed 0% of TTA (Control) was 1.8; that of the group fed 0.3% of TTA (TTA-L) was 1.7, and that of the group fed 0.6% of TTA (TTA-H) was 1.5. The mortality increased with increased dietary dose of TTA. The mitochondrial beta-oxidation capacity in the liver of fish fed the TTA diets was 1.5 to 2 times higher than that of the Control fish. TTA supplementation caused substantial changes in the fatty acid compositions of the phospholipids (PL), triacylglycerols (TAG) and free fatty acids (FFA) of gills, heart and liver. The percentages of n-3 fatty acids, particularly 22:6 n-3, increased in fish fed diets containing TTA, while the percentage of the saturated FAs 14:0 and 16:0 in the PL fractions of the gills and heart decreased. The sum of monounsaturated FAs in the PL and TAG fractions from liver was significantly higher in fish fed diets containing TTA. TTA itself was primarily incorporated into PL. Two catabolic products of TTA (sulphoxides of TTA) were identified, and these products were particularly abundant in the kidney. TTA supplementation had no significant effect on the activity of the membrane-bound enzyme Na(+),K(+)-ATPase.