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.

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

Very long chain fatty acids (VLCFAs) are lipids found in fish with a chain length longer than C22. They represent a minor lipid fraction composing of less than 1% of the total lipid. EPAX® EVOLVE 05 is a concentrate of VLCFAs providing roughly 10 times the amount found in fish. Here we report genotoxocity studies performed in cell culture and using a rat model. No genotoxicity was noted in a bacterial reverse mutation test (AMES test). An in vitro micronucleus assay was negative with a 4-hr test item incubation but a 24-hr incubation resulted in a positive signal. This prompted further study using an in vivo Sprague Dawley rat model. Test item exposure was demonstrated by plasma measurements from Sprague Dawley rats with peak absorption at 2-4 h post administration, as expected for fatty acids. The micronucleus assay showed no genotoxicity for fish oil containing VLCFAs. Together, the data shows that VLCFAs up to the test dose of 1200 mg/kg b.w. do not show genotoxicity.

Investigation of the functions of n-3 very-long-chain PUFAs in skin using in vivo Atlantic salmon and in vitro human and fish skin models.

The purpose of this study was to investigate the effect of dietary n-3 very-long-chain PUFA (n-3 VLC-PUFA) on the maturation and development of skin tissue in juvenile Atlantic salmon (Salmo salar) in vivo, as well as their effects on skin keratocyte and human skin fibroblast cell migration in vitro. Atlantic salmon were fed different dietary levels of n-3 VLC-PUFA from an initial weight of 6 g to a final weight of 11 g. Changes in skin morphology were analysed at two time points during the experiment, and the effects on skin tissue fatty acid composition were determined. Additionally, in vitro experiments using human dermal fibroblasts and primary Atlantic salmon keratocytes were conducted to investigate the effect of VLC-PUFA on the migration capacity of the cells. The results demonstrated that increased dietary levels of n-3 VLC-PUFA led to an increased epidermis thickness and more rapid scale maturation in Atlantic salmon skin in vivo, leading to a more mature skin morphology, and possibly more robust skin, at an earlier life stage. Additionally, human skin fibroblasts and salmon skin keratocytes supplemented with n-3 VLC-PUFA in vitro showed more rapid migration, indicating potentially beneficial effects of VLC-PUFA in wound healing. In conclusion, VLC-PUFA may have beneficial effects on skin tissue development, function and integrity.

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.

Metabolic effects of n-3 PUFA as phospholipids are superior to triglycerides in mice fed a high-fat diet: possible role of endocannabinoids.

BACKGROUND: n-3 polyunsaturated fatty acids, namely docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), reduce the risk of cardiovascular disease and can ameliorate many of obesity-associated disorders. We hypothesised that the latter effect will be more pronounced when DHA/EPA is supplemented as phospholipids rather than as triglycerides. METHODOLOGY/PRINCIPAL FINDINGS: In a 'prevention study', C57BL/6J mice were fed for 9 weeks on either a corn oil-based high-fat obesogenic diet (cHF; lipids ∼35% wt/wt), or cHF-based diets in which corn oil was partially replaced by DHA/EPA, admixed either as phospholipids or triglycerides from marine fish. The reversal of obesity was studied in mice subjected to the preceding cHF-feeding for 4 months. DHA/EPA administered as phospholipids prevented glucose intolerance and tended to reduce obesity better than triglycerides. Lipemia and hepatosteatosis were suppressed more in response to dietary phospholipids, in correlation with better bioavailability of DHA and EPA, and a higher DHA accumulation in the liver, white adipose tissue (WAT), and muscle phospholipids. In dietary obese mice, both DHA/EPA concentrates prevented a further weight gain, reduced plasma lipid levels to a similar extent, and tended to improve glucose tolerance. Importantly, only the phospholipid form reduced plasma insulin and adipocyte hypertrophy, while being more effective in reducing hepatic steatosis and low-grade inflammation of WAT. These beneficial effects were correlated with changes of endocannabinoid metabolome in WAT, where phospholipids reduced 2-arachidonoylglycerol, and were more effective in increasing anti-inflammatory lipids such as N-docosahexaenoylethanolamine. CONCLUSIONS/SIGNIFICANCE: Compared with triglycerides, dietary DHA/EPA administered as phospholipids are superior in preserving a healthy metabolic profile under obesogenic conditions, possibly reflecting better bioavalability and improved modulation of the endocannabinoid system activity in WAT.

Lipid content and fatty acid distribution in tissues from Portuguese dogfish, leafscale gulper shark and black dogfish.

The lipid characterization in tissues from the three deep-sea sharks leafscale gulper shark (Centrophorus squamosus), Portuguese dogfish (Centroscymnus coelolepis) and black dogfish (Centrocyllium fabricii) captured at Hatton Bank in the North Atlantic were examined. The objective was to determine the lipid content and the fatty acid composition in different tissues. In addition, the fatty acid composition in tissues and species was compared. The tissues examined were pancreas, heart, kidney, stomach, spleen and liver. The lipid content was high in liver (40-50%) and ranged from 1% to 5% in the other tissues. The dominant fatty acids were C16:0, C18:1 (n-9), C18:1 (n-7) and C22:6 (n-3) in all tissues. All tissues had a high content of unsaturated fatty acids.

Chemical characterisation of eggs from deep-sea sharks.

The chemical characterisation and nutritional value of eggs from the five deep-sea sharks leafscale gulper shark (Centrophorus squamosus), greater lantern shark (Etmopterus princeps), longnose velvet dogfish (Centroscymnus crepidater), Portuguese dogfish (Centroscymnus coelolepis) and black dogfish (Centrocyllium fabricii) captured at Hatton Bank in the North Atlantic were examined. The chemical composition was quite similar for all the eggs studied. The dominant fatty acid in all the eggs was the monounsaturated fatty acid C18:1, which varied from 27-39%. The eggs had a relatively high content of C16:0 (13.0-18.5%) and C22:6n-3 (10.3-15.1%). The two main lipid classes in the eggs were triacylglycerols (36-55%) and phospholipids (34-41%). The eggs had high amounts of vitamin A and E. The shark eggs were particularly rich in the amino acids aspartic acid, glutamic acid, leucine and arginine.

Proximate composition, fatty acid and lipid class composition of the muscle from deep-sea teleosts and elasmobranchs.

Proximate composition of muscle was determined for the following deep-sea fish species: roughhead grenadier (Macrourus berglax), mora/deep-sea cod (Mora moro), Portuguese dogfish (Centroscymnus coelolepis), black dogfish (Centroscyllium fabricii), leafscale gulper shark (Centrophorus squamosus), greater lantern shark (Etmopterus princeps), smalleyed rabbitfish/ghostshark (Hydrolagus affinis), birdbeak dogfish (Deania calcea) and two species of smooth head (Alepocephalus bairdii and Alepocephalus agassizii). The first eight species contained less than 1% fat in the muscle, while the last two contained 3.0% and 3.6% fat, respectively. Fatty acid and lipid class composition was determined for the first five fish species and showed that the dominant class of lipids was phospholipids. The lipids consisted mainly of polyunsaturated fatty acids (PUFA), and docosahexaenoic acid (DHA) was the dominant fatty acid. Roughhead grenadier and mora showed resemblance to cod (Gadus morhua) regarding protein content, fat content and fatty acid composition. However, the muscle from the deep-sea fish species did contain a higher proportion of arachidonic acid (20:4n-6) than cod muscle.

Fatty acid and lipid class composition in eyes and brain from teleosts and elasmobranchs.

The fatty acid and lipid class composition of the eyes and brain were determined for the following species: Atlantic cod (Gadus morhua), saithe (Pollachius virens), redfish (Sebastes marinus), Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), Portuguese dogfish (Centroscymnus coelolepis), black dogfish (Centroscyllium fabricii), and leafscale gulper shark (Centrophorus squamosus). Fatty acid analyses of eyes from teleosts in the present study indicated that the lean species contained high ratios of docosahexaenoic acid (DHA) versus eicosapentaenoic acid (EPA), and high ratios of n-3 fatty acids versus n-6 fatty acids, while these ratios were significantly lower for the fatty fish species. The lipid class analyses revealed that among both elasmobranchs and teleosts, phospholipid was the dominant class of lipids in the eyes of lean species, while triacylglycerol was the dominant class of lipids in fatty species. Analyses of the fatty acid composition of brains revealed that the deep-sea elasmobranchs, Portuguese dogfish, black dogfish, and leafscale gulper shark, contained a level of arachidonic acid (AA) that was higher than their level of EPA and about fivefold higher than what was found in the brains of teleosts. Such high levels of AA are not normally observed in fish brains; rather, they are generally observed in brains of higher vertebrates.