Development of a fish cell culture model to investigate the impact of fish oil replacement on lipid peroxidation

Fish oils are rich in omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), predominantly 20:5n-3 and 22:6n-3, whereas vegetable oils contain abundant C18-PUFA, predominantly 18:3n-3 or 18:2n-6. We hypothesized that replacement of fish oils with vegetable oils would increase the oxidative stability of fish lipids. Here we have used the FHM cell line to test this hypothesis. The FHM cells were readily able to synthesize 20:5n-3 and 24:6n-3 from 18:3n-3 but 22:6n-3 synthesis was negligible. Also, they were readily able to synthesize 20:3n-6 from 18:2n-6 but 20:4n-6 synthesis was negligible. Mitochondrial β-oxidation was greatest for 18:3n-3 and 20:5n-3 and the rates for 16:0, 18:2n-6, 22:6n-3 and 18:1n-9 were significantly lower. Fatty acid incorporation was predominantly into phospholipids (79-97%) with very little incorporation into neutral lipids. Increasing the fatty acid concentration in the growth medium substantially increased the concentrations of 18:3n-3 and 18:2n-6 in the cell phospholipids but this was not the case for 20:5n-3 or 22:6n-3. When they were subjected to oxidative stress, the FHM cells supplemented with either 20:5n-3 or 22:6n-3 (as compared with 18:3n-3 or saturated fatty acids) exhibited significantly higher levels of thiobarbituric reactive substances (TBARS) indicating higher levels of lipid peroxidation. The results are discussed in relation to the effects of fatty acid unsaturation on the oxidative stability of cellular lipids and the implications for sustainable aquaculture

Purification and properties of a glutathione peroxidase from Southern bluefin tuna ('Thunnus maccoyii') liver

A glutathione peroxidase (GPX) protein was purified approximately 1000-fold from Southern bluefin tuna (Thunnus maccoyii) liver to a final specific activity of 256 μmol NADPH oxidised min−¹ mg−¹ protein. Gel filtration chromatography and denaturing protein gel electrophoresis of the purified preparation indicated that the protein has a native molecular mass of 85 kDa and is most likely a homotetramer with subunits of approximately 24 kDa. The Km values of the purified enzyme for hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and glutathione were 12, 90, 90 and 5900 μM, respectively. The Km values for cumene hydroperoxide and t-butyl hydroperoxide were approximately 8-fold greater than the Km value for hydrogen peroxide. Thus, the SBT liver GPX has a considerably greater affinity for hydrogen peroxide than for the other two substrates. The pH optimum of the purified enzyme was pH 8.0. Immunoblotting experiments with polyclonal antibodies, raised against a recombinant human GPX, provided further evidence that the purified SBT enzyme is a genuine GPX

Dynamics of fatty acid metabolism in a cell line from southern bluefin tuna (Thunnus maccoyii)

Bluefin tunas are large predatorymarine fish of great commercial value but little is known of their specific nutritional requirements. The three species are farmed in sea cages in Australia, the Mediterranean, Mexico and Japan where they are fed small oily fish sourced from wild-catch fisheries. This may not be sustainable and, therefore, it is important to investigate the possible consequences of the replacement ofwild-catch fisheries products (fish oil and fish meal) with alternative oil and meal sources in feeds for these fish. To this endwe have studied fatty acid metabolism in a recently developed southern bluefin tuna (SBT, Thunnus maccoyii) cell line designated SBT-E1. The predominant fatty acids in the total lipid of the SBT-E1 cells were 16:0, 18:0 and 18:1n−9. There were also substantial amounts of 20:4n−6, 22:5n−3 and 22:6n−3 but only very limited amounts of 18:2n−6, 18:3n−3 or 20:5n−3. The fatty acid composition of the cells reflected that of the culture medium except that 20:4n−6, 22:5n−3 and 22:6n−3 were substantially more abundant in the cells than in the medium. Fatty acid esterification occurred predominantly into phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the two most abundant classes of lipids. The SBT-E1 cells showed very limited Δ6 fatty acyl desaturase (Fads) activity towards either 18:3n−3 or 18:2n−6 but substantial elongation of very long chain fatty acids (Elovl) activity towards 20:5n−3. The latter activity is usually attributable to an Elovl5 enzyme. Surprisingly though, there were much higher levels of Δ6 Fads compared with Elovl5 gene expression in the SBT-E1 cells, suggesting that a different Elovl enzyme may catalyse this reaction in SBT. The cells also showed substantial β-oxidation of 18:3n−3 and 20:5n−3 but much less activity towards 18:0, 18:1n−9 or 18:2n−6. These results may explain the high 22:6n−3 to 20:5n−3 ratios found in the SBT tissue lipids, especially in the phospholipids. The results are discussed in terms of the presumed nutritional requirements of bluefin tunas given their high trophic level in marine food webs