Effects of dietary alpha linolenic acid on biosynthesis of N-3 long chain polyunsaturated fatty acids in animals.

Abstract

Omega-3 (n-3) long chain polyunsaturated fatty acids (LCPUFA), particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), are important for normal health as well as growth and brain development in humans. These fatty acids can be consumed in the diet directly, or synthesised from short chain PUFA consumed in the diet. Fish, particularly in species with a high fat content like salmon, are a major source of these beneficial fatty acids in the human diet. Fish production from aquaculture continues to expand due to a growing human population and demand for fish. Currently there is a reliance on fish oil and fish meal derived exclusively from wild fish as the primary lipid and protein source in fish feeds. Depleted wild fish stocks have made this source of n-3 LCPUFA unsustainable and alternative sources of n-3 LCPUFA are required to fill the void. Most animal species can convert the plant derived 18 carbon (C18) n-6 linoleic acid (LA, 18:2 n-6) and n-3 α-linolenic acid (ALA, 18:3 n-3) to 20 and 22 carbon (C20-C22) LCPUFA by using a series of enzymes to extend and alter the saturation level. There are two types of enzymes responsible for desaturating and elongating fatty acids are desaturases and elongases. The genes associated with these processes appear to be regulated by the extremes of dietary PUFA intake but the extent is currently unclear. This thesis is aimed to examine the effect of dietary PUFA on tissue n-3 LCPUFA levels in animals (rat and fish) after the consumption of diets with increasing levels of ALA, and to investigate whether the expression of desaturases and elongases is involved in the regulation of lipid metabolism and therefore LCPUFA biosynthesis. Furthermore, this thesis also investigated the potential enzyme functions of barramundi Δ6 desaturase and elongase using a yeast heterologous system. Experiments showed that while high ALA diets consistently produced higher levels of n-3 LCPUFA in rat tissues than low ALA diets, mRNA abundance of the Δ6 desaturase (FADS2) and elongase 2 (ELOVL2) genes were increased only in animals fed the low PUFA reference diet compared to those fed diets with adequate to high PUFA levels. There was no correlation between the gene expression of desaturases, elongases or transcription factors and the levels of EPA, docosapentaenoic acid (DPA, 22:5 n-3) or DHA in rat blood, liver and other tissues as a result of feeding increasing levels of ALA. In barramundi however, while vegetable oils induced significant increases in mRNA abundance of FADS2 and ELOVL genes compared with those fed the fish oil-based commercial diet, the tissue EPA, DPA and DHA levels were not increased. It is therefore hypothesised that the enzyme activity of barramundi Δ6 desaturase was low and therefore limited the effectiveness of the enzymes in the LCPUFA pathway to produce EPA and DHA. Furthermore, a large amount of variation between individual fish in DHA levels among those fed the vegetable oil-based diets was found, and this may provide a possibility for a future breeding program of barramundi for better DHA production. Barramundi FADS2 and ELOVL genes were also cloned into yeast cells and performed functional expression of the two enzymes. Results revealed that the barramundi Δ6 desaturase also showed Δ8 desaturase activity and the elongase showed a broad range of fatty acid specificity with the greatest activity with EPA. In addition, a significant amount of the desaturation and elongation fatty acid products could be detected in the culture medium at various time points after the addition of fatty acid substrates, and that it was important to take the levels of fatty acids in the medium into account when it came to calculating enzyme activity.