Quantitation of alpha-linolenic acid elongation to eicosapentaenoic and docosahexaenoic acid as affected by the ratio of n6/n3 fatty acids

Conversion of linoleic acid (LA) and alpha-linolenic acid (ALA) to their higher chain homologues in humans depends on the ratio of ingested n6 and n3 fatty acids. In order to determine the most effective ratio with regard to the conversion of ALA to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), human hepatoma cells were incubated with varying ratios of [13C] labeled linoleic acid ([13C]LA)- and alpha-linolenic acid ([13C]ALA)-methyl esters. Regulative cellular signal transduction pathways involved were studied by determinations of transcript levels of the genes encoding delta-5 desaturase (D5D) and delta-6 desaturase (D6D), peroxisome proliferator-activated receptor  alpha (PPARα) and sterol regulatory element binding protein 1c (SREBP-1c). Mitogen-activated protein kinase kinase 1 (MEK1) and mitogen-activated protein kinase kinase kinase 1 (MEKK1) were also examined. Results: Maximum conversion was observed in cells incubated with the mixture of [13C]LA/ [13C]ALA at a ratio of 1:1, where 0.7% and 17% of the recovered [13C]ALA was converted to DHA and EPA, respectively. Furthermore, differential regulation of enzymes involved in the conversion at the transcript level, dependent on the ratio of administered n6 to n3 fatty acids in human hepatocytes was demonstrated. Formation of EPA and DHA was highest at an administered LA/ALA ratio of 1:1, although gene expression of PPARα, SREBP-1c and D5D involved in ALA elongation were higher in the presence of ALA solely. Also, our findings suggest that a diet-induced enhancement of the cell membrane content of highly unsaturated fatty acids is only possible up to a certain level. (Author's abstract)
Within the common Western diet, ALA intake averages 1.5 g, whereas the average daily uptake of the corresponding n6 fatty acid LA is about 10 times. With this 10:1 ratio of dietary n6 to n3 LCPUFA, the formation of pro-inflammatory/aggregatory  eicosanoids from LA is favored over those from n3 fatty acids showing anti-inflammatory/aggregatory effects. Controlling the synthesis of n3 LCPUFA, such as EPA and DHA from ALA, and the respective eicosanoids by consumption of the optimized ratio of n6 to n3 LCPUFA is of nutritional interest. LCPUFA and their derivatives are of interest as modulators of gene expression. They are reported to be ligands of the nuclear transcription factors peroxisome proliferator-activated receptors (PPARs) and suppressors of sterol regulatory element binding proteins (SREBP). Regulating the degradation of fatty acids by mitochondrial as well as by peroxisomal and microsomal fatty acid oxidation, PPARα plays a central role in fatty acid homeostasis. SREBPs as well as PPARs are, in addition to their ligand-induced regulation activity, substrates for several kinases such as mitogen-activated protein kinases (MAPK). Results from several studies have shown that lowering dietary LA intake, increases EPA in plasma phospholipids, indicating the rate of conversion of ALA to EPA being dependent on the ratio of n3/n6 fatty acids. It has also been postulated that bioconversion of ALA in humans is influenced by the absolute amounts of dietary ALA and LA and not by their ratio. The objective of this study was to quantify the conversion of [13C]alpha-linolenic acid to eicosapentaenoic acid and docosahexaenoic acid in the presence of varying n6/n3 fatty acid ratios in the hepatoma cell line HepG2. In addition, the transcript levels of genes encoding for  enzymes involved in the fatty acid conversion were also examined after incubation of the cells with different ratios of n6/n3 LCPUFAs. In this study, a human hepatoma cell line (HepG2) was used as a model system to investigate the impact of varying ratios of n6/n3 fatty acids on ALA. Incorporation of [13C]LA and [13C]ALA in hepatocytes increased with increasing administered amounts of these fatty acids. Percentage quantities of cellular recovered [13C]LA or [13C]ALA, relating to the  administered amount, however were lower in cells exposed to 100 μM of a single fatty acid ([13C]LA or [13C]ALA) than in cells exposed to mixtures of [13C]LA and [13C]ALA. These findings suggest a limited ability of the cells to incorporate PUFAs beyond a certain level. An increase in n3 dietary fatty acid administration from 1.1% to 5% of total fatty acids resulted in an increase in 20:5-n3 membrane phospholipid content. But further rise of dietary n3 fatty acids did not lead to an increased incorporation of 20:5-n3 into membrane phospholipids. The conversion of [13C]ALA to EPA/DHA was higher than the conversion of [13C]LA to AA. This effect might be attributed to a differential effect of fatty acids of the n6 series and the n3 series on desaturase activity and/or gene expression.  Despite high amounts of EPA formed in cells exposed to 100 μM [13C]ALA, conversion to DHA was lower compared to cells incubated with lower doses of [13C]ALA in the form of [13C]LA/[13C]ALA mixtures. The administration of 100 μM pure ALA alone caused a marked induction of PPARα and total recovery of [13C]ALA in cells exposed to pure [13C]ALA was considerably lower, compared to cells incubated with any of the mixtures of [13C]LA and [13C]ALA. It would appear that [13C]ALA was preferentially used as substrate for PPARα-mediated β-oxidation in cells incubated with pure [13C]ALA. The authors suggest that further investigations with regard to receptor-ligand binding and state of phosphorylation of PPARα or SREBP1c receptor proteins in the presence of defined ration of n6/n3 PUFAs are necessary to elucidate the mechanisms of gene expression. (Editor's comments)