Abstract
Abstract Objectives Alpha-linolenic acid (ALA) is an essential fatty acid found in plant-based oils, with health benefits attributed to its metabolic conversion to very-long chain polyunsaturated fatty acids. Other routes of cellular ALA metabolism exist, but much of our knowledge of ALA metabolism stems from dated analytical techniques. Essential fatty acids are present in cells primarily as fatty acyls, glycerolipids, and glycerophospholipids, but finer details of ALA metabolism remain unexplored. The current study used unbiased metabolomics to profile the ALA metabolites produced in liver cells after treatment with ALA. Methods H4IIE hepatoma cells were incubated with or without 60 μM ALA for 0, 15, 30, 60, 120, 180, 260, 480, and 720 minutes. Samples were extracted and prepared for HPLC/LC-QTOF-MS analysis. MS data acquisitions were completed in both positive and negative modes. Data were analyzed with 2-way ANOVA (ALA and time as factors) followed by Bonferroni FWER corrections for multiple comparisons. Differences with p < 0.05 were considered significant. Results Among the metabolites detected, differences in patterns of ALA-containing phospholipids (PLs) were observed with ALA treatment. Consistent with a high proportion of phosphatidylcholine (PC) in mammalian cell membranes, ALA-containing PCs such as PC(18:3/18:3), PC(18:3/18:4), lysoPC(18:3/0:0), and PC(18:3/18:1) were significantly higher in ALA-treated cells at most time points. In addition, several phosphatidylethanolamine (PE) species with ALA were significantly higher in ALA-treated cells, including PE(18:3/18:3), PE(18:3/18:2), PE(18:3/16:0), PE(18:3/20:5), and PE(18:3/22:6). To a lesser degree, ALA was also found in phosphatidylserine (PS)(18:3/20:5), lysoPS(18:3/0:0), lysophosphatidylglycerol (18:3/0:0) and phosphatidic acid (PA)(18:3/18:2) and PA(18:3/22:6), suggesting that ALA incorporation into PLs is not limited to PC, contrary to the current known pathways of ALA metabolism. Conclusions Based on these results, there appears to be a preference for incorporation of ALA into PC and PE; however, ALA is also incorporated into PS, PG, and PA. Given the role of different PLs, and functionality based on their fatty acyl composition, future studies will explore the functional outcomes of ALA incorporation into different PL classes. Funding Sources NSERC Discovery Grant
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