Ethanol Alters Dietary Fatty Acid Partitioning in the Larval Zebrafish: Metabolic Insights From a Novel HPLC Lipidomics Application
Authors: Quinlivan‐Repasi Vanessa H , Farber Steven A 
Published: November 2023

It is well known that consistent excess alcohol consumption promotes the accumulation of abnormally high amounts of triglyceride in hepatocytes. However, the relative contributions of dietary and newly synthesized fatty acids to alcoholic hepatic steatosis are still under investigation, and the effect of ethanol metabolism on metabolic channeling of dietary fatty acids is not well understood. To address this question in a quantitative manner relevant to human health, it was necessary to develop an animal model wherein the metabolism of individual fatty acids could be studied in the context of ethanol metabolism, as well as the digestion and adsorption of a physiologically relevant mixed‐lipid diet. Precise identification and quantitation of the complex lipid products of individual fatty acids against the background of the entire lipidome was also required. Our successful approach employs larval zebrafish to test the hypothesis that ethanol amplifies the bioactive potential of dietary fatty acids by increasing the rate of membrane turnover in intestinal enterocytes, resulting in an increase in the amount of dietary fatty acid directed to membrane phospholipid synthesis. We have optimized a procedure for ethanol treatment of larval zebrafish that recapitulates human acute alcoholic steatosis without affecting feeding behavior or increasing mortality. Following ethanol exposure, larvae are fed fluorescent saturated fatty acids in the context of a high‐fat and high‐cholesterol meal, and the incorporation of these fluorescent fatty acids into complex lipids is assayed by a novel high‐performance liquid chromatography (HPLC) platform that utilizes both fluorescent and charged aerosol detection. This approach facilitates the resolution and quantitation of both fluorescent and unlabeled lipid species belonging to all complex lipid classes in a single sample, which may be extracted from as little as one zebrafish larva or embryo. Preprandial ethanol treatment correlates with an increase in the incorporation of a fluorescent short‐chain fatty acid (BODIPY‐C5) into a single‐species phospholipid, an increase in the incorporation of a fluorescent medium‐chain fatty acid (BODIPY‐C12) into cholesterol ester and multiple phospholipids, and an increase in the esterification of cholesterol by a fluorescent long‐chain fatty acid (BODIPY‐C16). These results reveal that in addition to promoting hepatic lipid storage, acute ethanol exposure also alters the partitioning of dietary saturated fatty acids into complex lipids. Further studies will address the question of how cholesterol ester synthesis may be coupled to the uptake of dietary fatty acids, and how this process may be influenced by concurrent ethanol metabolism.Support or Funding InformationThis work was supported by the National Institutes of Health (R01DK093399), the Carnegie Institution for Science endowment, and the G. Harold and Leila Y. Mathers Charitable Foundation. Vanessa Quinlivan‐Repasi received support from the National Institutes of Health (F31AA023142).