Quantitation of the mass of fatty acid ethyl esters synthesized by Hep G2 cells incubated with ethanol

Fatty acid ethyl esters (FAEE), esterification products of fatty acids and ethanol, have been increasingly implicated as mediators of ethanol-induced organ damage, The first goal of this study was to determine the mass of FAEE synthesized by Hep G2 cells exposed to a given dose of ethanol, The second goal was to determine whether all fatty acids in cells are equally available for FAEE synthesis, Hep Ca cells and essential fatty acid deficient Hep Ca cells (Hep G2-EFD) were used to study the synthesis of FAEE upon exposure to ethanol, A two-pool fatty acid model was created: (1) a "previously incorporated pool" formed by incubating the cells with C-14-labeled fatty acids for 24 hr; and (2) a "newly incorporated pool" formed by incubating cells with H-3-labeled fatty acids for 0.5 hr, The FAEE production from each pool was then determined. The total production of FAEE within 3 hr by Hep G2 cells in culture was 150 to 250 pmol/mg cell protein. The fatty acids most recently incorporated into the cells were preferred as substrates for FAEE synthesis because a higher percentage of fatty acids from the newly incorporated pool was used for FAEE synthesis than from the previously incorporated pool. Furthermore, a dose-response relationship was observed between the amount of fatty acid in the newly incorporated pool and FAEE production, but not between the amount of fatty acid in the previously incorporated pool and FAEE synthesis, Taken together, the results indicate that a relatively small amount of endogenously synthesized FAEE is generated from specific intracellular pools of fatty acid since not all fatty acids are equally available for FAEE synthesis. This indicates that a endogenous FAEE are toxic, they exert their toxic effect at very low intracellular FAEE concentrations.