Analysis of amino acid supplementation effects on hepatocyte cultures using flux balance analysis.

When cultured hepatocytes are exposed to challenging environments such as plasma, they frequently suffer a decline in liver-specific functions. Media supplements are sought to reduce or eliminate this effect. A rational design approach for amino acid supplementation in hepatocyte culture has been developed in our prior work, and designed amino acid supplementation (DAA) was found to increase urea and albumin production. To fully characterize the metabolic state of hepatocytes under different amino acid supplementations, a number of metabolite measurements are performed in this work and used in a metabolic network flexibility analysis framework including thermodynamic constraints to determine the range of values for the intracellular fluxes. A metabolic objective prediction model is used to infer the metabolic objectives of the hepatocytes and to quantify the intracellular flux distribution for three different amino acid supplementations. The results illustrate that DAA leads to greater fluxes in the tricarboxylic acid cycle (TCA) cycle, urea cycle, and fatty acid oxidation concomitant with lower fluxes in intracellular lipid metabolism compared with empirical amino acid and no amino acid supplementation for hepatocytes during plasma exposure. It is also found that hepatocytes exhibit flexibility in their metabolic objectives depending on the composition of the amino acid supplementations. By incorporating both experimental data and thermodynamic constraints into the mathematical model, the proposed approach leads to identification of metabolic objectives and characterization of fluxes' variability and pathway changes due to different cultured conditions.