(235e) Design of Amino Acid Supplementation for Hepatocyte Culture Using Flux and Pathway Analysis

The complete metabolic network of a cell is very large and robust, consisting of a large number of interconnecting reactions and metabolites with sufficient flexibility to allow the cells to cope with the changing environment. Due to the complexity of the metabolic network, a variety of mathematical models and experimental techniques have been developed to facilitate the analysis of cell metabolism and manipulation of its behavior. Flux balance analysis (FBA) provides a set of mathematical tools that can be applied for the analysis of experimental data. FBA has been used to quantitatively interpret cell behavior across experimental conditions, such as the effect of genetic mutations [1], and prediction of the cellular behavior assuming a cellular objective [2].

In mammalian cells, the cellular objectives are not always clear [3], but a rational design approach can be used to drive cells towards a particular objective. We recently applied this idea to design an amino acid supplementation that increases urea production of rat hepatocytes while albumin production was improved as well [4]. Experimental validation of the theoretical results proved that such improvement was possible. However, the experimental data did not match our theoretical prediction mainly due to unaccounted constraints regarding amino acid transportation, thermodynamic constraints, etc.

In this work, we imposed additional pathway energy balances and reaction reversibility constraints on the mathematical model of hepatocyte metabolism. We applied metabolic network flexibility analysis (MNFA) to estimate the range of intracellular fluxes and found that incorporation of these constraints significantly reduces the feasible region of the flux space. Furthermore, we performed experiments to investigate the competition among amino acids sharing common cellular transporters. The experimental results provided additional bounds for amino acid transport that further constrain the feasible region of the mathematical model. Moreover, we developed a bi-level programming, metabolic objective prediction (MOP), to derive the hepatocyte objectives.

The results of the analysis reveal that the cells respond to variations in available nutrients by changing their metabolic objectives and pathway utilization. The combined computational and experimental approaches help our understanding of hepatocyte metabolism in response to amino acid supplementation that can be used to design the optimal amino acids supplementation during patient treatments for an extracorporeal bioartificial liver device.

Reference:

[1] Varma, A. and Palsson B. O. (1994). "Metabolic Flux Balancing - Basic Concepts, Scientific and Practical Use." Bio-Technology 12(10): 994-998.

[2] Sharma, N. S., Ierapetritou M. G., et al. (2005). "Novel quantitative tools for engineering analysis of hepatocyte cultures in bioartificial liver systems." Biotechnol Bioeng 92(3): 321-35.

[3] Nolan, R. P., A. P. Fenley, et al. (2006). "Identification of distributed metabolic objectives in the hypermetabolic liver by flux and energy balance analysis." Metab Eng 8(1): 30-45.

[4] Yang H, Roth C. M., Ierapetritou M. G. (2009). ?A Rational Design Approach for Amino Acid Supplementation in Hepatocyte Culture.? Biotechnol Bioeng.