This project focuses on a quantitative assessment of metabolic flux through TCA cycle related pathways in a hepatoma cell line, AS-30D cells. The two long term goals of the project are: 1. To further develop a quantitative modeling approach for the analysis of TCA cycle pathways utilizing carbon tracer analysis. 2. To develop a more complete understanding of the metabolic abnormalities characteristic of tumor cells as described in the truncated TCA cycle theory. The project has five specific aims. 1. To evaluate the role of acetoacetate as a precursor for cholesterol and as an oxidative energy source. This project assess the possible significance of acetoacetyl CoA synthetase in cholesterol synthesis of tumors. A relationship providing a quantitative estimate of flux to cholesterol via two pathways will be introduced. 2. To assess the physiological significance of pyruvate carboxylation in AS-30D cells. This flux was discovered using the C02 ratios technique. 3. To examine the hypothesis that TCA cycle malate is preferentially metabolized by malate dehydrogenase while externally supplied malate is metabolized by the mitochondrial malic enzyme. 4. To analyze the flux of glutamine to lipid via reductive carboxylation of a-ketoglutarate. Procedures are designed to evaluate whether this flux occurs in the cytoplasm or in the mitochondria. 5. To examine theoretical and experimental aspects of pre-steady state tracer kinetics in the TCA cycle when the system is in metabolic steady state. A key feature of this analysis is that the sum of the time constants for tracer flux through a pathway are determined from the 14C02 production profile. For all projects experimental methods utilize identical flasks of isolated cells, each with a different 14C labeled compound. All biochemical assays and based on published techniques. Theoretical work is performed using algebra and simple differential equations. The strength of this approach is in the combination of conventional biochemical methods and modeling methodology.
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