In the assessment of the metabolic status of man and experimental animal no measurement is more fundamental than the rate of gluconeogenesis. Yet, reliable methods for measuring this important indicator are not available. A common experimental procedure is to inject an animal with a radiolabeled carbon precursor of glucose (e.g., alanine) and measure the rate of appearance of radiolabel in glucose. This method requires a correction factor for isotope dilution as the radiolabeled carbon travels to glucose. The objective of the proposed research is to develop improved methods for estimating gluconeogenesis from labeled carbon. The path of carbon from gluconeogenic precursors, lactate or alanine, to glucose is complex because the gluconeogenic pathway interacts with the TCA cycle. To provide a framework for analyzing this complex interaction, I have developed a comprehensive mathematical model of TCA cycle flux and gluconeogenesis. The proposed work will apply relationships developed in this modeling process to a practical, medically important problem. The four specific aims of the project are: (1) To determine if the oxaloacetate pool is homogeneous in hepatic cells in vivo. (2) To evaluate the commonly used assumptions that the dilution of tracer at oxaloacetate is not changed by processes which stimulate gluconeogenesis, and that flux of glutamine into the TCA cycle is not significant. 3) To evaluate a new method for determining isotope dilution based on the modeling approach I have developed. (4) To determine if pryuvate carboxylase, the first step in gluconeogenesis, can be evaluated in vivo using a modified form of the 14Co2 ratios equation where urea carbon substitutes for CO2 and reflects hepatic 14CO2 production. To accomplish these specific aims studies will be carried out using isolated rat hepatocytes as well as intact animals (rats). The long term goal of this project is to improve the understanding of human hepatic carbon metabolism.