The biochemistry and physiology of hepatic ureagenesis has posed one of the more complex problems in metabolic regulation. The current proposal entails a comprehensive investigation of the mechanism(s) regulating hepatic ureagenesis in vivo or in liver perfusion, and delineates the determinants of mass isotopomers of [15N]urea production from 15N labeled precursor. We will address two primary hypotheses: (i) The preferential utilization of 5-15N of glutamine (Gln) for carbamoyl- phosphate (CP) synthesis may determine the rate of 15N incorporation into urea nitrogens depending upon acid base homeostasis and/or hormonal states; and (ii) Metabolites of arginine, i.e., nitric oxide (NO) and/or agmatine may have a key role in the synthesis of N-acetylglutamate (NAG), and therefore, the utilization of [5-15N]Gln for CP synthesis and [15N]urea production. An alternative hypothesis is that the partitioning of mitochondrial pyruvate metabolism between the pyruvate dehydrogenase (PDH) pathway (production of acetyl-CoA) and alanine formation via the mitochondrial alanine aminotransferase (MAAT) pathway, may determine the rate of NAG synthesis, and thereby, hepatic ureagenesis at various hormonal or acid-base states. We will use rats and/or liver perfusion as a model system and a perfusate which approximates the complex extracellular fluid taken up by the liver in vivo, with only one substrate labeled with 15N or 13C. We will determine the isotopic enrichment of the immediate nitrogenous precursor pools in urea synthesis, and identify primary site(s) of hormonal or acid-base regulation of [15N]urea mass isotopomers production from 15N labeled precursors by using Gas Chromatography-Mass Spectrometry (GC-MS) and/or Nuclear Magnetic Resonance (NMR). The data to be obtained will have considerable impact on our understanding of liver nitrogen metabolism in normal and disease states. We will provide a rigorous experimental framework for understanding the determinants of urea isotopomers production and delineate the mechanism(s) by which [H+] or hormones regulate hepatic nitrogen metabolism. A long term goal is to apply our experimental approach to human subjects in cases such as sepsis, hepatic encephalopathy and/or perturbed hormonal status, such as in diabetes.