REGULATION OF 15N UREA ISOTOPOMERS PRODUCTION

肝性尿失禁的生物化学和生理学研究提出了 代谢调节中更复杂的问题。目前的提案 需要对(S)调节机制进行全面调查 肝脏尿失禁在体内或在肝脏灌流中，并描绘了 由15N生产[15N]尿素的质量同分异构体的决定因素 标记的前体。我们将讨论两个主要假设：(I) 谷氨酰胺(Gln)的5-15N优先用于氨甲酰化- 磷酸盐(CP)合成可决定15N掺入速率 转化为尿素氮取决于酸碱平衡和/或激素 和(Ii)精氨酸的代谢物，即一氧化氮(NO)和/或 胍丁胺可能在N-乙酰谷氨酸的合成中起关键作用 (NAG)，因此，利用[5-15N]Gln合成CP和 [15N]尿素生产。另一种假设是， 线粒体丙酮酸代谢在丙酮酸之间的分配 脱氢酶(PDH)途径(乙酰辅酶A的产生)和丙氨酸 通过线粒体丙氨酸氨基转移酶(MAAT)途径形成， 可能决定NAG合成的速度，从而决定肝脏 在各种激素或酸碱状态下的尿失禁。 我们将使用大鼠和/或肝脏灌流作为模型系统，并 类似于所吸收的复杂细胞外液的灌流液 活体肝脏，只有一种底物标记15N或13C。 我们将测定即刻氮的同位素富集度。 尿素合成中的前驱体池，并确定其原生中心(S) [15N]尿素质量同分异构体的激素或酸碱调节 用气相色谱-质谱法从15N标记前驱体制备 光谱分析(GC-MS)和/或核磁共振(核磁共振)。 将获得的数据将对我们的 了解正常和疾病状态下的肝脏氮代谢。 我们将提供一个严格的实验框架来理解 尿素同分异构体生产的决定因素和描述 [H+]或激素调节肝脏氮的机制(S) 新陈代谢。长期目标是将我们的实验方法应用于 脓毒症、肝性脑病和/或 荷尔蒙状态紊乱，如糖尿病。

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.