REGULATION OF 15N UREA ISOTOPOMERS PRODUCTION

肝脏尿素生成的生物化学和生理学已经提出了一个 代谢调节中更复杂的问题。 现时的建议 需要全面调查监管机制 体内或肝脏灌注中的肝尿素生成，并描绘了 从15 N生产[15 N]尿素的质量同位素异构体的决定因素 标记的前体。 我们将讨论两个主要假设：（一） 5- 15 N谷氨酰胺（Gln）优先用于氨基甲酰基- 磷酸盐（CP）的合成可能决定15 N掺入的速率 转化为尿素氮，这取决于酸碱平衡和/或激素 状态;和（ii）精氨酸的代谢，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，[5- 15 N]Gln用于CP合成和 [15 N]尿素生产。 另一种假设是， 线粒体丙酮酸代谢在丙酮酸 脱氢酶（PDH）途径（产生乙酰辅酶A）和丙氨酸 通过线粒体丙氨酸氨基转移酶（MAAT）途径形成， 可以决定NAG合成的速率，从而， 各种激素或酸碱状态下的尿素生成。 我们将使用大鼠和/或肝脏灌注作为模型系统， 灌注液，其近似于吸收的复杂细胞外液 通过肝脏在体内，只有一个底物标记的15 N或13 C。 我们将测定直接含氮化合物的同位素丰度 前体库在尿素合成，并确定主要网站（S） [15 N]尿素质量同位素异构体激素或酸碱调节 通过使用气相色谱-质谱从15 N标记的前体生产 质谱（GC-MS）和/或核磁共振（NMR）。 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， 尿素同位素生产的决定因素，并描绘了 [H+]或激素调节肝氮的机制 新陈代谢. 长期目标是将我们的实验方法应用于 在例如败血症、肝性脑病和/或 激素紊乱，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.