PATHOPHYSIOLOGY OF BILIRUBIN BOUND COVALENTLY TO ALBUMIN

这项研究项目的主要目标是调查 BIL-ALB的病理生理意义 血清结合胆红素(BR)的很大一部分是 显然与白蛋白共价结合的是来自慢性阻塞性肺病患者的血清 肝胆疾病。具体目标：调查化学性质 和BIL-ALB的形成位置，并确定其他有机阴离子 在胆汁淤积的情况下，也与白蛋白共价结合。方法： BIL-ALB和其他BR组分将通过反相高效液相色谱法进行测定 方法。BIL-ALB将从黄褐斑混合血清中按顺序纯化 柱层析法。结合的BR物种的身份将是 薄层色谱法测定邻氨基苯甲酸乙酯的含量 偶氮衍生物和与甲醇钠进行甲醇分解的产物。 BIL-ALB蛋白水解液的纯化及Edman降解 Br标记的‘显色肽’将被用来识别氨基酸位置/S 是有约束力的。铬酸氧化和薄层色谱分离技术研究 酰亚胺将有助于确定BR-白蛋白键的性质。一个 将使用隔离的大鼠肝脏灌流模型，以及详细的动力学 体外体系中非酶促形成BIL-ALB的研究 确定BIL-ALB合成的部位--在肝脏还是在外周 发行量。白蛋白周转率因下列原因引起的任何变化 将研究BIL-ALB的形成。此外，共价结合 将寻找雌激素和苯二氮烯代谢物以白蛋白 胆汁淤积症患者。意义：这些研究应该更好地 对BR和BR的代谢、转运和蛋白结合的理解 其他存在肝胆疾病的有机阴离子，并会 从而对有机阴离子与蛋白质的结合进行更全面的研究。 在完成了由美国国立卫生研究院支持的两年实验室研究后 肝脏科院校培训资助金，候选人获委任为 1983年7月，消化科教职员工。耶鲁大学的肝脏 学习单元是这个项目的理想环境。联席赞助商是 在肝细胞功能和代谢方面经验丰富的研究人员。 戈登博士是国际知名的胆红素化学专家。 和新陈代谢。因此，设备和技术专长都是 太棒了。与生物化学等基础科学部门的互动 化学很容易就能找到

The major objective of this research project is to investigate the pathophysiological significance of BIL-ALB, recently confirmed by us to be a significant fraction of serum conjugated bilirubin (BR) that is apparently covalently bound to albumin is sera from patients with hepatobiliary diseases. Specific aims: To investigate the chemical nature and site of formation of BIL-ALB and to determine if other organic anions also bind covalently to albumin in the presence of cholestasis. Methods: BIL-ALB and other BR fractions will be measured by a reverse-phase HPLC method. BIL-ALB will be purified from pooled, jaundiced sera by sequential column chromatography. The identity of the conjugated BR species will be determined by thin-layer chromatography (TLC) of ethyl anthranilate azoderivatives and products of methanolysis with sodium methoxide. Proteolysis of BIL-ALB followed by purification and Edman-degradation of BR-tagged 'chromopeptides' will be used to identify the amino acid site/s of binding. Chromic acid oxidation and TLC separation of the liberated imides would aid in identifying the nature of the BR-albumin bond. An isolated perfused rat liver model will be used, along with detailed kinetic studies of any non-enzymatic formation of BIL-ALB in in vitro systems to determine the site of BIL-ALB synthesis- in the liver or in peripheral circulation. Any alteration in the turnover rate of albumin resulting from BIL-ALB formation will be studied. In addition, covalent binding of estrogen and benzodiazepene metabolites to albumin will be looked for in patients with cholestasis. Significance: These studies should better our understanding of the metabolism, transport and protein-binding of BR and other organic anions in the presence of hepatobiliary diseases and would lead to a more complete study of protein binding of organic anions. After completing 2 years of laboratory research supported by an NIH Institutional Training grant in Hepatology, the candidate was appointed to the Digestive Diseases Division faculty in July, 1983. The Yale Liver Study Unit is an ideal environment for this project. The co-sponsors are well experienced researchers in hepatocellular function and metabolism. Dr. E. Gordon is an internationally renowned expert in bilirubin chemistry and metabolism. Thus, both equipment and technical expertise are excellent. Interaction with basic science department such as Biochemistry and Chemistry is readily available