EXPLORATION OF ALBUMIN AS MODEL FOR MEASUREMENT OF XENOBIOTIC EXPOSURE IN HUMANS

许多含有羧酸官能团的化合物是 通过代谢转化为相应的酰基葡萄糖醛酸苷， UDP-葡萄糖醛酸转移酶。 在我们实验室进行的研究 已经表明羧酸的酰基葡糖苷酸非甾体 非甾体类药物（NSAID），用作模型羧酸 化合物是反应性的，并且在体内不可逆地与蛋白质结合， 体外 共价结合的加合物的产生可以是 导致严重超敏反应的原因是 使用许多含羧酸的化合物。 的 这些酰基葡萄糖醛酸苷与蛋白质结合的机制， 超敏反应的定义仍然不明确，尽管最近 我们实验室的研究提供了一个深入了解 分子水平上的化学事件。 因此，体外研究 由此托美汀的酰基葡糖苷酸，羧酸 含有NSAID，已显示可引起过敏 在服用药物的患者中发生反应，与人血清发生反应 白蛋白（HSA）在生理条件下。 HSA-托美汀加合物 被还原，羧甲基化和用胰蛋白酶消化。 通过HPLC纯化含有托美汀的胰蛋白酶肽， 通过Kratos上的液体二次离子质谱（LSIMS）分析 MS 50质谱仪由UCSF质谱仪操作 设施。 由此分析，确定了具体的改性 人血清白蛋白胰蛋白酶肽及其药物肽的化学性质 共价加合物是可能的。 进一步的质谱分析 对托美汀酰基葡糖苷酸修饰的胰蛋白酶肽进行了 使用Kratos概念IIHH通过串联质谱法（MS/MS） 在UCSFMass中运行的四扇区串联质谱仪 光谱设施由弗雷德·沃尔斯。 根据这些MS/MS分析， 修饰的肽氨基酸序列以及位点和类型 的共价修饰。 因此，从这些质量 光谱实验表明，托美丁酰基葡糖苷酸与 通过两种不同的机制与HSA的亲核蛋白残基结合。 一种机制涉及赖氨酸胺和丝氨酸的转酰化 羟基通过亲核取代的葡萄糖醛酸， 在蛋白质和药物之间形成酰胺和酯键， 分别 第二种机制，占大多数， 不可逆结合通过赖氨酸-胺反应的机制发生， 与直链醛形式的酰基迁移异构体的 酰基葡糖苷酸以席夫碱的方式。 所得亚胺 然后中间体重新排列成更稳定的产物， 通过葡糖醛酸赖氨酸键与蛋白质结合。 赖氨酸 发现199是修饰程度最大的残基。 的 目前和今后工作的重点是确定 酰基葡萄糖醛酸苷与HSA在体内的反应性，并研究 涉及哪种加合物。 述质谱 HSA-托美汀加合物的分析方法 在体外形成，这是通过一个非常富有成效的 与UCSF质谱设施的合作，将应用于 用于分析体内形成的酰基葡糖苷酸修饰的HSA。

Many compounds which contain the carboxylic acid functionality are converted metabolically to their corresponding acyl glucuronides via UDP-glucuronosyl transferase. Studies performed in our laboratory have shown that acyl glucuronides of carboxylic acid non-steroidal antiinflammatory drugs (NSAIDs), used as model carboxylic acid compounds, are reactive and bind irreversibly to protein, in vivo and in vitro. The generation of covalently bound adducts may be responsible for the severe hypersensitivity reactions involved with the use of a number of carboxylic acid-containing compounds. The mechanisms by which these acyl glucuronides bind to proteins and cause hypersensitivity reactions remains poorly defined, although recent studies in our laboratory have provided an insight into underlying chemical events at the molecular level. Thus, in vitro studies were performed whereby the acyl glucuronide of tolmetin, a carboxylic acid containing NSAID which has been shown to cause hypersensitivity reactions in patients taking the drug, was reacted with human serum albumin (HSA) under physiological conditions. HSA-tolmetin adducts were reduced, carboxymethylated and digested with trypsin. Tolmetin-containing tryptic peptides were purified by HPLC and analyzed by liquid secondary ion mass spectrometry (LSIMS) on a Kratos MS50 mass spectrometer operated by the UCSF Mass Spectrometry Facility. From this analysis, the determination of specific modified HSA tryptic peptides and the chemical nature of the drug-peptide covalent adducts were possible. Further mass spectrometric analyses were performed on tolmetin acyl glucuronide modified tryptic peptides by tandem mass spectrometry (MS/MS) using a Kratos concept IIHH four-sector tandem mass spectrometer operated in the UCSFMass Spectrometry Facility by Fred Walls. From these MS/MS analyses the modified peptide amino acid sequences as well as the sites and types of covalent modification were established. Thus, from these mass spectrometric experiments, tolmetin acyl glucuronide was shown to bind to nucleophilic protein residues of HSA by two distinct mechanisms. One mechanism involved the transacylation of lysine amines and serine hydroxyl groups by nucleophilic displacement of the glucuronic acid to form amide and ester linkages between the protein and drug, respectively. The second mechanism, which accounted for most of the irreversible binding, occurs by a mechanism where lysine-amines react with the straight-chain aldehyde form of acyl migration isomers of the acyl glucuronide in a Schiff's base fashion. The resulting imine intermediate then rearranges to a more stable product, where the drug is bound to the protein by a glucuronic acid lysine linkage. Lysine 199 was found to be the residue modified to the greatest extent. The focus of ongoing and future work is directed toward determining the reactivity of acyl glucuronides with HSA in vivo, and to investigate which types of adducts are involved. The mass spectrometric methodologies developed for the analysis fo HSA-tolmetin adducts formed in vitro, which were obtained through a very productive collaboration with the UCSFMass Spectrometry Facility, will be applied to the analysis of acyl glucuronide modified HSA formed in vivo.