Metabolomic and Isotopomer Analysis of Xenobiotic Stress

描述（由申请人提供） 目的是使用代谢组学结合质量同位素分析来研究两种相关化合物所产生的代谢和氧化应激，1，4-丁二醇（14 BD）和γ-丁内酯。 这些工业溶剂广泛用于化学工业、建筑材料和消费品中。 最后，它们是在年轻人中非常流行的危险滥用药物γ-羟基丁酸盐的前体。 研究人员概述了一种加速肝脏中γ-羟基丁酸酯处置的策略。 代谢组学与质量同位素分析的结合将为理解外源胁迫提供新的途径。 其目标是： 1.确定代谢组学方法鉴定的化合物是否为14 BD和GHB的代谢提供了新的见解。 研究人员将生成一个代谢信息数据库，该数据库是通过对对照组和14 BD或GHB暴露大鼠的血浆、尿液和肝脏进行质谱分析获得的。 多变量统计方法将减少未知峰数据集的维数，检测治疗之间的差异。 将对一些技术进行评估，包括主成分分析、Fisher判别分析和偏最小二乘法。 2.研究从（i）用未标记和均匀13 C标记的14 BD和GHB灌注的离体大鼠肝脏和（ii）输注这些化合物的大鼠血浆、尿液、肝脏和肾脏中提取的代谢物的浓度和质量同位素异构体分布的时间模式。 大鼠将是正常的或用干扰14 BD和/或GHB代谢的化合物（乙醇、甲基吡唑）预处理。 将使用一种新的软件工具Metran分析干预后的变化模式，该软件工具可从途径模型和同位素异构体数据中产生具有统计置信度的通量估计。还将通过目标1中描述的多变量统计分析数据，以识别可区分的同位素异构体，这可能为调节机制提供线索。 3.检验葡萄糖醛酸内酯、α-酮戊二酸前体或牛磺酸可加速肝和肾中GHB代谢的假设。 这将涉及对催化GHB转化为琥珀酸半醛的酶的机制的研究。 4.通过无创性技术测定肝脏谷胱甘肽的周转率来表征肝脏对氧化应激的反应。 这将通过施用低剂量的2 H2O和对乙酰氨基酚，然后测量尿对乙酰氨基酚-谷胱甘肽加合物的2 H-标记来实现。

DESCRIPTION (provided by applicant) The goal is to use metabolomics coupled to mass isotopomer analysis to study the metabolic and oxidative stress exerted by two related compounds, i.e.,1,4butanediol (14BD) and gamma-butyrolactone. These industrial solvents are used extensively in the chemical industry in building materials and in consumer products. Lastly, they are precursors of the dangerous drug of abuse gamma-hydroxybutyrate (GHB) which is very popular among young people. The investigators have outlined a strategy to accelerate the disposal of gamma-hydroxybutyrate in the liver. The coupling of metabolomics to mass isotopomer analysis will provide new avenues for understanding xenobiotic stress. The aims are: 1. To determine if compounds identified by the metabolomics approach provide new insight into the metabolism of 14BD and GHB. The investigators will generate a database of metabolic information obtained by mass spectrometric analyses of plasma, urine and liver of control and 14BD- or GHB- exposed rats. Multivariate statistical methods will reduce the dimensions of the data set of unknown peaks, detecting those that discriminate between treatments. A number of techniques will be evaluated, including Principal Component Analysis, Fisher Discriminant Analysis and Partial Least Squares. 2. To study the temporal patterns of concentration and mass isotopomer distribution of metabolites extracted from (i) isolated rat livers perfused with unlabeled and uniformly 13C-labeled 14BD and GHB and (ii) the plasma, urine, liver and kidney of rats infused with these compounds. The rats will be normal or pre-treated with compounds that interfere with the metabolism of 14BD and/or GHB (ethanol, methylpyrazole). The patterns of change in the profiles following an intervention will be analyzed with a new software tool, Metran, which produces flux estimates with statistical confidence from a pathway model and isotopomer data. The data will also be analyzed by multivariate statistics described in aim 1 to identify discriminating isotopomers, which may provide clues to regulatory mechanisms. 3. To test the hypotheses that the metabolism of GHB in liver and kidney can be accelerated by glucuronolactone, precursors of alpha-ketoglutarate or by taurine. This will involve an investigation of the mechanisms of the enzymes that catalyze the conversion of GHB to succinic semialdehyde. 4. To characterize the response of the liver to oxidative stress by a noninvasive technique to measure the turnover of glutathione in liver. This will be achieved by administering low doses of 2H20 and acetaminophen, followed by measuring the 2H-labeling of urinary acetaminophen-glutathione adduct.