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.
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