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1. Metabonomic analysis, via a combination of untargeted and targeted liquid chromatography-mass spectrometry (LC-MS) and untargeted 1H NMR spectroscopy-based metabolite profiling, was performed on aqueous (AQ) and organic liver extracts from control (SCID) and chimeric humanized (PXB) mice dosed with troglitazone at 0, 300 and 600 mg/kg/day for seven days. 2. LC-MS analysis of AQ liver extracts showed a more 'human-like' profile for troglitazone metabolites for PXB, compared with SCID, mice. 3. LC-MS detected differences in endogenous metabolites, particularly lipid species in dosed mice, including elevated triacylglycerols and 1-alkyl,2-acylglycerophosphates as well as lowered diacylglycerophosphocholines and 1-alkyl,2-acylglycerophosphocholines for PXB compared with SCID mouse liver extracts. Following drug administration changes in the relative proportions of the ions for various unsaturated fatty acids were observed for both types of mouse, some of which were specific to PXB or SCID mice. 4. 1H NMR spectroscopy revealed that AQ PXB mouse liver extracts had elevated amounts of inosine, fumarate, creatine, aspartate, trimethylamine N-oxide, glycerophosphocholine, phosphocholine, choline, glutamine, glutamate, acetate, alanine and lactate relative to SCID mice and decreased histidine, glycogen, α- and β-glucose, taurine, and glutathione. Increased uracil and tyrosine concentrations were detected for PXB mice on troglitazone administration. 5. Metabonomic profiling thus showed clear differences between humanized and SCID mice, including after administration of troglitazone. [ABSTRACT FROM AUTHOR]

A protocol for the preparation of tissue extracts for the targeted analysis of ca. 150 polar metabolites, including those involved in central carbon metabolism is described, using a reversed-phase ion pair U(H)PLC-MS method. Data collection enabled by multiple-reaction monitoring provides highly specific, sensitive acquisition of metabolic intermediates with a wide range of physicochemical properties and pathway coverage. Technical aspects are discussed for method transfer along with the basic principles of sample sequence setup, data analysis, and validation. General comments are given to help the assessment of data quality and system performance.

Alcoholism is a complex disorder that, in man, appears to be genetically influenced, although the underlying genes and molecular pathways are not completely known. Here, the intragastric alcohol feeding model in rodents was used together with high mass accuracy LC-MS(n) analysis to assess the metabonomic changes in nonpolar metabolite profiles for livers from control and alcohol-treated rats and mice. Ion signals with a peak area variance of less than 30% (based on repeat analysis of a pooled quality control sample analyzed throughout the batch) were submitted to multivariate statistical analysis (using principal components analysis, PCA). PCA revealed robust differences between profiles from control and alcohol-treated animals from both species. The major metabolites seen to differ between control and alcohol-treated animals were identified using high accuracy MS(n) data and verified using external search engines ( http://www.lipidmaps.org ; http://www.hmdb.ca; http://www.genome.jp/kegg/ ) and authentic standards. The main metabolite classes to show major changes in the alcoholic liver-derived samples were fatty acyls, fatty acid ethyl esters, glycerolipids, and phosphatidylethanol homologues. Significant metabolites that were up-regulated by alcohol treatment in both rat and mouse livers included fatty acyls, metabolites such as octadecatrienoic acid and eicosapentaenoic acid, a number of fatty acid ethyl esters such as ethyl arachidonate, ethyl docosahexaenoic acid, ethyl linoleate, and ethyl oleate and phosphatidylethanol (PEth) homologues (predominantly PEth 18:0/18:2 and PEth 16:0/18:2; PEth homologues are currently considered as potential biomarkers for harmful and prolonged alcohol consumption in man). A number of glycerophospholipids resulted in both up-regulation (m/z 903.7436 [M + H](+) corresponding to a triglyceride) and down-regulation (m/z 667.5296 [M + H](+) corresponding to a diglyceride). Metabolite profiles were broadly similar in both mouse and rat models. However, there were a number of significant differences in the alcohol-treated group particularly in the marked down-regulation of retinol and free cholesterol in the mouse compared to the rat. Unique markers for alcohol treatment included ethyl docosahexaenoic acid. Metabolites were identified with high confidence using predominantly negative ion MS(n) data for the fatty acyl components to match to www.lipidmaps.org MS and MS/MS databases; interpreting positive ion data needed to take into account possible adduct ions which may confound the identification of other lipid classes. The observed changes in lipid profiles were consistent with alcohol-induced liver injury in humans.