(89c) Targeted Metabolomics of Cofactors In Liver Drug Metabolism

Metabolomics, the comprehensive quantification of small molecules in biological samples, offers a powerful complement to genomics and proteomics in systems biology. In conjunction with mathematical models, the metabolome of a cell or tissue can be used to quantitatively and comprehensively characterize the effects of disease or other biological and chemical stresses on the engagements of metabolic pathways. Quantifying pathway engagements is particularly useful when a clear phenotype and/or mechanism remain to be established for a complex disease (e.g. cancer) or chemical insult (e.g. adverse drug reaction). In this work, we apply targeted metabolomics to characterize the metabolic burden in liver cells treated with combinations of drug chemicals. Drug metabolizing enzymes in the liver depend on the availability of cofactors such as ATP/ADP, NADH/NAD+, NADPH/NADP+, and GSSG/GSH (oxidized and reduced glutathione) to continuously operate xenobiotic functionalization (phase I) and conjugation (phase II) reactions critical for safe elimination. The goal of our analysis is to investigate the hypothesis that drug chemicals not known to directly interact (e.g. through mutual inhibition of corresponding phase I enzymes) could generate an unexpected metabolic burden through cofactor depletion.

In recent years, coupled liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a promising analytical platform to selectively detect and quantify a variety of non-volatile small molecule metabolites without requiring costly chemical derivatization of the target analytes. However, MS/MS analysis of cofactors, due to the presence of labile bonds susceptible to oxidation or reduction, is highly sensitive to the buffers and conditions used for sample preparation and chromatographic separation. To date, development of robust protocols for single platform analysis of intracellular cofactors remains insufficiently addressed. 

In this paper, we describe a streamlined, single platform methodology for the extraction, detection, and quantification of intracellular cofactors and other small molecule metabolites in mammalian cells. To avoid the use of detergents in sample preparation, a series of methanol/chloroform extractions were performed on freeze-quenched cell pellets. The extracts were analyzed on a hybrid triple quadruple linear ion trap mass spectrometer (3200 QTRAP, AB Sciex, Foster City, CA) coupled to a binary pump liquid chromatography system (1200 Series, Agilent Technologies, Santa Clara, CA). Compound detection was optimized in Multiple Reaction Monitoring (MRM) mode for each target metabolite. Chromatograph separation was achieved on a C-18 reverse phase column (Synergy Fusion, Phenomenex, Torrance, CA). To account for ion suppression and other matrix effects, the method of standard additions was used for establishing correlations, i.e. standard curves, between peak areas and metabolite concentrations.

The extraction and LC-MS/MS analysis protocols were used to determine differences in intracellular cofactor concentrations between untreated control liver cells (HepG2 hepatocarcinoma) and cells treated with combinations of anti-diabetic agents (troglitazone and metformin). Our results suggest that metformin exerts a stronger effect on intracellular cofactor concentrations compared to troglitazone. Interestingly, the effects were differential across the various cofactors. A significant difference in the ATP concentration relative to the control was observed only when both anti-diabetic agents were administered, consistent with our observations on cell growth.