Metabolomic assays of the concentration and mass isotopomer distribution of gluconeogenic and citric acid cycle intermediates

We developed gas chromatography-mass spectrometry assays for the relative concentration and for the mass isotopomer distribution of gluconeogenic and citric acid cycle intermediates in tissues. The assay involves (i) spiking the sample with one or more internal standards, (ii) chloroform-methanol extraction at -25 degrees C (iii) Folch wash of the extract, (iv) treatment of the water-methanol phase with methoxylamine, (v) evaporation and trimethylsilyl derivatization, and (vi) ammonia positive chemical ionization gas chromatography-mass spectrometry. For metabolomic computations, indices of concentrations for all compounds assayed are calculated as (Area of analyte)/(Area of reference compound). The assay was applied to a study of the effect of mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, on the profile of gluconeogenic intermediates in rat livers perfused with pyruvate. Crossover analysis of concentrations indices, compared to a control group, yielded very similar profiles as previous enzymatic assays, and correctly identified the site of action of mercaptopicolinate. Principal component analysis distinguished between control and drug treated samples. A loadings plot was used to identify the site of action of the drug in the metabolic pathway. Since metabolite concentrations do not address the flux through a pathway, perfusions with [1,4-C-13(2)] succinate dimethylester were conducted to assess fluxes around PEPCK. This allowed a dynamic metabolomics analysis which indicated that considerable flux through the pathway remained in the presence of mercaptopicolinate. This study illustrates the power of dynamic metabolomics to complement concentration based metabolomic studies.