Abstract
As part of a study of gluconeogenesis investigated by a combination of metabolomics and mass isotopomer analysis, we developed mass spectrometric assays for the concentration and 13C-labeling pattern of gluconeogenic and citric acid cycle intermediates in rat liver. Since all the intermediates to be assayed are water-soluble, they are extracted by the Folch-wash technique: (i) chloroform-methanol (2:1) extraction of the frozen powdered tissue at −25°C, (ii) breaking phases with ice-cold water, (iii) addition of methoxylamine to the water-methanol phase to protect keto groups, (iv) evaporation, (v) TMS derivatization, and (vi) ammonia positive chemical ionization GC-MS analysis. Internal standardization is achieved by spiking the tissue powder with a mixture of [U-13C6]citrate, [U-13C4]succinate and 3-hydroxy-[2H5]glutarate. Data are expressed as relative concentrations calculated as (area analyte)/(area standard), which are suitable for crossover and principal component analyses, when comparing a control group with an intervention group. The assay was applied to a study of the effect of 0.3 mM mercaptopicolinate, an inhibitor of PEP carboxykinase, on the profile of gluconeogenic intermediates in rat livers perfused with 2 mM pyruvate. The crossover plots generated were virtually identical to those obtained in the past using enzymatic assays. Supported by NIH Roadmap grant R33DK070291.
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