Assay of the activity of malonyl‐CoA decarboxylase (MCD) by gas chromatography‐mass spectrometry (GC‐MS)

Abstract

We developed a GC-MS assay for the activity of MCD in tissue homogenates. Extracts of rat liver (0.25 mg tissue equivalent/assay) are incubated at 37°C with 0.4 mM [U-13C3]malonyl-CoA to form [1,2-13C2]acetyl-CoA by the action of MCD. The reaction mixture contains 2 mM ADP to prevent the hydrolysis of [1,2-13C2]acetyl-CoA by acetyl-CoA hydrolase present in the tissue extracts. After stopping the reaction with methanol, an internal standard of [2H3, 1-13C]acetyl-CoA is added before reacting the two forms of acetyl-CoA with thiophenol. The product acetylthiophenol is analyzed by ammonia positive chemical ionization GC-MS, monitoring 170 (M + NH4+) to 174 (M + NH4+ + 4). The assay was applied to a study of the kinetics of MCD in rat liver, yielding Km and Vmax of 171 μM and 2.5 μmol·min 1·(g liver) 1, respectively. Also, 10 μM CBM-301940, a potent MCD inhibitor (Chugai Pharma), decreased MCD activity 7-fold. Lastly, liver MCD activities (μmol·min 1·g 1 ± S.D.) in three groups of rats that were fed, one-day fasted or two-day fasted were 1.66 ± 0.53, 2.35 ± 0.38 (p < 0.05), and 2.72 ± 0.62 (p < 0.05) respectively. Supported by NIH Roadmap grant R33DK070291.