Analysis of branched-chain α-keto acid dehydrogenase complex activity in rat tissues using α-keto[1-13C]isocaproate as substrate

We have developed a highly sensitive nonradioisotope method for the measurement of branched-chain α-keto acid dehydrogenase complex (BCKDC) activity using α-keto[1-13C]isocaproate ([1-13C]KIC) as substrate. The enzyme reaction was performed in a 10-ml test tube. After incubation of the reaction mixture (1 ml), the reaction was terminated by acidification, followed by the addition of internal standard (3.5 μmol K2CO3) for calculation of 13CO2. The 13CO2 released to the gas phase in the test tube was calculated from the molar ratio of 13CO2/12CO2 assayed by gas chromatography isotope ratio mass spectrometry. A distinct linear relationship between 13CO2 production rate and BCKDC activity was obtained when using purified BCKDC. The detection limit of 13CO2 in the assay method was at least 0.167 nmol, which is similar to that in the previously reported radiochemical method. The enzyme assay was found to be linear with respect to reaction time and quantity of enzyme used up to approximately 60 nmol 13CO2 produced. We measured BCKDC activities in several rat tissues, including skeletal muscle. The activities of BCKDC obtained are comparable to those reported previously. These results suggest that the novel BCKDC assay method using [1-13C]KIC is useful for measurement in tissues with low BCKDC activity such as skeletal muscle.