Analysis of Branched‐Chain α‐Keto Acid Dehydrogenase Complex Activity in Rat Tissues Using α‐Keto[1‐13C]isocaproate as Substrate.

Abstract

The branched‐chain α‐keto acid dehydrogenase complex (BCKDC) is the rate‐limiting enzyme in the catabolism of branched‐chain amino acids (BCAA). The radiochemical method has been used to measure the low level of the BCKDC activity in tissues such as skeletal muscle. In the present report, we have developed a highly sensitive, non‐radioisotope method for the measurement of BCKDC activity using α‐keto[1‐13C]isocaproate ([1‐13C]KIC) as substrate. The 13CO2 was released from [1‐13C]‐KIC in the reaction catalyzed by BCKDC. Amounts of 13CO2 were 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 novel assay method was at least 0.167 nmol. We measured BCKDC activities in several rat tissues including skeletal muscle such as low levels of BCKDC activity. 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 the measurement in tissues with low BCKDC activity such as skeletal muscle.