Analysis of Branched‐Chain α‐Keto Acid Dehydrogenase Complex (BCKDC) Activity and Activity State in Rat Skeletal Muscle by Isotope Ratio Mass Spectrometry (IRMS) using 13C‐Labeled Stable Isotope Substrate

Abstract

The 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 introduced a non‐radioisotope method, using the α‐keto[1‐13C]‐isocaproate ([1‐13C]‐KIC) as stable isotope substrate. 13CO2 was released from [1‐13C]‐KIC in the reaction catalyzed by BCKDC. The ratio of 13CO2/12CO2 was measured by isotope ratio mass spectrometry. The detection limit of 13CO2 was estimated a few times lower than that of the radiochemical method. Using the purified BCKDC from bovine kidney, the production of 13CO2 was found to be linear with respect to reaction time (r2 = 0.9978) and quantity (r2 = 0.9920) of the enzyme. The muscle BCKDC activity measured by the new method was almost the same as that by the radiochemical method reported previously. Thus, we concluded that the novel assay method for the BCKDC activity is applicable to tissues containing the low level of the enzyme activity.