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

作者：Matsumoto Hideki; Akita Keiichi; Sakai Ryosei; Shimomura Yoshiharu^*

来源：Analytical Biochemistry, 2010, 399(1): 1-6.

DOI：10.1016/j.ab.2009.12.017

• Branched-chain amino acid
• Branched-chain alpha-keto acid dehydrogenase complex
• alpha-Ketoisocaproate
• Isotope ratio mass spectrometry

We have developed a highly sensitive nonradioisotope method for the measurement of branched-chain alpha-keto acid dehydrogenase complex (BCKDC) activity using alpha-keto[1-C-13] isocaproate ([1-C-13]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 mu mol K2CO3) for calculation of (CO2)-C-13. The (CO2)-C-13 released to the gas phase in the test tube was calculated from the molar ratio of (CO2)-C-13/(CO2)-C-12 assayed by gas chromatography isotope ratio mass spectrometry. A distinct linear relationship between (CO2)-C-13 production rate and BCKDC activity was obtained when using purified BCKDC. The detection limit Of (CO2)-C-13 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 (CO2)-C-13 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-C-13]KIC is useful for measurement in tissues with low BCKDC activity such as skeletal muscle.

• 出版日期2010-4-1