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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 13 C‐Labeled Stable Isotope Substrate
Author(s) -
Matsumoto Hideki,
Akita Keiichi,
Sakai Ryosei,
Shimomura Yoshiharu
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.551.1
Subject(s) - chemistry , isotope , substrate (aquarium) , specific activity , dehydrogenase , mass spectrometry , enzyme , catabolism , kinetic isotope effect , skeletal muscle , stable isotope ratio , detection limit , enzyme assay , chromatography , radiochemistry , metabolism , isotopes of carbon , biochemistry , organic chemistry , deuterium , biology , ecology , total organic carbon , physics , endocrinology , quantum mechanics
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‐ 13 C]‐isocaproate ([1‐ 13 C]‐KIC) as stable isotope substrate. 13 CO 2 was released from [1‐ 13 C]‐KIC in the reaction catalyzed by BCKDC. The ratio of 13 CO 2 / 12 CO 2 was measured by isotope ratio mass spectrometry. The detection limit of 13 CO 2 was estimated a few times lower than that of the radiochemical method. Using the purified BCKDC from bovine kidney, the production of 13 CO 2 was found to be linear with respect to reaction time (r 2 = 0.9978) and quantity (r 2 = 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.