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Analysis of Branched‐Chain α‐Keto Acid Dehydrogenase Complex Activity in Rat Tissues Using α‐Keto[1‐ 13 C]isocaproate as Substrate.
Author(s) -
Matsumoto Hideki,
Akita Keiichi,
Sakai Ryosei,
Kobayashi Tsuyoshi,
Shimomura Yoshiharu
Publication year - 2010
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.24.1_supplement.lb262
Subject(s) - substrate (aquarium) , skeletal muscle , chemistry , dehydrogenase , catabolism , specific activity , enzyme , detection limit , metabolism , mass spectrometry , isotope , amino acid , enzyme assay , chromatography , stereochemistry , biochemistry , biology , ecology , physics , quantum mechanics , endocrinology
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‐ 13 C]isocaproate ([1‐ 13 C]KIC) as substrate. The 13 CO 2 was released from [1‐ 13 C]‐KIC in the reaction catalyzed by BCKDC. Amounts of 13 CO 2 were calculated from the molar ratio of 13 CO 2 / 12 CO 2 assayed by gas chromatography isotope ratio mass spectrometry. A distinct linear relationship between 13 CO 2 production rate and BCKDC activity was obtained when using purified BCKDC. The detection limit of 13 CO 2 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‐ 13 C]KIC is useful for the measurement in tissues with low BCKDC activity such as skeletal muscle.