A radioisotopic method for the determination of acetoacetyl Coenzyme A with 3-hydroxy-3-methylglutaryl Coenzyme A synthase |
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Authors: | W T Hron H M Miziorko L A Menahan |
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Institution: | 1. Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 USA;2. Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 USA |
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Abstract: | A simple and sensitive assay for the quantitative determination of acetoacetyl-CoA (AcAc-CoA) in liver and heart is described. The method is based on incorporation of 14C]acetyl-CoA into acid-stable nonvolatile material in the presence of avian HMG-CoA synthase. The specificity of this procedure for the measurement of AcAc-CoA was demonstrated by pretreating tissue extracts with 3-hydroxyacyl-CoA dehydrogenase or CoA transferase from Escherichia coli to deplete. AcAc-CoA prior to assay. Acid-stable nonvolatile 14C activity measured in the assay was proportional to the amount of tissue extract added. Satisfactory recovery of AcAc-CoA added at the initial extraction step further validated this procedure. This radioactive assay for acetoacetyl-CoA using a highly purified avian 3-hydroxy-3-methylglutaryl-CoA synthase has the advantages of both extreme specificity for AcAc-CoA as substrate and high sensitivity, facilitating the determination of this metabolite under a variety of physiological conditions. |
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