A radiochemical assay for acetoacetyl-CoA synthetase

A sensitive radiochemical assay is described for the assay of acetoacetyl-CoA synthetase activity in cytosolic extracts. Enzyme activity is measured by the incorporation of 14C from acetoacetate into acetyl carnitine as mediated by acetoacetyl-CoA synthetase, endogenous acetoacetyl-CoA thiolase, and exogenous carnitine acetyl transferase. Separation of 14C-labeled reactants from 14C-labeled acetyl carnitine is achieved by cation-exchange chromatography. The assay is sensitive with less than 10 pmol of product readily detected. Acetoacetyl-CoA synthetase activity was measured in human fibroblasts, 0.12 nmol min-1 mg cytosolic protein-1, and was found to be more than two orders of magnitude below the activity level of acetoacetyl-CoA synthetase in rat liver cytosol, 18.4 nmol min-1 mg cytosolic protein-1. An HPLC method is also described for the purification of [3-14C]acetoacetate.