The synthesis and characterisation of 2-methylacetoacetyl coenzyme A and its use in the identification of the site of the defect in 2-methylacetoacetic and 2-methyl-3-hydroxybutyric aciduria.

(1) 2-Methylacetoacetyl coenzyme A was prepared, purified by ion exchange chromatography as judged by TLC and HPLC and a number of its properties characterised. (2) An assay of 3-oxothiolase in homogenates of cultured human fibroblasts using 2-methylacetoacetyl CoA as substrate is described. (3) This enzyme activity was shown to be absent in fibroblasts from two patients with 2-methylacetoacetic and 2-methyl-3-hydroxybutyric aciduria. (4) These patients also showed decreased activity (42% normal) with acetoacetyl CoA, and indicated that the defective thiolase could also utilize this substrate in normals. The residual activity with acetoacetyl CoA in the patients' fibroblasts resembled the cytosolic acetoacetyl CoA-specific thiolase in properties. We suggest that the enzyme defective in the patients was the mitochondrial acetoacetyl CoA thiolase involved in ketone body utilization in extrahepatic tissues.