3-hydroxy-3-methylglutaric aciduria: a new assay of 3-hydroxy-3-methylglutaryl-coa lyase using high performance liquid chromatography.

A new assay has been developed for 3-hydroxy-3-methylglutaryl-CoA lyase, the final enzyme in the leucine degradative pathway. The assay was performed by incubating lysates of fibroblasts with [glutaryl-3-14C](D,L)-3-hydroxy-3-methyl-glutaryl coenzyme A. The products were analysed by high performance liquid chromatography with continuous liquid scintillation counting. This provided simultaneous identification and quantification of one of the enzymatic products, [3-14C]acetoacetic acid. The mean 3-hydroxy-3-methylglutaryl-CoA lyase activity in fibroblasts from five controls was 732 +/- 81 (SD) pmol/min X mg protein. Using this assay, we have studied skin fibroblasts cultured from a patient with 3-hydroxy-3-methylglutaric aciduria and found 3% of normal 3-hydroxy-3-methylglutaryl-CoA lyase activity. The activities in skin fibroblasts cultured from the parents were 46 and 53% of control activity which is consistent with heterozygocity. Kinetic studies of 3-hydroxy-3-methylglutaryl-CoA lyase in skin fibroblasts cultured from two normal subjects yielded Km values of 14.4 and 18.8 mumol/l for 3-hydroxy-3-methylglutaryl-CoA.