Detection of heterozygotes in maple-syrup-urine disease: measurements of branched-chain alpha-ketoacid dehydrogenase and its components in cell cultures.

To detect heterozygotes for maple-syrup-urine disease (MSUD), activities of branched-chain-alpha-ketoacid (BCKA) dehydrogenase and its components in skin fibroblasts of two obligatory heterozygotes and amnion cells of a fetus at risk were measured. Intact heterozygous cells were found to decarboxylate [1-14C] alpha-ketoisovalerate at rates equal to or only slightly lower than normal subjects. The inability to differentiate heterozygotes from normals with the intact cell assay confirms earlier studies with intact leukocytes using [1-14C]leucine as substrate. By contrast, measurements of BCKA dehydrogenase activity with disrupted cell suspensions showed MSUD heterozygotes with 30%--60% of normal activity. Moreover, biphasic kinetics in heterozygous cells were observed with increasing substrate concentrations. The altered biphasic kinetics probably reflect expression of the normal allele in the early hyperbolic portion of the curve of the mutant allele in the later secondary rise at high substrate concentrations. Assays of component activities showed concordant E1 decarboxylase deficiency in both heterozygous- and homozygous-affected cells, whereas the E3, dihydrolipoyl dehydrogenase-component, activity was normal. The above results taken together appear to provide an approach to detection of the heterozygote in MSUD.