Heterogeneity in maple syrup urine disease: aspects of cofactor requirement and complementation in cultured...

Fibroblast strains derived from six patients with maple syrup urine disease have been investigated for their requirements of the cofactors NAD, CoASH, Mg++ and TPP in comparison with 10 normal control strains. The reconstitution of the decarboxylase function of branched chain alpha-keto acid (BCKA) dehydrogenase complex in lysed cells was studied with respect to the substrates alpha-keto-isocaproic acid, alpha-keto-isovaleric acid, and alpha-keto-beta-methylvaleric acid (KIC, KIVA, MEVA). The enzyme activity of all normal control strains for the substrates KIC and KIVA was not reconstituted by TPP + Mg++ alone, but CoASH + NAD could reconstitute the enzyme activity with KIC and KIVA in different degrees. Only two control strains were tested with MEVA as substrate, and these showed in contrast that TPP + Mg++ could partly reconstitute the enzyme activity. In contrast to the relative homogeneity in the reconstitution profiles of normal strains, the five classical and one intermittent MSUD strains showed heterogeneity in cofactor requirements. Complementation analysis using heterokaryons prepared from fibroblasts of four patients with classical MSUD and one patient with intermittent MSUD showed, in contrast to experiments with normal controls, a partial amelioration of the defect in two combinations; it is suggested that the defect in these strains is located at different functional subunits of the multienzyme complex.