SummaryIn a homozygous affected patient with maple syrup urine disease, pharmacologic doses of thiamine lowered urinary excretion of branched chain α-ketoacids and stimulated branched chain α-ketoacid dehydrogenase (BCKAD) in his peripheral blood leukocytes. Supplementation of his branched chain aminoacid restricted diet with 100 mg/day of thiamine eliminated recurrent episodes of ketoacidosis. These clinical responses were studiedin vitrousing mitochondrial inner membranes prepared from his cultured skin fibroblasts and those from another thiamine-responsive patient from Canada. BCKAD in both mutant cell lines had similarities to normal enzyme including: identical apparent Kmvalue for thiamine pyrophosphate; similar heat inactivation profiles which were slowed by the presence of thiamine pyrophosphate; and stimulation above basal activity by thiamine pyrophosphate. Differences in the enzymes included: decreased apparent Vmaxfor thiamine pyrophosphate; increased lability at 37°; and failure to respond to added NAD+CoASH, and Mg2+.We propose that “excess” thiamine led to increased available thiamine pyrophosphate which stabilized the branched chain α-ketoacid dehydrogenase, decreased biologic turnover, increased enzyme specific activity and producedin vivotolerance to branched chain aminoacids in these patients with maple syrup urine disease.SpeculationBy studying the partially purified normal and mutant branched chain α-ketoacid dehydrogenases from cultured human fibroblasts, directin vitroeffects of thiamine pyrophosphate can be measured and related toin vivoclinical responses. This should improve and extend the treatment and management of patients with maple syrup urine disease and provide a method for study of other mutant human enzymes located in the mitochondrial membrane.