title: A simple and rapid enzymatic assay for the branched-chain alpha-ketoacid dehydrogenase complex using high-performance liquid chromatography.
abstract: Maple syrup urine disease (MSUD) is caused by a congenital defect of the branched-chain alpha-ketoacid dehydrogenase complex (BCKADC), and is one of the target disorders in newborn screening. However, it is not always easy to confirm the diagnosis; conventional methods of enzyme assay require cell culture, isolation of mitochondria, or radioisotope-labelled reagents, and disease-causing mutations can exist in any of the genes encoding the three enzyme subunits. To realize a practical test for diagnostic confirmation, we developed a simple and rapid enzymatic assay for BCKADC. In this procedure, the production of isovaleryl-CoA from 2-ketoisocaproic acid was measured using high-performance liquid chromatography. Detection of the BCKADC product was significantly reproducible depending on concentration of the substrates. We applied the assay to two patients with MSUD and demonstrated pathologically low levels of residual activity in both subjects. These results indicate that our method is a practical and sensitive assay for BCKADC, and that it can be a useful adjunct in newborn screening for MSUD.
authors.affiliation: Department of Pediatrics, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. ugoch@mac.com
authors.forename: G
authors.initials: G
authors.lastname: Tajima
authors.forename: H
authors.initials: H
authors.lastname: Yofune
authors.forename: A D
authors.initials: AD
authors.lastname: Bahagia Febriani
authors.forename: Y
authors.initials: Y
authors.lastname: Nishimura
authors.forename: H
authors.initials: H
authors.lastname: Ono
authors.forename: N
authors.initials: N
authors.lastname: Sakura
mesh_terms: D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) / Q000032:analysis*; D000293:Adolescent; D002624:Chemistry, Clinical / Q000379:methods*; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D007658:Ketone Oxidoreductases / Q000032:analysis*; D008214:Lymphocytes; D008375:Maple Syrup Urine Disease / Q000175:diagnosis*; D009154:Mutation; D010421:Pentanoic Acids / Q000737:chemistry; D012680:Sensitivity and Specificity; D013997:Time Factors; D014633:Valine / Q000737:chemistry
chemical_list: D010421:Pentanoic Acids; C017517:2-methyl-3-oxovaleric acid; C032737:isovaline; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D014633:Valine