A simple and rapid enzymatic assay for the branched‐chain α‐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.

Propionyl-CoA carboxylase – A review

“…It can be measured in phytohemagglutinin-stimulated lymphocytes by high performance liquid chromatography (HPLC) by measuring amount of methylmalonyl-CoA produced from unlabeled propionyl-CoA [26]. In several types of tissues, enzyme assays look for the production of methylmalonyl-CoA or succinyl-CoA by non-radiometric assays using HPLC or radiometric assays following carboxylation by addition of radiolabeled CO2 [27–29]. Activity is inhibited by avidin and so the enzyme is difficult to isolate after death [30].…”

Living donor liver transplantation from a heterozygous parent for classical maple syrup urine disease

“…Because of the limited deceased donor availability in Japan, LDLT from one of the parents was considered. BCKADC activities of the patient and her parents were measured using lysates of lymphocytes isolated from peripheral blood specimen . Mother showed a lower activity than normal control group, but father's activity was not inferior to a normal range (Table ).…”

Identification of metabolic pathways involved in the biosynthesis of flavor compound 3-methylbutanal from leucine catabolism by Carnobacterium maltaromaticum LMA 28

“…α-Ketoacid dehydrogenase activity was determined according to the method described by Tajima et al (2004). The basic reaction mixture consisted of 7.5 mM α-ketoisocaproic acid, 7.5 mM CoASH (coenzyme A trilithium salt), 1 mM thiamine pyrophosphate, 35 mM magnesium chloride, 2.5 mM NAD + , and 50 mM Tris-HCl buffer (pH 7.5).…”