title: Establishment of a practical enzymatic assay method for determination of isovaleryl-CoA dehydrogenase activity using high-performance liquid chromatography.
abstract: BACKGROUND
  Isovaleric acidemia (IVA) is one of the various target disorders for tandem mass spectrometry (MS/MS) newborn screening. In the diagnosis of IVA, no enzymatic assay method for isovaleryl-CoA dehydrogenase (IVD) activity has been reported whereby the production of enoyl-CoA species was directly detected. We established a direct assay method to detect 3-methylcrotonyl-CoA (MC-CoA) production using high-performance liquid chromatography (HPLC).
  
  
  METHODS
  Isovaleryl-CoA dehydrogenase crude enzyme was prepared by sonicating lymphocytes in peripheral blood. Aliquots were incubated with isovaleryl-CoA, flavin adenine dinucleotide, and phenazine methosulfate. 3-Methylcrotonyl-CoA produced in the samples was separated by HPLC and detected using an ultraviolet spectrophotometer.
  
  
  RESULTS
  The detection of MC-CoA was reproducible depending upon the concentration of the substrates, the incubation time, and the number of cells contained in the crude enzyme solution. We applied this assay to three patients diagnosed with IVA and showed that neither of them had detectable residual activity. Only a few hours were required from the initial blood sampling to the end of the assay.
  
  
  CONCLUSIONS
  These results demonstrate that this method for detecting MC-CoA production, using HPLC, is a practical assay for determining IVD activity. It can be a useful confirmatory test for IVA cases detected through MS/MS screening of newborns.
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: Go
authors.initials: G
authors.lastname: Tajima
authors.forename: Nobuo
authors.initials: N
authors.lastname: Sakura
authors.forename: Hiroko
authors.initials: H
authors.lastname: Yofune
authors.forename: Andi
authors.initials: A
authors.lastname: Dwi Bahagia Febriani
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Nishimura
authors.forename: Akiko
authors.initials: A
authors.lastname: Sakamoto
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Ono
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Shigematsu
authors.forename: Masao
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D050769:Isovaleryl-CoA Dehydrogenase; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D044925:Oxidoreductases Acting on CH-CH Group Donors; D050769:Isovaleryl-CoA Dehydrogenase