Establishment of a practical enzymatic assay method for determination of isovaleryl-CoA dehydrogenase activity using high-performance liquid chromatography.
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.
Tajima
Go
G
Department of Pediatrics, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. ugoch@mac.com
Sakura
Nobuo
N
Yofune
Hiroko
H
Dwi Bahagia Febriani
Andi
A
Nishimura
Yutaka
Y
Sakamoto
Akiko
A
Ono
Hiroaki
H
Shigematsu
Yosuke
Y
Kobayashi
Masao
M
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
D044925:Oxidoreductases Acting on CH-CH Group Donors; D050769:Isovaleryl-CoA Dehydrogenase