title: Assay for methylmalonyl coenzyme A mutase activity based on determination of succinyl coenzyme A by ultrahigh-performance liquid chromatography tandem mass spectrometry.
abstract: Methylmalonic acidemia (MMA) is an inherited metabolic disease. In this condition, metabolism from methylmalonyl coenzyme A (CoA) to succinyl-CoA is inhibited because of either low methylmalonyl-CoA mutase (MCM) activity or adenosylcobalamin deficiency owing to altered vitamin B12 metabolism. A high-precision assay for detecting MCM activity would facilitate not only MMA diagnosis but also the ability to determine the severity of MMA. We developed an MCM assay method based on ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) that involves the determination of succinyl-CoA, which is formed in an enzyme reaction, using peripheral lymphocytes. Using 0.05, 0.5, and 5 μmol/L succinyl-CoA, the intra-assay coefficient of variation (CV) was less than 5.2% and the inter-assay CV was less than 8.7%. The MCM activities of five healthy individuals and four patients were investigated with this assay. The MCM activities of the patients were very low in relation to those of healthy individuals. Together, these results show that the UPLC-MS/MS method is useful for a detailed MCM activity assay.
authors.affiliation: Department of Hospital Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, 467-8603, Japan.
authors.forename: Kana
authors.initials: K
authors.lastname: Gotoh
authors.forename: Yoko
authors.initials: Y
authors.lastname: Nakajima
authors.forename: Go
authors.initials: G
authors.lastname: Tajima
authors.forename: Yuji
authors.initials: Y
authors.lastname: Hotta
authors.forename: Tomoya
authors.initials: T
authors.lastname: Kataoka
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kawade
authors.forename: Naruji
authors.initials: N
authors.lastname: Sugiyama
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ito
authors.forename: Kazunori
authors.initials: K
authors.lastname: Kimura
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Maeda
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D000328:Adult; D000592:Amino Acid Metabolism, Inborn Errors / Q000175:diagnosis / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008765:Methylmalonyl-CoA Mutase / Q000378:metabolism*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D055815:Young Adult
chemical_list: D000214:Acyl Coenzyme A; C015357:methylmalonyl-coenzyme A; C012046:succinyl-coenzyme A; D008765:Methylmalonyl-CoA Mutase