title: An interference-free two-step enzyme assay with UPLC-tandem mass spectrometric product measurement for the clinical diagnosis of uridine diphosphate galactose-4-epimerase deficiency.
abstract: We present a robust clinical assay for the measurement of red blood cell uridine diphosphate galactose-4-epimerase enzyme activity for the diagnostic confirmation of patients positive for a newborn screen for inherited galactosemia in whom galactose-1-phosphate uridyltransferase activity is normal. Previous assays required the use of ion-pairing reagents and frequent need for system maintenance that was not appropriate for heavy clinical use where patient results should be quickly available. We have designed a two-step enzyme assay which converts stable-isotope-labeled UDP-galactose to isotope-labeled-UDP-glucose which is converted in the second reaction to the final product of [(13)C6]-UDP-glucuronic acid. Measurement conditions t remove potential interference from endogenous UDP-glucose and UDP-galactose. We also report a significant ion suppression effect of the red cell preparation for which we have optimized assay sample volume to minimize this effect.
authors.affiliation: Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 19104, United States.
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 19104, United States.
authors.forename: Gail A Ditewig
authors.initials: GA
authors.lastname: Meyers
authors.affiliation: Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 19104, United States; University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, United States. Electronic address: bennettmi@chop.edu.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Bennett
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D004912:Erythrocytes / Q000201:enzymology; D005693:Galactosemias / Q000097:blood* / Q000175:diagnosis / Q000201:enzymology; D006801:Humans; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014534:UDPglucose 4-Epimerase / Q000097:blood* / Q000172:deficiency; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
chemical_list: D014531:Uridine Diphosphate Galactose; D014534:UDPglucose 4-Epimerase; D014532:Uridine Diphosphate Glucose
keywords: Epimerase enzyme assay; Galactosemia; UPLC–tandem mass spectrometry