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

作者：Chen Jie; Meyers Gail A Ditewig; Bennett Michael J^*

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2014, 959: 5-9.

DOI：10.1016/j.jchromb.2014.03.029

• Galactosemia
• Epimerase enzyme assay
• UPLC-tandem mass spectrometry

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-UDPglucose which is converted in the second reaction to the final product of [C-13(6)]-UDP-glucuronic acid. Measurement conditions t remove potential interference from endogenous UDP-glucose and UDPgalactose. 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.

• 出版日期2014-5-15