Newborn screening for galactosemia by a second‐tier multiplex enzyme assay using UPLC‐MS/MS in dried blood spots

Abstract

Galactosemia is one of the most important inherited metabolic disorders detected by newborn screening tests. Abnormal results during screening should be confirmed by enzyme activity assays. Recently, we developed a multiplex enzyme assay for galactosemia in erythrocytes using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In this study, we proposed a second-tier multiplex enzyme assay for galactosemia that can be directly applied to dried blood spots (DBSs). Supernatants from two rehydrated-punched 3.2-mm DBSs were incubated with a reaction mixture containing [¹³C6]galactose, [¹³C2]galactose-1-phosphate, and UDP-glucose as substrates for three galactose-metabolizing enzymes. After a 4-hour incubation, the end products from the combined reaction mixture, [¹³C6]galactose-1-phosphate, UDP-[¹³C2]galactose, and UDP-galactose, were simultaneously measured using UPLC-MS/MS. Substrates, products, and internal standards from the mixture of the three enzyme reactions were clearly separated in the UPLC-MS/MS system, with an injection cycle time of 10 min. Intra- and inter-assay imprecisions of the UPLC-MS/MS were 8.4-14.8% and 13.2-15.7% CV, respectively. Enzyme activities in DBSs from 37 normal individuals and 10 patients with enzyme deficiencies were analyzed. DBSs from galactosemia patients showed consistently lower enzyme activities as compared to those of normal individuals. In conclusion, multiplex enzyme assays using UPLC-MS/MS can be successfully applied to DBS analysis. This method allows a fast and effective second-tier test for newborns showing abnormal screening results.

Liquid Chromatography–Tandem Mass Spectrometry Enzyme Assay for UDP-Galactose 4′-Epimerase: Use of Fragment Intensity Ratio in Differentiation of Structural Isomers

“…This contrasts with the range found for control subjects of 17.1-40.1 mol ⅐ g Ϫ1 Hb ⅐ h Ϫ1 with a HPLC method (18 ). A similar but not identical discrepancy was seen with a multiplex GALE ultraperformance liquid chromatography-MS/MS-based enzyme assay compared to a radiometric assay (15,16 ). Our method revealed a K m for UDPGal of 0.05 mmol/L.…”

Use of Tandem Mass Spectrometry for Newborn Screening of 6 Lysosomal Storage Disorders in a Korean Population

“…The use of tandem mass spectrometry (MS/MS) in newborn screening programs has improved the detection of inborn errors of metabolism, and this technique can be applied to a wide range of metabolites [3-6]. MS/MS screening for LSDs was first described by Li et al [7]; they performed direct multiplex assays for Fabry, Gaucher, Krabbe, Niemann-Pick A/B, and Pompe diseases by using dried blood spots (DBS).…”

Tandem Mass Spectrometry Newborn Screening for Inborn Errors of Intermediary Metabolism: Abnormal Profile Interpretation

“…Confirmatory assessment should include OA analysis in urine, analysis of amino acids and acylcarnitines in plasma or serum as shown in Tables (3)(4)(5). When the IMD has no specific metabolic profile, determining enzymatic activity and/or establishing the gene defect become essential for confirmatory diagnosis.…”

“…Detected diseases include defects of intermediary metabolism like aminoacidopathies (AAs), organic acidemias (OAs), and fatty acid oxidation disorders (FAODs) [4]. Nowadays the analytical power of MS/MS has been also used for the screening of carbohydrate metabolism disorders, like galactosemia and lysosomal storage diseases [5,6].…”