Multiplex Enzyme Assay for Galactosemia Using Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry

作者：Ko Dae Hyun; Jun Sun Hee; Park Hyung Doo; Song Sang Hoon; Park Kyoung Un; Kim Jin Q; Song Young Han; Song Junghan^*

来源：Clinical Chemistry, 2010, 56(5): 764-771.

DOI：10.1373/clinchem.2009.139618

BACKGROUND: Galactosemia is one of the most important inherited disorders detected by newborn screening tests. Abnormal results in screening tests should be confirmed by enzyme activity assays, but existing methods are time and labor intensive. We developed a novel multiplex enzyme assay for galactosemia using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).
METHODS: [(13)C6]-galactose, [(13)C2]-galactose-1-phosphate, and UDP-glucose were used as substrates for 3 galactose-metabolizing enzymes. The end products from the combined reaction mixtures, [(13)C6]-galactose-1-phosphate, UDP-[(13)C2]-galactose, and UDP-galactose, were simultaneously measured using UPLC-MS/MS. Linearity, imprecision, ion suppression, and the effects of substrate were evaluated to determine assay performance. Enzyme activities from 35 healthy individuals, 8 patients with enzyme deficiency, and 18 mutant cells were analyzed.
RESULTS: Substrates, products, and internal standards from the mixture of 3 enzyme reactions were clearly separated by using UPLC-MS/MS, with an injection cycle time of 10 min. Ion suppression was 0.1%-2.5%, the interassay imprecision of UPLC-MS/MS was 3.3%-10.6% CV, and the linearity of each system was good (R(2) = 0.994-0.999). Patient samples and mutated cells showed consistently low enzyme activities compared with those of normal individuals and wild-type cells.
CONCLUSIONS: This method allows for a high-throughput and reproducible multiplex enzyme assay for galactosemia in erythrocytes.

• 出版日期2010-5