Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seongnam-si, Korea.
Clin Chem. 2010 May;56(5):764-71. doi: 10.1373/clinchem.2009.139618. Epub 2010 Mar 18.
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).
[(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.
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.
This method allows for a high-throughput and reproducible multiplex enzyme assay for galactosemia in erythrocytes.
半乳糖血症是新生儿筛查试验中检测到的最重要的遗传性疾病之一。筛查试验中的异常结果应通过酶活性测定来确认，但现有方法既费时又费力。我们开发了一种使用超高效液相色谱-串联质谱（UPLC-MS/MS）的新型半乳糖血症多重酶联测定法。
使用[(13)C6]-半乳糖、[(13)C2]-半乳糖-1-磷酸和 UDP-葡萄糖作为 3 种半乳糖代谢酶的底物。将来自组合反应混合物的终产物[(13)C6]-半乳糖-1-磷酸、UDP-[(13)C2]-半乳糖和 UDP-半乳糖，使用 UPLC-MS/MS 同时进行测量。评估线性度、精密度、离子抑制和底物的影响，以确定测定性能。对半乳糖血症患者、酶缺乏症患者和 18 株突变细胞的 35 名健康个体、8 名患者和 18 株突变细胞的酶活性进行了分析。
通过使用 UPLC-MS/MS 对 3 种酶反应混合物的混合物中的底物、产物和内标进行了清晰的分离，注射周期时间为 10 分钟。离子抑制率为 0.1%-2.5%，UPLC-MS/MS 的批间精密度为 3.3%-10.6%CV，每个系统的线性度良好（R(2) = 0.994-0.999）。与正常个体和野生型细胞相比，患者样本和突变细胞的酶活性始终较低。
该方法可对半乳糖血症患者红细胞中的半乳糖血症进行高通量、可重复的多重酶联测定。
