The Manton Center for Orphan Disease Research, Division of Genetics, Department of Pediatrics, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA.
Clin Chem. 2010 May;56(5):772-80. doi: 10.1373/clinchem.2009.140459. Epub 2010 Mar 26.
The diagnosis of galactosemia usually involves the measurement of galactose-1-phosphate uridyltransferase (GALT) activity. Traditional radioactive and fluorescent GALT assays are nonspecific, laborious, and/or lack sufficient analytical sensitivity. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay for GALT enzyme activity measurement.
Our assay used stable isotope-labeled alpha- galactose-1-phosphate ([(13)C(6)]-Gal-1-P) as an enzyme substrate. Sample cleanup and separation were achieved by reversed-phase ion-pair chromatography, and the enzymatic product, isotope-labeled uridine diphosphate galactose ([(13)C(6)]-UDPGal), was detected by MS/MS at mass transition (571 > 323) and quantified by use of [(13)C(6)]-Glu-1-P (265 > 79) as an internal standard.
The method yielded a mean (SD) GALT enzyme activity of 23.8 (3.8) mumol x (g Hgb)(-1) x h(-1) in erythrocyte extracts from 71 controls. The limit of quantification was 0.04 micromol x (g Hgb)(-1) x h(-1) (0.2% of normal control value). Intraassay imprecision was determined at 4 different levels (100%, 25%, 5%, and 0.2% of the normal control values), and the CVs were calculated to be 2.1%, 2.5%, 4.6%, and 9.7%, respectively (n = 3). Interassay imprecision CVs were 4.5%, 6.7%, 8.2%, and 13.2% (n = 5), respectively. The assay recoveries at the 4 levels were higher than 90%. The apparent K(m) of the 2 substrates, Gal-1-P and UDPGlc, were determined to be 0.38 mmol/L and 0.071 mmol/L, respectively. The assay in erythrocytes of 33 patients with classical galactosemia revealed no detectable activity.
This LC-MS/MS-based assay for GALT enzyme activity will be useful for the diagnosis and study of biochemically heterogeneous patients with galactosemia, especially those with uncommon genotypes and detectable but low residual activities.
半乳糖血症的诊断通常涉及半乳糖-1-磷酸尿苷酰转移酶（GALT）活性的测量。传统的放射性和荧光 GALT 测定是非特异性的、费力的和/或缺乏足够的分析灵敏度。我们开发了一种基于液相色谱-串联质谱（LC-MS/MS）的 GALT 酶活性测定方法。
我们的测定法使用稳定同位素标记的 alpha-半乳糖-1-磷酸（[(13)C(6)]-Gal-1-P）作为酶底物。通过反相离子对色谱实现样品净化和分离，通过 MS/MS 在质量转换（571 > 323）处检测酶产物，同位素标记的尿苷二磷酸半乳糖（[(13)C(6)]-UDPGal），并使用[(13)C(6)]-Glu-1-P（265 > 79）作为内标进行定量。
该方法在 71 名对照者的红细胞提取物中产生了平均（SD）GALT 酶活性为 23.8（3.8）µmol x（g Hgb）（-1）x h（-1）。定量限为 0.04 µm ol x（g Hgb）（-1）x h（-1）（正常对照值的 0.2%）。在 4 个不同水平（正常对照值的 100%、25%、5%和 0.2%）测定日内精密度，计算 CV 分别为 2.1%、2.5%、4.6%和 9.7%（n = 3）。在 5 次测定中，批间精密度 CV 分别为 4.5%、6.7%、8.2%和 13.2%（n = 5）。在 4 个水平下，测定的回收率均高于 90%。Gal-1-P 和 UDPGlc 的 2 种底物的表观 Km 分别确定为 0.38 mmol/L 和 0.071 mmol/L。在 33 名经典半乳糖血症患者的红细胞中，未检测到可检测的活性。
这种基于 LC-MS/MS 的 GALT 酶活性测定方法将有助于半乳糖血症患者的诊断和研究，特别是那些具有罕见基因型和可检测但残留活性低的生化异质性患者。
