Quantification of Galactose-1-Phosphate Uridyltransferase Enzyme Activity by Liquid Chromatography–Tandem Mass Spectrometry

Abstract

Background 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. Method Our assay used stable isotope-labeled α-galactose-1-phosphate ([13C6]-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 ([13C6]-UDPGal), was detected by MS/MS at mass transition (571 > 323) and quantified by use of [13C6]-Glu-1-P (265 > 79) as an internal standard. Results The method yielded a mean (SD) GALT enzyme activity of 23.8 (3.8) µmol · (gHgb)−1 · h−1 in erythrocyte extracts from 71 controls. The limit of quantification was 0.04 µmol · (g Hgb)−1 · 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 Km 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. Conclusions 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.

Laboratory diagnosis of galactosemia: a technical standard and guideline of the American College of Medical Genetics and Genomics (ACMG)

“…Newly developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) based assays are more sensitive and therefore require smaller sample volume. 37 For mutational analysis of the GALT gene, 1-2 ml whole blood collected in lavender-top (EDTA) tube is preferable. However, DNA extraction yields good results even from heparinized blood.…”

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

“…The lysis buffer used to prepare erythrocyte hemolysates were generated for each experiment and contained 12.8 mmol/L NaH 2 PO 4 (pH 7) and 3.7 mmol/L dithiothreitol (10 ). Glycine buffer and NaH 2 PO 4 buffer were stored at 4°C.…”

“…The blood samples were collected in sodium-heparin tubes, washed to obtain an erythrocyte pellet, and stored at Ϫ80°C as previously described (10 ). One volume of washed erythrocyte pellet was lysed with 3 volumes of lysis buffer.…”

False-Positive Newborn Screen Using the Beutler Spot Assay for Galactosemia in Glucose-6-Phosphate Dehydrogenase Deficiency

“…Multiple fluorescent and radioactive enzyme assays have been developed to screen for classical galactosemia (Li et al 2010). However, the semiquantitative fluorescent Beutler spot assay is commonly used for diagnosis and mass screening in the USA (Fig.…”