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Ultra fast and sensitive liquid chromatography tandem mass spectrometry based assay for galactose-1-phosphate uridylyltransferase and galactokinase deficiencies.
Mol Genet Metab. 2011 Jan; 102(1):33-40.MG

Abstract

The diagnosis of transferase and galactokinase deficiency galactosemia usually involves the measurement of erythrocyte galactose-1-phosphate uridylyltransferase (GALT) and galactokinase (GALK) enzyme activity, respectively. The current gold standard assays for these enzymes are radioactive assays, which are laborious and/or incapable of measuring low enzyme activities. To further our knowledge of genotype-phenotype relationships, we had developed an assay for GALT activity alone using LC-MS/MS. In this study we generated a robust and sensitive LC-MS/MS based GALT and GALK assay using a novel normal phase chromatographic condition. We improved upon our earlier assay by drastically reducing the instrument run time and eliminating the use of an ion pairing reagent. Stable isotope labeled substrates were utilized in the GALT and GALK assays. The enzymatic products ([(13)C(6)]-uridine diphosphate galactose in GALT assay and [(13)C(6)]-galactose-1-phosphate in GALK assay) were quantified in a 3 min LC-MS/MS run. The assays were sensitive enough to allow for the quantification of enzyme activities as low as 0.2% and 0.3% of normal control values in the GALT and GALK assays, respectively. Thirty-three samples from non-galactosemic patients were assayed to have erythrocyte GALT activity of 23.4±4.2 and GALK activity of 1.8±0.47 (mean±SD) μmol⋅(g Hgb)(-1) h(-1). Erythrocyte GALT activities in a cohort of 16 patients with classic or severe galactosemia were measured: 4 patients had GALT activity less than 1% of normal control values and the remaining 12 had no detectable GALT activity. No GALK activity was detected in a GALK deficient sample we analyzed. Lastly, we tested the feasibility of adapting this LC-MS/MS based GALT/GALK assay as a newborn screening (NBS) test.

Authors+Show Affiliations

The Manton Center for Orphan Disease Research, Division of Genetics, Department of Pediatrics, Children's Hospital Boston, Harvard Medical School, Boston, MA, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

20863731

Citation

Li, Yijun, et al. "Ultra Fast and Sensitive Liquid Chromatography Tandem Mass Spectrometry Based Assay for Galactose-1-phosphate Uridylyltransferase and Galactokinase Deficiencies." Molecular Genetics and Metabolism, vol. 102, no. 1, 2011, pp. 33-40.
Li Y, Ptolemy AS, Harmonay L, et al. Ultra fast and sensitive liquid chromatography tandem mass spectrometry based assay for galactose-1-phosphate uridylyltransferase and galactokinase deficiencies. Mol Genet Metab. 2011;102(1):33-40.
Li, Y., Ptolemy, A. S., Harmonay, L., Kellogg, M., & Berry, G. T. (2011). Ultra fast and sensitive liquid chromatography tandem mass spectrometry based assay for galactose-1-phosphate uridylyltransferase and galactokinase deficiencies. Molecular Genetics and Metabolism, 102(1), 33-40. https://doi.org/10.1016/j.ymgme.2010.08.018
Li Y, et al. Ultra Fast and Sensitive Liquid Chromatography Tandem Mass Spectrometry Based Assay for Galactose-1-phosphate Uridylyltransferase and Galactokinase Deficiencies. Mol Genet Metab. 2011;102(1):33-40. PubMed PMID: 20863731.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Ultra fast and sensitive liquid chromatography tandem mass spectrometry based assay for galactose-1-phosphate uridylyltransferase and galactokinase deficiencies. AU - Li,Yijun, AU - Ptolemy,Adam S, AU - Harmonay,Lauren, AU - Kellogg,Mark, AU - Berry,Gerard T, Y1 - 2010/09/21/ PY - 2010/08/25/received PY - 2010/08/26/accepted PY - 2010/9/25/entrez PY - 2010/9/25/pubmed PY - 2011/4/26/medline SP - 33 EP - 40 JF - Molecular genetics and metabolism JO - Mol. Genet. Metab. VL - 102 IS - 1 N2 - The diagnosis of transferase and galactokinase deficiency galactosemia usually involves the measurement of erythrocyte galactose-1-phosphate uridylyltransferase (GALT) and galactokinase (GALK) enzyme activity, respectively. The current gold standard assays for these enzymes are radioactive assays, which are laborious and/or incapable of measuring low enzyme activities. To further our knowledge of genotype-phenotype relationships, we had developed an assay for GALT activity alone using LC-MS/MS. In this study we generated a robust and sensitive LC-MS/MS based GALT and GALK assay using a novel normal phase chromatographic condition. We improved upon our earlier assay by drastically reducing the instrument run time and eliminating the use of an ion pairing reagent. Stable isotope labeled substrates were utilized in the GALT and GALK assays. The enzymatic products ([(13)C(6)]-uridine diphosphate galactose in GALT assay and [(13)C(6)]-galactose-1-phosphate in GALK assay) were quantified in a 3 min LC-MS/MS run. The assays were sensitive enough to allow for the quantification of enzyme activities as low as 0.2% and 0.3% of normal control values in the GALT and GALK assays, respectively. Thirty-three samples from non-galactosemic patients were assayed to have erythrocyte GALT activity of 23.4±4.2 and GALK activity of 1.8±0.47 (mean±SD) μmol⋅(g Hgb)(-1) h(-1). Erythrocyte GALT activities in a cohort of 16 patients with classic or severe galactosemia were measured: 4 patients had GALT activity less than 1% of normal control values and the remaining 12 had no detectable GALT activity. No GALK activity was detected in a GALK deficient sample we analyzed. Lastly, we tested the feasibility of adapting this LC-MS/MS based GALT/GALK assay as a newborn screening (NBS) test. SN - 1096-7206 UR - https://www.unboundmedicine.com/medline/citation/20863731/Ultra_fast_and_sensitive_liquid_chromatography_tandem_mass_spectrometry_based_assay_for_galactose_1_phosphate_uridylyltransferase_and_galactokinase_deficiencies_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1096-7192(10)00317-3 DB - PRIME DP - Unbound Medicine ER -