Direct non-radioactive assay of galactose-1-phosphate:uridyltransferase activity using high performance liquid chromatography

Martijn Lindhout, M Estela Rubio-Gozalbo, Jaap A. Bakker, Jorgen Bierau*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

14 Citations (Web of Science)

Abstract

Background Galactose-1-phosphate uridyltransferase (GALT) catalyses the conversion of galactose-1-phosphate (Gal-1-P) and UDP-glucose (UDP-Glc) into glucose-6-phosphate and UDP-galactose (UDP-Gal) Complete, or near complete, deficiency of GALT causes classic galactosaemia. The diagnosis is confirmed by measuring GALT activity in erythrocytes The most commonly used assays require radio labelled substrates or indirect coupled assays Methods: GALT activity was measured in erythrocyte lysates using optimal concentrations of the substrates galactose-1-phosphate and UDP-Glc UDP-Gal and UDP-Glc were separated using reversed-phase high performance liquid chromatography with UV detection Clinical validity was assessed using blood samples from galactosaemic patients Results UDP-Gal and UDP-Glc were separated with HPLC. The assay was linear with incubation times up 80 min and between 0 and 425 nmol haemoglobin Within-day and between-day imprecision at 50, 75 and 100% enzyme activity was
Original languageEnglish
Pages (from-to)980-983
JournalClinica Chimica Acta
Volume411
Issue number13-14
DOIs
Publication statusPublished - 4 Jul 2010

Keywords

  • Galactosaemia
  • Galactose-1-phosphate uridyltransferase

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