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

BackgroundGalactose-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.MethodsGALT 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.ResultsUDP-Gal and UDP-Glc were separated with HPLC. The assay was linear with incubation times up 80 min and between 0 and 42.5 nmol haemoglobin. Within-day and between-day imprecision at 50, 75 and 100% enzyme activity was < 1.4% and < 2.4%, respectively. Mean GALT activity in 33 individuals was 601 ± 79 nmol UDP-Gal/(µmol Hb · h) (range 492–697). Patients with classical galactosaemia were easily detected by their extremely low activity.ConclusionsWe have developed a reliable and convenient direct method to measure GALT activity in human erythrocytes using HPLC with UV detection.