Direct non-radioactive assay of galactose-1-phosphate:uridyltransferase activity using high performance liquid chromatography.
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 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/(micromol Hb.h) (range 492-697). Patients with classical galactosaemia were easily detected by their extremely low activity.


CONCLUSIONS
We have developed a reliable and convenient direct method to measure GALT activity in human erythrocytes using HPLC with UV detection.
Lindhout
Martijn
M
Department of Clinical Genetics, Maastricht University Medical Centre, Maastricht, The Netherlands.
Rubio-Gozalbo
M Estela
ME
Bakker
Jaap A
JA
Bierau
Jörgen
J
D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D057075:Enzyme Assays / Q000379:methods* / Q000592:standards; D005260:Female; D005693:Galactosemias / Q000097:blood / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008297:Male; D012016:Reference Values; D015203:Reproducibility of Results; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000097:blood / Q000378:metabolism*; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D014531:Uridine Diphosphate Galactose; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase; D014532:Uridine Diphosphate Glucose