Galactose 1-Phosphate Uridyl Transferase

Principle Of Method: The enzyme measured catalyses the reaction.

Galactose 1-phosphate + UDP-glucose ® UDP-galactose + glucose 1-phosphate

Two different assay methods are used, one for red cells and the other for fibroblasts, chorionic villi and amniotic cells. In the former the glucose 1-phosphate formed in the reaction is converted to glucose 6-phosphate by endogenous phosphoglucomutase. Endogenous glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase result in oxidation of the glucose 6-phosphate with reduction of added NADP to NADPH (2 molecules per molecule glucose 1-phosphate). The reaction is stopped by dilution with phosphate buffer and the NADPH measured fluorimetrically.

In the latter method, labelled [14C]-galactose 1-phosphate is used as substrate with UDP-glucose. At the end of the incubation the unreacted galactose 1-phosphate is degraded to galactose by addition of alkaline phosphatase and the reaction mixture is spotted on to small pieces of DEAE chromatography paper. The [14C]-galactose is washed off with water while the UDP [14C]-galactose formed remains bound to the paper, and is determined by liquid scintillation counting.

Uses And Limitations Of The Method: The red cell method is used to diagnose classical galactosaemia using a heparinised blood specimen. It can also be used to detect heterozygotes and variants such as those carrying the “Duarte” allele. The method is not suitable for prenatal diagnosis of galactosaemia.

The main use for the isotope method is for prenatal diagnosis of galactosaemia using chorionic villi or amniotic cells. It may sometimes be helpful in making a diagnosis in patients with equivocal results from the routine red cell assay. In such cases cultured fibroblasts are assayed.

Specimen Requirements: 0.5ml heparinised blood (orange top tube) is required, unseparated and sent to the laboratory at ambient temperature. For testing for homozygotes (ie affected patients) delivery by first class post is sufficient, but for testing heterozygotes it is recommended that the blood is sent by courier or Royal Mail Special Delivery.

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