Test ID: GALTP    
Galactose-1-Phosphate Uridyltransferase Biochemical Phenotyping, Erythrocytes

Isoelectric focusing is used to resolve the isoenzymes of galactose-1-phosphate uridyltransferase (GALT). The band patterns, when used in conjunction with a quantitative GALT result, can be used to predict the GALT phenotype of an individual.

In isoelectric focusing, a pH gradient is established across an agarose gel by adding a select mixture of amphoteric molecules to the gel and applying an electric field to the gel. Each protein (isoenzyme) has its own unique isoelectric point, a pH at which the net charge of the protein is equal to zero. Therefore, if a protein is applied to the gel, it will migrate through the pH gradient in the gel until it reaches its isoelectric point. There the protein will stop and "focus" into distinct bands.

In this procedure, an RBC hemolysate is focused on a 5% agarose gel containing ampholytes of a 5 to 7 pH range. The isoenzyme bands are then visualized by applying a substrate mixture that results in a series of reactions (shown below). The final product, nicotinamide adenine dinucleotide phosphate (NADPH; reduced form), is stained a blue-violet color when it reacts with phenazine methosulfate (PMS) and 3-(4-5 dimethylthiazol-2-yl)I-2,5-diphenyltetrazolium bromide (MTT). (Shin YS, Niedermeier HP, Endres W, et al: Agarose gel isoelectrofocusing of UDP-galactose pyrophosphorylase and galactose-1-phosphate uridyltransferase: developmental aspect of UDP-galactose pyrophosphorylase. Clin Chim Acta 1987;166:27-35, modified to acrylamide as described by Leclerc P, Forest JC: Electrophoretic determination of isoamylases in serum with commercially available reagents. Clin Chem 1982;28:37-40)

Thursday; 9 a.m.