|Values are valid only on day of printing.|
An aqueous mixture containing CLR H2O, uridine diphosphate (UDP)-glucose, (13)C(2)-labeled galactose-1-phosphate, and UDP-n-acetylglucosamine (internal standard) is added to hemolysate aliquot. The mixture is then vortexed briefly and incubated. The enzyme galactose-1-phosphate uridyltransferase (GALT) (EC 126.96.36.199) will convert (13)C(2)-labeled galactose-1-phosphate to (13)C(2)-labeled UDP-galactose during the incubation.
(13)C(2)-galactose-1-phosphate + UDP-glucose -> (13)C(2)-UDP-galactose + glucose-1-phosphate
After incubation the reaction is quenched and extracted. The mixture is then centrifuged. The top acetonitrile extract layer is then transferred to a 96-well (Nunc, polypropylene) plate. Then extract is injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) API 3200 system for chromatographic separation and measurement of analytes. The MS/MS is operated in the multiple reaction monitoring (MRM) negative mode to follow the precursor to product species transitions for (13)C(2)-labeled UDP-galactose (567.0 to 322.9 m/z) and internal standard of UDP-n-acetylglucosamine (606.2 to 384.7 m/z). The ratio of the extracted peak area of (13)C(2)labeled UDP-galactose to its internal standard UDP-n-acetylglucosamine as determined by liquid chromatography-tandem mass spectrometry is used to calculate the concentration of product analyte in the sample. The concentration of the product is then normalized using the calculated hemoglobin concentration to determine the patient's enzyme level in nmol/h/mg of hemoglobin.(Unpublished Mayo method)
A PCR-based assay utilizing Sequenom Mass Array platform is used to test for the presence of the following 14 mutations in the GALT gene: -119_-116delGTCA, D98N, S135L, T138M, M142K, F171S, Q188R, L195P, Y209C, K285N, N314D, Q344K, c.253-2A>G, and 5 kb deletion.(Unpublished Mayo method)
Monday, Wednesday, Friday; 7 a.m. set up (specimen must arrive the day prior)