|Values are valid only on day of printing.|
Ninety mcL of 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 30 mcL hemolysate aliquot. The mixture is then vortexed briefly and incubated while rotating at 225 RPM and 37 degrees C for 15 minutes. The enzyme galactose-1-phosphate uridyltransferase (GALT) (EC 220.127.116.11) will convert (13)C(2)-labeled galactose-1-phosphate to (13)C(2)-labeled UDP-galactose during a 15 minute incubation at 37 degrees C.
(13)C(2)-galactose-1-phosphate + UDP-glucose -> (13)C(2)-UDP-galactose + glucose-1-phosphate
After incubation the reaction is quenched and extracted with 250 mcL of acetonitrile, vortexing for 30 seconds on a multi-tube vortexer. The mixture is then centrifuged at 3,000 rpm for 15 minutes in an ambient centrifuge. The top acetonitrile extract layer is then transferred to a 96-well (Nunc, polypropylene) plate. Then 10 mcL 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using the calculated hemoglobin concentration to determine the patient's enzyme level in nmol/h/mg of hemoglobin.(Unpublished Mayo method)
Monday, Wednesday, Friday; 7 a.m. set up (specimen must be received the day prior)