Variant forms of galactosemia can now be readily identified, and both GALK and GALE can be assayed simultaneously, saving considerable additional efforts.
2] Nonstandard abbreviations: GALT, galactose-1-phosphate uridyltransferase; GALK, galactokinase; GALE, UDP-galactose 4-epimerase; UPLC-MS/MS, ultraperformance liquid chromatography-tandem mass spectrometry.
To evaluate the linearity of the reaction as a function of the amount of enzymes in the assay, we measured the GALK, GALT, and GALE enzyme activities in the erythrocyte samples at 2- to 20-fold, 5- to 40-fold, and 5- to 40-fold dilutions, respectively.
To validate the capability of our system, we analyzed 8 patients with confirmed galactosemia (4 with GALE deficiency, 3 with GALT deficiency, and 1 with GALK deficiency).
These mutant constructs were transfected into 293 (for GALK and GALT) or ldlD (for GALE) cells using the Lipofectamin 2000 system (Invitrogen) according to the manufacturer's recommendations.
To compare our revised method with a traditional radiometric assay (9), 19 whole bloods with various enzyme activities were selected and tested for their GALT and GALE activities and 33 samples for GALK activities.
In terms of GALK and GALT enzyme assay, no signal in the channels of products ([[sup.
We chose a 3-fold dilution of erythrocytes for all subsequent GALK assays, and a 20fold dilution for GALT and GALE assays.
GALK and GALT enzyme assays showed a linear response up to 240 min; GALE showed a linear response up to 120 min.
Passing and Bablok regression analysis revealed that the UPLC-MS/MS method and the radiometric assay compared favorably for GALK (r = 0.
The treatment center directors commented that in their experience true GALK and GALE deficiencies are associated with very high Gal values, usually >3.
4] Nonstandard abbreviations: NB, newborn; GALT, galactose-1-phosphate uridyltransferase; Gal, galactose; GALK, galactokinase; GALE, galactose-4-epimerase; D/G galactosemia, Duarte/classic galactosemia; Gal-1-P, galactose 1-phosphate; PA DOH, Pennsylvania Department of Health.