The GALE reaction catalyzes the reversible conversion of uridine diphosphate galactose (UDPGal) to uridine diphosphate glucose (UDPGlc).
The RPMI media, L-glutamine, and glycine (culture grade) were from Fisher Scientific; fetal bovine serum and HEPES from Invitrogen; and UDPGal ([greater than or equal to] 97%), UDPGlc ([greater than or equal to] 98%), and all other chemicals and reagents from Sigma-Aldrich.
The calibrators had 2 variables: one was the total concentration of UDPGal and UDPGlc (UDPGal and UDPGlc) and the other was the percentage of UDPGal and UDPGlc that was accounted for by UDPGal (Table 1).
Three volumes of 17.3 mmol/L [NAD.sup.+] solution, 4 volumes of 4.0 mmol/L UDPGal solution, and 15 volumes of glycine buffer (0.5 mol/L, pH 8.7) were added to a 1.5-mL microcentrifuge tube and mixed well.
We examined the assay conditions for optimal substrate concentration by running the assay at different UDPGal concentrations (0.0,0.1,0.25,0.5,1, and 1.5 mmol/L) and optimal [NAD.sup.+] concentration.
We evaluated recovery of the assay by spiking a known amount of UDPGlc into reaction mixtures in which the enzyme substrate, UDPGal, was added after the reaction mixture was inactivated by boiling the samples for 3 min.
Six calibrators were prepared, which consisted of aqueous solutions containing the internal standard (IS) [[sup.13][C.sub.6]]-Glu-1-P at a constant concentration of 2.0 [micro]mol/L and uridine diphosphate galactose (UDPGal) concentrations of 12.5, 2.5, 0.5, 0.1, 0.02, and 0.004 [micro]mol/L.
A product ion scan of UDPGal revealed 1 major ion of m/z 323 (negative ion of uridine monophosphate), which proved to be the strongest ion for [[sup.13][C.sub.6]]-UDPGal as well.
The MS/MS transitions of 259 > 79 and 565 > 323 were employed for the detection of unlabeled Glu-1-P and UDPGal, respectively.
1D (565 > 323), which is a combination of UDPGlc and UDPGal, owing to the fact that they have the same retention time and fragmentation pattern.
In an independent study, a known amount of UDPGal in solution was spiked into a GALT assay sample after the reaction was quenched.