Improved UPLC tandem mass spectrometry assay for uridine diphosphate galactose-4-epimerase deficiency

摘要

This two step enzyme assay was shown to be an accurate and very sensitive method for the measurement of UDP-galactose-4-epimerase activity in erythrocytes. The unique second step of enzyme assay convert UDP-glucose to UDP-glucuronic acid which is separated from UDPglucose during the run on the LC column and has its different MRM transition. This resolves the challenge of traditional LC/MS/MS methods of attempting to separate UDP-glucose and UDP-galactose. The isomers of UDP-glucose and UDP-galactose not only have the same MRM transitions but also have the same retention times unless anion pairing regent such as TEA is used. The use of TEA is not acceptable in clinical practice as it results in the need for excessively frequent instrument cleaning. The mass of the uniformly labeled UDP-galactose substrate is 6 units higher than that of unlabeled UDP-galactose. That means that the intermediate product UDP-glucose and the final product of this assay UDP-glucuronic acid are also uniformly labeled. This assures that there is no interference from endogenous unlabeled UDP-galactose and UDPglucose in our red blood cell pellets and also eliminates any potential natural abundance conflicts. The calibration curve were prepared in UDP-glucose which went through the second enzyme step with the rest of the blank and patient samples converting to UDP-glucuronic acid. This eliminates any possible effect of variation in the activity of UDP-glucose dehydrogenase. Unlike the GALT assay, the GALE assay was found to be very sensitive to a matrix effect. As shown in Figure 3, The GALE activity decreases when hemoglobin concentration increases. Diluting the red blood cell lysate is crucial to obtaining accurate results. We recommend diluting the washed RBC 20 times where the hemoglobin concentration is about 20 g/L. Then use 10 μl of the hemolysate to start the assay. This sample preparation remove the matrix effect.