Determination of beta4-galactosyltransferase-7 activity using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.
OBJECTIVE
Patients with Ehlers-Danlos syndrome were described to contain reduced activities of beta4-galactosyltransferase-7 (beta4Gal-T7). Therefore, measurement of beta4Gal-T7 activity can help to characterize defects in proteoglycan biosynthesis in patients with connective tissue diseases.


METHODS
We developed a sensitive and specific method to assay beta4Gal-T7 which is based on the transfer of galactose from UDP-galactose to the synthetic peptide Bio-BIK-F-Xyl.


RESULTS
Calibration curves exhibited consistent linearity in the range of 10-2000 microg/L Bio-BIK-F-Xyl-Gal, corresponding to a beta4Gal-T7 activity of 3.5-659 microU/L. The limit of detection and the lower limit of quantification were 3.70 microg/L (1.22 microU/L) and 4.50 microg/L Bio-BIK-F-Xyl-Gal (1.48 microU/L beta4Gal-T7 activity), respectively. Interassay imprecision (CV) was 8.1-13.1% in the range from 15.9 to 659 microU/L, and mean recovery was 85.3% (range 61.7-106.3%).


CONCLUSIONS
This sensitive, robust and interference-free LC-MS/MS assay allows an accurate determination of beta4Gal-T7 activity in human body fluids.
Kuhn
Joachim
J
Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstrasse 11, Bad Oeynhausen, Germany. jkuhn@hdz-nrw.de
Kleesiek
Knut
K
Götting
Christian
C
D000818:Animals; D002138:Calibration; D018929:Cell Culture Techniques; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004535:Ehlers-Danlos Syndrome / Q000097:blood / Q000201:enzymology; D005260:Female; D005690:Galactose / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism*; D006801:Humans; D008297:Male; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014531:Uridine Diphosphate Galactose / Q000378:metabolism
D014531:Uridine Diphosphate Galactose; D005700:Galactosyltransferases; C023244:UDP-galactose xylose galactosyltransferase; D005690:Galactose