Measurement of fibrosis marker xylosyltransferase I activity by HPLC electrospray ionization tandem mass spectrometry.
BACKGROUND
Xylosyltransferase I (XT-I), the key enzyme in the biosynthesis of glycosaminoglycan chains in proteoglycans, has increased activity in the blood serum of patients with connective tissue diseases. Therefore, the measurement of serum XT-I activity is useful to monitor disease activity in these patients.


METHODS
We developed an HPLC electrospray ionization tandem mass spectrometry method to assay XT-I activity in serum by use of a synthetic peptide (Bio-BIK-F) as the XT-I substrate. On the basis of XT-I-mediated transfer of D-xylose from UDP-D-xylose to the synthetic peptide to form Bio-BIK-F-Xyl, we determined XT-I activity in human serum samples.


RESULTS
Multiple calibration curves for the analysis of Bio-BIK-F-Xyl exhibited consistent linearity and reproducibility in the range of 0.20-20 mg/L, corresponding to XT-I activity of 1.14-114 mU/L under assay conditions. The mean (SD, range) XT-I activity values in 30 blood donor sera were 18.4 (3.0, 8.7-24.8) mU/L. The limit of detection and lower limit of quantification were 8.5 microg/L (0.05 mU/L) and 163 microg/L Bio-BIK-F-Xyl (0.93 mU/L XT-I activity), respectively. Interassay imprecision (CV) was 5.4%-26.1% in the range of 0.64 to 129 mU/L, and mean recovery was 107% (range, 96%-129%). Method comparison with the radiochemical assay showed a moderate correlation (r = 0.79). The Passing-Bablok regression line was: radiochemical assay = 0.045 LC-MS/MS + 0.061 mU/L, S(y/x) = 0.186.


CONCLUSIONS
This simple and robust LC-MS/MS assay permits the rapid and accurate determination of XT-I activity in human serum.
Kuhn
Joachim
J
Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany. jkuhn@hdz-nrw.de
Prante
Christian
C
Schön
Sylvia
S
Götting
Christian
C
Kleesiek
Knut
K
D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000097:blood; D002138:Calibration; D002460:Cell Line; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D003240:Connective Tissue Diseases / Q000175:diagnosis* / Q000473:pathology; D005260:Female; D005355:Fibrosis; D006801:Humans; D007313:Insecta / Q000166:cytology; D008297:Male; D008875:Middle Aged; D010430:Pentosyltransferases / Q000032:analysis / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D000097608:UDP Xylose-Protein Xylosyltransferase
D015415:Biomarkers; D010430:Pentosyltransferases; D000097608:UDP Xylose-Protein Xylosyltransferase