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 μg/L (0.05 mU/L) and 163 μg/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.