Measurement of Fibrosis Marker Xylosyltransferase I Activity by HPLC Electrospray Ionization Tandem Mass Spectrometry

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 52; no. 12; pp. 2243 - 2249
• Main Authors: Kuhn, Joachim, Prante, Christian, Schon, Sylvia, Gotting, Christian, Kleesiek, Knut
• Format: Journal Article
• Language: English
• Published: England Am Assoc Clin Chem 01.12.2006; Oxford University Press
• Subjects: Adolescent; Adult; Aged; Analytical chemistry; Animals; Biomarkers - analysis; Biomarkers - blood; Biosynthesis; Blood; Calibration; Cell Line; Cell Line, Tumor; Chromatography; Chromatography, High Pressure Liquid; Connective Tissue Diseases - diagnosis; Connective Tissue Diseases - pathology; Enzymes; Female; Fibrosis; Humans; Insecta - cytology; Ionization; Ions; Liquid chromatography; Male; Mass spectrometry; Middle Aged; Pentosyltransferases - analysis; Pentosyltransferases - blood; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; UDP Xylose-Protein Xylosyltransferase
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2006.071167

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. 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. 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. This simple and robust LC-MS/MS assay permits the rapid and accurate determination of XT-I activity in human serum.