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This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: clinchem.2006.071167v1 52/12/2243    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by Kuhn, J. Articles by Kleesiek, K. Search for Related Content PubMed PubMed Citation Articles by Kuhn, J. Articles by Kleesiek, K. Related Collections Proteomics and Protein Markers

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

Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein–Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany.

^aAddress correspondence to this author at: Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein–Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstraße 11, 32545 Bad Oeynhausen, Germany. Fax 49-5731-972013; e-mail jkuhn{at}hdz-nrw.de.

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.

The following articles in journals at HighWire Press have cited this article:

				C. Gotting, J. Kuhn, and K. Kleesiek Serum Xylosyltransferase Activity in Diabetic Patients as a Possible Marker of Reduced Proteoglycan Biosynthesis Diabetes Care, October 1, 2008; 31(10): 2018 - 2019. [Abstract] [Full Text] [PDF]

				C. Ponighaus, M. Ambrosius, J. C. Casanova, C. Prante, J. Kuhn, J. D. Esko, K. Kleesiek, and C. Gotting Human Xylosyltransferase II Is Involved in the Biosynthesis of the Uniform Tetrasaccharide Linkage Region in Chondroitin Sulfate and Heparan Sulfate Proteoglycans J. Biol. Chem., February 23, 2007; 282(8): 5201 - 5206. [Abstract] [Full Text] [PDF]