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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Schlattmann, Peter / Tate, Jillian R. / Tsongalis, Gregory J.

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Determination of globotriaosylceramide in plasma and urine by mass spectrometry

Ralf Krüger1 / Kai Bruns1, a / Silke Grünhage1 / Heidi Rossmann1 / Jörg Reinke2 / Michael Beck2 / Karl J. Lackner1

1Institute of Clinical Chemistry and Laboratory Medicine, Medical Center of the Johannes Gutenberg University, Mainz, Germany

2Department of Pediatrics/Villa Metabolica, Medical Center of the Johannes Gutenberg University, Mainz, Germany

aPresent address: Sanofi-Aventis Deutschland GmbH, Industriepark Hoechst, Frankfurt, Germany.

Corresponding author: Dr. Ralf Krüger, Institute of Clinical Chemistry and Laboratory Medicine, Medical Center of the Johannes Gutenberg University, Langenbeckstr. 1, 55131 Mainz, Germany

Citation Information: Clinical Chemistry and Laboratory Medicine. Volume 48, Issue 2, Pages 189–198, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2010.048, December 2009

Publication History

Published Online:


Background: Fabry disease is an X-chromosomally inherited lysosomal storage disorder leading to accumulation of glycosphingolipids, mainly globotriaosylceramide (ceramide-trihexoside, Gb3). Concentrations of Gb3 in plasma and urine have been used to diagnose Fabry disease and to monitor enzyme replacement therapy with recombinant α-galactosidase.

Methods: Gb3 was purified from plasma or urine by combined liquid extraction/protein precipitation and solid-phase extraction, and was detected by flow-injection analysis electrospray mass spectrometry (MS) using multi-reaction-monitoring. Calibration was performed via standard addition using C17-Gb3 as internal standard. The most abundant isoforms were monitored for calculation of total Gb3.

Results: A MS-based assay for quantification of Gb3 in plasma and urine was established and validated. Intra- and inter-assay coefficient of variation (CV) of the method were ≤12%. However, at low concentrations the CV was 16%. The linear range covers roughly two orders of magnitude, down to 0.54 mg/L in plasma and 0.07 mg/L in urine. Careful adjustment of tuning parameters was necessary to obtain identical isoform intensities and quantitative results on different mass spectrometers. Gb3 concentrations in healthy controls were <4 mg/L in EDTA-plasma and <10 μg/mmol creatinine in urine. Significantly increased Gb3 concentrations were found in plasma and urine from male and female patients with Fabry disease.

Conclusions: An improved MS protocol for Gb3 quantification has been developed, validated, and shown to be suitable for diagnosis and monitoring of Fabry patients.

Clin Chem Lab Med 2010;48:189–98.

Keywords: Fabry disease; Gb3 (globotriaosylceramide); glycosphingolipid; liquid chromatography-tandem mass spectrometry; lysosomal storage disorder

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