title: Determination of globotriaosylceramide in plasma and urine by mass spectrometry.
abstract: 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 alpha-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 <or=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 microg/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.
authors: Institute of Clinical Chemistry and Laboratory Medicine, Medical Center of the Johannes Gutenberg University, Mainz, Germany. krueger@zentrallabor.klinik.uni-mainz.de
authors: Ralf
authors: R
authors: Krüger
authors: Kai
authors: K
authors: Bruns
authors: Silke
authors: S
authors: Grünhage
authors: Heidi
authors: H
authors: Rossmann
authors: Jörg
authors: J
authors: Reinke
authors: Michael
authors: M
authors: Beck
authors: Karl J
authors: KJ
authors: Lackner
mesh_terms: D002138:Calibration; D002853:Chromatography, Liquid; D056947:Enzyme Replacement Therapy; D000795:Fabry Disease / Q000097:blood / Q000175:diagnosis / Q000628:therapy / Q000652:urine; D005260:Female; D006801:Humans; D016464:Lysosomal Storage Diseases / Q000201:enzymology / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008297:Male; D012015:Reference Standards; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry; D014281:Trihexosylceramides / Q000097:blood* / Q000652:urine*; D000519:alpha-Galactosidase / Q000378:metabolism
chemical_list: D014281:Trihexosylceramides; C018549:globotriaosylceramide; D000519:alpha-Galactosidase