title: Quantification of globotriaosylsphingosine in plasma and urine of fabry patients by stable isotope ultraperformance liquid chromatography-tandem mass spectrometry.
abstract: BACKGROUND
  Biochemical markers that accurately reflect the severity and progression of disease in patients with Fabry disease and their response to treatment are urgently needed. Globotriaosylsphingosine, also called lysoglobotriaosylceramide (lysoGb3), is a promising candidate biomarker.
  
  
  METHODS
  We synthesized lysoGb3 and isotope-labeled [5,6,7,8,9] (13)C5-lysoGb3 (internal standard). After addition of the internal standard to 25 μL plasma or 400 μL urine from patients with Fabry disease and healthy controls, samples were extracted with organic solvents and the lysoGb3 concentration was quantified by UPLC-ESI-MS/MS (ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry). Calibration curves were constructed with control plasma and urine supplemented with lysoGb3. In addition to lysoGb3, lyso-ene-Gb3 was quantified. Quantification was achieved by multiple reaction monitoring of the transitions m/z 786.4 > 282.3 [M+H](+) for lysoGb3, m/z 791.4 > 287.3 [M+H](+) for [5,6,7,8,9] (13)C5-lysoGb3, and 784.4 > 280.3 [M+H](+) for lyso-ene-Gb3.
  
  
  RESULTS
  The mean (SD) plasma lysoGb3 concentration from 10 classically affected Fabry hemizygotes was 94.4 (25.8) pmol/mL (range 52.7-136.8 pmol/mL), from 10 classically affected Fabry heterozygotes 9.6 (5.8) pmol/mL (range 4.1-23.5 pmol/mL), and from 20 healthy controls 0.4 (0.1) pmol/mL (range 0.3-0.5 pmol/mL). Lyso-ene-Gb3 concentrations were 10%-25% of total lysoGb3. The urine concentration of lysoGb3 was 40-480 times lower than in corresponding plasma samples. Lyso-ene-Gb3 concentrations in urine were comparable or even higher than the corresponding lysoGb3 concentrations.
  
  
  CONCLUSIONS
  This assay for the quantification of lysoGb3 and lyso-ene-Gb3 in human plasma and urine samples will be an important tool in the diagnosis of Fabry disease and for monitoring the effect of enzyme replacement therapy in patients with Fabry disease.
authors.affiliation: Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Henrik
authors.initials: H
authors.lastname: Gold
authors.forename: Mina
authors.initials: M
authors.lastname: Mirzaian
authors.forename: Nick
authors.initials: N
authors.lastname: Dekker
authors.forename: Maria
authors.initials: M
authors.lastname: Joao Ferraz
authors.forename: Johan
authors.initials: J
authors.lastname: Lugtenburg
authors.forename: Jeroen D C
authors.initials: JD
authors.lastname: Codée
authors.forename: Gijs A
authors.initials: GA
authors.lastname: van der Marel
authors.forename: Herman S
authors.initials: HS
authors.lastname: Overkleeft
authors.forename: Gabor E
authors.initials: GE
authors.lastname: Linthorst
authors.forename: Johanna E M
authors.initials: JE
authors.lastname: Groener
authors.forename: Johannes M
authors.initials: JM
authors.lastname: Aerts
authors.forename: Ben J H M
authors.initials: BJ
authors.lastname: Poorthuis
mesh_terms: D000328:Adult; D002138:Calibration; D002247:Carbon Isotopes; D002853:Chromatography, Liquid / Q000379:methods*; D000795:Fabry Disease / Q000175:diagnosis*; D006017:Glycolipids / Q000032:analysis*; D006801:Humans; D007553:Isotope Labeling; D008875:Middle Aged; D015203:Reproducibility of Results; D013107:Sphingolipids / Q000032:analysis*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002247:Carbon Isotopes; D006017:Glycolipids; D013107:Sphingolipids; C063288:globotriaosyl lysosphingolipid