title: Mass spectrometric quantification of glucosylsphingosine in plasma and urine of type 1 Gaucher patients using an isotope standard.
abstract: Deficiency of glucocerebrosidase (GBA) leads to Gaucher disease (GD), an inherited disorder characterised by storage of glucosylceramide (GlcCer) in lysosomes of tissue macrophages. Recently, we reported marked increases of deacylated GlcCer, named glucosylsphingosine (GlcSph), in plasma of GD patients. To improve quantification, [5-9] (13)C5-GlcSph was synthesised for use as internal standard with quantitative LC-ESI-MS/MS. The method was validated using plasma of 55 GD patients and 20 controls. Intra-assay variation was 1.8% and inter-assay variation was 4.9% for GlcSph (m/z 462.3). Plasma GlcSph levels with the old and new methods closely correlate (r=0.968, slope=1.038). Next, we analysed GlcSph in 24h urine samples of 30 GD patients prior to therapy. GlcSph was detected in the patient samples (median 1.20nM, range 0.11-8.92nM), but was below the limit of quantification in normal urine. Enzyme replacement therapy led to a decrease of urinary GlcSph of GD patients, coinciding with reductions in plasma GlcSph and markers of Gaucher cells (chitotriosidase and CCL18). In analogy to globotriaosylsphingsone in urine of Fabry disease patients, additional isoforms of GlcSph differing in structure of the sphingosine moiety were identified in GD urine samples. In conclusion, GlcSph can be sensitively detected by LC-ESI-MS/MS with an internal isotope standard. Abnormalities in urinary GlcSph are a hallmark of Gaucher disease allowing biochemical confirmation of diagnosis.
authors.affiliation: Department of Medical Biochemistry, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Mina
authors.initials: M
authors.lastname: Mirzaian
authors.affiliation: Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Patrick
authors.initials: P
authors.lastname: Wisse
authors.affiliation: Department of Medical Biochemistry, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Ferraz
authors.affiliation: Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Henrik
authors.initials: H
authors.lastname: Gold
authors.affiliation: Department of Medical Biochemistry, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Wilma E
authors.initials: WE
authors.lastname: Donker-Koopman
authors.affiliation: Department of Medical Biochemistry, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Marri
authors.initials: M
authors.lastname: Verhoek
authors.affiliation: Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Herman S
authors.initials: HS
authors.lastname: Overkleeft
authors.affiliation: Department of Medical Biochemistry, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Rolf G
authors.initials: RG
authors.lastname: Boot
authors.affiliation: Department of Medical Biochemistry, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Gertjan
authors.initials: G
authors.lastname: Kramer
authors.affiliation: Department of Medical Biochemistry, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Nick
authors.initials: N
authors.lastname: Dekker
authors.affiliation: Department of Medical Biochemistry, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands. Electronic address: j.m.f.g.aerts@lic.leidenuniv.nl.
authors.forename: Johannes M F G
authors.initials: JM
authors.lastname: Aerts
mesh_terms: D015415:Biomarkers / Q000097:blood / Q000652:urine; D002247:Carbon Isotopes; D016022:Case-Control Studies; D019742:Chemokines, CC / Q000097:blood; D056947:Enzyme Replacement Therapy; D005776:Gaucher Disease / Q000097:blood / Q000175:diagnosis* / Q000188:drug therapy* / Q000652:urine; D005962:Glucosylceramidase / Q000172:deficiency / Q000627:therapeutic use*; D006596:Hexosaminidases / Q000097:blood; D006801:Humans; D015588:Observer Variation; D011609:Psychosine / Q000031:analogs & derivatives* / Q000097:blood / Q000652:urine; D012015:Reference Standards; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D015415:Biomarkers; C111792:CCL18 protein, human; D002247:Carbon Isotopes; D019742:Chemokines, CC; D011609:Psychosine; C035742:sphingosyl beta-glucoside; D006596:Hexosaminidases; C086400:chitotriosidase; D005962:Glucosylceramidase
keywords: Gaucher disease; Glucocerebrosidase; Glucosylsphingosine; LC-MS/MS; Mass spectrometry; Quantification