title: A Novel Rapid MALDI-TOF-MS-Based Method for Measuring Urinary Globotriaosylceramide in Fabry Patients.
abstract: Fabry disease is an X-linked lysosomal storage disorder caused by deficiency of α-galactosidase A, resulting in the accumulation of glycosphingolipids in various organs. Globotriaosylceramide (Gb3) and its isoforms and analogues have been identified and quantified as biomarkers of disease severity and treatment efficacy. The current study aimed to establish rapid methods for urinary Gb3 extraction and quantitation. Urine samples from 15 Fabry patients and 21 healthy control subjects were processed to extract Gb3 by mixing equal volumes of urine, methanol containing an internal standard, and chloroform followed by sonication and centrifugation. Thereafter, the lower phase was analyzed by MALDI-TOF MS and the relative peak areas of the internal standard and four major species of Gb3 determined. The results showed high reproducibility with intra- and inter-assay coefficients variation of 9.9% and 13.7%, respectively. The limit of detection was 0.15 ng/μL and the limit of quantitation was 0.30 ng/μL. Total urinary Gb3 levels in both genders of classic Fabry patients were significantly higher than in healthy controls (p < 0.0001). Gb3 levels in Fabry males were higher than in Fabry females (p = 0.08). We have established a novel assay for urinary total Gb3 that takes less than 15 min from start to finish. Graphical Abstract ᅟ.
authors.affiliation: Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT, England.
authors.forename: Fahad J
authors.initials: FJ
authors.lastname: Alharbi
authors.affiliation: Queen Elizabeth Medical Centre, University Hospitals Birmingham NHS Foundation Trust, Birmingham, B15 2TH, England.
authors.forename: Tarekegn
authors.initials: T
authors.lastname: Geberhiwot
authors.affiliation: Department of Haematology, Lysosomal Storage Disorders Unit, Royal Free Hospital, London, NW3 2QG, England.
authors.forename: Derralynn A
authors.initials: DA
authors.lastname: Hughes
authors.affiliation: Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT, England. D.G.Ward@bham.ac.uk.
authors.forename: Douglas G
authors.initials: DG
authors.lastname: Ward
mesh_terms: D015415:Biomarkers / Q000652:urine; D000795:Fabry Disease / Q000652:urine*; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000191:economics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014281:Trihexosylceramides / Q000652:urine*
chemical_list: D015415:Biomarkers; D014281:Trihexosylceramides; C018549:globotriaosylceramide
keywords: Biomarker; Fabry disease; Gb3; MALDI-TOF-MS