title: Quantification of α-galactosidase activity in intact leukocytes.
abstract: BACKGROUND
  Mutations of the α-galactosidase (α-Gal) A gene in Fabry disease lead to a severe disturbance in glycosphingolipid catabolism. The atypical clinical picture of Fabry disease hampers diagnosis, resulting in a delayed start of therapy. Current tests utilize leukocyte lysates to evaluate the activity of α-Gal A. It has never been investigated whether cell homogenisation is necessary.
  
  
  METHODS
  Isolated leukocyte subsets were incubated with the α-Gal substrate methylumbelliferyl-α-D galactopyranosid (MU-Gal) and substrate conversion was measured by fluorimetry. Specificity of the reaction was evaluated using the α-Gal inhibitor deoxygalactonojirimycin (DGJ). The novel procedure was compared to the standard method. A reference population and Fabry patients were tested.
  
  
  RESULTS
  Substrate conversion in intact leukocytes was a function of substrate concentration, cell number and time and could be inhibited by DGJ. Monocytes showed the highest enzyme activity among leukocyte populations. The novel procedure highly correlated with the standard method. Both Fabry hemizygotes and heterozygotes showed reduced substrate conversion.
  
  
  CONCLUSIONS
  We here present a novel sensitive, fast and simple procedure for the evaluation of α-Gal activity suitable to identify enzyme deficiencies in Fabry patients. Furthermore, we show for the first time that leukocyte subtypes have different α-Gal activities.
authors.affiliation: Institute of Immunology, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Markus A
authors.initials: MA
authors.lastname: Hölzl
authors.forename: Miriam
authors.initials: M
authors.lastname: Gärtner
authors.forename: Johannes J
authors.initials: JJ
authors.lastname: Kovarik
authors.forename: Johannes
authors.initials: J
authors.lastname: Hofer
authors.forename: Hanno
authors.initials: H
authors.lastname: Bernheimer
authors.forename: Gere
authors.initials: G
authors.lastname: Sunder-Plassmann
authors.forename: Gerhard J
authors.initials: GJ
authors.lastname: Zlabinger
mesh_terms: D000795:Fabry Disease / Q000175:diagnosis*; D006801:Humans; D007962:Leukocytes / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D008722:Methods; D009000:Monocytes; D012107:Research Design / Q000592:standards*; D000519:alpha-Galactosidase / Q000032:analysis / Q000378:metabolism*
chemical_list: D000519:alpha-Galactosidase