title: Measurement of lysosomal glucocerebrosidase activity in mouse liver using a fluorescence-activated cell sorter assay.
abstract: Lysosomal acid beta-glucocerebrosidase hydrolyzes glucocerebroside to glucose ceramide. Patients diagnosed with Gaucher disease, however, lack this enzyme, leading to the accumulation of glucocerebroside in tissue macrophages within multiple organs. Such patients can receive enzyme replacement therapy during which a human placental-derived or recombinant form of acid beta-glucocerebrosidase is targeted to the macrophages. As part of evaluating the effectiveness of such therapies, currently available methodologies for measuring acid beta-glucocerebrosidase activity are primarily conducted in cultured cell lines or tissue culture. However, these in vitro assays are limited by their ability to evaluate the efficacy of in vivo acid beta-glucocerebrosidase replacement therapy in animal models. In particular, there is an unmet need to simultaneously define cellular localization and evaluate enzyme activity following treatment in vivo. In addition, results of commonly used fluorescent-based assays for enzyme activity are difficult to compare day to day and/or across laboratories due to the variability inherent in flow cytometric measurement. In this article, we describe a reproducible and consistent quantitative method for the combined measurement of fluorescein intensity from enzyme-substrate conversion and cell localization by phenotype-specific phycoerythrin-antibody staining. Following infusion of recombinant human acid beta-glucocerebrosidase in mice, nonparenchymal cells are prepared from the livers of treated and control animals. Acid beta-glucocerebrosidase activity is measured in molecules of equivalent soluble fluorophore units within Kupffer cell populations as defined by phenotype-specific monoclonal antibodies. This assay should be applicable to investigations of other Gaucher disease treatments in both human and animal models.
authors.affiliation: Department of Preclinical Studies, Genzyme Corporation, Framingham, MA 01701, USA. katherine.chan@pharma.novartis.com
authors.forename: Katherine Watt
authors.initials: KW
authors.lastname: Chan
authors.forename: James
authors.initials: J
authors.lastname: Waire
authors.forename: Betsy
authors.initials: B
authors.lastname: Simons
authors.forename: Ken
authors.initials: K
authors.lastname: Karey
authors.forename: John
authors.initials: J
authors.lastname: Fung
authors.forename: Diane
authors.initials: D
authors.lastname: Copeland
authors.forename: Laura
authors.initials: L
authors.lastname: Andrews
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005962:Glucosylceramidase / Q000008:administration & dosage / Q000378:metabolism*; D007294:Inositol / Q000008:administration & dosage / Q000031:analogs & derivatives*; D008099:Liver / Q000166:cytology* / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D015203:Reproducibility of Results
chemical_list: D007294:Inositol; D005962:Glucosylceramidase; C011423:conduritol epoxide