2. Academic Validation
  3. Validation and assessment of preanalytical factors of a fluorometric in vitro assay for glucocerebrosidase activity in human cerebrospinal fluid

Validation and assessment of preanalytical factors of a fluorometric in vitro assay for glucocerebrosidase activity in human cerebrospinal fluid

  • Sci Rep. 2020 Dec 16;10(1):22098. doi: 10.1038/s41598-020-79104-5.
Linn Oftedal 1 2,  Jodi Maple-Grødem 1 3,  Marthe Gurine Gunnarsdatter Førland 1 4,  Guido Alves 1 3 5,  Johannes Lange 6 7
Affiliations

Affiliations

  • 1 The Norwegian Centre for Movement Disorders, Stavanger University Hospital, Stavanger, Norway.
  • 2 Department of Psychiatry, Centre for Age-Related Medicine, Stavanger University Hospital, Stavanger, Norway.
  • 3 Department of Chemistry, Bioscience and Environmental Engineering, University of Stavanger, Stavanger, Norway.
  • 4 Centre for Organelle Research, University of Stavanger, Stavanger, Norway.
  • 5 Department of Neurology, Stavanger University Hospital, Stavanger, Norway.
  • 6 The Norwegian Centre for Movement Disorders, Stavanger University Hospital, Stavanger, Norway. Johannes.lange@sus.no.
  • 7 Centre for Organelle Research, University of Stavanger, Stavanger, Norway. Johannes.lange@sus.no.
PMID: 33328543 DOI: 10.1038/s41598-020-79104-5
Abstract

Lysosomal dysfunction is an emerging feature in the pathology of Parkinson's disease and Dementia with Lewy bodies. Mutations in the GBA gene, encoding the Enzyme Glucocerebrosidase (GCase), have been identified as a genetic risk factor for these synucleinopathies. As a result, there has been a growing interest in the involvement of GCase in these diseases. This GCase activity assay is based on the catalytic hydrolysis of 4-methylumbelliferyl β-D-glucopyranoside that releases the highly fluorescent 4-methylumbelliferyl (4-MU). The final assay protocol was tested for the following parameters: Lower limit of quantification (LLOQ), precision, parallelism, linearity, spike recovery, number of freeze-thaw events, and sample handling stability. The GCase activity assay is within acceptable criteria for parallelism, precision and spike recovery. The LLOQ of this assay corresponds to an enzymatic activity of generating 0.26 pmol 4-MU/min/ml. The enzymatic activity was stable when samples were processed and frozen at - 80 °C within 4 h after the lumbar puncture procedure. Repetitive freeze-thaw events significantly decreased Enzyme activity. We present the validation of an optimized in vitro GCase activity assay, based on commercially available components, to quantify its enzymatic activity in human cerebrospinal fluid and the assessment of preanalytical factors.

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