Validation and assessment of preanalytical factors of a fluorometric in vitro assay for glucocerebrosidase activity in human cerebrospinal fluid.
Sci Rep
; 10(1): 22098, 2020 12 16.
Article
en En
| MEDLINE
| ID: mdl-33328543
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 ß-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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Enfermedad de Parkinson
/
Cuerpos de Lewy
/
Alfa-Sinucleína
/
Glucosilceramidasa
Tipo de estudio:
Diagnostic_studies
/
Etiology_studies
/
Prognostic_studies
/
Risk_factors_studies
Límite:
Humans
Idioma:
En
Revista:
Sci Rep
Año:
2020
Tipo del documento:
Article
País de afiliación:
Noruega