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

Linn Oftedal,Marthe Gurine Gunnarsdatter Førland,Guido Alves,Jodi Maple-Grødem,Johannes Lange

doi:10.1038/s41598-020-79104-5

Linn Oftedal, Marthe Gurine Gunnarsdatter Førland + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-79104-5

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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.

Highlights

Lysosomal dysfunction is an emerging feature in the pathology of Parkinson’s disease and Dementia with Lewy bodies
The notion that cerebrospinal fluid (CSF) β-glucocerebrosidase activity is reduced in Parkinson’s disease (PD) patients independent of their GBA mutation carrier status suggests that GCase activity could be a valuable biomarker for idiopathic forms of PD and Dementia with Lewy bodies (DLB) and as an objective outcome measure in clinical trials[13], and further studies are warranted
We modified and optimized the assay described by van Dijk et al.[11] to determine GCase activity in CSF of PD patients

Summary

Introduction

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. Abbreviations CSF Cerebrospinal fluid GCase Glucocerebrosidase 4-MU 4-Methylumbelliferyl LLOQ Lower limit of quantification PD Parkinson’s disease DLB Dementia with Lewy bodies GD Gaucher disease LP Lumbar puncture. The most commonly known genetic risk factor for development of these synucleinopathies are mutations in the GBA gene[2] This gene encodes the enzyme glucocerebrosidase (GCase) that catalyzes the hydrolytic cleavage of g lycosphingolipids[3]. The notion that CSF β-glucocerebrosidase activity is reduced in PD patients independent of their GBA mutation carrier status suggests that GCase activity could be a valuable biomarker for idiopathic forms of PD and DLB and as an objective outcome measure in clinical trials[13], and further studies are warranted

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