Factors Influencing the Measurement of Lysosomal Enzymes Activity in Human Cerebrospinal Fluid

Abstract

Measurements of the activities of lysosomal enzymes in cerebrospinal fluid have recently been proposed as putative biomarkers for Parkinson's disease and other synucleinopathies. To define the operating procedures useful for ensuring the reliability of these measurements, we analyzed several pre-analytical factors that may influence the activity of β-glucocerebrosidase, α-mannosidase, β-mannosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, cathepsin D and cathepsin E in cerebrospinal fluid. Lysosomal enzyme activities were measured by well-established fluorimetric assays in a consecutive series of patients (n = 28) with different neurological conditions, including Parkinson's disease. The precision, pre-storage and storage conditions, and freeze/thaw cycles were evaluated. All of the assays showed within- and between-run variabilities below 10%. At −20°C, only cathepsin D was stable up to 40 weeks. At −80°C, the cathepsin D, cathepsin E, and β-mannosidase activities did not change significantly up to 40 weeks, while β-glucocerebrosidase activity was stable up to 32 weeks. The β-galactosidase and α-fucosidase activities significantly increased (+54.9±38.08% after 4 weeks and +88.94±36.19% after 16 weeks, respectively). Up to four freeze/thaw cycles did not significantly affect the activities of cathepsins D and E. The β-glucocerebrosidase activity showed a slight decrease (−14.6%) after two freeze/thaw cycles. The measurement of lysosomal enzyme activities in cerebrospinal fluid is reliable and reproducible if pre-analytical factors are accurately taken into consideration. Therefore, the analytical recommendations that ensue from this study may contribute to the establishment of actual values for the activities of cerebrospinal fluid lysosomal enzymes as putative biomarkers for Parkinson's disease and other neurodegenerative disorders.

Novel CSF biomarkers to discriminate FTLD and its pathological subtypes

“…S1), except for lysosomal activities, which change only after two freeze–thaw cycles 31. Longer storage time was associated with decreased levels of MFG‐E8 ( P  < 0.0001), as well as the activities of catalase ( P  < 0.0001) and all lysosomal enzymes ( P  < 0.01).…”

“…Most of the samples of the validation cohort had already undergone more than two freeze–thaw cycles, which affect the overall activity values of tHexA and β HexA 31. The number of samples available in which the activity of such enzymes could be optimally measured (TDP = 12, FTLD‐Tau = 6) was too small and thus the “TDP vs. Tau” model could not be validated in an independent cohort.…”

Lysosomal Dysfunction and α‐Synuclein Aggregation in Parkinson's Disease: Diagnostic Links

“…Several other lysosomal hydrolases and proteases are present in CSF, and their activity as well as their protein level can be measured using biochemical or proteomic approaches. An interrogation of the recently released public database of CSF proteins identified with high‐resolution mass spectrometry evidenced how the CSF proteome contains numerous proteins that are considered to be localized in the lysosome .…”

“…In particular, follow‐up studies should focus on comparing CSF lysosomal enzymes and protein levels between (1) large independent patient cohorts; (2) patients with various neurodegenerative diseases, such as dementia with Lewy bodies, multiple system atrophy, and progressive supranuclear palsy, when compared with controls; (3) pathologically confirmed and genotyped patient cohorts; and (4) patients at different disease stages. The importance of strict preanalytical conditions for comparing lysosomal enzyme activity between patients and control groups is underscored by changes in CSF GCase activity after several freeze/thaw cycles and at −20 ° C storage conditions . Thus, standard operating procedures for the collection of CSF is warranted.…”

Oxidative stress in two tropical species after exposure to diesel oil

“…A similar standardization was done by Limaye et al (2003) and Persichetti et al (2014); being a conventional practice in medicine tests that want to identify departures from normal or basal status. Before such standardization, the variation of the control treatment…”