GBA Activity Measurement in DBS Samples

GBA activity was measured by modifying the method proposed by Chamoles et al.^{15 (link)} One 3 mm diameter blood spot (approximately 3.2 μl) from each DBS sample was punched into 96-well polystyrene plates (Corning, Corning, NY, USA) using a DBS Puncher. Each plate contained at least two blank filter papers. We prepared 0.2 mol l⁻¹ citrate-phosphate buffer (pH 5.2, containing 0.9% taurodeoxycholate and 0.15% Triton-X 100) as elution buffer. Fifty microliters of 5 mmol l⁻¹ 4-methyl-umbelliferyl-β-d-glucopyranoside distilled in the elution buffer as a substrate was added to each well. Plates were sealed with an adhesive film (Corning) and incubated (400 r.p.m. shaking, 37 °C, 20 h; Labsystems iEMS, Finland). The seal was removed and the reaction was stopped with 200 μl glycine-carbonate buffer (0.17 mol l⁻¹, pH 10.5). Samples (200 μl) were transferred to a new 96-well polystyrene plate to avoid interference of the filter paper with fluorescence. Fluorescence was detected in a Wallac 1420 victor^{2 (link)} microplate reader (excitation 355 nm, emission 460 nm; Perkin Elmer, Waltham, MA, USA). After correcting readings with blanks, we used the 4-MU calibration curve to convert fluorescence readings into moles of 4-MU. Enzymatic activity was expressed as micromoles of 4-MU hydrolyzed substrate per liter of blood per hour. Reagents used above were obtained from Sigma (St Louis, MO, USA).