Sodium taurodeoxycholate

GBA activity was measured as previously described [20 (link)] Briefly, one 3.2 mm-diameter punch from each DBS sample was extracted in 200 ;L 0.2 M citrate phosphate buffer, pH 5.2 containing 1% Triton^® X-100 (Sigma, St. Louis, MO) and 1% sodium taurodeoxycholate (≥97% purity, Sigma, St. Louis MO) in a 96-well plate. Substrate solution (12.5 mM 4-MUG, Sigma, St. Louis, MO) was prepared either with or without inhibitor (0.5 mM Conduritol B Epoxide, Toronto Research Chemicals). Inhibited or uninhibited substrate solution was mixed 2:1 with DBS extract and incubated for 20 h at 37 °C. To stop the reactions, 100 μL of 0.5 M EDTA (pH 11.5) was added to each well. An eight point 4-MU standard curve (0 – 0.67 μM) was prepared on each plate in duplicate. The plate was read in a fluorometer at 355 nm excitation and 460 nm emission wavelengths. GBA activity was determined by subtracting the background activity measured in the inhibited reaction from that in the uninhibited reaction. Disease cut-off (1.71 μmol/L/h) was established as described in methods section 2.6. The limit of blank (LOB=0.16 μmol/L/h) and limit of detection (LOD=0.41 μmol/L/h) for the fluorescence assay were established previously [20 (link)].