Na2hpo4

CSF samples were thawed on ice, centrifuged briefly at 1000×g and diluted 1:2 in assay buffer (0.1 M citric acid (#84841.290, VWR Chemical, USA) and 0.2 M Na₂HPO₄ (#28026.260, VWR Chemicals, USA), pH 5, supplemented with 2 mg/ml taurodeoxycholic acid (TDC, #336840010, Acros organics, Belgium)) prior to the assay. The GCase substrate, 4-methylumbelliferyl β-d-glucopyranoside (#J66630.MD, Sigma Aldrich, USA), was dissolved at a concentration of 0.5 mM in assay buffer. 4-methylumbelliferyl (4-MU, #A10337, Alfa Aesar, USA) served as a calibrator.

The suspension of caCP was prepared by wet chemical precipitation by dissolving calcium acetate (Ca(C₂H₃O₂)₂, Acros Organics BV, Geel, Belgium 99%) and disodium hydrogen phosphate (Na₂HPO₄, VWR International Ltd., Radnor, PA, USA, AnalaR). The Ca/P ratio was 5:3. The suspensions’ pH value was kept at 11 with a calculated content of sodium carbonate (anhydrous, VWR International Ltd., Radnor, PA, USA ≥99.5% ACS) to gain carbonated caCP nanoparticles. During the preparation process, the suspensions were stirred vigorously over 4 h at around 1400 rpm. The formed precipitates were cleaned thoroughly with distilled water and kept in an oven for 4 h at 150 °C. The resulting white nanopowders were gathered and prepared for further characterization and composite preparation.

At P67 ± 2, mice underwent terminal anaesthesia via intraperitoneal injection of 100 µl sodium pentobarbitone. This was followed by transcardial perfusion using 0.9 % heparinized saline (until the fluid exiting the cut right atrium was entirely clear), followed by 20–30 ml of 4 % paraformaldehyde dissolved in 0.1 M phosphate-buffered saline (PBS, 0.1 M NaH₂–PO₄·2H₂O, 0.1 M Na₂HPO₄·12H₂O, 0.15 M NaCl, all reagents purchased from VWR International, Poole, UK). Brains were rapidly removed from the skull and post-fixed in 4 % paraformaldehyde (PFA) for ~23 h, then cryoprotected by immersion in 30 % sucrose for ~72 h, before freezing them on dry ice and storing them at −80 °C until sectioning. A 1 in 4 series of coronal sections (20 µm) were cut using a cryostat (−22 °C, Bright Instruments Ltd., Huntingdon, UK) and the free-floating sections stored (−20 °C) in antifreeze solution (0.1 M NaH₂PO₄·H₂O, 0.05 M Na₂HPO₄, 0.15 Mm NaCl, 50 % v/v ethanediol, 1 % w/v polyvinylpyrrolidone, 0.1 % w/v NaN₃, VWR International).

Aliquots were thawed and 500 μL of serum was diluted to a final volume containing 50% (v/v) serum, 40% (v/v) dd ¹H₂O (18.2 MΩ), 10% (v/v) 1 M PO₄³⁻ pH 7.4 buffer (Na₂HPO₄, VWR International Ltd., Radnor, PA, USA and NaH₂PO₄, Sigma-Aldrich, Gillingham, UK) in deuterium oxide (²H₂O, Sigma-Aldrich) and 1.2 mM sodium azide (NaN₃, Sigma-Aldrich). Samples were vortexed for 1 min, centrifuged at 13,000× g at 4 °C for 2 min and 600 μL transferred into 5 mm outer diameter NMR tubes (Bruker, Coventry, UK).
Urine & saliva samples were thawed at room temperature before addition of 500 μL to 500 μL of 1 M phosphate buffer (Na₂HPO₄ and NaH₂PO₄) at pH7.4 with 20% ²H₂O, 200 μM TSP and 2.4 mM sodium azide. The samples were vortexed for 30 s prior to 5 min centrifugation at 21,500× g and 4 °C before transferring 600 μL of sample to Bruker SampleJet 5 mm (outer diameter) NMR tubes. The final concentration in the NMR tube was 50% urine or saliva, 10% ²H₂O, 1.2 mM sodium azide and 100 μM TSP.