4.4. Measurement of Bone Markers
BLOOD
Subjects were evaluated at baseline and after 12 months of treatment. Blood samples were centrifuged at 1200 g for 15 min at 4 °C, and serum samples were separated, divided into aliquots, and stored at −80 °C until analysis. Parameters of bone metabolism, such as OC and BAP, were measured by an electrochemiluminescence assay (ECLIA) (Roche Diagnostics, Mannheim, Germany). The serum level of TRACP-5b was measured using a fragment-absorbed immunocapture enzymatic assay (Quidel Corporation, San Diego, CA, USA). A complete 24 h urine collection was conducted at the beginning and end of the intervention and aliquots were stored at −80 °C until analysis. Urinary DPD concentrations were measured by high-performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan).