Serum calcium, vitamin D, and biochemical markers of bone metabolism
Prior to each intervention, serum calcium was measured with the use of atomic absorption spectrometry (AAnalyst 300, PerkinElmer Instruments), and serum 25(OH)D was measured with the use of HPLC. At baseline and the end of each intervention period, fasting blood and urine were collected in the morning to assess serum calcium, 25(OH)D, and biomarkers of bone metabolism: serum IGF-1, IGF-binding protein-3 (IGFBP-3), and their ratio, procollagen type 1 N-terminal propeptide (P1NP), cross-linked C-telopeptide of type II collagen (CTx-II), sclerostin, osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), and their ratio, as well as urinary N-terminal telopeptide (NTx), normalized to creatinine. PTH was also assessed at baseline. Bone metabolism biomarkers and PTH were assessed with the use of commercial ELISA kits. Urinary creatinine was measured with the use of an enzymatic colorimetric assay (Roche Diagnostics Ltd.).