Total 25(OH)D and VDBP measurements

Each of serum sample was aliquoted into two tubes and stored at −80℃ until the analyses of total 25(OH)D and VDBP levels were performed. Serum total 25(OH) D levels were analyzed using the Elecsys^® Vitamin D total II assay and Cobas e602 analyzer (Roche Diagnostics, Mannheim, Germany). Bioavailable 25(OH) D concentrations were calculated using equations reported in previous studies and based on albumin level, total 25(OH)D and VDBP derived from the medical records [2,3]. VDBP concentrations were measured using the Human Vitamin D BP Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA), according to protocol of manufacturer.