Measurements

TRACP5b was measured manually with an enzyme immunoassay (N-Test TRACP-5b Nittobo, Nittobo Medical Co., Ltd, Tokyo). It is of note that there are two commercial assay reagents for TRACP5b,^14,15 both of which have comparable analytical performance. However, the above one used in this study is supposed to be unaffected by TRACP5a or an isoform of TRACP derived from macrophages. ¹⁵

BAP and PTH were measured using a chemiluminescent enzyme immunoassay with a DXI autoanalyzer (Beckman-Coulter, Tokyo). Calcium was measured by indirect potentiometry and IP by the timed endpoint molybdate UV method with a DXC autoanalyzer (Beckman-Coulter). Blood was collected into a vacuum blood collection tube containing a serum-separating agent. After the blood was centrifuged for 10 min at room temperature, serum was aliquoted and cryopreserved at −80°C. All samples were transferred to the Ariake Research Laboratory (Beckman Coulter, Inc., Tokyo, Japan) and measured collectively. Detailed information on the measurements is described elsewhere.^12,13 Since serum concentrations of calcium (Ca) is influenced by serum albumin, values of adjusted calcium (aCa: mmol/L) were calculated as total calcium (mmol/L) + 0.02 (40–albumin [g/L]). ¹⁶