Osteomark

BMD at the L2 to L4 lumbar spine (L-BMD) and total hip (H-BMD) were measured using dual-energy X-ray absorptiometry (GE Healthcare, WI, USA), and a quality assurance test was carried out on every measurement to detect machine drift. The interassay variance of the L-BMD measurement in our laboratory was 0.5 ± 0.5% (CV ± SD). Serum 25-hydroxyvitamin D (25 [OH] D) levels were measured using ECLIA. The serum level of calcium was corrected (cCa) based on the serum albumin level using the following formula: cCa = (Calcium + 4)-albumin. Serum levels of bone-derived alkaline phosphatase (BAP; Ostase CLEA; Beckman Coulter, Atlanta, GA, USA) and urinary excretion of type I collagen-cross-linked N-telopeptides (NTx; Osteomark; Creative Diagnostics, NY, USA) were also measured. The urinary excretion of pentosidine and plasma levels of total homocysteine were measured using an HPLC system after hydrolysis of the urine samples^{3 (link),4 (link)}. Plasma levels of total homocysteine were measured using an HPLC system. All chemical parameters mentioned above were measured at the central laboratory (LSI Medience, Tokyo, Japan).

Serum level of NOx (NO₂⁻ and NO₃⁻) was measured by colorimetric assay kit (Cayman Chemical, MI, USA) using Griess reagent. The inter- and intra-assay variances in the laboratory were 2.7% and 2.1%, respectively. In addition, we had checked intra-assay variance of serum samples obtained from 2 different time points in 10 participants. The coefficient of variation (CV) was 14.2 ± 9.2% (mean ± standard deviation). BMD at L2 to L4 lumbar spine (L-BMD) was determined by dual energy X-ray absorptiometry (DPX series, GE Healthcare, IL, USA). Serum levels of parathyroid hormone (PTH) and 25-hydroxyvitamin D (25[OH]D) were measured by electrochemiluminescence immunoassay (ECLIA). As for BTMs, serum level of bone-derived alkaline phosphatase (BAP; Ostase CLIA, Beckman Coulter, CA, USA) and urinary excretion of type I collagen cross-linked N-telopeptides (NTx; Osteomark, Creative Diagnostics, NY, USA) were measured. Serum homocysteine level was determined by using high-performance liquid chromatography (HPLC). Urinary excretion of pentosidine was measured by HPLC system after hydrolysis of urine samples [16 (link), 17 (link)]. All the biochemical parameters listed above were assayed at the central laboratory (LSI Medience, Tokyo, Japan).