Srivastava A K, Mohan S, Singer F R, Baylink D J
Musculoskeletal Disease Center, Jerry L. Pettis Veterans Medical Center and Department of Medicine, Loma Linda University, Loma Linda, CA 92357, USA.
Bone. 2002 Jul;31(1):62-9. doi: 10.1016/s8756-3282(02)00793-7.
We isolated and characterized a peptide fragment corresponding to amino acid sequence 14-28 of human osteocalcin in urine from Paget's disease, and developed a polyclonal antibody reactive to this peptide in urine. We used this antibody to measure urinary fragments of osteocalcin and compared to efficacy of the urinary osteocalcin assay with a serum osteocalcin (sOC) assay (ELISA-Osteo, Cis-Bio International) to monitor the short-term changes in bone turnover in response to alendronate treatment. The synthetic peptide-based urinary osteocalcin (uOC) radioimmunoassay (RIA) showed an analytical sensitivity of 6.25 ng/mL, standard curve range of 3.12-400 ng/mL, and mean intra- (n = 20) and interassay (n = 30) coefficient of variation (CV) of <15%. Urine osteocalcin concentrations in postmenopausal osteoporotic patients were approximately 90% higher than in normal premenopausal controls. Series of 24 h urine and matched serum samples were collected at baseline, 30 days, and 90 days after treatment of postmenopausal osteoporotic patients with daily dose of 10 mg alendronate. We measured urinary osteocalcin (uOc) levels and urinary N-telopeptide (uNTx, Ostex) in urine samples and serum N-telopeptide (sNTx), C-telopeptide (sCTx, Osteometer), serum osteocalcin (sOC) as well as bone-specific alkaline phosphatase (sALP) (Alkphose-B, Metra Biosystems) in serum samples. The percent change data obtained between baseline and 30 days (n = 18) posttreatment suggested a rapid decline in uOC concentration (-27%, p < 0.01) in response to alendronate treatment, as compared with a marginal and nonsignificant decrease in sOC (-7.2%, p = 0.417) or sALP (-3.4%, p = 0.689), two specific markers of bone formation. As expected, due to the coupling of bone formation and bone resorption, the concentration of all markers showed a 30%-45% decline compared with baseline values after 90 days (n = 16) of treatment. Correlation of markers after a 30 day treatment with alendronate revealed a higher correlation (r = 0.61, p < 0.01) between uOC and uNTx, as compared with sOC (r = 0.03, p = 0.447) or sALP (r = -0.14, p = 0.295) with uNTx. Similarly, correlation coefficients with r values between 0.48 and 0.55 (p < 0.05) were observed between uOC, sNTx, and sCTx, whereas no significant correlation was observed between sOC and sNTx or sCTx. These results provide indirect evidence that fragments measured by the urine assay probably originated from bone resorption, and suggest that the uOC assay could be used to assess short-term changes in bone metabolism with regard to osteocalcin.
我们从佩吉特病患者的尿液中分离并鉴定了一段与人类骨钙素氨基酸序列14 - 28相对应的肽片段，并制备了一种对尿液中该肽有反应的多克隆抗体。我们使用该抗体来测量尿液中的骨钙素片段，并将尿液骨钙素检测方法与血清骨钙素（sOC）检测方法（ELISA - Osteo，Cis - Bio International）的效果进行比较，以监测阿仑膦酸盐治疗后骨转换的短期变化。基于合成肽的尿液骨钙素（uOC）放射免疫分析（RIA）显示分析灵敏度为6.25 ng/mL，标准曲线范围为3.12 - 400 ng/mL，批内（n = 20）和批间（n = 30）变异系数（CV）均值<15%。绝经后骨质疏松患者的尿液骨钙素浓度比正常绝经前对照组高约90%。对每日服用10 mg阿仑膦酸盐治疗的绝经后骨质疏松患者，在基线、治疗后30天和90天收集了24小时尿液及配对血清样本。我们测量了尿液样本中的尿液骨钙素（uOc）水平和尿液N - 端肽（uNTx，Ostex）以及血清样本中的血清N - 端肽（sNTx）、C - 端肽（sCTx，Osteometer）、血清骨钙素（sOC）以及骨特异性碱性磷酸酶（sALP）（Alkphose - B, Metra Biosystems）。治疗后30天（n = 18）与基线之间获得的百分比变化数据表明，与骨形成的两个特异性标志物sOC（-7.2%，p = 0.417）或sALP（-3.4%，p = 0.689）的轻微且无统计学意义的下降相比，阿仑膦酸盐治疗后uOC浓度迅速下降（-27%，p < 0.01）。正如预期的那样，由于骨形成与骨吸收的耦合，治疗90天（n = 16）后所有标志物的浓度与基线值相比均下降了30% - 45%。阿仑膦酸盐治疗30天后标志物的相关性显示，与uNTx相比，uOC与uNTx之间的相关性更高（r = 0.61，p < 0.01），而sOC（r = 0.03，p = 0.447）或sALP（r = -0.14，p = 0.295）与uNTx之间的相关性较低。同样，在uOC、sNTx和sCTx之间观察到相关系数r值在0.48至0.55之间（p < 0.05），而在sOC与sNTx或sCTx之间未观察到显著相关性。这些结果提供了间接证据，表明尿液检测所测量的片段可能源自骨吸收，并表明uOC检测可用于评估骨钙素相关的骨代谢短期变化。
