Gomez B, Ardakani S, Evans B J, Merrell L D, Jenkins D K, Kung V T
Metra Biosystems, Inc., Mountain View, CA 94043, USA.
Clin Chem. 1996 Aug;42(8 Pt 1):1168-75.
The pyridinium cross-links of collagen, pyridinoline (Pyd) and deoxypyridinoline (Dpd), provide structural integrity and rigidity to collagen fibrils in bone. During bone degradation (resorption), the cross-links are released into the circulation and eventually excreted in urine. Pyridinium cross-link measurements in urine have been shown to be sensitive and specific indicators of resorption by both established HPLC and newer enzyme immunoassay (EIA) techniques. We have developed a monoclonal antibody that preferentially binds to the non-peptide-bound free forms of Pyd & Dpd. We have incorporated the antibody conjugated to alkaline phosphatase in a competitive EIA by using Pyd-coated microtiter strip wells. After a 3-h incubation of sample and antibody-enzyme conjugate, color is developed for 1 h with p-nitrophenyl phosphate as the substrate. The intraassay (n = 52) CVs were 3.0-7.6%, and interassay (n = 8) CVs were 6.1-7.4%. Comparisons of the assay (y) with HPLC (x) and a polyclonal antibody-based EIA (x') gave regression equations of y = 0.46x + 4, r = 0.96, and y = 0.56x' + 8, r = 0.96. The EIA detected increased Pyd & Dpd concentrations in urine from postmenopausal women and patients with osteoporosis, hyperthyroidism, hyperparathyroidism, and Paget disease of bone. EIA concentrations also reflected the reduction in Pyd&Dpd excretion resulting from estrogen replacement in surgically menopausal women. Measurement of pyridinium cross-links with this simple EIA appears to provide an accurate index of the rate of resorption and may be useful for metabolic bone disease assessment and monitoring the effects of antiresorptive therapy.
胶原蛋白的吡啶交联物，即吡啶啉（Pyd）和脱氧吡啶啉（Dpd），赋予骨中胶原纤维结构完整性和刚性。在骨降解（吸收）过程中，这些交联物释放进入循环系统，最终经尿液排出。已证明，采用成熟的高效液相色谱法（HPLC）和更新的酶免疫测定法（EIA），尿液中吡啶交联物的测量是骨吸收敏感且特异的指标。我们已研制出一种单克隆抗体，它优先结合非肽结合的游离形式的Pyd和Dpd。我们通过使用包被有Pyd的微量滴定板孔，将与碱性磷酸酶偶联的抗体用于竞争性EIA中。样品与抗体 - 酶偶联物孵育3小时后，以对硝基苯磷酸酯为底物显色1小时。批内（n = 52）变异系数为3.0 - 7.6%，批间（n = 8）变异系数为6.1 - 7.4%。该测定法（y）与HPLC（x）以及基于多克隆抗体的EIA（x'）比较，得到回归方程分别为y = 0.46x + 4，r = 0.96，以及y = 0.56x' + 8，r = 0.96。该EIA检测到绝经后女性以及患有骨质疏松症、甲状腺功能亢进、甲状旁腺功能亢进和骨佩吉特病患者尿液中Pyd和Dpd浓度升高。EIA浓度也反映了手术绝经后女性雌激素替代导致的Pyd和Dpd排泄减少。用这种简单的EIA测量吡啶交联物似乎能提供骨吸收速率的准确指标，可能有助于代谢性骨病的评估以及监测抗吸收治疗的效果。
