Garnero P, Grimaux M, Seguin P, Delmas P D
INSERM Unit 234, Hôpital Edouard Herriot, Lyon, France.
J Bone Miner Res. 1994 Feb;9(2):255-64. doi: 10.1002/jbmr.5650090215.
Three monoclonal antibodies recognizing the 5-13, 25-37, and 43-49 sequence of the human osteocalcin were used in competitive and two-site radioimmunoassays (RIA) to characterize specifically various immunoreactive forms of circulating human osteocalcin. The intact molecule accounts for 36% of total in normals (2.6 nM), 46% in patients with osteoporosis (3.1 nM), and 26% in chronic renal failure (6.9 nM). Four fragment were detected in addition to the intact molecule in the serum of healthy adults and patients with metabolic bone disease. N-terminal, mid, and mid C-terminal fragments were present in minute amounts (each accounting for 5-14% of the total circulating osteocalcin immunoreactivity). In contrast, the N-terminal midfragment, probably resulting from the cleavage around amino acids 43-44, represents about 30% (2 nM) of the total osteocalcin immunoreactive level in normals and patients with osteoporosis and up to 50% (13 nM) in patients with chronic renal failure. This large N-terminal midfragment, representing 75-80% of the intact osteocalcin level, is not lower when the plasma assay is performed immediately after sampling (within 20 minutes at 4 degrees C with proteinase inhibitors), indicating that it circulates in vivo. In addition, this fragment was detected in the supernatant of osteoblastic cells, representing about 28% of the intact peptide. Levels of N-terminal midfragment were not changed after treatment of patients with metabolic bone disease (Paget's disease, reflex sympathetic dystrophy, fibrous dysplasia, and osteoporosis) by bisphosphonate, suggesting that it is not released during bone resorption. The osteocalcin level measured with the two-site immunoradiometric assay specific for the intact molecule or with a conventional bovine RIA was rapidly decreased after incubation of serum at room temperature (-20 and -15%, respectively, after 3 h), whereas the total level of intact osteocalcin plus N-terminal midfragment was not changed. Intact osteocalcin loss can be partially avoided by proteinase inhibitors and by incubating serum at 4 degrees C. In conclusion, we characterized multiple immunoreactive forms of osteocalcin that circulate in addition to the intact molecule, none of them being specifically altered in osteoporosis. The N-terminal midfragment circulates in a large amount, probably resulting from cleavage of the intact molecule in the circulation and/or at peripheral sites. These fragments can also be generated in vitro by proteolytic degradation of the intact molecule. To obtain reliable intact osteocalcin values but also reliable levels measured with conventional competitive RIA, careful control of the sampling conditions is warranted.
三种识别人类骨钙素5 - 13、25 - 37和43 - 49序列的单克隆抗体被用于竞争性和双位点放射免疫分析（RIA），以特异性地表征循环中的人类骨钙素的各种免疫反应形式。完整分子在正常人中占总量的36%（2.6 nM），在骨质疏松症患者中占46%（3.1 nM），在慢性肾衰竭患者中占26%（6.9 nM）。除了完整分子外，在健康成年人和代谢性骨病患者的血清中还检测到四种片段。N端、中间和中间C端片段含量极少（各占循环骨钙素免疫反应性总量的5 - 14%）。相比之下，可能由氨基酸43 - 44周围的裂解产生的N端中间片段，在正常人和骨质疏松症患者中约占骨钙素免疫反应性总水平的30%（2 nM），在慢性肾衰竭患者中高达50%（13 nM）。这个大的N端中间片段占完整骨钙素水平的75 - 80%，在采样后立即（4℃下20分钟内，加入蛋白酶抑制剂）进行血浆分析时并不降低，表明它在体内循环。此外，在成骨细胞的上清液中检测到了这个片段，约占完整肽的28%。代谢性骨病（佩吉特病、反射性交感神经营养不良、纤维发育不良和骨质疏松症）患者经双膦酸盐治疗后，N端中间片段水平未改变，这表明它不是在骨吸收过程中释放的。用针对完整分子的双位点免疫放射分析或传统的牛RIA测量的骨钙素水平，在室温下孵育血清后迅速下降（3小时后分别下降20%和15%），而完整骨钙素加N端中间片段的总水平未改变。蛋白酶抑制剂和在4℃下孵育血清可部分避免完整骨钙素的损失。总之，我们表征了除完整分子外循环中的多种骨钙素免疫反应形式，在骨质疏松症中它们均未发生特异性改变。N端中间片段大量循环，可能是由循环中和/或外周部位的完整分子裂解产生的。这些片段也可通过完整分子的蛋白水解降解在体外产生。为了获得可靠的完整骨钙素值以及用传统竞争性RIA测量的可靠水平，有必要仔细控制采样条件。
