Diaz Diego E M, Nacher M, Rapado A, Serrano S, Bosch J, Aubia J, de la Piedra C
Biochemistry Laboratory, Fundación Jiménez Díaz, Madrid, Spain.
Eur J Clin Invest. 1998 Jan;28(1):48-58. doi: 10.1046/j.1365-2362.1998.00246.x.
The aim of this work was to study the immunoreactive forms of bone Gla protein (BGP) present in conditioned media of human osteoblast cultures (BGP released from osteoblast) and in the sera of healthy adult control subjects and patients with bone pathologies (chronic renal failure on haemodialysis, Paget's disease of bone and post-menopausal osteoporosis).
The technical procedure used was a combination of high-performance liquid chromatography (HPLC) and different BGP assays with several specificities to analyse BGP levels in the different HPLC fractions. Aliquots of conditioned media or sera were purified through a Sephadex G-50m column and by HPLC (C4 reverse-phase column) in a 25-40% acetonitrile gradient. Two-minute fractions were collected and divided into three aliquots in order to determine osteocalcin content using three different assays: (a) ELSA-OST-NAT IRMA, which only detects intact osteocalcin; (b) ELSA-OSTEO IRMA, which detects intact osteocalcin and N-terminal fragments; and (c) OSCA Test RIA, which detects intact osteocalcin, C-terminal and other fragments.
We found different immunoreactive forms of osteocalcin in the culture medium of human osteoblasts and in sera from control subjects and patients for the bone pathologies studied. We did not find great qualitative differences between the immunoreactive osteocalcin profile found in the culture medium from human osteoblasts and the sera from healthy control subjects. However, the different bone pathologies show different characteristic patterns of immunoreactive forms of osteocalcin.
An interesting finding has been the detection, both in sera and in osteoblast culture media, of several immunoreactive forms of intact osteocalcin that eluted from HPLC at different acetonitrile percentages, and therefore correspond to different molecular forms.
本研究旨在探讨人成骨细胞培养条件培养基中存在的骨钙素（BGP，即成骨细胞释放的骨钙素）以及健康成年对照者和患有骨病（血液透析的慢性肾衰竭、佩吉特骨病和绝经后骨质疏松症）患者血清中的免疫反应形式。
所采用的技术方法是将高效液相色谱法（HPLC）与具有多种特异性的不同骨钙素检测方法相结合，以分析不同HPLC馏分中的骨钙素水平。条件培养基或血清的等分试样通过Sephadex G - 50m柱并经HPLC（C4反相柱）在25 - 40%乙腈梯度下进行纯化。收集两分钟的馏分并分成三等分，以便使用三种不同的检测方法测定骨钙素含量：（a）ELSA - OST - NAT IRMA，仅检测完整的骨钙素；（b）ELSA - OSTEO IRMA，检测完整的骨钙素和N端片段；（c）OSCA Test RIA，检测完整的骨钙素、C端和其他片段。
我们在人成骨细胞培养基以及所研究骨病的对照者和患者血清中发现了不同的骨钙素免疫反应形式。我们发现人成骨细胞培养基中的免疫反应性骨钙素谱与健康对照者血清之间没有很大的定性差异。然而，不同的骨病显示出不同的骨钙素免疫反应形式特征模式。
一个有趣的发现是，在血清和成骨细胞培养基中均检测到了几种完整骨钙素的免疫反应形式，它们在不同乙腈百分比下从HPLC中洗脱，因此对应于不同的分子形式。
