Immunoreactive osteocalcin forms in conditioned media from human osteoblast culture and in sera from healthy adult control subjects and patients with bone pathologies

Abstract

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.