CHARACTERIZATION OF IMMUNOREACTIVE FORMS OF HUMAN OSTEOCALCIN GENERATED IN-VIVO AND IN-VITRO

Three monoclonal antibodies recognizing the 5-13, 25-37, and 43-49 seq uence of the human osteocalcin were used in competitive and two-site r adioimmunoassays (RIA) to characterize specifically various immunoreac tive forms of circulating human osteocalcin. The intact molecule accou nts for 36% of total in normals (2.6 nM), 46% in patients with osteopo rosis (3.1 nM), and 26% in chronic renal failure (6.9 nM). Four fragme nt were detected in addition to the intact molecule in the serum of he althy adults and patients with metabolic bone disease. N-terminal, mid , and mid C-terminal fragments were present in minute amounts (each ac counting for 5-14% of the total circulating osteocalcin immnoreactivit y). In contrast, the N-terminal mid-fragment, 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 wit h osteoporosis and up to 50% (13 nM) in patients with chronic renal fa ilure. This large N-terminal midfragment, representing 75-80% of the i ntact osteocalcin level, is not lower when the plasma assay is perform ed immediately after sampling (within 20 minutes at 4 degrees C with p roteinase inhibitors), indicating that it circulates in vivo. In addit ion, this fragment was detected in the supernatant of osteoblastic cel ls, representing about 28% of the intact peptide. Levels of N-terminal midfragment were not changed after treatment of patients with metabol ic bone disease (Paget's disease, reflex sympathetic dystrophy, fibrou s dysplasia, and osteoporosis) by bisphosphonate, suggesting that it i s not released during bone resorption. The osteocalcin level measured with the two-site immunoradiometric assay specific for the intact mole cule or with a conventional bovine RIA was rapidly decreased after inc ubation of serum at room temperature (-20 and -15%, respectively, afte r 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 os teocalcin that circulate in addition to the intact molecule, none of t hem being specifically altered in osteoporosis. The N-terminal midfrag ment circulates in a large amount, probably resulting from cleavage of the intact molecule in the circulation and/or at peripheral sites. Th ese fragments can also be generated in vitro by proteolytic degradatio n 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.