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Development and Evaluation of Three Immunofluorometric Assays That Measure Different Forms of Osteocalcin in Serum

¹ Department of Biotechnology, University of Turku, FIN-20520 Turku, Finland.

² Centre for Biotechnology, University of Turku and Åbo Akademi University, FIN-20520 Turku, Finland.

³ Department of Orthopaedics, Malmö University Hospital, S-20502 Malmö, Sweden.

⁴ Institute of Biomedicine, Department of Anatomy, University of Turku, FIN-20520 Turku, Finland.
^a Address correspondence to this author at: University of Texas Health Science Center at San Antonio, Department of Medicine, Division of Endocrinology, 7703 Floyd Curl Dr., San Antonio, TX 78284-7877. Fax 210-567-6693; e-mail kakonen{at}uthscsa.edu

Background: Circulating human osteocalcin (hOC) has been used as a marker of bone formation. Our aim was to validate three immunofluorometric assays (IFMAs), measuring different forms of hOC.

Methods: The two-site IFMAs were based on previously characterized monoclonal antibodies. Assay 2 recognized intact hOC, assays 4 and 9 measured the NH₂-terminal mid-fragment and the intact hOC. In addition, assay 9 required hOC to be -carboxylated.

Results: A 76–79% increase of serum immunoreactive hOC was found in the postmenopausal group compared with the premenopausal group with all IFMAs. With EDTA-plasma samples, the observed increases were lower (49–65%). The hOC concentration in the postmenopausal group receiving hormone replacement therapy was 42–44% lower than that in the postmenopausal control group in both serum and EDTA-plasma samples. The depressed carboxylation in warfarin-treated patients was accompanied by lower results in assay 9. The ratio of assay 9 to assay 4 totally discriminated the warfarin-treated patients from the controls. Assay 9 showed the smallest decreases in measured hOC after storage of serum or plasma for 4 weeks at 4 °C, followed by assay 4 and assay 2. Results from the last assay were <17% of their initial values after 4 weeks of storage. No diurnal variation was observed with assay 9 as opposed to the two other IFMAs.

Conclusion: The three assays with their distinct specificity profiles (intact vs fragmented and carboxylated vs decarboxylated hOC) may provide valuable tools for investigating the significance of different hOC forms in various bone-related diseases.

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