Development and evaluation of three immunofluorometric assays that measuredifferent forms of osteocalcin in serum

Background: Circulating human osteocalcin (hOC) has been used as a marker o f 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 monoclon al antibodies. Assay 2 recognized intact hOC, assays 4 and 9 measured the N H, terminal mid-fragment and the intact hOC. In addition, assay 9 required hOC to be gamma-carboxylated. Results: A 76-79% increase of serum immunoreactive hOC was found in the pos tmenopausal group compared with the premenopausal group with all IFMAs. Wit h EDTA-plasma samples, the observed increases were lower (49-65%). The hOC concentration in the postmenopausal group receiving hormone replacement the rapy was 42-44% lower than that in the postmenopausal control group in both serum and EDTA-plasma samples. The depressed carboxylation in warfarin-tre ated patients was accompanied by lower results in assay 9. The ratio of ass ay 9 to assay 4 totally discriminated the warfarin-treated patients from th e controls. Assay 9 showed the smallest decreases in measured hOC after sto rage of serum or plasma far 4 weeks at 4 degrees C, followed by assay 4 and assay 2. Results from the last assay were <17% of their initial Values aft er 4 weeks of storage. No diurnal variation was observed with assay 9 as op posed to the two Other IFMAs. Conclusion: The three assays with their distinct specificity profiles (inta ct vs fragmented and carboxylated vs decarboxylated hOC) may provide valuab le tools for investigating the significance of different hOC forms in vario us bone-related diseases. (C) 2000 American Association for Clinical Chemistry.