Theoretical Modeling of the o-Cresolphthalein Complexone Assay for Bone Calcium Resorption
O-cresolphthalein complexone (Ocpc) is a widely-used colorimetric indicator used to assess the Ca2+ concentration of biological and non-biological samples. To minimize interference by the ~ 0.8 - 1 mM Mg2+ present in culture media and biological samples, Ocpc has been used with 8-Hydroxyquinoline (8HQ) which preferentially binds to Mg2+ over Ca2+. The Ocpc + 8HQ assay could be useful in in-vitro bioassays to estimate changes in Ca2+ concentration from the ~2 mM normally present in cell culture media. While several formulations of the Ocpc+8HQ assay have been suggested, their utility for such applications is unclear. The overall objectives of this project were (1) to develop a theoretical model of the Ocpc + 8HQ assay to assess Ca2+ release, and (2) to validate and apply this model to media in a bone resorption assay. Such a model can provide a quantitative understanding of how the different molecules of the assay system interact and contribute to the final output signal (OD). Models of increasing complexity were developed, ranging from 1.1. Model I - the reaction between Ocpc and Ca2+ to form the colored complex Caocpc 1.2. Model II - including interactions with 8HQ and 1.3. Model III - including interactions with Mg2+. Next, the model was 2.1. Validated by comparing the linear region slopes of the predicted and experimental calibration curves, and then 2.2. Applied to a bone resorption scenario by computing the sensitivity and specificity of the assay using relevant cell culture and bone resorption assay parameters. These theoretical models contribute to understanding the effect of the different complexes formed on the output signal. The results indicate the utility and limitations of an Ocpc + 8HQ assay for analysing Ca2+ from bioassays in culture medium. The modeling approach may be useful for other assays with target and interferent molecules.