Kinetics of cortisol and cortisone binding to corticosteroid binding globulin and albumin in vivo

Cortisol is a major endogenous glucocorticoid that regulates numerous physiological processes. In plasma, cortisol and its inactive metabolite cortisone bind to corticosteroid-binding globulin (CBG) and albumin, leaving only the unbound fraction available for receptor activation and metabolism. Changes in ligand or protein concentrations alter unbound fractions. Existing binding equations are difficult to extend to multi-ligand, multi-protein systems and do not readily capture competitive endogenous binding interactions. The goal of this study was to develop a plasma protein binding model that quantitatively describes binding species and predicts unbound concentrations across physiological states. Total and unbound cortisol and cortisone, CBG and albumin were measured in plasma from healthy premenopausal women (n=13) at baseline and after 7 days of 30 mg hydrocortisone treatment. Reversible 1:1 binding models were implemented in COPASI and MATLAB/Simulink, and dissociation constants (Kd) were estimated by fitting binding models to observed unbound concentrations. A model describing simultaneous binding of cortisol and cortisone to CBG and albumin yielded in vivo Kd values for cortisol:CBG, cortisone:CBG, cortisol:albumin, and cortisone:albumin of 0.0130 {micro}M, 0.169 {micro}M, 172 {micro}M, and 519 {micro}M, respectively. Model predictions agreed with observed unbound cortisol and cortisone, and bootstrap resampling confirmed stable Kd estimates. This work provides a quantitative framework for predicting unbound cortisol and cortisone across physiological and disease states by accounting for both changes in ligand and protein concentrations. This enables extrapolation without reparameterization and supports exploration of conditions such as pregnancy, adrenal insufficiency, and liver disease, informing interpretation of altered cortisol concentrations in these populations. Significance statementThis work establishes a framework to predict in vivo cortisol and cortisone binding. The developed model was applied to predict unbound cortisol and cortisone concentrations in physiological and pathophysiological states and can be integrated into pharmacokinetic models. Our analysis demonstrates that cortisol and cortisone binding affinities estimated in the native plasma environment differ from those measured using purified proteins. These differences have important implications for predicting and analyzing unbound cortisol concentrations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/718600v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@4a6bc1org.highwire.dtl.DTLVardef@1e87515org.highwire.dtl.DTLVardef@5ed98eorg.highwire.dtl.DTLVardef@11d0a3c_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Authement, A. (2026) https://BioRender.com/zl1bg0k