On the validity of free hormone measurements

The influence of equilibrium perturbation on the measurement of free or non-protein-bound hormones in serum or plasma is described. The free analyte concentration in the sample will alter after dilution or addition of binding reagents. The extent of this perturbation is not constant. It depends on the binding properties of the endogenous carrier proteins which vary from sample to sample and from sample to reference standard. As examples, the different changes of the free thyroxine and free cortisol concentrations after dilution are demonstrated both experimentally and by theoretical modeling of the hormone-protein distribution. It is shown how this systematical error can be minimized in the design of free hormone assays.