Improved ultrafiltration method for determining unbound cortisol in plasma.

We describe a modified ultrafiltration method for measuring unbound cortisol in diluted or undiluted plasma or serum. After equilibration at 37 degrees C with purified [3H]cortisol, plasma or serum with or without buffer was placed in the ultrafiltration cell and two successive 0.2-mL fractions of protein-free ultrafiltrate were obtained. Under our conditions, free ligand concentration was independent of flow rate. The second fraction (the first is discarded) was used for determining the proportion of unbound cortisol. The assay is rapid (less than 2 h), practical (no more than 1.5 mL of plasma or serum is necessary), and reproducible (CV: 4.5% within assay and 5.2% in different assays). Samples from normal men and women (blood taken at 0800 and 1600 hours), from pregnant women, and from patients with Cushing's disease and adrenal insufficiency gave results that agreed with those obtained by equilibrium dialysis.