A simplified ultrafiltration method for determination of serum free cortisol

We describe the suitability of the Amicon MPS-1 centrifugal ultrafiltration device and the YMB membrane for measuring free cortisol in serum. The method combines two independent assays: total cortisol and the ultrafiltrate fraction of added [3H]cortisol. The unbound fraction is determined in 0.25-0.30 ml of ultrafiltrate collected from 0.6 to 1 ml of serum that has been equilibrated with [3H]cortisol at 37 degrees C for 20 min. The assay is rapid (less than 1 h), practical ( no more than 0.6 ml of serum is necessary) and repeatable (CV: 3.8% within-assay and 12.2% in different assays). Error introduced in free cortisol measurement due to dilution effects in dialysis is systematically defined, and the effect of tracer purity on the ultrafiltration method is examined. Dialyzed sera from normal men and women, from patients with Cushing's disease and adrenal insufficiency, and from pregnant women gave ultrafiltration results that accurately duplicated those obtained by previous dialysis.