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A simple method for measuring the free, non-protein-bound steroid fraction in plasma by equilibrium dialysis is described. The alteration occurring in the volume of the inner phase (undiluted plasma) is corrected by the difference in weight before and after dialysis. Total cortisol was determined by radioimmunoassay. Although it is not possible to differentiate between values for total cortisol after ACTH stimulation (242.2+/-shing's syndrome (n: 15)), and women treated with oestrogens (211.8+/-42.0 ng/ml (n: 20)), there were significant differences for free cortisol (ACTH stimulation: 29.3+/-5.6 ng/ml; Cushing's syndrome: 31.5+/-8.6 ng/ml; women under increased oestrogenic activity: 10.0+/-2.1 ng/ml; pregnant women: 13.7+/-5.1 ng/ml plasma). Compared with normal values for healthy women and men (9.3+/-1.4 ng/ml), women with increased oestrogenic activity showed slight elevations of free cortisol. Compared to cortisol, the percentage of dialysable testosterone and oestradiol was lower in women than in men (1.57 vs. 2.08% for testosterone and 1.68 vs. 2.15% for oestradiol). In healthy men the concentration of free steroid was 13.1+/-1.0 ng free testosterone/100 ml plasma (0.44+/-0.045 pg free oestradiol/ml plasma), in healthy women 0.64+/-0.07 ng/100 ml (0.98+/-0.10 pg/ml) and in women receiving oestrogens 0.37+/-0.04 ng/100 ml (0.01+/-0.015 pg/ml). When the method described here for determing the free fractions of cortisol (n: 45), oestradiol (n: 18) and testosterone (n: 18) at 37 degrees C is compared with the method of centrifugal filtration, the correlation was r: 0.80 r: 0.86 and r: 0.91, respectively. In practice, equilibrium dialysis with undiluted plasma is simple, fast and can be applied to all steroid hormones. It allows direct measurements of non-protein-bound steroids under nearly physiological conditions.
Kley et al. (Sun,) studied this question.