Estimation of the percentage of free steroid in undiluted serum by centrifugal ultrafiltration-dialysis

A new method is described for the estimation of the percentages of free steroids in undiluted serum or plasma. Samples were incubated with ³H-steroid and [¹⁴C]glucose, and duplicate aliquots of the mixture were subjected to centrifugal ultrafiltration through a dialysis membrane at 37°C. The percentages of free estradiol, testosterone, and progesterone in serum were estimated by comparing the ratio of ³H-steroid to [¹⁴C]glucose in the ultrafiltrate with the corresponding ratio in the serum retained by the dialysis membrane. In addition to being highly reproducible, the present method affords several advantages over existing methods. The use of [¹⁴C]glucose to monitor the movement of unbound serum components obviates measurement of the ultrafiltrate volume, and because the ultrafiltrate remains in contact with the dialysis membrane, small molecular weight components are able to pass through the dialysis membrane in either direction during centrifugal ultrafiltration. This latter feature, which could be described as 'isodialysis', allows the estimation of the percentage of free steroid under conditions which closely approximate the in vivo situation.