Centrifugal ultrafiltration-dialysis for non-protein-bound oestradiol in blood: importance of the support.

The percentage of non-protein-bound oestradiol in serum or other fluid measured by centrifugal ultrafiltration-dialysis was critically dependent on the supporting medium used to collect the ultrafiltrate. When either Whatman No. 1 filter paper discs, Whatman GF/D glass fibre discs or Celite were used, the ratios of 3H-steroid to [14C]glucose on the support were found to change with centrifugation time. This occurred when serum or protein-free filtrates were analysed and was eliminated when silanised Celite was employed as the support. It was also observed that when serum was analysed, the [3H]/[14C] ratio inside the cell increased linearly with centrifugation time and this was ascribed to the small changes in volume which occurred inside the cell as ultrafiltrate accumulated on the support. It was concluded that the correct time to sample the ratio of [3H]/[14C] inside was before centrifugation.