An improved ultrafiltration method for free thyroxine and triiodothyronine in serum

We describe an ultrafiltration technique for rapidly and directly determining free triiodothyronine or free thyroxine, or both. After equilibrating serum at 37 degrees C with purified tracer of high specific activity, we placed 0.15 mL of serum in 2.8 mL of phosphate buffer (0.1 mol/L, pH 7.4) in the ultrafiltration cell and obtained successive 0.2- and 0.6-mL fractions of protein-free ultrafiltrate. Under our conditions free ligand concentration was independent of flow rate. After purifying the second fraction with protein-coated charcoal, we could determine the proportion of free triiodothyronine or free thyroxine. Samples from normal adult men and women, including women who were taking oral contraceptives or were pregnant, and from hypo- and hyperthyroid patients gave results that agreed with those obtained by equilibrium dialysis. Speed is the main advantage of the method: one technologist can complete the procedure in 2 h and, using a multi-micro-ultrafiltration system, can process many samples in one day. For laboratories where index-type reactions are performed routinely and direct free triiodothyronine or free thyroxine is determined only on selected specimens, this method is superior to dialysis. It is also very convenient for rapidly purifying tracers, to at least 97% radiochemical purity, with 94% recovery and no dilution.