Unequal concentrations of free T3 and free T4 after ultrafiltration.

To the Editor: Ultrafiltration is a standard method for separating free T3 and free T4 from serum proteins and proteinbound hormone (1, 2 ). It is expected that free T3 and free T4 in aqueous solutions will accompany water as water moves across semipermeable membranes, a process that results in equal concentrations of free T3 and free T4 in the aqueous solutions on opposite sides of semipermeable membranes after ultrafiltration. The movements of free thyroid hormones through semipermeable membranes have not been compared to the movement of water, nor have the concentrations of free hormones on opposite sides been determined in the absence of hormone-binding proteins. We report experiments with 4 different ultrafiltration devices to determine the movements of H2O, IT3, and I-T4 across semipermeable membranes in a biologically relevant protein-free aqueous solution. A pool of normal human serum was obtained (Equitech-Bio). Serum thyroxine-binding globulin, transthyretin, albumin, total protein, total T4, dialyzable (free) T4, and thyroidstimulating hormone were within their respective reference intervals (Quest Diagnostics). Serum pH was controlled at 7.4 by adding HEPES acid to a final concentration of 54 mmol/L (3 ). Ultrafiltrate was prepared from 50 mL of this serum by applying 25 psi of nitrogen gas pressure to a stirred ultrafiltration device with a regenerated cellulose membrane, 10-kDa molecular weight cutoff (MWCO) (Millipore) at 37 °C. Ultrafiltration was continued until the volumes of retentate and ultrafiltrate were equal. Radiolabeled water (H2O), triiodothyronine (I-T3), and thyroxine (I-T4) were obtained from PerkinElmer Life Sciences. Before each experiment, I-T3 and I-T4 were repurified as previously described (3 ). These freshly repurified radiolabeled analytes (I-T3 and I-T4) and the H2O were added to different aliquots of the same serum ultrafiltrate at room temperature. The borosilicate glassware used in this study (Fisher Scientific) was found to adsorb 0.7% of I-T3 and 0.4% of I-T4 in the absence of serum proteins with a previously described method (3 ). The 4 ultrafiltration devices studied (Millipore) were Centricon YM10, YM-30, and YM-100 (10-kDa, 30kDa, and 100-kDa MWCO), and Amicon Ultra-4 (10-kDa MWCO). All devices used regenerated cellulose membranes. Devices of each type were applied to aliquots of serum ultrafiltrate containing H2O, I-T3, or I-T4 by ultrafiltration at 37 °C in a temperature controlled centrifuge at 3000g (5702RH, Eppendorf). Samples were ultrafiltered until 25%, 50%, or 75% of the aqueous solution had passed through the membrane, using a separate device for each analyte and percentage. Each experiment was performed twice in triplicate. The radioactivity (cpm/200 L) was determined for each retentate and ultrafiltrate solution. The radioactive stock solutions were used as experimental controls. In each experiment, the progress of ultrafiltration to 25%, 50%, and 75% was determined gravimetrically, by weighing each retentate and ultrafiltrate compartment. Ultrafiltration to 25%, 50%, and 75% was defined as the percentage of water mass that