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Letters to the Editor |
1 Department of Biochemistry
2 Departments of Internal Medicine, and Pathology, Loma Linda University, School of Medicine, Loma Linda, CA
aAddress correspondence to this author at: Department of Biochemistry, Loma Linda University School of Medicine, Mortensen Hall, Rm. 209, Loma Linda, CA 92354. Fax 909-558-7916; e-mail bwilcox{at}llu.edu.
To the Editor:
Ultrafiltration is a standard method for separating free T3 and free T4 from serum proteins and protein-bound 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 3H2O, 125I-T3, and 125I-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 thyroid-stimulating 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 (3H2O), triiodothyronine (125I-T3), and thyroxine (125I-T4) were obtained from PerkinElmer Life Sciences. Before each experiment, 125I-T3 and 125I-T4 were repurified as previously described (3). These freshly repurified radiolabeled analytes (125I-T3 and 125I-T4) and the 3H2O 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 125I-T3 and <0.4% of 125I-T4 in the absence of serum proteins with a previously described method(3).
The 4 ultrafiltration devices studied (Millipore) were Centricon YM-10, YM-30, and YM-100 (10-kDa, 30-kDa, 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 3H2O, 125I-T3, or 125I-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 µL) 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 crossed the membrane. After ultrafiltration, the loss of radiolabeled compounds (cpm/200 µL) was determined for each retentate and ultrafiltrate.
The concentration of 3H2O was evenly distributed between retentates and ultrafiltrates in all 4 ultrafiltration devices (Fig. 1
, A–D). The concentrations of 125I-T3 and 125I-T4 were unevenly distributed between retentates and ultrafiltrates in all 4 ultrafiltration devices (Fig. 1
, A–D). In all devices tested, the losses of 3H2O were trivial. By contrast, the losses of 125I-T3 decreased as ultrafiltration progressed, ranging from a high of 75% to insignificant. The losses of 125I-T4 decreased as ultrafiltration progressed, ranging from a high of 56% to insignificant. It is important to note that 3H2O, 125I-T3, and 125I-T4 were dissolved in normal human serum ultrafiltrate, void of serum proteins, containing normal concentrations of serum electrolytes and free thyroid hormones.
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The application of various ultrafiltration devices to radiolabeled water, T3, and T4 yielded striking inconsistencies (Fig. 1
, A–D). The differences in hormone concentrations on opposite sides of semipermeable membranes and the magnitudes of hormone losses during ultrafiltration were unexpected.
A recent study by the authors documented inconsistency in the retention of serum proteins by these same devices (4). The present study confirms the findings of the previous study; ultrafiltration is complex, poorly characterized, and incompletely understood. The movement of water, T3, and T4 across semipermeable membranes could not be predicted based on the reported MWCO. These inconsistencies complicate the interpretation of previous free thyroid hormone measurements involving uncharacterized ultrafiltration.
Acknowledgments
Grant funding/support: This work was partially supported by the Department of Biochemistry at Loma Linda University School of Medicine and Mortensen Chair. No extramural funds were used for this study.
Financial disclosures: Jerald C. Nelson was formerly Senior Medical Director of Quest Diagnostics Nichols Institute, San Juan Capistrano. He has no current affiliation with Quest Diagnostics. He is a consultant to Antech Diagnostics.
References
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, retentate and
, ultrafiltrate).