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Technical Briefs |
1 Departments of Biochemistry and 2 Internal Medicine and Pathology, Loma Linda University School of Medicine, Loma Linda, CA
aAddress correspondence to this author at: Loma Linda University Medical Center, 11234 Anderson St., Rm. 1568, Loma Linda, CA 92354; fax 909-558-0490; e-mail jcnelson{at}llu.edu
Abstract
Background: Direct equilibrium dialysis and direct ultrafiltration free thyroxine (T4) assays rely on semipermeable membranes to exclude T4-binding serum proteins from dialysates and ultrafiltrates. The presence of these proteins in dialysates or ultrafiltrates will yield spuriously high free T4 values when free T4 is quantified by RIA.
Methods: We used a nonanalog free T4 RIA that detects and quantifies dialyzable and ultrafilterable serum free T4 to detect T4-binding serum proteins. Two equilibrium dialysis devices and 3 ultrafiltration devices were used to illustrate this application. Displacements of [125I]T4 from anti-T4 by various concentrations of T4-depleted thyroxine-binding globulin, albumin, and serum total protein were compared to displacements by various concentrations of free T4.
Results: Both dialysis devices excluded detectable T4-binding serum proteins from dialysates. Two of 3 ultrafiltration devices excluded detectable T4-binding serum proteins from ultrafiltrates. One did not, and its ultrafiltrate yielded spurious free T4 values that correlated directly with serum protein concentrations.
Conclusion: The presence or absence of T4-binding proteins in dialysates and ultrafiltrates and the spurious free T4 values that these proteins cause can be documented using a nonanalog free T4 RIA.
Direct equilibrium dialysis and direct ultrafiltration free thyroxine (T4) methods use semipermeable membranes to separate free T4 from T4-binding serum proteins. Nonanalog free T4 RIAs measure this free T4. These measurements are based on [125I]T4 binding to anti-T4 and its displacement from anti-T4 by unlabeled free T4 (1)(2)(3)(4). When T4-binding serum proteins (T4BSPs) are present they bind [125I]T4 and displace [125I]T4 from anti-T4, yielding spurious results from these free T4 assays (see Figure 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol53/issue5). The spurious free T4 results caused by T4BSPs and the protein concentrations at which they occur have not been reported. This study used T4-depleted serum proteins to obtain spurious free T4 results and to determine the protein concentrations at which they occurred.
A nonanalog free T4 RIA (Nichols Institute Diagnostics) and a total T4 RIA (Coat-A-Count, Diagnostic Products) were used. A pool of normal human serum with thyroxine-binding globulin (TBG; 17 mg/L), transthyretin (0.27 g/L), albumin (42 g/L), total protein (78 g/L), total T4 (94 nmol/L, 73 µg/L), and dialyzable (free) T4 (12.9 pmol/L, 10 ng/L) was obtained from Equitech-Bio. Anti-T4, anti-T3, anti-IgG, and salicylates were undetectable. Adapting the method of Grundy et al. (5), we used Amberlite IRA-410 anion exchange resin (Alfa Aesar) to strip 1 serum aliquot of T4. Another serum aliquot was ultrafiltered. Concentrations of these T4-depleted serum total proteins varied from 5 x 10–7 to 0.05 g/L. In addition, proteins concentrated by ultrafiltration were diluted with the corresponding ultrafiltrate to concentrations that varied from 40–160 g/L. Highly purified human serum albumin (>99%, Sigma-Aldrich) and TBG (>95%, Cortex Biochem) were dissolved in T4-depleted human serum dialysate. Concentrations of albumin varied from 2 x 10–2 to 200 g/L and of TBG from 6 x 10–5 to 60 mg/L. Total and free T4 were undetectable in these protein solutions.
The dialysis devices tested were a vertical membrane device using 10 mL of retentate and 10 mL of dialysate (Fisher Scientific) and a horizontal membrane device using 200 µL of retentate and 2400 µL of dialysate (Nichols Institute Diagnostics). According to the manufacturers, both devices use a regenerated cellulose membrane with a 12–14 kDa molecular weight cutoff (MWCO; Spectra/Por, Fisher Scientific). Membranes were washed with deionized water before use. The dialysis buffer used was reported previously (1). To test for proteins in dialysates, T4-depleted human serum (40–160 g/L) was dialyzed for 18 h at 37 °C. During dialysis, mean (SD) pH was controlled to 7.4 (0.1) at 37 °C by the HEPES contained in the dialysis buffer (6). The final HEPES ion concentration was 54 mmol/L (1).
The ultrafiltration devices tested were the Centricon YM-10, Centricon YM-30, and Amicon Ultra-4 (Millipore). According to the manufacturer the regenerated cellulose membranes in these devices had MWCOs of 10 kDa, 30 kDa, and 10 kDa, respectively.
All devices were prerinsed with deionized water. Centrifugation was carried out in a temperature-controlled (37 °C), fixed-angle rotor centrifuge at 3000g. Before ultrafiltration, T4-depleted serum pH was controlled to 7.4 (0.1) at 37 °C, by addition of 40 µL of 1200 mmol/L HEPES acid (Fisher Biotech) per milliliter of serum. Serum pH stability was obtained by 15 min of vortex-mixing at room temperature while a continuous stream of moist air passed across the serum. The final HEPES ion concentration was 54 mmol/L (1).
A 4th ultrafiltration device, the Centricon YM-100 device (Millipore), was used as a positive control. This device had a regenerated cellulose membrane with a MWCO of 100 kDa. T4 binding serum proteins (T4BSPs) are expected to enter the ultrafiltrates obtained with this device because their molecular weights are 54–66 kDa. The dialysate of T4-depleted human serum was used as the negative control.
Sodium levothyroxine (for injection) was obtained in 500-µg vials (Bedford Laboratories) and dissolved in 5 mL of sodium chloride, USP, 9 g/L (Abbott Laboratories). This T4 solution (125 µmol/L, 100 mg/L) was diluted with T4-depleted human serum dialysate to concentrations ranging from 2.6 x 10–5 to 26 nmol/L (2 x 10–5 to 20 µg/L).
Free T4 is often adsorbed from aqueous solutions onto solid surfaces. This adsorption was determined using the method of Holm et al. (7) for the test tubes (borosilicate glass) and screw-capped vials (borosilicate glass) used in the present study (Fisher Scientific). [125I]T4 (Perkin-Elmer Life Sciences) was purified using Sephadex G-25 (Sigma-Aldrich) column chromatography (8). The Sephadex columns were equilibrated to 0.01 mol/L PBS (1 PBS tablet dissolved in 200 mL of water to obtain: 10 mmol/L phosphate buffer, 2.7 mmol/L potassium chloride, and 137 mmol/L sodium chloride; Sigma-Aldrich), at pH 5.4 and room temperature. A stock solution of [125I]T4 was added to the column and eluted with 100 mmol/L sodium hydroxide (Sigma-Aldrich). Fractions were collected in 13 x 100 mm glass test tubes using an automated fraction collector (LKB). Gamma radiation was quantified using a multiwell, automated gamma counter (Gamma 4000, Beckman-Coulter). Adsorption was <0.4% in test tubes and vials.
A theoretical lower limit for the detection of displacements was calculated as the mean minus 2 SD of the zero control data. This limit was 0.001 mg/L for T4-depleted TBG, 0.025 g/L for T4-depleted albumin, and 0.0015 g/L for T4-depleted total protein, compared to 0.8 pmol/L (0.6 ng/L) for free T4 (see Fig. 2 in the online Data Supplement).
As a percentage of normal serum concentration, T4-depleted serum total protein concentrations and T4-depleted TBG concentrations were similarly effective in displacing [125I]T4, and both were more effective than T4-depleted albumin (Table 1
). We used T4-depleted serum total proteins, which contain all T4BSPs, to test for spurious free T4 results in dialysates and ultrafiltrates.
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When T4-depleted serum total proteins at concentrations ranging from 40 to 160 g/L were applied to the 2 dialysis devices tested, no spurious free T4 determinations were obtained. One of the 3 ultrafiltration devices yielded spurious free T4 results (Fig. 1
), which correlated with the concentrations of serum proteins in retentates (r = 0.94; P = 0.047). This result was unexpected because the designated MWCO was 10 kDa and the molecular weights of T4BSPs are 54–66 kDa. Spurious free T4 determinations were obtained with the positive control, as expected (Fig. 1
).
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Previous studies have measured albumin in serum dialysates or ultrafiltrates. Weeke et al. (9)(10) measured albumin in serum ultrafiltrates with a microalbuminuria RIA with a sensitivity of 0.001 g/L. They calculated that a leakage of 0.0015% of undiluted serum albumin would induce an error of 
5% in free T4 determinations. With the free T4 RIA method, the presence of 0.06% (0.025 g/L) serum albumin resulted in spurious free T4 values of 3% (0.5 pmol/L).
Tikanoja et al. (11) measured albumin in serum ultrafiltrates using an albumin RIA with a detection limit of 8.0 x 10–4 g/L. They used a cutoff value for protein leakage of 0.005% of serum proteins, citing Weeke et al. (9). Ultrafiltration devices that allowed <0.005% (0.002 g/L) of albumin into ultrafiltrates were regarded as acceptable. Only 1 device of 4 met this criterion. Again, this compares to the free T4 RIA method for which 0.06% (0.025 g/L) of serum albumin resulted in spurious free T4 values of 3% (0.5 pmol/L).
Holm et al. (12) measured albumin in both serum dialysates and serum ultrafiltrates using a double antibody sandwich ELISA with a detection limit of 2.8 x 10–6 g/L. They found no detectable albumin in serum dialysates but found detectable albumin in the ultrafiltrates obtained with each of 5 different devices.
The previous studies measured protein concentrations by use of albumin assays that were more sensitive for detecting albumin than the free T4 RIA method used in the present study (0.001 g/L, 8.0 x 10–4 g/L, and 2.8 x 10–6 g/L, compared to 0.025 g/L). The present method, however, detects the spurious free T4 values caused by any and all T4BSPs as a consequence of binding radiolabeled T4.
Uncertainty about spurious free T4 determinations as a consequence of T4BSP interference is unnecessary. The laboratory that applies such a free T4 RIA to the quantification of T4 in serum dialysates or serum ultrafiltrates can use the same RIA to rule in or rule out these spurious free T4 values.
Acknowledgments
Grant/funding support: This work was partially supported by the Loma Linda University School of Medicine and Mortensen Chair. No extramural funds were used for this study.
Financial disclosures: Jerald C. Nelson is a consultant to Antech Diagnostics.
Acknowledgements: We thank Nichols Institute Diagnostics for providing some of the test kits and reagents used in this study.
References
The following articles in journals at HighWire Press have cited this article:
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  B. Yue, A. L. Rockwood, T. Sandrock, S. L. La'ulu, M. M. Kushnir, and A. W. Meikle Free Thyroid Hormones in Serum by Direct Equilibrium Dialysis and Online Solid-Phase Extraction-Liquid Chromatography/Tandem Mass Spectrometry Clin. Chem., April 1, 2008; 54(4): 642 - 651. [Abstract] [Full Text] [PDF]
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K. S. Fritz, R. M. Weiss, J. C. Nelson, and R. B. Wilcox Unequal Concentrations of Free T3 and Free T4 after Ultrafiltration Clin. Chem., July 1, 2007; 53(7): 1384 - 1385. [Full Text] [PDF]
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