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Clinical Chemistry 53: 911-915, 2007. First published March 15, 2007; 10.1373/clinchem.2006.083915
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(Clinical Chemistry. 2007;53:911-915.)
© 2007 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

A Direct Free Thyroxine (T4) Immunoassay with the Characteristics of a Total T4 Immunoassay

Kristofer S. Fritz1, R. Bruce Wilcox1 and Jerald C. Nelson2,a

Departments of1 Biochemistry and 2 Internal Medicine and Pathology, Loma Linda University School of Medicine, Loma Linda, CA.

aAddress correspondence to this author at: 11234 Anderson Street, Room 1568, Loma Linda, CA 92354. Fax 909-558-0490; e-mail jcnelson{at}llu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Direct free thyroxine (T4) measurements have been linked to both T4-binding serum protein concentrations and protein-bound T4 concentrations. Whether this is evidence of a relationship to total T4 concentrations has not been reported.

Methods: We compared an analog-based direct free T4 immunoassay and a total T4 immunoassay. Each assay was applied to the fractions of serum T4 obtained by ultrafiltration and equilibrium dialysis. Both were applied to serum-based solutions in which free T4, T4-binding proteins, protein-bound T4, and total T4 were systematically varied, held constant, or excluded.

Results: Neither the free T4 assay nor the total T4 assay detected dialyzable or ultrafilterable serum T4. Both assays detected and reported the T4 retained with serum proteins. Both free and total T4 results were related to the same total T4 concentrations in the presence and absence of T4-binding proteins. Both results were similarly related to total T4 concentrations when free T4 was held constant while total T4 was varied. Both were similarly related to a total T4 concentration that was held constant while free T4 progressively replaced protein-bound T4. These free T4 results, like total T4 results, were unresponsive to a 500-fold variation in dialyzable T4 concentrations.

Conclusion: New experiments extend the characterization of a longstanding and incompletely characterized analog-based free T4 immunoassay. These free T4 measurements relate to total T4 concentrations in the same way that total T4 measurements do.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interrelationships among serum free thyroxine (T4),1 the proteins that bind T4, protein-bound T4, and total T4 are variable (1)(2)(3)(4)(5)(6). Protein-bound and total T4 concentrations vary (correlate) directly with free T4 concentrations when serum T4-binding proteins are constant. When free T4 is constant, protein-bound T4 and total T4 concentrations vary directly with concentrations of T4-binding proteins. There are reports of direct analog-based free T4 results that vary directly with serum concentrations of T4-binding protein (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) or vary as total T4 concentrations vary (while free T4 is held constant) (19)(20). These relationships imply that these analog-based direct free T4 assays detect total T4 concentrations.

This possibility led us to ask the following questions about the direct analog-based free T4 immunoassay. What form of T4 does the assay detect, and what concentrations of T4 does it measure? Which form of T4 does it detect after equilibrium dialysis and ultrafiltration? Do the free T4 values correlate with total T4 concentrations when total T4 is varied (while free T4 is constant) or when total T4 is constant (while free T4 is varied)?


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
t4 immunoassays
We studied a manual analog-based free T4 immunoassay (Coat-A-Count, Diagnostic Products Corp.) that uses a radiolabeled T4 analog, immobilized T4 antibody, a single incubation, and 21-fold dilution with a reagent solution. The total T4 immunoassay (Coat-A-Count, Diagnostic Products Corp.) uses radiolabeled free T4 (nonanalog), immobilized T4 antibody, and a single incubation; it is a manual method that dilutes the solutions it analyzes 41-fold with a reagent solution. Both assays were applied to the same experimental T4 solutions. The nonanalog free T4 immunoassay (Nichols Institute Diagnostics) applied to equilibrium dialysates and ultrafiltrates (21)(22)(23)(24)(25)(26)(27)(28)(29)(30) uses radiolabeled free T4, immobilized T4 antibody, and a single incubation; it is a manual method that dilutes the solutions it analyzes 1.0625-fold with a reagent solution.

We performed each assay according to its manufacturer’s instructions. Each T4 result reported was a mean of triplicates, and each experiment was repeated for confirmation. We detected and quantified gamma radiation by use of a Gamma 4000 multiwell automated gamma counter (Beckman-Coulter).

normal human serum
We obtained normal human serum from 16 healthy male volunteers, ages 21 to 55 years. These sera were pooled. Serum collection was approved by the institutional review board, and serum samples were given anonymous identifiers. In this pool, serum thyroid-stimulating hormone (TSH), total T4, free T4, thyroxine-binding globulin (TBG), transthyretin (TTR), and albumin were within their respective reference intervals, and test results for anti-T4, anti-T3, and anti-IgG antibodies and for salicylates were negative (testing performed at Quest Diagnostics; data not presented).

t4-depleted normal human serum
We stripped an aliquot of the serum pool of T4 using Amberlite IRA-410 anion exchange resin (Alfa Aesar) (31). No residual total T4 was detected by the total T4 RIA, and no dialyzable T4 was detected by the nonanalog free T4 immunoassay. TBG, TTR, and albumin concentrations remained normal after T4 depletion. We estimated the affinity of serum proteins for T4 as the free fraction of serum T4 (the ratio of dialyzable free T4 to total T4). To measure the free fraction after T4 depletion, T4 was restored to its original concentration. There was no significant change in affinity after T4 depletion (data not presented).

sodium levothyroxine
We obtained sodium levothyroxine, for injection, in 500-µg vials (Bedford Labs). It was dissolved at room temperature in 5 mL of 9 mL/L NaCl, USP grade (Abbott Labs). This produced a stock solution containing 125 µmol/L (10 000 µg/dL) sodium levothyroxine.

equilibrium dialysis
We obtained dialysis devices and dialysate buffer from Nichols Institute Diagnostics. The chemical composition of this buffer has been reported (27). Serum samples were dialyzed for 18 h at 37 °C in an Isotemp Incubator, model 630D (Fisher Scientific). A moisture-saturated atmosphere was maintained during dialysis by enclosing dialysis cells in containers with open-water reservoirs.

ultrafiltration
We ultrafiltered large volumes of serum (up to 50 mL) under 20 psi nitrogen gas pressure at 37 °C using a stirred ultrafiltration device with a regenerated cellulose membrane that has a nominal molecular weight cutoff of 12 to 14 kDa (Millipore). We ultrafiltered small volumes of serum (up to 2 mL) using a Centricon YM-10 ultrafiltration device with a regenerated cellulose membrane that has a 10-kDa molecular weight cutoff (Fisher Scientific). Centrifugation at 3000g was carried out at 37 °C in a temperature-controlled Eppendorf 5702RH centrifuge (Fisher Scientific). All ultrafiltration devices were prewashed twice with deionized water.

serum ph control
When dialysis was applied, the pH of serum dialysate and retentate was controlled to mean (SD) 7.4 (0.1) during equilibrium dialysis at 37 °C by the HEPES acid in dialysate buffer (27). At equilibrium, the final HEPES ion concentration was calculated to be 54 mmol/L.

When ultrafiltration was applied, whole serum pH was controlled to 7.4 (0.1) at 37 °C, before ultrafiltration, by adding 40 µL of 1200 mmol/L HEPES acid (Fisher Biotech) per mL serum. Serum pH stability was obtained during 15 min of vortex mixing while passing a continuous stream of moist air across the serum. The final HEPES ion concentration was 54 mmol/L.

free t4 adsorption to container surfaces
Free T4 can adsorb onto solid surfaces from aqueous solutions. We tested the borosilicate glass vials and test tubes used (Fisher Scientific) for free T4 adsorption by a modification of the procedure reported by Holm et al. (32). Radiolabeled free T4 ([125I]T4; Perkin-Elmer Life Sciences) was freshly repurified by column chromatography before use, using Sephadex G-25 (Sigma-Aldrich) (33)(34). Columns were equilibrated to 10 mmol/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. Stock [125I]T4 was added to the column and eluted with 100 mmol/L sodium hydroxide (Sigma-Aldrich). We collected fractions in 13 x 100-mm glass test tubes using an automated fraction collector (LKB) and quantified gamma radiation. The test tubes adsorbed <0.4% of free [125I]T4 in the absence of serum proteins.

We also tested the 2-mL screw-capped glass vials used to store test samples. They also adsorbed <0.4% of free [125I]T4 in the absence of serum proteins. Samples were stored at –80 °C before assay.

experimental strategies
We applied the direct free T4 immunoassay and the total T4 immunoassay to the following solutions:


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  		Table 1. Serum fractions and T4 values.


Figure 2
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  		Figure 2. Assay responses to replacement of protein-bound T4 by free T4, while holding T4 constant.

(A), dialyzable T4 values by nonanalog free T4 RIA; (B), direct free T4 values by analog-based free T4 RIA; and (C), total T4 values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neither the analog-based direct free T4 immunoassay nor the total T4 immunoassay detected ultrafilterable or dialyzable serum T4. Both assays detected and quantified the T4 retained with serum proteins during dialysis and ultrafiltration. The nonanalog free T4 immunoassay was the only assay that detected and quantified ultrafilterable and dialyzable serum T4 (Table 1Up ).

When T4 was added to normal human serum dialysate at concentrations of 39 to 309 nmol/L (3 to 24 µg/dL), the direct free T4 values were 9 to 81 pmol/L (0.7 to 6.3 ng/dL) and the total T4 values were 30 to 257 nmol/L (2.3 to 20 µg/dL; Table 2 ).


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  		Table 2. Results when T4 was added to serum dialysate.

When T4 was added to T4-depleted normal human serum at concentrations of 39 to 309 nmol/L (3 to 24 µg/dL), the direct free T4 values were 6 to 71 pmol/L (0.5 to 5.5 ng/dL) and the total T4 values were 40 to 257 nmol/L (3.1 to 20 µg/dL; Table 3 ).


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  		Table 3. Results when T4 was added to T4-depleted serum.

When serum protein concentrations, protein bound T4 concentrations, and total T4 concentrations were varied from 25% to 200% of whole serum concentrations while free T4 was constant, the direct free T4 values varied from 2.6 to 34.8 pmol/L (0.2 to 2.7 ng/dL) and the total values varied from 21.9 to 139 nmol/L (1.7 to 10.8 µg/dL; Fig. 1 ; SDs reported by error bars).


Figure 1
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  		Figure 1. Comparison of analog-based free T4 estimates to total T4 determinations.

Free T4 concentration was held constant at 20 pmol/L (1.6 ng/dL). The concentrations of serum proteins, protein bound T4, and total T4 were varied from 25%, 50%, 100%, and 200% of those in whole serum.

When free T4 progressively replaced protein bound T4 while total T4 was constant, both analog-based direct free T4 measurements and total T4 measurements were closely related to total T4 concentration (Figs. 2BUp and C). Neither the analog-based direct free T4 assay nor the total T4 assay detected or followed the variation in dialyzable T4 concentrations when they varied from 16.7 to 8940 pmol/L (1.3 to 693 ng/dL; Fig. 2AUp ). The mean total T4 result was 77.5 nmol/L (6.0 µg/dL; Fig. 2CUp ). The mean free T4 result was 18.6 pmol/L (1.4 ng/dL; Fig. 2BUp ).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The primary reason for carrying out free T4 measurements is to differentiate patients with unusual or abnormal serum free T4 concentrations from patients with unusual or abnormal T4 binding to serum proteins. When T4 binding to serum proteins is normal (or constant), total T4 measurements will be proportional to free T4 concentrations, and both will be categorically similar (low, normal, or high) (see Table 3Up ). This is not the situation when free T4 concentrations are constant (or similar) and T4 binding serum protein concentrations (or affinities) are unusual or abnormal. Under these conditions, dialyzable T4 concentrations will have a different relationship to total T4 concentrations (16)(19)(35)(36).

The data obtained with these experiments document a direct analog-based free T4 immunoassay that reports free T4 values with the characteristics of total T4 values, but each assay’s calibration is strikingly different. The direct free T4 immunoassay did not follow normal free T4 concentrations in the presence of varied total T4 concentrations (Fig. 1Up ) or abnormal free T4 concentrations when total T4 was normal (Fig. 2Up ). The information provided by this direct free T4 immunoassay is qualitatively similar to the information provided by total T4 immunoassays. There is no evidence that this free T4 immunoassay will be more useful than a total T4 immunoassay.


   Acknowledgments
 
Grant/funding support: None declared.

Financial disclosures: This research was supported with funds from the Department of Biochemistry, Loma Linda University School of Medicine, Loma Linda, CA.

Acknowledgements: The authors thank Nichols Institute Diagnostics, San Clemente, CA, for providing some of the test packages and reagents used in this study. 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.


   Footnotes
 
1 Nonstandard abbreviations: T4, thyroxine; TSH, thyroid-stimulating hormone; TBG, thyroxine-binding globulin; and TTR, transthyretin.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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