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A direct free thyroxine ([T.sub.4]) immunoassay with the characteristics of a total [T.sub.4] immunoassay.

Interrelationships among serum free thyroxine ([T.sub.4]) [3] the proteins that bind [T.sub.4], protein-bound [T.sub.4], and total [T.sub.4] are variable (1-6). Protein-bound and total [T.sub.4] concentrations vary (correlate) directly with free [T.sub.4] concentrations when serum [T.sub.4]-binding proteins are constant. When free [T.sub.4] is constant, protein-bound [T.sub.4] and total [T.sub.4] concentrations vary directly with concentrations of [T.sub.4]-binding proteins. There are reports of direct analog-based free [T.sub.4] results that vary directly with serum concentrations of [T.sub.4]-binding protein (7-18) or vary as total [T.sub.4] concentrations vary (while free [T.sub.4] is held constant) (19, 20). These relationships imply that these analog-based direct free [T.sub.4] assays detect total [T.sub.4] concentrations.

This possibility led us to ask the following questions about the direct analog-based free [T.sub.4] immunoassay. What form of [T.sub.4] does the assay detect, and what concentrations of [T.sub.4] does it measure? Which form of [T.sub.4] does it detect after equilibrium dialysis and ultrafiltration? Do the free [T.sub.4] values correlate with total [T.sub.4] concentrations when total [T.sub.4] is varied (while free [T.sub.4] is constant) or when total [T.sub.4] is constant (while free [T.sub.4] is varied)?

Materials and Methods


We studied a manual analog-based free [T.sub.4] immunoassay (Coat-A-Count, Diagnostic Products Corp.) that uses a radiolabeled [T.sub.4] analog, immobilized [T.sub.4] antibody, a single incubation, and 21-fold dilution with a reagent solution. The total [T.sub.4] immunoassay (Coat-A-Count, Diagnostic Products Corp.) uses radiolabeled free [T.sub.4] (nonanalog), immobilized [T.sub.4] 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 [T.sub.4] solutions. The nonanalog free [T.sub.4] immunoassay (Nichols Institute Diagnostics) applied to equilibrium dialysates and ultrafiltrates (21-30) uses radiolabeled free [T.sub.4], immobilized [T.sub.4] 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 [T.sub.4] 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).


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 [T.sub.4], free [T.sub.4], thyroxine-binding globulin (TBG), transthyretin (TTR), and albumin were within their respective reference intervals, and test results for anti-[T.sub.4], anti-[T.sub.3], and anti-IgG antibodies and for salicylates were negative (testing performed at Quest Diagnostics; data not presented).


We stripped an aliquot of the serum pool of [T.sub.4] using Amberlite IRA-410 anion exchange resin (Alfa Aesar) (31). No residual total [T.sub.4] was detected by the total [T.sub.4] RIA, and no dialyzable [T.sub.4] was detected by the nonanalog free [T.sub.4] immunoassay. TBG, TTR, and albumin concentrations remained normal after [T.sub.4] depletion. We estimated the affinity of serum proteins for [T.sub.4] as the free fraction of serum [T.sub.4] (the ratio of dialyzable free [T.sub.4] to total [T.sub.4]). To measure the free fraction after [T.sub.4] depletion, [T.sub.4] was restored to its original concentration. There was no significant change in affinity after [T.sub.4] depletion (data not presented).


We obtained sodium levothyroxine, for injection, in 500-[micro]g 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 [micro]mol/L (10 000 [micro]g/dL) sodium levothyroxine.


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 [degrees]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.


We ultrafiltered large volumes of serum (up to 50 mL) under 20 psi nitrogen gas pressure at 37 [degrees]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[degrees]C in a temperature-controlled Eppendorf 5702RH centrifuge (Fisher Scientific). All ultrafiltration devices were prewashed twice with deionized water.


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 [degrees]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 [degrees]C, before ultrafiltration, by adding 40 [micro]L 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 [T.sub.4] can adsorb onto solid surfaces from aqueous solutions. We tested the borosilicate glass vials and test tubes used (Fisher Scientific) for free [T.sub.4] adsorption by a modification of the procedure reported by Holm et al. (32). Radiolabeled free [T.sub.4] ([[sup.125]I][T.sub.4]; 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 [[[sup.125]I][T.sub.4] 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 [[[sup.125]I][T.sub.4] 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 [[[sup.125]I][T.sub.4] in the absence of serum proteins. Samples were stored at -80 [degrees]C before assay.


We applied the direct free [T.sub.4] immunoassay and the total [T.sub.4] immunoassay to the following solutions:

* Fractions of serum [T.sub.4] obtained by equilibrium dialysis and ultrafiltration.

* Varied concentrations of sodium levothyroxine added to normal human serum dialysate (see Materials and Methods) at concentrations of 39 to 309 nmol/L (3 to 24 [micro]g/ dL).

* Varied concentrations of sodium levothyroxine added to [T.sub.4]-depleted normal human serum at concentrations of 39 to 309 nmol/L (3 to 24 [micro]g/dL). The dialyzable [T.sub.4] in these solutions (direct equilibrium dialysis) was 7.7 to 125 pmol/L (0.6 to 9.7 ng/dL).

* Varied concentrations of serum proteins, protein-bound [T.sub.4], and total [T.sub.4] while free [T.sub.4] concentration remained constant. Ultrafiltration was applied to normal serum. One aliquot of retentate was undiluted. Other aliquots were diluted 2-fold, 4-fold, and 8-fold with the corresponding ultrafiltrate. Thus, serum proteins, protein-bound [T.sub.4], and total [T.sub.4] were varied from a low of 25% of native serum concentrations to a high of 200% of native serum concentrations, while free [T.sub.4] was constant. We confirmed the variation in total [T.sub.4] concentrations by measuring total [T.sub.4], the variation in protein concentrations by measuring total protein and TTR (Quest Diagnostics; data not presented), and the constancy of free [T.sub.4] concentration by measuring dialyzable [T.sub.4] (data not presented). [T.sub.4] depleted serum proteins were varied in the same way.

* Varied concentrations of serum proteins and protein-bound [T.sub.4] progressively replaced with free [T.sub.4] while total [T.sub.4] was held constant. We applied equilibrium dialysis to normal serum. We measured the total [T.sub.4] in retentate using the total [T.sub.4] immunoassay (Table 1) and added sodium levothyroxine solution to the dialysate to match the concentration of total [T.sub.4] in the retentate. The retentate was progressively diluted with [T.sub.4]-enriched dialysate until dialyzable [T.sub.4] concentrations reached a plateau. We measured dialyzable [T.sub.4] by use of nonanalog free [T.sub.4] immunoassay (see Methods and Materials). High dialyzable [T.sub.4] concentrations were diluted with immunoassay zero calibrator, as needed, to bring them into the interval quantified by the nonanalog free [T.sub.4] immunoassay (2.6 to 129 pmol/L). These dialyzable [T.sub.4] measurements varied from 16.7 to 8940 pmol/L (1.3 to 693 ng/dL) (Fig. 2A).


Neither the analog-based direct free [T.sub.4] immunoassay nor the total [T.sub.4] immunoassay detected ultrafilterable or dialyzable serum [T.sub.4]. Both assays detected and quantified the [T.sub.4] retained with serum proteins during dialysis and ultrafiltration. The nonanalog free [T.sub.4] immunoassay was the only assay that detected and quantified ultrafilterable and dialyzable serum [T.sub.4] (Table 1).


When [T.sub.4] was added to normal human serum dialysate at concentrations of 39 to 309 nmol/L (3 to 24 [micro]g/dL), the direct free [T.sub.4] values were 9 to 81 pmol/L (0.7 to 6.3 ng/dL) and the total [T.sub.4] values were 30 to 257 nmol/L (2.3 to 20 [micro]g/dL; Table 2).

When [T.sub.4] was added to [T.sub.4]-depleted normal human serum at concentrations of 39 to 309 nmol/L (3 to 24 [micro]g/dL), the direct free [T.sub.4] values were 6 to 71 pmol/L (0.5 to 5.5 ng/dL) and the total [T.sub.4] values were 40 to 257 nmol/L (3.1 to 20 [micro]g/dL; Table 3).

When serum protein concentrations, protein bound [T.sub.4] concentrations, and total [T.sub.4] concentrations were varied from 25% to 200% of whole serum concentrations while free [T.sub.4] was constant, the direct free [T.sub.4] 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 [micro]g/dL; Fig. 1; SDs reported by error bars).

When free [T.sub.4] progressively replaced protein bound [T.sub.4] while total [T.sub.4] was constant, both analog-based direct free [T.sub.4] measurements and total [T.sub.4] measurements were closely related to total [T.sub.4] concentration (Figs. 2B and C). Neither the analog-based direct free [T.sub.4] assay nor the total [T.sub.4] assay detected or followed the variation in dialyzable [T.sub.4] concentrations when they varied from 16.7 to 8940 pmol/L (1.3 to 693 ng/dL; Fig. 2A). The mean total [T.sub.4] result was 77.5 nmol/L (6.0 [micro]g/dL; Fig. 2C). The mean free [T.sub.4] result was 18.6 pmol/L (1.4 ng/dL; Fig. 2B).


The primary reason for carrying out free [T.sub.4] measurements is to differentiate patients with unusual or abnormal serum free [T.sub.4] concentrations from patients with unusual or abnormal [T.sub.4] binding to serum proteins. When [T.sub.4] binding to serum proteins is normal (or constant), total [T.sub.4] measurements will be proportional to free [T.sub.4] concentrations, and both will be categorically similar (low, normal, or high) (see Table 3). This is not the situation when free [T.sub.4] concentrations are constant (or similar) and [T.sub.4] binding serum protein concentrations (or affinities) are unusual or abnormal. Under these conditions, dialyzable [T.sub.4] concentrations will have a different relationship to total [T.sub.4] concentrations (16, 19, 35, 36).


The data obtained with these experiments document a direct analog-based free [T.sub.4] immunoassay that reports free [T.sub.4] values with the characteristics of total [T.sub.4] values, but each assay's calibration is strikingly different. The direct free [T.sub.4] immunoassay did not follow normal free [T.sub.4] concentrations in the presence of varied total [T.sub.4] concentrations (Fig. 1) or abnormal free [T.sub.4] concentrations when total [T.sub.4] was normal (Fig. 2). The information provided by this direct free [T.sub.4] immunoassay is qualitatively similar to the information provided by total [T.sub.4] immunoassays. There is no evidence that this free [T.sub.4] immunoassay will be more useful than a total [T.sub.4] immunoassay.

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.

Received November 29, 2006; accepted February 5, 2007. Previously published online at DOI: 10.1373/clinchem.2006.083915


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[3] Nonstandard abbreviations: [T.sub.4], thyroxine; TSH, thyroid-stimulating hormone; TBG, thyroxine-binding globulin; and TTR, transthyretin.


[1] Departments of Biochemistry and

[2] Internal Medicine and Pathology, Loma Linda University School of Medicine, Loma Linda, CA.

* Address correspondence to this author at: 11234 Anderson Street, Room 1568, Loma Linda, CA 92354. Fax 909-558-0490; e-mail
Table 1. Serum fractions and [T.sub.4] values.


Immunoassays Retentate Ultrafiltrate

Total [T.sub.4], nmol/L 139 ND
Analog free [T.sub.4], pmol/L 34.7 ND
Nonanalog free [T.sub.4], pmol/L 129 20.6

 Equilibrium dialysis

Immunoassays Retentate Dialysate

Total [T.sub.4], nmol/L 77 ND
Analog free [T.sub.4], pmol/L 17 ND
Nonanalog free [T.sub.4], pmol/L 129 19

ND, not detected: total [T.sub.4] <12.87 nmol/L
or free [T.sub.4] <1.29 pmol/L.

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

[T.sub.4] added, Analog-based free Total [T.sub.4],
nmol/L [T.sub.4], pmol/L nmol/L

39 9 30
77 21 60
116 33 104
154 44 130
193 54 176
232 66 196
270 79 229
309 81 257
Mean 48 148

For [T.sub.4], 1 [micro]g/dL = 12.87 nmol/L.

Table 3. Results when [T.sub.4] was added to [T.sub.4] depleted serum.

[T.sub.4] added, Analog-based Total
nmol/L free [T.sub.4], [T.sub.4],
 pmol/L nmol/L

39 6 40
77 14 76
116 23 112
154 32 139
193 39 169
232 51 199
270 58 241
309 71 257
Mean 37 154

For [T.sub.4], 1 [micro]g/L = = 12.87 nmol/L.
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Title Annotation:Endocrinology and Metabolism
Author:Fritz, Kristofer S.; Wilcox, R. Bruce; Nelson, Jerald C.
Publication:Clinical Chemistry
Date:May 1, 2007
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