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

2007 ◽  
Vol 53 (5) ◽  
pp. 911-915 ◽  
Author(s):  
Kristofer S Fritz ◽  
R Bruce Wilcox ◽  
Jerald C Nelson
Keyword(s):  
Serum Protein ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Equilibrium Dialysis ◽  
Free Thyroxine ◽  
Free T4 ◽  
Serum T4 ◽  
Presence And Absence

Abstract 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.

scholarly journals Quantifying Spurious Free T4 Results Attributable to Thyroxine-Binding Proteins in Serum Dialysates and Ultrafiltrates

2007 ◽  
Vol 53 (5) ◽  
pp. 985-988 ◽  
Author(s):  
Kristofer S Fritz ◽  
R Bruce Wilcox ◽  
Jerald C Nelson
Keyword(s):  
Serum Protein ◽  
Total Protein ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Equilibrium Dialysis ◽  
Free T4 ◽  
Serum Total Protein ◽  
Semipermeable Membranes ◽  
Serum Free ◽  
Thyroxine Binding Globulin

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.

The Transport of Thyroid Hormones

1983 ◽  
Vol 65 (4) ◽  
pp. 337-342 ◽  
Author(s):  
R. Hoffenberg ◽  
D. B. Ramsden
Keyword(s):  
Thyroid Hormones ◽  
Binding Proteins ◽  
Serum Proteins ◽  
High Capacity ◽  
Free Fraction ◽  
Total Serum ◽  
Free T4 ◽  
Specific Serum ◽  
Target Organs ◽  
Serum T4

1. Hormones have to be transported from their sites of synthesis to their target organs. For lipophilic hormones, such as steroids and thyroid hormones, transport is accomplished by binding to specific serum proteins, in the case of thyroxine (T4) and tri-iodothyronine (T3) to thyroxine-binding globulin (TBG) and prealbumin (PA). Normally about 70% of circulating T4 and 75–80% of T3 is bound to TBG, about 20% of T4 and 10% of T3 to PA and 10–15% of each to albumin, which has a low affinity but high capacity for both hormones [1, 2]. Apart from facilitating transport, binding to serum protein prevents excessive loss of hormone into the urine by glomerular filtration or flooding into cells, and may provide a readily available reservoir in times of need. The union between binding proteins and their ligands is reversible, so that a small proportion of hormone is non-protein-bound or ‘free’, in equilibrium with that which is protein-bound. For T4 this free fraction is normally 0.02-0.04% of the total serum T4 concentration, for T3 about 0.3% [3, 4]. 2. The major binding proteins of T4 and T3 will briefly be described and the nature of free T4 and T3 considered.

CORTICOSTERONE BINDING BY MOUSE SERA DURING FOETAL AND POST-NATAL DEVELOPMENT

1977 ◽  
Vol 84 (1) ◽  
pp. 177-190 ◽  
Author(s):  
Lia Savu ◽  
Emmanuel Nunez ◽  
Max-Fernand Jayle
Keyword(s):  
Amniotic Fluid ◽  
Binding Sites ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Equilibrium Dialysis ◽  
Normal Adult ◽  
Scatchard Analysis ◽  
Mammalian Development ◽  
Single Class ◽  
Mean Values

ABSTRACT The binding properties of corticosterone binding globulin (CBG) of mouse sera have been studied by equilibrium dialysis and electrophoretic techniques, at different stages of foetal and post-natal development. Scatchard analysis has demonstrated in all cases a single class of high affinity saturable binding sites for corticosterone. Remarkable increases of the binding capacities were observed in the foetal and pregnant sera, as compared to normal adult and immature levels. The mean values of n1M1 × g−1 of serum proteins (concentration of binding sites, n1 × moles of binding proteins M1) were 21 10−8 in 14–19 day pregnant females, 17 10−8 in the amniotic fluid, 4.2 10−8 in 14–19 day embryos, and only 0.8 10−8 in the normal adult female. Neonatal mice, aged 0–6 days exhibited no CBG activities. The association constants showed values of 2.5–4.1 108 m−1 when measured with foetal sera, and of 1.2–2.1 108 m−1 with pregnant or control adult sera and with the amniotic fluid, at 25°C. Comparative electrophoretic, thermal denaturation and competition studies with foetal and pregnant plasma CBG's are also reported. The results are discussed in relation to the origin of CBG in the foetal serum, and also with respect to similar studies in the rat, guinea pig and man. The possible biological implications of serum steroid binding proteins in mammalian development are briefly outlined.

Effect of Sodium Ethylmercurithiosalicylate (Thimerosal) on Serum Binding of Thyroid Hormones

1973 ◽  
Vol 51 (2) ◽  
pp. 156-159 ◽  
Author(s):  
Diego Bellabarba ◽  
Raymonde Tremblay
Keyword(s):  
Thyroid Hormones ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Free Fraction ◽  
Inhibitory Effect ◽  
Free T4 ◽  
Thyroxine Binding Globulin ◽  
Free T3 ◽  
Serum Binding Protein ◽  
Barbital Buffer

Sodium ethylmercurithiosalicylate (Thimerosal, Merthiolate) has been found to interfere with the binding of thyroid hormones to serum proteins. Dialysis studies showed that this compound, added to serum in concentrations varying from 90 to 360 mg/100 ml, caused an increase of the dialyzable or free fraction of thyroxine (T4) and triiodothyronine (T3). The increase was higher for the free T4 (3.8- to 18-fold) than for the free T3 fraction (2.3- to 5-fold). Electrophoretic studies on the distribution of tracer amounts of labeled T4 among the serum binding proteins revealed that the inhibitory effect of sodium ethylmercurithiosalicylate was exerted mainly on thyroxine binding globulin (TBG). In presence of this compound (180 mg/100 ml of serum) the percentage of tracer T4 bound to TBG was reduced from 53% to 9%. These findings were also confirmed by examining the binding of tracer amounts of labeled T4 and T3 in a serum diluted in barbital buffer, which inhibits the hormonal binding to thyroxine binding prealbumin and albumin. In presence of sodium ethylmercurithiosalicylate a significant displacement of both T4 and T3 from the serum binding protein (TBG) was observed.

Indirect estimation of thyroid hormone-binding proteins to calculate free thyroxine index: comparison of nonisotopic methods that use labeled thyroxine ("T-uptake")

1995 ◽  
Vol 41 (1) ◽  
pp. 41-47 ◽  
Author(s):  
J D Faix ◽  
H N Rosen ◽  
F R Velazquez
Keyword(s):  
Thyroid Hormone ◽  
Binding Proteins ◽  
Free Thyroxine ◽  
Reference Ranges ◽  
Hormone Binding ◽  
Free T4 ◽  
Nonthyroidal Illness ◽  
Free Thyroxine Index ◽  
Uptake Method ◽  
Hypothyroid Patients

Abstract There are many alternative ways of estimating free thyroxine (T4) when thyrotropin screening results are abnormal. In addition to free T4 immunoassays, the menu of most automated immunoassay instruments includes a nonisotopic version of the original triiodothyronine (T3)-uptake assay called "T-uptake." We evaluated the ability of five such assays (Access, ES-300, IMx, Magnum Opus, and Stratus) to accurately estimate the free thyroxine index (FTI) in euthyroid, hyperthyroid, and hypothyroid patients with abnormal concentrations of thyroid hormone-binding proteins, and in patients with nonthyroidal illness. For comparison, we calculated a similar FTI, using either T3-uptake or direct measurement of thyroxine-binding globulin (TBG). Euthyroid reference ranges were comparable. Of euthyroid patients with increased TBG, 12-32% and 5-20% had increased or suppressed FTI, respectively, depending on the T-uptake method used. Except for IMx, 6-35% of hypothyroid patients with increased TBG had inappropriately increased FTI. Patients with nonthyroidal illness had comparable results regardless of the method used, and T-uptake methods were variably affected by known inhibitors of thyroid hormone binding. The most reliable T-uptake method appeared to be the IMx, which, despite claims that it measures all thyroid hormone-binding proteins, correlated best with TBG concentrations.

Dependence of Free Thyroxine Estimates Obtained with Equilibrium Tracer Dialysis on the Concentration of Thyroxine-Binding Globulin

1992 ◽  
Vol 38 (7) ◽  
pp. 1294-1300 ◽  
Author(s):  
J C Nelson ◽  
R B Wilcox ◽  
M R Pandian
Keyword(s):  
Pregnant Women ◽  
Normal Control ◽  
Equilibrium Dialysis ◽  
Free Fraction ◽  
Free Thyroxine ◽  
Tracer Method ◽  
Free T4 ◽  
Nonthyroidal Illness ◽  
Thyroxine Binding Globulin ◽  
In Pregnancy

Abstract Some equilibrium dialysis determinations of free thyroxine (T4) vary directly with thyroxine-binding globulin (TBG) concentration. This apparent TBG dependence has been limited to methods involving radiolabeled T4 added to the dialysis system (tracer dialysis). In this study we compared tracer dialysis with direct dialysis for determining free T4 and obtained the following results (mean +/- SD) for patients with hypothyroxinemia of nonthyroidal illness (23.8 +/- 10.7 vs 24.2 +/- 10.9 pmol/L, P greater than 0.8), patients with congenital TBG deficiency (11.4 +/- 2.2 vs 16.2 +/- 7.1 pmol/L, P greater than 0.05), normal control subjects (32.7 +/- 6.5 vs 18.5 +/- 5.8 pmol/L, P less than 0.001), and pregnant women (31.2 +/- 8.7 vs 12.1 +/- 2.6 pmol/L, P less than 0.001). Direct dialysis determinations were independent of TBG and total T4. Tracer determinations were greater than direct determinations in normals, a discrepancy that increased in pregnancy. Tracer determinations correlated significantly with total T4 and TBG concentrations (P less than 0.001). TBG and total T4 dependence in the tracer method was attributable to small overestimations of the free fraction of T4. Similar overestimations multiplied by increasing total T4 concentrations resulted in greater errors. Relative to results for normal sera, the tracer method overestimated free T4 when total T4 was increased and underestimated free T4 when total T4 was decreased.

scholarly journals Studies on the Accuracy of a Quantitative Kit Radioimmunoassay for Free Thyroxine

1983 ◽  
Vol 20 (5) ◽  
pp. 280-284 ◽  
Author(s):  
R G Symons ◽  
T J Wilke ◽  
M L Wellby
Keyword(s):  
Protein Concentration ◽  
Serum Proteins ◽  
Quantitative Estimation ◽  
Equilibrium Dialysis ◽  
Free Thyroxine ◽  
Thyroid Function Tests ◽  
Serum Dilution ◽  
Clinical Symptomatology ◽  
Free Thyroxine Index ◽  
Serum Binding Protein

Experiments designed to determine the accuracy of the Corning Immophase Free T4 assay revealed that there was a marked perturbation of thyroxine binding to the serum proteins during the assay; binding of 125I-T4 to the immobilised T4 antibody in the absence of merthiolate increased considerably with buffer dilution of serum; and the free thyroxine concentration declined significantly during pregnancy. These changes in measured fT4 were not observed with an equilibrium dialysis-radioimmunoassay procedure. The assay was precise, easily performed, and as effective as the free thyroxine index (FTI) in diagnosing thyroid disease. We conclude that the assay does not provide an accurate quantitative estimation of serum fT4 concentration in samples with elevated TBG concentration and that the kinetic principles on which the assay is based are altered as serum binding-protein concentration is reduced (serum dilution). Both FTI and fT4 (Corning) data in pregnant patients should be interpreted with caution and with reference to clinical symptomatology and other thyroid function tests.

scholarly journals Clinical and Laboratory Evaluation of Four Methods of Assessing Free Thyroxine Status in Thyroid Clinic Patients

1980 ◽  
Vol 17 (6) ◽  
pp. 334-338 ◽  
Author(s):  
J A Fyffe ◽  
L Ayoub ◽  
H N Cohen ◽  
J G Turner ◽  
J A Thomson ◽  
...  
Keyword(s):  
Thyroid Function ◽  
Equilibrium Dialysis ◽  
Cost Benefit ◽  
Free Thyroxine ◽  
Laboratory Evaluation ◽  
Therapeutic Trial ◽  
Diagnostic Efficiency ◽  
Clinical Value ◽  
Free T4 ◽  
Free Thyroxine Index

The clinical value of four laboratory methods of assessing free thyroxine status was compared in 82 consecutive patients newly referred to a thyroid clinic with suspected thyroid dysfunction. The methods of determining free thyroxine used were: (1) free thyroxine index using a thyroid hormone uptake test (FTI (THUT)); (2) a free thyroxine index using thyroxine binding globulin (FTI (TBG)); (3) a kinetic radioimmunoassay (Immophase); and (4) an equilibrium dialysis method. The definitive thyroid status was evaluated by a combination of clinical assessment (including Wayne index), routine tests of thyroid function (total T4, T3, TSH, and TRH tests where appropriate), and by therapeutic trial in one case. The diagnostic efficiency of the tests was markedly dependent upon the method of determining the reference range for euthyroid patients. Best efficiency for each test was achieved using an amended range after excluding outliners. Test efficiency was then 97·6 % for FTI (THUT) and the kinetic RIA and 96·8% for FTI (TBG). Misclassification by one or more of these tests occurred in only four patients (mild hypothyroid, euthyroid on phenytoin, euthyroid on oral contraceptive and valium, T3 hyperthyroid). In contrast, free T4 by equilibrium dialysis was much less efficient (86·6%) and was technically the most complex. Overall the kinetic T4 RIA provided similar diagnostic information to the indirect indices. However, further studies of cost benefit in settings other than a thyroid clinic are required to assess whether this method might replace total T4 and/or FTI as a first-line test of thyroid function.

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