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.

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.

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.

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.

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.

SERUM PROTEIN-BOUND IODINE AND SERUM THYROXINE DURING PERPHENAZINE THERAPY

1967 ◽  
Vol 55 (1) ◽  
pp. 136-145 ◽  
Author(s):  
J. Mølholm Hansen ◽  
K. Siersbæk-Nielsen
Keyword(s):  
Serum Protein ◽  
Serum Proteins ◽  
Normal Serum ◽  
Thyroxine Binding Globulin ◽  
Serum Thyroxine ◽  
Iodine Compound

ABSTRACT Four out of 19 patients treated with perphenazine who were investigated in 1962, and 5 out of 31 patients who were investigated in 1966 had a raised PBI. The increase was most marked in the patients investigated in 1962. Measurements of dialysable thyroxine revealed that the increase was not due to an increase in thyroxine-binding globulin. All 50 patients had normal serum thyroxine as estimated by Murphy's method. Most probably the increase in PBI was due to the fact that the perphenazine tablets contain an iodine compound, which in some subjects may become bound to the serum proteins.

Effect of albumin concentration on the assay of serum free thyroxin by equilibrium radioimmunoassay with labeled thyroxin analog (Amerlex Free T4).

1983 ◽  
Vol 29 (2) ◽  
pp. 321-325 ◽  
Author(s):  
N Amino ◽  
K Nishi ◽  
K Nakatani ◽  
H Mizuta ◽  
K Ichihara ◽  
...  
Keyword(s):  
Serum Albumin ◽  
Pregnant Women ◽  
Binding Proteins ◽  
Solid Phase ◽  
Albumin Concentration ◽  
Serum Concentrations ◽  
Serum Samples ◽  
Free T4 ◽  
Serum Free

Abstract Serum free thyroxin (FT4) in normal nonpregnant and pregnant subjects was measured by radioimmunoassay (RIA) with Amerlex FT4 RIA (Amersham International) and LiquiSol FT4 RIA (Damon Diagnostics) kits. Amerlex FT4 values in serum from pregnant women were lower than those in serum from nonpregnant women, but LiquiSol FT4 values were similar in serum from both groups. Amerlex FT4 values were directly correlated with the concentrations of albumin in serum and inversely correlated with those of thyroxin-binding globulin, but not with prealbumin concentrations. No significant correlations were observed between LiquiSol FT4 values and serum concentrations of thyroxin-binding proteins. Amerlex FT4 values were normal in patients with excess, deficient, or decreased thyroxin-binding globulin. Albumin added to serum samples increased Amerlex FT4 values but not LiquiSol FT4 values. Albumin inhibited the binding of labeled thyroxin analog to the solid-phase thyroxin antibody. These data indicate that the albumin concentration influences FT4 values as measured by an RIA involving a thyroxin analog and that Amerlex FT4 values should be carefully interpreted when the patient has an abnormal concentration of serum albumin.

Radioimmunoassay for Serum Triiodothyronine: Evaluation of Simple Techniques to Control Interference from Binding Proteins

1974 ◽  
Vol 20 (9) ◽  
pp. 1150-1154 ◽  
Author(s):  
Victor S Fang ◽  
Samuel Refetoff
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Cross Reactivity ◽  
Heat Inactivation ◽  
Nonspecific Binding ◽  
High Concentration ◽  
Assay Sensitivity ◽  
Thyroxine Binding Globulin ◽  
Fixed Concentration

Abstract Simple techniques for controlling interference from binding proteins in serum, such as thyroxine-binding globulin, in radioimmunoassay for triiodothyronine (T3) have been evaluated for their efficacy, and their effect on assay sensitivity and on recovery of added T3. Ethanol precipitation of serum proteins decreased the assay sensitivity, nonspecific binding was increased, and recoveries of added T3 were inconsistent. Heat-inactivation of thyroxine-binding globulin or use of 8-anilino-1naphthalene sulfonic acid (ANS) to displace T3 from thyroxine-binding globulin produced comparable recovery rates. The heat-inactivation method slightly decreased the sensitivity of the assay and prolonged the procedure, whereas use of ANS is simple, and the assay sensitivity is maintained. When sera contain a high concentration of thyroxine-binding globulin, a fixed concentration of ANS (175 µg/100 µl of serum) might be too low to displace T3 from all its binding sites, but a concentration of ANS greater than 200 µg/100 µl of serum interferes with T3 quantitation by competitively binding to the antibodies. The cross-reactivity of thyroxine to T3-antibodies varies with the antiserum. Thyroxine-binding globulin appears to be the only protein in serum that competes with the antibody for T3 binding.

A Reassessment of 8-Anilino-1-Naphthalene Sulphonic Acid as a Thyroxine Binding Inhibitor in the Radioimmunoassay of Thyroxine

1977 ◽  
Vol 14 (1-6) ◽  
pp. 330-334 ◽  
Author(s):  
S. E. Evans ◽  
W. A. Burr ◽  
T. C. Hogan
Keyword(s):  
Polyethylene Glycol ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Sulphonic Acid ◽  
Thyroxine Binding Globulin ◽  
Double Antibody ◽  
Resin Separation

The effectiveness of 8-anilino-1-naphthalene sulphonic acid (ANS) in the radioimmunoassay (RIA) of thyroxine (T4) as an inhibitor of the binding of T4 to serum T4-binding proteins is assessed. The optimum ANS concentration is dependent upon the antiserum and the method used for separating free and bound T4. If T4 binding to serum proteins is not completely inhibited, resin separation methods may yield low values, while polyethylene glycol and double-antibody methods may produce high values for T4 concentration. Even with optimum ANS concentration gross errors in measurement of T4 may occur in patients with high thyroxine-binding globulin (TBG) concentrations.

rT3 metabolism in patients with nephrotic syndrome and normal GFR compared with normal subjects

1991 ◽  
Vol 260 (4) ◽  
pp. E641-E650 ◽  
Author(s):  
E. M. Kaptein ◽  
M. T. Hoopes ◽  
M. Parise ◽  
S. G. Massry
Keyword(s):  
Nephrotic Syndrome ◽  
Equilibrium Dialysis ◽  
Free Fraction ◽  
Thyroid Stimulating Hormone ◽  
Normal Serum ◽  
Production Rates ◽  
Free T4 ◽  
Hormone Production ◽  
Transfer Rates ◽  
Serum Free

To evaluate reverse 3,3',5'-triiodothyronine (rT3) metabolism in nephrotic syndrome, serum rT3 kinetic studies from 10 nephrotics (mean urinary protein losses 7.0 g/day) with normal glomerular filtration rates (GFR; creatinine clearance 107 ml/min) were compared with 9 normal healthy subjects. Serum disappearance data were analyzed in a three-pool model, including rapidly (liver and kidney) and slowly (muscle, skin, and brain) equilibrating pools exchanging with serum, with all losses from the rapidly equilibrating pool. Serum free thyroxine (T4), determined by equilibrium dialysis, and parathyroid hormone levels were unaltered; total T4, T3, and rT3, and free rT3, albumin, and transferrin levels were significantly decreased; and free fractions of T4 and rT3 and thyroid-stimulating hormone (TSH) levels were increased. Despite reduced rT3 binding in serum, fractional transfer rates from serum to extravascular sites and serum clearance rates of total rT3 were unaltered. Free hormone clearance, serum appearance, and maximum hormone production rates were decreased. Total hormone transfer rates between serum and tissue pools and rT3 mass in serum and both tissue pools were reduced. Binding in the slowly equilibrating pool was decreased, and binding in both rapidly and slowly equilibrating pools was correlated with the free fraction of rT3 (r = -0.79, P = 0.007, and r = -0.70, P less than 0.025, respectively), with a shift of rT3 from the slow to the rapid pool. These findings suggest that binding of rT3 and T4 to serum carrier proteins is reduced, the transfer process for rT3 from serum to extravascular sites is decreased by factors in addition to reduced serum binding, degradation of rT3 is impaired, and decreased slow-pool binding may reflect reduced rT3 binding to serum-derived proteins in interstitial fluid. Furthermore, rT3 production rates are reduced, despite normal serum free T4 levels, accounting for low serum free rT3 concentrations. Total rT3 levels are decreased because of decrements in both serum binding and production rates.

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.

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