Unbound T4 Research Articles

ВЛИЯНИЕ НАРУШЕННЫХ УСЛОВИЙ РЕПАРАТИВНОЙ РЕГЕНЕРАЦИИ КОСТНОЙ ТКАНИ НА ДИНАМИКУ ГОРМОНОВ ЩИТОВИДНОЙ ЖЕЛЕЗЫ

Managing bone tissue regeneration is a key problem in traumatology and orthopedics. The researches proved that thyroid hormones affect hemostasis and reparative regeneration of bone tissue favorably and promote osteogenesis and bone consolidation.The aim of this research is to study the influence of disturbed reparative regeneration on the concentration of thyroid hormones in blood in the dynamics. Experimental research on rabbits (n=12) with modelling of disturbed reparative regeneration revealed disorder in thyroid hormones synthesis and different directions of T3 and unbound T3 production on the 35th day when the maxi-mum decrease of T3 (2times) and unbound T3 (1.5times) compared to normal values was registered. The analysis of production of T4 and unbound T4 revealed suppression of the values from the 1st to 50th day and gradual increase in unbound T4 from the 50th day. The most significant manifestation of thyroid gland reaction on the disturbed reparative regeneration of shin bones was the decrease in T3 and peripheral conversion of thyroid hormones up to 50th day with formation of false joint in these terms.

Analytical Application of Flow Immunosensor in Detection of Thyroxine and Triiodothyronine in Serum.

In this study, an immunosensor based on kinetic exclusion analysis (KinExA) was used for thyroxine (T4) and triiodothyronine (T3) estimation. A KinExA™ 3200 instrument was used for this analysis, which is an automated flow fluorimeter designed to separate free unbound antibody binding sites in reaction mixtures of antibody, antigen, and antibody-antigen complex. A T3-BSA- and T4-BSA-coated polymethyl methacrylate (PMMA) bead microcolumn is generated inside the flow cell of the instrument. A sample mixture containing T3 and T4 with their respective monoclonal antibodies and their complexes are drawn past the microbead column. The unbound T3 or T4 monoclonal antibody binding sites are captured by their respective T3 and T4 antigens coated on the PMMA beads as bovine serum albumin conjugates. Fluorescently labeled secondary antibodies bind to the T3 or T4 antigen-antibody complex to generate fluorescence intensity for analysis. The limit of detection for the T3 and T4 assays was found to be 0.06 and 1.9 ng mL-1 with acceptable precision values. The convenience of the automated KinExA format may be valuable in medical diagnostic laboratories.

Early Life Exposure To Phthalate Diesters: Associations With Thyroid Function And Cognitive Function

Background: Epidemiological studies concerning the relations of exposure to phthalates, thyroid function and cognitive function among children could be limited. Objectives: We conducted the longitudinal study to examine the associations of prenatal and postnatal exposure to phthalates, thyroid function and cognitive function in children 2-9 years old. Methods: We recruited 430 pregnant women in their trimester in Taichung, Taiwan from 2001-2002. A total of 110, 79, and 76 children were followed up at ages 2, 5, and 8, respectively. The serum levels of thyroxine (T4), unbound T4 (FT4), triiodothyronine (T3), thyroid-stimulating hormone (TSH), and thyroxine-binding globulin (TBG) in children were measured at three different time points using radioimmunoassay. We also evaluated the children's cognitive function at three followed-up visits using the Bayley and Wechsler tests for assessing neurocognitive function and intelligence (IQ). Urine samples from mothers during pregnancy and from children at each follow-ep visit were analyzed for seven metabolite concentrations of widely used phthalate esters (such as MMP, MEP, MBP, MBzP, MEHP, MEHHP, and MEOHP). We constructed a linear mixed model to examine these associations after adjusted for covariates. Results: We found significant inverse associations between the children's levels of urinary MEHHP, MEOHP, and the sum of the three metabolites of di(2-ethylhexyl) and their serum levels of FT4 (i.e. Beta= -0.04, 95% CI: -0.07, -0.01 for MEOHP). Urinary MMP, MEP, MBP, MBzP, MEHP, and MEOHP levels in children were significantly negatively related to IQ scores (i.e. Beta= -1.85, 95% CI: -3.40, -0.30 for MEOHP). We found a marginally significant positive relationship between the children's serum levels of FT4 and their IQ scores (Beta=5.81, 95% CI: -2.74, 14.36 for MEOHP). Conclusions: Early life exposure to phthalate diesters could be associated with decreasing thyroid function and cognitive development in the young children.

A patient-specific model of the negative-feedback control of the hypothalamus-pituitary-thyroid (HPT) axis in autoimmune (Hashimoto's) thyroiditis

The purpose of modelling the negative-feedback control mechanism of the hypothalamus-pituitary-thyroid (HPT) axis in autoimmune (Hashimoto's) thyroiditis is to describe the clinical course of euthyroidism, subclinical hypothyroidism and overt hypothyroidism for patients. Thyroid hormone thyroxine (T4) and triiodothyronine (T3) levels are controlled by negative-feedback control through thyroid-stimulating hormone (TSH). T4, like other hormones, can be bound or unbound; the unbound T4 (FT4) is used as a marker for hypothyroidism. Autoimmune thyroiditis is a disease in which the thyroid-infiltrating lymphocytes attack autoantigens in follicle cells, destroying them over a long time. To describe the operation of the feedback control, we developed a mathematical model involving four clinical variables: TSH, FT4, anti-thyroid peroxidase antibodies and the thyroid gland's functional size. The first three variables are regularly measured while the last variable is determined through relationships between the other three variables. The problem of two different time scales for circulating hormones and thyroid damage is addressed using singular perturbation theory. Analysis of the mathematical model establishes stability and conditions under which the diseased state can maintain the slow movement toward diseased state equilibrium. Although we have used four variables in modelling the feedback control through the HPT axis, the predicted clinical course given any set of parameters is shown to depend on the steady-state levels of TSH and FT4. This observation makes possible the development of the clinical charts based only on the levels of TSH, time and potential steady-state values. To validate the model predictions, a dataset obtained from a Sicilian adult population has been employed.

Estimation of Serum Ferritin Level in Female Patients with Telogen Effluvium

Background: Telogen Effluvium (TE) is the most common cause of diffuse hair loss in adult females. TE may be a squeal of various metabolic alterations such as pregnancy, malnutrition especially iron deficiency anemia in premenopausal women. Hemoglobin concentration can be used to screen for iron deficiency, whereas serum ferritin concentration can be used to confirm iron deficiency. In this study we tried to put a light spot on the role of serum ferritin level in female patients with TE. Methods: A case control study was done including 100 female patients less than 40 years (their age ranged from 18 to 40 years) divided into 2 groups. The patient group containing 80 female patients with telogen effluvium the control group containing 20 normal females without hair loss. A full history was done with full clinical examination. Blood samples were collected from all studied groups and examined for: serum ferritin level (by Ferritin ELISA -EIA- 1872 test kits), Unbound T4 (by FT4 RIA KITS) and Complete blood counts (Haemoglobin level, RBCs, HCT values, MCV, MCH, MCHC). Results: The results of this study showed normal serum ferritin level in female patients suffering from telogen effluvium as well as normal serum Hg level, HCT value, RBCs count and Blood Indices. Insignificant relation was found between control and study groups using SPSS statistical data analysis. Conclusion: There was no closely linked relationship between iron metabolism and TE.

Sequential Evaluations of Trace Elements in Patients Receiving Parenteral Nutrition

To evaluate alterations of serum trace element (TE) levels in critically ill patients receiving parenteral nutrition (PN) including the recommended doses of TEs. Measurements of serum levels of TEs such as zinc, iron, copper, manganese and iodine were performed on the day before, and two and four weeks after, administration of PN. Forty-six patients who received PN from October 2007 to August 2009, were enrolled. All patients received 100µmol (6.5mg) zinc, 35µmol (2mg) iron, 5µmol (0.3mg) copper, 1µmol (0.05mg) manganese and 1µmol (0.13mg) iodine as the recommended daily doses. There were no significant differences in the serum levels of iron, copper and manganese among the three measurement points. On the other hand, there were significant differences in the serum levels of zinc (p<0.001), triiodothyronine (T3) (p=0.010) and unbound T3 (p=0.006) among the three points, but not for thyroxine (T4) and unbound T4. The recommended doses of TEs are justified for critically ill patients in terms of constant serum levels, except for zinc and iodine.

Familial Dysalbuminemic Hyperthyroxinemia: A Persistent Diagnostic Challenge

Familial dysalbuminemic hyperthyroxinemia (FDH)1 is a well-characterized condition associated with increased circulating total thyroxine (T4) concentrations and normal physiological thyroid function. It is caused by mutations in the ALB (albumin) gene that increase the affinity of albumin for T4 by approximately 60-fold. When measured by a technique that minimally disturbs the equilibria between T4 and its serum binding proteins, such as equilibrium or symmetric dialysis (SyD) performed in a near-physiological medium, the free T4 (FT4) value is characteristically within the reference interval. Assays that rely on the competition of a T4 analog with unbound T4 in the sample can give spuriously high results in FDH patients, because albumin binding of the T4 analog is enhanced by the FDH mutation. “Two step” methods, in which the T4 analog never comes into contact with serum albumin owing to a wash step immediately after capture, avoid this problem. Such assay methods are expected to give FT4 results within the reference interval in FDH patients, but this expectation has been questioned (1)(2). Thyroid-function tests, including 1- and 2-step methodologies, were examined in 4 affected individuals from different families who had their FDH diagnoses proved genetically …

Simultaneous determination of unbound thyroid hormones in human plasma using high performance frontal analysis with electrochemical detection

A direct injection HPLC method in combination with high-performance frontal analysis (HPFA) and electrochemical detection (ECD) was developed for the simultaneous and sensitive determination of unbound thyroid hormones (thyroxine, triiodothyronine, and reverse triiodothyronine) in human plasma. The present on-line HPLC/HPFA system consists of an HPFA column, an extraction column and an analytical HPLC column connected through a column-switching device, and the eluent from the analytical column was monitored by ECD. The calibration lines showed good linearity (rsq. > 0.999) within 7.4–148.2 pM for T4 and 1.5–74.1 pM for T3 and rT3. Unbound T4 and T3 concentrations determined by the present system were 16.4 ± 2.4 pM ( n = 15) and 7.14 ± 1.04 pM ( n = 15), which were in agreement with those determined by the EIA method. The unbound rT3 concentration was 2.30 ± 0.27 pM ( n = 15). The CV% values of intra-day and inter-day assays ( n = 15) were less than 14.9% for T4, 14.5% for T3 and 13.2% for rT3. The present system was also applied to a competitive binding study of these thyroid hormones in human plasma.

Thyroid hormones and their effects: a new perspective

ABSTRACTThe thyroid hormones are very hydrophobic and those that exhibit biological activity are 3′,5′,3,5‐Ltetraiodothyronine (T4), 3′,5,3‐L‐triiodothyronine (T3), 3′,5′,3‐L‐triiodothyronine (rT3) and 3,5,‐Ldiiodothyronine (3,5‐T2). At physiological pH, dissociation of the phenolic ‐OH group of these iodothyronines is an important determinant of their physical chemistry that impacts on their biological effects. When non‐ionized these iodothyronines are strongly amphipathic. It is proposed that iodothyronines are normal constituents of biological membranes in vertebrates. In plasma of adult vertebrates, unbound T4 and T3 are regulated in the picomolar range whilst protein‐bound T4 and T3 are maintained in the nanomolar range. The function of thyroid‐hormone‐binding plasma proteins is to ensure an even distribution throughout the body. Various iodothyronines are produced by three types of membrane‐bound cellular deiodinase enzyme systems in vertebrates. The distribution of deiodinases varies between tissues and each has a distinct developmental profile. Thyroid hormones have many effects in vertebrates. It is proposed that there are several modes of action of these hormones.(1) The nuclear receptor mode is especially important in the thyroid hormone axis that controls plasma and cellular levels of these hormones.(2) These hormones are strongly associated with membranes in tissues and normally rigidify these membranes.(3) They also affect the acyl composition of membrane bilayers and it is suggested that this is due to the cells responding to thyroid‐hormone‐induced membrane rigidification. Both their immediate effects on the physical state of membranes and the consequent changes in membrane composition result in several other thyroid hormone effects. Effects on metabolism may be due primarily to membrane acyl changes. There are other actions of thyroid hormones involving membrane receptors and influences on cellular interactions with the extracellular matrix. The effects of thyroid hormones are reviewed and appear to be combinations of these various modes of action. During development, vertebrates show a surge in T4 and other thyroid hormones, as well as distinctive profiles in the appearance of the deiodinase enzymes and nuclear receptors. Evidence from the use of analogues supports multiple modes of action. Re‐examination of data from the early 1960s supports a membrane action. Findings from receptor ‘knockout’ mice supports an important role for receptors in the development of the thyroid axis. These iodothyronines may be better thought of as ‘vitamone’ ‐like molecules than traditional hormonal messengers.

Thyroid hormones and their effects: a new perspective.

The thyroid hormones are very hydrophobic and those that exhibit biological activity are 3',5',3,5-L-tetraiodothyronine (T4), 3',5,3-L-triiodothyronine (T3), 3',5',3-L-triiodothyronine (rT3) and 3,5',-L-diiothyronine (3,5-T2). At physiological pH, dissociation of the phenolic -OH group of these iodothyronines is an important determinant of their physical chemistry that impacts on their biological effects. When non-ionized these iodothyronines are strongly amphipathic. It is proposed that iodothyronines are normal constituents of biological membranes in vertebrates. In plasma of adult vertebrates, unbound T4 and T3 are regulated in the picomolar range whilst protein-bound T4 and T3 are maintained in the nanomolar range. The function of thyroid-hormone-binding plasma proteins is to ensure an even distrubtion throughout the body. Various iodothyronines are produced by three types of membrane-bound cellular deiodinase enzyme systems in vertebrates. The distribution of deiodinases varies between tissues and each has a distinct developmental profile. Thyroid hormones. (1) the nuclear receptor mode is especially important in the thyroid hormone axis that controls plasma and cellular levels of these hormones. (2) These hormones are strongly associated with membranes in tissues and normally rigidify these membranes. (3) They also affect the acyl composition of membrane bilayers and it is suggested that this is due to the cells responding to thyroid-hormone-induced membrane rigidificataion. Both their immediate effects on the physical state of membranes and the consequent changes in membrane composition result in several other thyroid hormone effects. Effects on metabolism may be due primarily to membrane acyl changes. There are other actions of thyroid hormones involving membrane receptors and influences on cellular interactions with the extracellulara matrix. The effects of thyroid hormones are reviewed and appear to b combinations of these various modes of action. During development, vertebrates show a surge in T4 and other thyroid hormones, as well as distinctive profiles in the appearance of the deiodinase enzymes and nuclear receptors. Evidence from the use of analogues supports multiple modes of action. Re-examination of data from th early 1960s supports a membrane action. Findings from receptor 'knockout' mice supports an important role for receptors in the development of the thyroid axis. These iodothyronines may be better thought of as 'vitamone'-like molecules than traditional hormonal messengers.

Hepatic uptake of 3,5,3'-triiodothyronine: electrochemical driving forces.

We used the multiple indicator dilution technique to assess the electrochemical forces driving uptake of 3,5,3'-triiodo-L-thyronine (T3) across the basolateral plasma membrane in the single-pass perfused rat liver. With the use of 4 g/dl albumin solutions, the influx and efflux clearances were 0.020 +/- 0.005 and 0.0049 +/- 0.0017 (SE) ml.s-1.g liver-1, respectively, indicating that the total T3 concentration at equilibrium should be about four times greater in cytoplasm than in plasma. However, when the influx and efflux clearances were divided by the unbound (free) T3 concentration in the perfusate and cytosol, they were not different (3.76 +/- 0.26 vs. 4.30 +/- 0.38 ml.s-1.g liver-1), indicating that the uptake process does not generate a gradient of unbound T3 across the plasma membrane. To further test whether T3 uptake is driven by the electrical potential difference across the plasma membrane, liver cells were depolarized by isosmotic replacement of perfusate chloride with gluconate. There was no effect on uptake or efflux. To test whether uptake is coupled to influx of sodium, perfusate sodium was replaced with choline. Although there was a modest decline in both the influx and efflux clearances, there was no change in their ratio, as would be expected for sodium-coupled active transport. These results indicate that uptake of T3 across the basolateral hepatocyte membrane occurs by passive diffusion. We found no evidence to support concentrative, active transport by either electrogenic or sodium-coupled mechanisms.

Triiodothyronine Uptake Measurement in Serum by Time-Resolved Fluorescence Immunofluorometry

A solid phase competition-type fluoroimmunoassay for triiodothyronine (T3) uptake in serum is described. In the assay, exogenous free T3 binds to the unoccupied binding sites on serum thyroxine binding proteins while the remaining unbound T3 competes with immobilized T3 for binding to a soluble biotinylated anti-T3 monoclonal antibody. The bound biotinylated antibody is quantitated by the addition of streptavidin labeled with the europium chelator 4,7-bis(chlorosulfophenyl-1,10 phenanthroline-2,9-bicarboxylic acid (BCPDA) in the presence of excess europium. The fluorescence signal of the final complex, which is directly proportional to the number of unoccupied binding sites on thyroxine binding proteins, is then measured on the dried solid-phase with a pulsed-laser time-resolved fluorometer. The assay requires a 10 microliters serum sample and a total incubation time of 90 minutes. The coefficients of variation for within-run and between-run assays ranged from 2.0 to 5.7%. Results obtained by the present method compared well with those determined by two commercial radioimmunoassays (r greater than 0.9).

Hereditary and environmental influences on the variation of thyroid hormones in normal male twins.

Heritability of the variation of the plasma total and unbound T4 (free T4), T3, T4-binding globulin (TBG), and TSH concentrations was investigated in 15 monozygotic and 15 dizygotic male twin pairs. The variability in plasma of total (58%) and free T4 (72%) concentrations was significantly less (P less than 0.01) in the twin pairs than in unrelated men. Half of the variability of T3 (P less than 0.05), TBG (P less than 0.05), and TSH (P less than 0.05) was affected by influences shared by the twin pairs in both monozygotic and dizygotic twin pairs. The heritability index for variability of the plasma total T4 and free T4 was greater than 28% (P less than 0.05), and it was 25% or less for T3 and TBG. Variation in TBG accounted for less than 20% (P less than 0.001) of the variation in thyroid hormone concentrations. The results indicate that familial factors, which are affected by genetic and/or environmental factors, influence the variation of plasma TBG, TSH, T4, free T4, and T3 concentrations among normal men. Genetic factors influenced the variation in plasma T4 and free T4 levels.

70 Familial Congenital Hypothroildism Secondary to Transplacental Thyroid Inhibitory Autoantibodies

Each of three children in a family was found to be hypothyroid at birth as part of the neonatal hypothyroid screening program. Their mother had developed Graves disease at age 8 years but became hypothyroid at age 25 - 3 years before the 1st child. Serum obtained 6 years after the 1st child was born, inhibited TSH binding to human thyroid cell membranes in vitro. At birth each child was found to have increased TSH concentration. In retrospect thyroid microsomal autoantibodies were present at 1:6400 at 2 months of age in first boy and at 1:6400 at 3 days of age in third child. The oldest two children have been followed 3 months off thyroxine therapy and continue to have normal thyroid function (T4, unbound T4, T3 and TSH). In patients with congenital hypothyroidism in whom the mother has autoimmune thyroiditis or there is a family history of thyroiditis, the possibility of a transient condition secondary to transplacental immunoglobulins should be sought.

Circulating antitriiodothyronine autoantibodies in two euthyroid patients: apparent lack of interference in total T3 radioimmunoassay based on second antibody or solid phase separation techniques

Two clinically euthyroid patients were noted to have low total T3 levels as assessed by RIA using either dextran-charcoal (DC) or polyethylene glycol (PEG) for separation of bound from unbound T3, in spite of normal free T3, total and free T4 and basal and TRH-stimulated TSH concentrations. The presence of circulating substances binding T3 was suggested by high nonspecific binding in total T3 RIA system using either DC or PEG separation. The presence of anti-T3 autoantibodies was then suspected and confirmed by the presence of [125]-T3 bound to patients' gammaglobulins, precipitated with rabbit anti-human immunoglobulins. Serum T3 concentration determined by extracting T3 from patients' sera with methanol was 166 and 226 ng/dl. Similar or even lower values were unexpectedly obtained in RIA systems with solid phase or second antibody (anti-rabbit) separation and with competitive protein binding assay. To face this paradoxical finding, simulated experiments were carried out by incubating T3- and T4-free sera added with various amounts of stable T3 and T4 in the presence of goat anti-T3 or anti-T4 serum. These samples were then radioimmunoassayed. The DC separation caused a consistent underestimation of the actual T3 and T4 concentration. The second antibody separation caused a T3 and T4 overestimation for actual levels below 200 ng/dl and 10 micrograms/dl, respectively, while at the higher T3 or T4 concentrations, an overlap or, even, an underestimation of actual T3 or T4 levels were found. These data provide evidence that, with second antibody or solid phase separation methods, there could be an apparent lack of interfering effect of endogenously occurring antibodies.

Production and Metabolism of Thyroid Hormones in Protein-Deficient and Food-Restricted Pregnant Rats

Thyroxine (T4) and triiodothyronine (T3) concentrations in the plasma and their metabolic clearance and production rates were studied in pregnant protein-deficient and food-restricted rats on the 20th day of gestation. Total T3 levels in the plasma were significantly reduced in malnourished dams, with those of food-restricted dams being consistently lower than those of protein-deficient dams. There were no changes in plasma total T4 concentrations as a result of dietary treatment. Unbound T4 and T3 in the plasma were significantly reduced in both protein-deficient and food-restricted dams. Maternal protein deficiency significantly lowered fetal T4, whereras food restriction caused an elevation. Metabolic clearance rates of T3 and T4 and production rate of T4 were unaffected by diet, while the production rate of T3 was significantly increased in protein deficient dams. Thyroxine to triiodothyronine ratios in malnourished dams were elevated suggesting depressed peripheral conversion of T4 to T3. Alternative factors which may lead to these effects are discussed.

Effect of ionic strength and ionic composition of assay buffers on the interaction of thyroxine with plasma proteins.

When plasma proteins are diluted with buffer the ionic strength and ionic composition of that buffer affects the interactions between thyroxine (T4) and its plasma protein-binding sites. Increases in phosphate, chloride or barbiturate ion concentration from 50 to 200 mmol/l caused a significant decrease in the affinity of plasma proteins for T4, and a concurrent increase in the concentration of unbound T4. These results cannot be completely accounted for by changes in ionic strength since at the same ionic strength different anions caused quantitatively different effects on unbound T4 concentration. The degree of depression of T4 binding by the three anions studied was in the order barbiturate greater than chloride greater than phosphate. The results of a systematic study on the composition of diluent buffer systems indicated that when a 50 mM-sodium phosphate-100 mM-NaCl buffer (pH 7-4) was used as a plasma diluent, there were unlikely to be gross changes in the T4-binding properties of plasma proteins with dilution.

Rapid Radioimmunoassay of Triiodothyronine

Abstract The method used in this report is based on the immunological production of a specific antisera to triiodothyronine (T3) in rabbits. Conducted at 4 °C, the radioimmunoassay requires three to five days to complete. Hoping to speed this assay, we investigated the influence of time, temperature, and pH. Incubation at 37 °C and pH 8.0 for 30 min did not deleteriously affect either precision, specificity, reproducibility, or accuracy. Bound and unbound T3 were separated in 10 min by adding polyethylene glycol ("Carbowax 6000") at 0-4 °C. T3 was prevented from binding to thyroxine-binding globulin by the addition of 8-anilino-1-naphthalene sulfonic acid. To minimize the differences in protein concentration between standards and unknowns, we added T3-free serum to all standards. Thus, a simplified and convenient method is described for measurement of T3 that is suitable for clinical use because it is simpler and faster than previously reported methods.