T4 Assay Research Articles

Thyroid function in pregnancy

Advances in understanding the physiology of thyroid function in normal pregnancy have highlighted the importance of the consequences of abnormal function on obstetric outcome and foetal well-being. Pubmed search was done using the terms thyroid and pregnancy. Areas of agreement are the following: gestational normative reference ranges for thyroid function tests are required for proper interpretation of any abnormalities. Measurement of thyroid-stimulating antibodies and antithyroid peroxidase antibodies is useful for diagnosis of thyroid disease in pregnancy. Treatment of Graves' hyperthyroidism should be done with propylthiouracil for first trimester only, then carbimazole or methimazole. Patients on levothyroxine require an increase in dosage during gestation. Areas of controversy are the following: total thyroxine (TT4) versus Free T4 (FT4) assays in pregnancy. Screening for thyroid function in early gestation: should it be routinely performed on everyone? What tests are appropriate? Growing points are the following: physiology of thyroxine delivery to the foetus. Establishment of gestational thyroid hormone reference ranges. Evaluation of strategies to screen thyroid function in early pregnancy. Areas timely for developing research are the following: placental thyroid hormone physiology, thyroid hormone therapy and screening thyroid function.

Micro Open-Sandwich ELISA to Rapidly Evaluate Thyroid Hormone Concentration from Serum Samples

Thyroxine (T4) is the most commonly measured thyroid hormone for the diagnosis of thyroid function. To elucidate a rapid and sensitive assay for T4, we made a microfluidics-based noncompetitive immunodetection chip system using anti-T4 antibody fragments obtained from a phage display library. Based on the open-sandwich ELISA principle that detects antigen-dependency of the interaction between the two antibody variable regions V(H) and V(L), we could detect less than 1 ng/ml of T4. The assay was also successfully applied to evaluate total T4 concentration in the serum of healthy individuals. This would be the first micro open-sandwich ELISA constructed with antibody fragments directly selected from immunized mice. The system will be applied to the sensitive detection of many diagnostic markers.

Measurement of thyroid hormones in donkey (Equus asinus) blood and milk: validation of ELISA kits and evaluation of sample collection, handling and storage

Donkey's milk is well tolerated by human infants with cow's milk allergy and is useful in the treatment of human immune-related diseases and in the prevention of atherosclerosis. Thyroid hormones (TH) stimulate lactation and active triiodothyronine (T3) in colostrum and milk could take paracrine action supporting lactogenesis in the mother, and play physiological roles for the suckling offspring (systemic or within the gastrointestinal tract). The aims were to measure TH concentrations in donkey blood and milk, validate ELISA methods, evaluate the effects of sample collection and post-collection handling and the stability of TH in milk and blood serum and plasma samples. In milk and blood samples obtained from lactating jennies total concentrations of TH were assayed using competitive-type ELISA kits. Good validation results were obtained for both TH concentrations in blood serum and plasma and T3 in milk samples extracted with cold (-20°C) ethanol alkalinized (pH 9·0) with NH4OH. In most of the milk extract samples, thyroxine (T4) concentrations resulted below the sensitivity threshold. Intra- and inter-assay coefficients of variations of TH concentrations in different blood and milk samples were below 10%. Parallelism tests gave displacement lines parallel to those of the calibrators for both TH in blood serum and plasma and for T3 in milk extracts. Mean recovery rates were between 95% and 123%, but the concentration values approaching the highest calibrators were overestimated. Therefore, serum and plasma samples for T3 assay must be previously diluted with buffer. Both TH concentrations in blood serum and plasma and T3 in milk did not change during storage for up to 6 months at -20°C. In conclusion, the ELISA methods tested in the present study are suitable for determination of both TH concentrations in donkey blood samples, and for T3 measurement in milk, after extraction with cold alkaline ethanol.

Effect of Laboratory Practices on the Incidence Rate of Congenital Hypothyroidism

Newborn screening (NBS) laboratories in the United States expanded their programs to include primary congenital hypothyroidism (CH) in the 1970s. An increase in the national CH-incidence rate since 1987 has been reported. Our goal was to analyze national data reported by state NBS programs and laboratories from 1991 to 2000 to determine the extent to which changing laboratory methods might have contributed to the reported rise in CH-incidence rate. We used generalized estimating equations to analyze the association between the rate of confirmed cases of CH per 100000 live births and the initial screening method (thyroxine [T4] or thyrotropin [TSH] assay), the T4- and TSH-assay methods, the screening-test cutoff value used to report abnormal T4- or thyrotropin-assay results, and the performance of a second screen on >or=80% of newborns in the state. We then evaluated the association of CH rate with year after adjusting for any screening methodology or parameter that was significant in the univariate analysis. During 1991-2000, laboratories that used a TSH assay for initial screening reported a 24% higher incidence rate of CH than those that used a T4 assay. The assay type also affected the incidence rate. Screening for T4 by enzyme immunometric assay (EIA) or fluoroimmunoassay (FIA) methods resulted in 38% and 24% higher incidence rates of CH, respectively, compared with the radioimmunoassay (RIA) method, whereas screening for TSH by the FIA method resulted in a 20% higher incidence rate of CH than did screening with radiochemical methods. During the decade studied, many laboratories changed their T4-assay method from RIA to either FIA or EIA; this particular change seemed to have the greatest impact on the CH-incidence rate. Although the use of different laboratory methods and screening practices by NBS laboratories affected the incidence rate of CH, after adjusting for screening methodologies and parameters, an increasing incidence rate still persisted during the decade studied. Thus, there seem to be additional unknown factors that contributed to the reported increase in incidence rate.

Non-invasive measurement of thyroid hormone in feces of a diverse array of avian and mammalian species

We developed and validated a non-invasive thyroid hormone measure in feces of a diverse array of birds and mammals. An I 131 radiolabel ingestion study in domestic dogs coupled with High Pressure Liquid Chromatography (HPLC) analysis, showed that peak excretion in feces occurred at 24–48 h post-ingestion, with I 131-labelled thyroid hormone metabolites excreted primarily as triiodothyronine (T3) and relatively little thyroxine (T4), at all excretion times examined. The immunoreactive T3 profile across these same HPLC fractions closely corresponded with the I 131 radioactive profile. By contrast, the T4 immunoreactive profile was disproportionately high, suggesting that T4 excretion included a high percentage of T4 stores. We optimized and validated T3 and T4 extraction and assay methods in feces of wild northern spotted owls, African elephants, howler monkeys, caribou, moose, wolf, maned wolf, killer whales and Steller sea lions. We explained 99% of the variance in high and low T3 concentrations derived from species-specific sample pools, after controlling for species and the various extraction methods tested. Fecal T3 reflected nutritional deficits in two male and three female howler monkeys held in captivity for translocation from a highly degraded habitat. Results suggest that thyroid hormone can be accurately and reliably measured in feces, providing important indices for environmental physiology across a diverse array of birds and mammals.

Autoantibodies against thyroid hormones and their influence on thyroxine determination with chemiluminescence immunoassay in dogs

Autoantibodies against thyroxin (T4AA) and triiodothyronine (T3AA) are present in dogs with autoimmune thyroiditis and have been reported to interfere with immunoassays. The objectives of this study were to determine the frequency of autoantibodies and to determine whether interference occurs by T4AA, using a non-immunological method (high performance liquid chromatography, HPLC) for thyroxin (T4) measurement. Based on clinical symptoms, T4 and thyroid stimulating hormone (TSH) concentration, 1,339 dogs were divided into six groups: Group 1: hypothyroid (n = 149); Group 2: subclinical thyroiditis (n = 110); Group 3: suspicious for non thyroidal illness (n = 691); Group 4: biochemical euthyroid (n = 138); Group 5: hypothyroid dogs under substitution therapy (n = 141); Group 6: healthy dogs (n = 110). The incidence of T4AA and T3AA, determined using radiometric assay, was low (0.5% and 3.8%) and higher in hypothyroid dogs compared to dogs suspicious for hypothyroidism (Group 2-4) (p<0.05). T4AA was not detected in dogs with normal T4 and elevated TSH. T4 concentrations of T4AA positive samples determined using HPLC were comparable to results obtained by chemiluminescence immunoassay. These findings indicate that the probability of interference of T4AA leading to falsely elevated T4 concentration in the T4 assay seems to be low.

The Blood-Brain Barrier Thyroxine Transporter Organic Anion-Transporting Polypeptide 1c1 Displays Atypical Transport Kinetics

Organic anion-transporting polypeptide (Oatp) 1c1 is a high-affinity T(4) transporter expressed in brain barrier cells. Oatp1c1 transports a variety of additional ligands including the conjugated sterol estradiol 17beta-glucuronide (E(2)17betaG). Intriguingly, published data suggest that E(2)17betaG inhibition of Oatp1c1-mediated T(4) transport exhibits characteristics suggestive of atypical transport kinetics. To determine whether Oatp1c1 exhibits atypical transport kinetics, we first performed detailed T(4) and E(2)17betaG uptake assays using Oatp1c1 stably transfected HEK293 cells and a wide range of T(4) and E(2)17betaG concentrations (100 pm to 300 nm and 27 nm to 200 mum, respectively). Eadie-Hofstee plots derived from these detailed T(4) and E(2)17betaG uptake experiments display a biphasic profile consistent with atypical transport kinetics. These data along with T(4) and E(2)17betaG cis-inhibition dose-response measurements revealed shared high- and low-affinity Oatp1c1 binding sites for T(4) and E(2)17betaG. T(4) and E(2)17betaG recognized these Oatp1c1 binding sites with opposite preferences. In addition, sterols glucuronidated in the 17 or 21 position, exhibited preferential substrate-dependent inhibition of Oatp1c1 transport, inhibiting Oatp1c1-mediated E(2)17betaG transport more strongly than T(4) transport. Together these data reveal that Oatp1c1-dependent substrate transport is a complex process involving substrate interaction with multiple binding sites and competition for binding with a variety of other substrates. A thorough understanding of atypical Oatp1c1 transport processes and substrate-dependent inhibition will allow better prediction of endo- and xenobiotic interactions with the Oatp transporter.

Developmental Perchlorate Exposure: Gilbert Responds

Developmental Perchlorate Exposure: Gilbert Responds

Effects of chronic estradiol treatment on the thyroid gland structure and function of ovariectomized rats

BackgroundEstrogen therapy is widely used nowadays in women to treat many postmenopausal symptoms but it may have some undesirable effects due to multiple organs affection. So, the aim of this study was to determine the effects of chronic estradiol treatment on the structure and function of the thyroid gland in ovarictomized rats as a model simulating menopause.FindingsThirty adult female Wistar rats divided into three groups were used in this study; the first group was sham-operated, while the second and third groups were ovariectomized. The first and second groups were injected with olive oil while the third group was injected with estradiol dipropionate daily for three months, after that; hormonal assay for T3, T4, TSH and specimens of the thyroid were taken and processed to be examined by light and electron microscopy. The results of this study revealed that serum levels of T3 and T4 decreased in ovariectomized animals and significantly increased after estradiol treatment, while TSH increased in ovariectomized animals and decreased with estradiol treatment. Histological and morphometric study in ovariectomized group revealed marked accumulation of colloid in follicular lumens with decreased epithelial height in addition to increased connective tissue amount. After estradiol treatment the follicles became smaller in size, having small amount of colloid with increased epithelial height in addition to decreased connective tissue content. Ultrastructural study supported these results in addition to the presence of large amount of intracytoplasmic colloid vesicles after estradiol treatment.ConclusionLow estrogen level may lead to mild thyroidal hypofunction while estradiol treatment may lead to hyperactivity so it should be used very cautiously in the treatment of postmenopausal symptoms to avoid its undesirable stimulatory effect on the thyroid.

Interference in free thyroxine immunoassay

We report two patients who presented with non-specific symptoms of tiredness that prompted their clinician to order a thyroid function test. In both cases, free thyroxine (T4) was reported to be high, with a normal thyroid-stimulating hormone (TSH). Repeated blood tests from the same laboratory using the same assay method yielded similar results. Due to discrepancies between the clinical and laboratory findings, we decided to repeat the blood tests at another laboratory using a different assay method. This revealed normal free T4 and TSH levels, suggesting that heterophile antibodies were interfering with the free T4 assay, leading to clinical confusion.

Apport des techniques radio-immunologiques pour une meilleure prise en charge de l’enfant malnutri au Sénégal

Malnutrition in children continues to be a major health burden in Senegal. The main objective of this study was to discuss the existence of a thyroid disease induced by the various deficiencies, among which iodine, in these children and to assess the effects of the treatment on the thyroid function. Materials and methods Sixty eight children divided in a random way in two groups according to the addition or not of marine algae hypnea to the traditional nutritional supplementation were included in the study. To appreciate at the same time the nutritional and biological state and to follow the evolution under treatment, the anthropometric parameters were the ratios: weight/age, weight/size, and height/age. Radioimmunoassays of free T3, free T4 and immunoradiometric assays of TSH were carried out in undernourished children before and after supplementation enriched or not in algae. Other blood parameters were measured among them protides, magnesium, and hemoglobin. Results They showed two types of biological abnormalities: low T3 syndrome (8.6%), compensated functional hypothyroidism (9%). The treatment induced neither iodine overload, nor thyroid disease. Conclusion In the program of nutritional rehabilitation, the isotopic techniques allow us to establish the low prevalence (17.6%) of the disturbances of the thyroid function (low T3 syndrome and compensated functional hypothyroidism, thus providing the differential diagnosis), and to confirm the absence of any thyroid disease induced by the iodine supply of the marine algae hypnea.

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

Direct free thyroxine (T(4)) measurements have been linked to both T(4)-binding serum protein concentrations and protein-bound T(4) concentrations. Whether this is evidence of a relationship to total T(4) concentrations has not been reported. We compared an analog-based direct free T(4) immunoassay and a total T(4) immunoassay. Each assay was applied to the fractions of serum T(4) obtained by ultrafiltration and equilibrium dialysis. Both were applied to serum-based solutions in which free T(4), T(4)-binding proteins, protein-bound T(4), and total T(4) were systematically varied, held constant, or excluded. Neither the free T(4) assay nor the total T(4) assay detected dialyzable or ultrafilterable serum T(4). Both assays detected and reported the T(4) retained with serum proteins. Both free and total T(4) results were related to the same total T(4) concentrations in the presence and absence of T(4)-binding proteins. Both results were similarly related to total T(4) concentrations when free T(4) was held constant while total T(4) was varied. Both were similarly related to a total T(4) concentration that was held constant while free T(4) progressively replaced protein-bound T(4). These free T(4) results, like total T(4) results, were unresponsive to a 500-fold variation in dialyzable T(4) concentrations. New experiments extend the characterization of a longstanding and incompletely characterized analog-based free T(4) immunoassay. These free T(4) measurements relate to total T(4) concentrations in the same way that total T(4) measurements do.

A new bioluminescent cellular assay to measure the transcriptional effects of chemicals that modulate the alpha-1 thyroid hormone receptor

Interactions of environmental pollutants with the thyroid endocrine axis have received much attention especially because thyroid hormones (THs) play a major role in mammalian brain development. In order to screen for compounds that act on the triiodothyronine (T3) signaling pathway, we developed a new reporter gene assay expressing luciferase under the control of the TH receptor (TR). PC12 cells expressing the α1-isoform of TR of avian origin were stably transfected with a luciferase gene controlled by the SV40 promoter, and enhanced by a four-spaced direct repeat (DR4) thyroid response element (TRE). The resulting PC-DR-LUC cells were used to optimize a T3 assay in multiwell microplates. This assay was highly sensitive (30 pM T3) and reproducible, and responded as expected to TH analogues. Several halogenated phenolic (3,3′,5,5′-tetrabromobisphenol A, 3,3′,5,5′-tetrachlorobisphenol A, 4-hydroxy-2′,3,4′,5,6′-pentachlorobiphenyl) and phenol (pentachlorophenol, 2,4,6-triiodophenol) compounds suspected of being thyroid-disrupting environmental chemicals induced partial agonistic and/or complex competitive/uncompetitive antagonistic responses in PC-DR-LUC cells at micromolar concentrations. A cell viability test indicated that these effects were not related to cytotoxicity of the chemicals. These results suggest that the PC-DR-LUC assay could be a valuable tool for the large-scale screening for thyroid receptor agonists and antagonists in vitro, and for detecting thyroid disruptors in the environment.

Can thyroid dysfunction explicate severe menopausal symptoms?

SummaryMany of the menopausal manifestations look like those accredited to thyroid hyperfunction or hypofunction. Can thyroid dysfunction explicate severe menopausal symptoms? The study comprised 350 women with different menopausal symptoms. All women had serum TSH, T3 and free T4 estimated. Women with thyroid dysfunction were appropriately treated and other women were treated with ERT. The study showed that 21 women (6%) had hypothyroidism and 18 (5.1%) had hyperthyroidism. Marked improvement in the menopausal-like symptoms occurred after treatment of the thyroid dysfunction. Elderly women with severe or resistant menopausal symptoms can be offered TSH, T3 and T4 assays to rule out the thyroid disturbances before attempting hormone replacement therapy.

Measurement of total thyroxine concentration in serum from dogs and cats by use of various methods

To compare results obtained from assay of total thyroxine (T4) concentration in serum of dogs and cats by use of 4 methods. Serum samples obtained from 98 dogs and 100 cats and submitted by veterinarians to an endocrine testing laboratory. Total T4 concentration was determined in each sample by use of 4 assay methods. Assay methods included a radioimmunoassay (RIA) marketed for use in dogs, an RIA for use in humans, a chemiluminescent enzyme immunoassay for use in humans, and an in-house ELISA. Total T4 concentrations obtained by use of all methods were significantly correlated. Bias-plot comparison revealed similar good overall agreement. Total T4 concentrations determined by use of the RIA marketed for use in dogs were generally lower than concentrations measured by use of the other methods. Clinical comparisons were made by evaluation of the T4 results in the context of the reference range recommended by each laboratory. A difference was found for clinical comparisons on the basis of T4 assay method when used to identify dogs as possible hypothyroid suspects. This difference was related more to the reference range used than to the absolute T4 value. The number of hyperthyroid-suspect cats with T4 values greater than the reference range was the same for each of the 4 assay methods. Total T4 concentrations determined in dogs and cats by use of 4 commonly used methods provided similar and consistent results.

THYROID DYSFUNCTION IN WOMEN WITH MENOPAUSAL SYMPTOMS

Background ; Many of the menopausal manifestations resemble those attributed to thyroid hypofunction or evenhyperfunction. This study aimed at recognizing the role that might be played by the thyroid disturbances in suchsymptoms.Materials & Methods ; The study comprised 135 women with menopausal symptoms attending the gynecologicoutpatient clinic and the endocrinology outpatient clinic at Mansoura University hospitals. All women had serum TSH,T3 and T4 tested.Results ; Among the menopausal women in this study, II women (8.1%) sufferred thyroid dysfunction; 6 women(4.4%) with thyroid hypofunction suffered cognitive and moodal changes and neurological manifestations asparaesthesia and muscle cramps. The other 5 women (3.7%) with thyroid hyperfunction, mostly suffered hot flushesand depression. Nevertheless, all the differences were statistically insignificant.Conclusion ; From these preliminary results, it is believed that postmenopausal women with severe or resistantmenopausal symptoms should be offered TSH, T3 and T4 assays to rule out the thyroid disturbances before attemptinghormone replacement therapy.

Evaluation of thyroid function in neonatal and adult rats: The neglected endocrine mode of action

Although known to regulate growth and development, cellular metabolism, the use of oxygen, and basal metabolic rate, thyroid hormones have been only minimally evaluated in neonatal rodents at critical times of development. Despite some modulation of metabolic rate by other hormones, such as testosterone, growth hormone, and norepinephrine, 3,5,3'-triiodothyronine (T3) and 3,5,3',5'-tetraiodothyronine (T4) are the most important metabolic rate modulators. Endpoints used for thyroid function assessment in neonatal and adult rats include thyroid-stimulating hormone (TSH), T3, and T4 levels and histopathology. In rodents, decreased serum levels of T3 and T4 and increased serum TSH levels, with sustained release of TSH and resultant follicular cell hypertrophy/hyperplasia, are typical hormonal and histopathological findings attributable to compounds altering thyroid function. Hypothyroidism early in the neonatal period can affect reproductive endpoints in both male and female rats, with the critical period of exposure being the first two weeks postnatal. Hypothyroidism has been shown to reduce gonadotrophin levels and delay pubertal spermatogenesis in male rats and to block gonadotropin-induced first ovulation in immature female rats by decreasing FSH and luteinizing hormone (LH) serum concentrations. Inclusion of evaluations of TSH, T3, and T4 assays in multigeneration and developmental neurotoxicity protocols may assist in risk assessments.

Practical dosimetry of 131I in patients with thyroid carcinoma.

Radioiodine treatments of patients with well-differentiated thyroid carcinoma have generally been safe and beneficial. Safety can be ensured while efficacy is increased through practical methods of dosimetry that measure body retention of 131I. Prescriptions for therapeutic 131I can be decreased when the retention level is high and increased when the level is low. Assays of serum free T4 will alert the physician to possible increased radiation to blood and bone marrow, and appreciable concentrations of free T4 are indications to reduce the therapeutic 131I. Carcinomas > or = 1 cm in diameter that are not visible on diagnostic scintigraphy are unlikely to respond to the commonly prescribed mCi of 131I. Biologic responses to commonly prescribed levels of therapeutic 131I, as seen in toxic changes of normal tissues and in indices of tumor size, will be the final dosimeters. With lower levels of prescribed diagnostic 131I, stunning should not impair dosimetry. Thus, readily obtained measurements make dosimetry a practical method for improving carcinoma therapy with 131I.

DEPISTAGE NEONATAL DE L'HYPOTHYROIDIE CONGENITALE PAR DOSAGE DE LA TSH ET DE LA T4

Neonatal screening for congenital hypothyroidism was indertaken in 1996 in 8 health centers of Rabat using combined assays of TSH and T4 in eluats of blood spots on filter paper. 3414 assays have been achieved, 22 newborns were recontrolled to have an elevated level of TSH (>20 mU/l) and/or a low level of T4 (<45 nmol/l). The rate of recall amounted to 0,65%. 3 cases of hypothyroidism have been confirmed. The incidence is 1/1138. The substitutive treatment has been implemented in the first month of life for the 2 newborns and at the 73rd day of life for the third.

Desenvolvimento de ensaio imunofluorométrico para a medida da globulina ligadora de tiroxina (thyroxine-binding globulin, TBG) e sua aplicação em casos de deficiência desta proteína

A globulina ligadora de tiroxina (thyroxine-binding globulin, TBG) é a principal transportadora de hormônios tiroidianos no soro. Variações na concentração sérica de TBG determinam variações proporcionais nas concentrações séricas totais de T4 e T3, sem implicar alterações de função, desde que a fração livre permaneça normal. Várias condições clínicas comuns levam a alterações significativas nos níveis de TBG, sendo as variações mais importantes devidas a defeitos genéticos. Como a TBG é codificada por gene localizado no cromossomo X, os defeitos se manifestam mais facilmente no sexo masculino. Descrevemos o desenvolvimento de ensaio imunofluorométrico para a medida de TBG com base em anticorpos monoclonais, sendo um desenvolvido em nosso laboratório e outro comercial. O método apresenta sensibilidade de 0,8mg/l e coeficientes de variação intra e interensaio inferiores a 10%. O estudo comparativo com método comercial mostrou alta correlação (r = 0,93; n = 48), sendo os valores normais obtidos de 10mg/l a 29mg/l. Estudamos também 20 indivíduos portadores de deficiência congênita de TBG, 19 homens e uma mulher, que apresentavam valores normais de TSH e baixos de T4 total; em todos eles os níveis de TBG foram indetectáveis. Já os níveis de T4 livre medidos por método indireto em 16 desses indivíduos mostraram-se elevados em todos, ao passo que, quando medidos por método direto pós-diálise nos quatro restantes, mostraram-se normais. Nossos resultados reforçam a necessidade prática da disponibilidade de ensaio para a medida de TBG para esclarecimento e definição diagnóstica de alguns casos especiais, principalmente quando o ensaio de T4 livre direto, pós-diálise, não é disponível.