Synthesis Of Thyroxine Research Articles

Effects of Experimental Increases and Decreases in Thyroxine Levels on the Extent of Cataleptic Freezing Reactions in Rats

Numerous clinical observations have provided evidence for a tight connection between impairments in the functions of the hypothalamo-hypophyseal-thyroid system and nervous and mental disorders. The aim of the present work was to compare the effects of experimental decreases and increases in blood thyroxine levels on the extents of two types of pathological freezing reaction in male Wistar rats--spontaneous catalepsy and catalepsy evoked by pinches at the nape of the neck (pinch-induced catalepsy). Chronic administration of the thyroxine synthesis inhibitor propylthiouracil (5 mg/kg/day for 28 days) significantly decreased the blood hormone level and sharply increased the proportion of animals showing spontaneous catalepsy and the immobility time, but had no effect on the extent of pinch-induced catalepsy. At the same time, chronic administration of thyroxine (0.1 mg/kg/day for 28 days), which produced significant increases in blood hormone levels, had no effect on the extent of spontaneous catalepsy but significantly increased the proportion of animals showing pinch-induced catalepsy and the duration of this type of catalepsy. It is concluded that both insufficiency and excess of thyroid hormones have cataleptogenic actions, but enhance different types of catalepsy.

Congenital Iodine Deficiency and Hypothyroidism Impair LTP and Decrease C- fos and C- jun Expression in Rat Hippocampus

Iodine is essential for the synthesis of triiodothyronine (T 3) and thyroxine (T 4). Iodine deficiency leads to inadequate thyroid hormone. Thyroid hormones deficiency during brain development affects cognitive functions, such as attention, learning, and memory. However, the mechanism underlying these deficits is unclear. To investigate the role of iodine deficiency and hypothyroidism in synaptic plasticity, this study examined the induction of long-term synaptic plasticity (LTP) and expression of immediate early (IEA) gene proteins in rat hippocampus following congenital iodine deficiency or hypothyroidism. Through gestation and lactation, iodine-deficient or hypothyroid dam rats were administered with either iodine-deficient diet or methimazole-added drinking water. Exposure was terminated on postnatal day (PN) 30. In hippocampus of pup rats, the induction of LTP in area CA1 was determined on PN60 and the expression of c- fos and c- jun proteins was examined on PN20, PN30 and PN60. Compared to control pups, both treated groups have shown: (1) significantly lower concentrations of serum FT 3 and FT 4, (2) much smaller population spike (PS) amplitude ( p < 0.01) and field-excitatory postsynaptic potential (f-EPSP) slope induced by high-frequency stimulation (HFS) ( P < 0.01), and (3) significantly lower integrated optical density (IOD) total of c- fos and c- jun expression ( P < 0.05 or P < 0.01). In summary, iodine deficiency and hypothyroidism during critical periods of brain development impair LTP induction and decrease the expression of c- fos and c- jun proteins in hippocampus.

An approach to the structure of thyroglobulin. Hormone-forming sequences in porcine thyroglobulin.

The mixture of CNBr peptides obtained by treatment of porcine thyroglobulin with cyanogen bromide was separated into three fractions by Sephadex G-200 gel filtration in 1 M propionic acid. When one of these fractions was reduced and S-alkylated, a hormone-containing CNBr peptide was purified by filtration on Biogel A-1.5 m and ion-exchange chromatography on DEAE-Sephadex. Similarly, two other hormone-containing CNBr peptides were separated from another reduced and S-alkylated fraction by Sephacryl S-200 gel filtration and DEAE-Sephadex chromatography. The three purified CNBr peptides have the same Mr (15000), differ in amino acid composition and contain 60-70% of the hormones present in the initial thyroglobulin. Fragmentation of these peptides into smaller hormone peptides was carried out by trypsin digestion followed by gel filtration. This resulted in the purification of eight discrete hormone-containing tryptic peptides whose homogeneity was established by the study of their N-terminal and C-terminal sequences. At least four sites for the synthesis of thyroxine and two sites for the synthesis of triiodothyronine have been identified in the three CNBr peptides. A possible additional site was recovered in the form of the peptides seryl-triiodothyronine and probably seryl-thyroxine. To gain information on the number of hormone-forming sites in thyroglobulin iodinated in vivo and on the distribution of these sites in the different hormone-containing peptides isolated, estimation of thyroxine and triiodothyronine content of batches of thyroglobulin of iodine content comprised between 0.73% and 1.6% was carried out, together with the estimation of the hormone content of all the CNBr peptides separated from two preparations of thyroglobulin of different iodine contents (0.73% and 1.23%). It was shown that the number of thyroxine and triiodothyronine residues increased linearly with increasing thyroglobulin iodine content in the range 0.73-1.6% and reached six residues of thyroxine and two residues of triiodothyronine/mol protein for a thyroglobulin iodine content of 1.6% with no indication of saturation. This result is at variance with previous findings on porcine thyroglobulin iodinated in vivo which suggested that the thyroxine content reached a plateau of three to four residues/molecule for an iodine content comprised between 0.7% and 1.1%.

EFFECTS OF OZONATION ON THE SPECIATION OF DISSOLVED IODINE IN ARTIFICIAL SEAWATER

Iodine in the form of iodide is required for synthesis of tri-iodothyronine and thyroxine in fish. Iodine chemical speciation in aliquots of raw artificial seawater mix was measured before, during, and after exposure for fixed time periods to air only and to concentrations of ozone required to achieve oxidation-reduction potentials typical of a protein skimmer (400 mV) and an ozone contact chamber (800 mV). Chemical species of iodine were also measured in tank water from a large, recirculating, ozonated aquarium system that has a low-grade incidence of thyroid lesions (e.g., thyroiditis, hyperplasia, adenoma, and adenocarcinoma) in its fish. With increasing exposure to ozone, concentrations of iodide and dissolved organic iodine (DOI) decreased, whereas iodate levels increased. As a result of exposure to 400 mV, iodide concentration dropped to less than half the amount found in raw artificial seawater mix. After exposure to 800 mV, initial iodide levels decreased by 67%, and DOI became undetectable, whereas iodate concentration increased by 155%, with no remarkable change in total iodine concentration. These results indicate ozone-induced conversions from iodide to iodate, and DOI to iodide or iodate (or both). Iodide and DOI were not detectable in the aquarium system's water samples. Ozonation of artificial seawater may alter the relative concentrations of iodine species in a closed tank system, so that iodide supplementation of the diet or tank water of captive teleosts and elasmobranchs living in ozonated seawater is advisable.

Iodine deficiency and development of brain.

Iodine is a trace element essential for the synthesis of triodothyronine (T3) and thyroxine (T4). Inadequate intake of iodine leads to insufficient production of these hormones, which play a vital role in the process of early growth and development of most organs, especially the brain. The neurological sequele of iodine deficiency are mediated by thyroid hormone deficiency, varying from minimal brain function to a syndrome of severe intellectual disability. All the basic processes of neurogenesis: cellular proliferation, differentiation, migration, and selective cell death are impaired during period of brain growth spurt. Evidence suggests alterations in synaptology, neurons, myelin sheaths, glial cells, and morphology of cerebrum and cerebellum in severe iodine deficiency. Foetal thyroid ontogenesis occurs after the first trimester. Until then foetus is dependent on maternal T4. A thyroid dependent event important for subsequent brain development occurs in the beginning of the third trimester of pregnancy.

Hypothyroidism.

Hypothyroidism.

KClO 4 inhibits thyroidal activity in the larval lamprey endostyle in vitro

An in vitro experimental system was devised to assess the direct effects of the goitrogen, potassium perchlorate (KClO 4), on radioiodide uptake and organification by the larval lamprey endostyle. Organification refers to the incorporation of iodide into lamprey thyroglobulin (Tg). Histological and biochemical evidence indicated that endostyles were viable at the termination of a 4 h in vitro incubation. A single iodoprotein, designated as lamprey Tg, was identified in the endostylar homogenates by polyacrylamide gel electrophoresis and Western blotting. Lamprey Tg was immunoreactive with rabbit anti-human Tg serum and had an electrophoretic mobility similar to that of reduced porcine Tg. When KClO 4 was added to the incubation medium, both iodide uptake and organification by the endostyle were significantly reduced relative to controls as determined by gamma counting, and gel-autoradiography and densitometry, respectively. Western blotting showed that KClO 4 significantly lowered the total amount of lamprey Tg in the endostyle. Based on the results of this in vitro investigation, we conclude that KClO 4 acts directly on the larval lamprey endostyle to inhibit thyroidal activity. These data support a previous supposition from in vivo experimentation that KClO 4 acts directly on the endostyle to suppress the synthesis of thyroxine and triiodothyronine, resulting in a decrease in the serum levels of these two hormones.

Iodination of human thyroglobulin (Tg) alters its immunoreactivity. I. Iodination alters multiple epitopes of human Tg.

Human Tg, the site of synthesis of thyroid hormones, thyroxine (T4) and triiodothyronine (T3), is one of the major autoantigens in autoimmune thyroiditis. The degree of iodination of Tg may have a major impact on its immunological properties by changing its antigenicity with respect to antibody binding. We have previously prepared a panel of MoAbs that bind to different epitopes of the Tg molecule. In the present study, we show that iodination alters the conformation of Tg molecule in such a way that it is recognized differently by different MoAbs. Monoclonal antibody 137C1 recognizes Tg regardless of its iodine content. Monoclonal antibody 42C3 recognizes Tg only if the Tg is iodinated either in vitro or in vivo. Monoclonal antibody 133B1 recognizes both in vivo iodinated Tg and non-iodinated Tg, but this MoAb did not recognize Tg following in vitro iodination. Monoclonal antibody 41A5 recognizes intact Tg and tryptic peptides of normal (in vivo) iodinated and non-iodinated Tg, but did not bind the tryptic peptides of artificially (in vitro) iodinated Tg. From the results of these experiments, we conclude that iodination of Tg by either in vivo or in vitro methods changes its conformation in such a way that some natural epitopes are 'lost' and some 'new' epitopes are generated. The generation of new epitopes may be important in the generation of autoimmune responses leading to autoimmune disease.

Metamorphosis in summer flounder: manipulation of thyroid status to synchronize settling behavior, growth, and development

In the aquaculture of summer flounder ( Paralichthys dentatus), the inherent variation in growth and settling behavior during metamorphosis may lead to cannibalism and necessitate increased labor due to grading. Our goal was to use thyroid hormone manipulation to synchronize settling behavior and produce a uniformly sized cohort of juvenile summer flounder. Premetamorphic flounder were treated with exogenous thiourea (TU, 30 ppt, an inhibitor of thyroxine (T4) synthesis). Following removal of TU, metamorphosing flounder were exposed to either exogenous T4 (T4-Na salt, 100 ppb, dissolved in DMSO) or DMSO only. A control (no TU, then DMSO only, when appropriate) was used for comparison. Settling behavior in the control was prolonged, with the Peak Settlement Interval (defined as the interval beginning on the day the first 20% settled until the day 80% had settled) requiring 9 days, and a settling rate of 4.1±0.2 fish/day. Settling behavior was synchronized by thyroid hormone treatment. The Peak Settlement Interval was reduced (5 days) and settling rate increased (8.5±0.6 fish/day) by TU treatment. Treatment with TU followed by T4 further reduced the Peak Settlement Interval (3 days) and increased settling rate (11.2±0.6 fish/day). Development rate (DR) and growth rate (GR) were reduced by TU treatment, but following TU with T4 treatment restored DR and GR to control levels. However, TU treatment eliminated the larger, potentially cannibalistic juveniles found in control tanks. Both treatments reduced variance in GR, whereas only TU-T4 reduced variance in DR. Percent survival was unaffected by treatment. In aquaculture, the effective synchronization of settling behavior by thyroid hormone manipulations should reduce the labor costs associated with grading. We recommend a treatment regime that could aid in the production of more uniform cohorts of juvenile summer flounder.

Iodine-Induced hypothyroidism.

Iodine is an essential element for thyroid hormone synthesis. The thyroid gland has the capacity and holds the machinery to handle the iodine efficiently when the availability of iodine becomes scarce, as well as when iodine is available in excessive quantities. The latter situation is handled by the thyroid by acutely inhibiting the organification of iodine, the so-called acute Wolff-Chaikoff effect, by a mechanism not well understood 52 years after the original description. It is proposed that iodopeptide(s) are formed that temporarily inhibit thyroid peroxidase (TPO) mRNA and protein synthesis and, therefore, thyroglobulin iodinations. The Wolff-Chaikoff effect is an effective means of rejecting the large quantities of iodide and therefore preventing the thyroid from synthesizing large quantities of thyroid hormones. The acute Wolff-Chaikoff effect lasts for few a days and then, through the so-called "escape" phenomenon, the organification of intrathyroidal iodide resumes and the normal synthesis of thyroxine (T4) and triiodothyronine (T3) returns. This is achieved by decreasing the intrathyroidal inorganic iodine concentration by down regulation of the sodium iodine symporter (NIS) and therefore permits the TPO-H202 system to resume normal activity. However, in a few apparently normal individuals, in newborns and fetuses, in some patients with chronic systemic diseases, euthyroid patients with autoimmune thyroiditis, and Graves' disease patients previously treated with radioimmunoassay (RAI), surgery or antithyroid drugs, the escape from the inhibitory effect of large doses of iodides is not achieved and clinical or subclinical hypothyroidism ensues. Iodide-induced hypothyroidism has also been observed in patients with a history of postpartum thyroiditis, in euthyroid patients after a previous episode of subacute thyroiditis, and in patients treated with recombinant interferon-alpha who developed transient thyroid dysfunction during interferon-a treatment. The hypothyroidism is transient and thyroid function returns to normal in 2 to 3 weeks after iodide withdrawal, but transient T4 replacement therapy may be required in some patients. The patients who develop transient iodine-induced hypothyroidism must be followed long term thereafter because many will develop permanent primary hypothyroidism.

Sodium Iodide Symporter (NIS) Gene Expression in Human Placenta

The placenta must allow the passage of iodide from the maternal to the fetal circulation for synthesis of thyroxine by the fetal thyroid. The thyroid sodium iodide symporter (NIS) was cloned in 1996 and, although widely distributed among epithelial tissues, early studies failed to detect it in placenta. We demonstrated NIS mRNA in human placenta and in the human choriocarcinoma cell line, JAr. NIS protein was localized to trophoblasts, with a tendency to apical distribution, in sections of human placenta immunostained with a monoclonal antibody against hNIS. We conclude that NIS is expressed in placenta and may mediate placental iodide transport.

Congenital hypothyroidism: etiologies, diagnosis, and management.

Congenital hypothyroidism is a common preventable cause of mental retardation. The overall incidence is approximately 1:4000; females are affected about twice as often as males. Approximately 85% of cases are sporadic, while 15% are hereditary. The most common sporadic etiology is thyroid dysgenesis, with ectopic glands more common than aplasia or hypoplasia. While the pathogenesis of dysgenesis is largely unknown, some cases are now discovered to be the result of mutations in the transcription factors PAX-8 and TTF-2. Loss of function mutations in the thyrotropin (TSH) receptor have been demonstrated to cause some familial forms of athyreosis. The most common hereditary etiology is the inborn errors of thyroxine (T4) synthesis. Recent mutations have been described in the genes coding for the sodium/iodide symporter, thyroid peroxidase (TPO), and thyroglobulin. Transplacental passage of a maternal thyrotropin receptor blocking antibody (TRB-Ab) causes a transient form of familial congenital hypothyroidism. The vast majority of infants are now diagnosed after detection through newborn screening programs using a primary T4-backup TSH or primary TSH test. Screening test results must be confirmed by serum thyroid function tests. Thyroid scintigraphy, using 99mTc or 123I, is the most accurate diagnostic test to detect thyroid dysgenesis or one of the inborn errors of T4 synthesis. Thyroid sonography is nearly as accurate, but it may miss some cases of ectopic glands. If maternal antibody-mediated hypothyroidism is suspected, measurement of maternal and/or neonatal TRB-Ab will confirm the diagnosis. The goals of treatment are to raise the serum T4 as rapidly as possible into the normal range, adjust the levothyroxine dose with growth to keep the serum T4 (or free T4) in the upper half of the normal range and the TSH normal, and maintain normal growth and development while avoiding overtreatment. An initial starting dose of 10-15 microg/kg per day is recommended; this dose will decrease on a weight basis over time. Serum T4 (or free T4) and TSH should be monitored every 1-2 months in the first year of life and every 2-3 months in the second and third years.

A negative iodine balance is found in healthy neonates compared with neonates with thyroid agenesis.

We studied the effects of the presence or absence of the thyroid gland on the iodine metabolism and excretion in term Dutch newborns by performing a retrospective study of the urinary iodine excretion in 193 term newborns with abnormal congenital hypothyroidism screening results. Thirty-six euthyroid newborns with decreased thyroxine-binding globulin levels were compared with 157 hypothyroid patients, 54 due to thyroid agenesis and 103 due to thyroid dysgenesis. A significant difference in the urinary iodine excretion was observed between the agenesis group (mean: 28 micrograms/24 h) and the euthyroid newborns (mean: 46 micrograms/24 h, P=0.001). In conclusion, healthy, euthyroid, term newborns excreted more iodine in their urine than newborns with thyroid agenesis. These results strongly indicated the existence of a temporarily negative iodine balance: the excretion of iodine prevailed over the intake and the newborn's thyroidal iodine, stored during pregnancy, could be used for thyroxine synthesis in the postnatal period. Since healthy term neonates were able to maintain adequate plasma free thyroxine concentrations under normal TSH stimulation, the prenatally acquired iodine stores could be considered sufficiently high to compensate for the transient postnatal losses.

A comparative review of the structure and biosynthesis of thyroglobulin

Thyroglobulin, the major iodoglycoprotein of the thyroid ( M r 669 kDa) has a sedimentation coefficient of 19 S and an isoelectric point (p I) of 4.4–4.7. The protein has been isolated and purified from saline extracts of the gland of several animal species, by methods such as ammonium sulfate fractionation, DEAE-cellulose chromatography and Sepharose 4B/6B gel-filtration. DEAE-cellulose chromatography of thyroglobulin from many species, by linear gradient, yielded a complex elution pattern, while camel thyroglobulin showed only a major and minor peak. As an iodoprotein, the protein has 0.1–2.0% iodine. The amino acid and iodoamino acid composition of thyroglobulins, in general, is similar. However, a high thyroxine content (15 mol/mol protein) has been noted for buffalo species. Asparagine or aspartic acid has been reported as the major N-terminal amino acid for thyroglobulins of several animal species whereas glutamic acid is the sole N-terminal amino acid for buffalo thyroglobulin. As a glycoprotein, thyroglobulin contains 8–10% total carbohydrate with galactose, mannose, fucose, N-acetyl glucosamine and sialic acid residues. The carbohydrate in the protein is distributed as two distinct units, A and B. In addition, human thyroglobulin has carbohydrate unit C. The occurrence of sulfate and phosphate as Gal-3-SO 4 and Man-6-PO 4, respectively, has been reported in few species. The quaternary structure of native thyroglobulin is comprised of two equal sized subunits of 330 kDa. However, the protein appears to contain 4–8 non-identical units in few species. The synthesis of thyroid hormones occurs in the matrix of the protein and is regulated by pituitary thyrotropin. The role of tyrosine residues 5 and 130 in thyroxine synthesis has been well documented.

ChemInform Abstract: Synthesis of Diaryl Ethers Through the Copper‐Promoted Arylation of Phenols with Arylboronic Acids. An Expedient Synthesis of Thyroxine.

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Synthesis of diaryl ethers through the copper-promoted arylation of phenols with arylboronic acids. An expedient synthesis of thyroxine

Abstract Diaryl ethers are readily synthesized in high yield at room temperature through the copper(II)-promoted coupling of arylboronic acids and phenols. The reaction is tolerant of a wide range of substituents on both coupling partners. These reaction conditions permit the racemization-free arylation of phenolic amino acids, methodology that has been applied to an efficient synthesis of thyroxine.

Anti-Thyroid Isoflavones from Soybean: Isolation, Characterization, and Mechanisms of Action

The soybean has been implicated in diet-induced goiter by many studies. The extensive consumption of soy products in infant formulas and in vegetarian diets makes it essential to define the goitrogenic potential. In this report, it was observed that an acidic methanolic extract of soybeans contains compounds that inhibit thyroid peroxidase- (TPO) catalyzed reactions essential to thyroid hormone synthesis. Analysis of the soybean extract using HPLC, UV-VIS spectrophotometry, and LC-MS led to identification of the isoflavones genistein and daidzein as major components by direct comparison with authentic standard reference isoflavones. HPLC fractionation and enzymatic assay of the soybean extract showed that the components responsible for inhibition of TPO-catalyzed reactions coeluted with daidzein and genistein. In the presence of iodide ion, genistein and daidzein blocked TPO-catalyzed tyrosine iodination by acting as alternate substrates, yielding mono-, di-, and triiodoisoflavones. Genistein also inhibited thyroxine synthesis using iodinated casein or human goiter thyroglobulin as substrates for the coupling reaction. Incubation of either isoflavone with TPO in the presence of H 2O 2 caused irreversible inactivation of the enzyme; however, the presence of iodide ion in the incubations completely abolished the inactivation. The ic 50 values for inhibition of TPO-catalyzed reactions by genistein and daidzein were ca. 1–10 μM, concentrations that approach the total isoflavone levels ( ca. 1 μM) previously measured in plasma from humans consuming soy products. Because inhibition of thyroid hormone synthesis can induce goiter and thyroid neoplasia in rodents, delineation of anti-thyroid mechanisms for soy isoflavones may be important for extrapolating goitrogenic hazards identified in chronic rodent bioassays to humans consuming soy products.

A concise synthesis of thyroxine (T 4) and 3,5,3′-Triiodo- l-thyronine (T 3)

The mono- and di-iodo derivatives of 1-oxaspiro[2,5]bicycloocta-4,7-diee-6-one, 8 and 9, reacted readily with 3,5-diiodo- l-tyrosine at pH 8.0 to give 3,5,3′-triiodo- l-thyronine (T 3) and thyroxine in 70% and 94% yields respectively. In turn, 8 and 9 were prepared by the sodium bismuthate oxidation of their corresponding iodinated p-hydroxybenzyl alcohol derivatives, 6 and 7 in 32% and 37% yields.

Synthesis of thyroxine: biomimetic studies

The biomimetic oxidative coupling of the ethyl ester of N-acetyl-3,5-diiodotyrosine (1) to yield the ethyl ester of N-acetylthyroxine (2) has been investigated. A putative mechanism involving phenolic coupling to yield an intermediate aryloxydienone (7) followed by an E2 elimination for loss of the side chain has been proposed. Oxidative couplings with analogous 4-substituted 3,5-diiodophenols indicate that a number of mechanisms are possible; these include quinone methide intermediates and SN2 substitutions in the intermediate aryloxydienones. Rearomatization of the intermediate aryloxydienones is a strong driving force for the loss of the side chains. The results indicate that 3,5-diiodo-4-aryloxydienones are good leaving groups in E2 and SN2 mechanisms. The synthetic method provides a facile synthesis of thyroxine analogues from readily available 4-substituted 3,5-diiodophenols. Keywords: diiodotyrosine, phenolic coupling, phenoxyl radicals, thyroxine.

Dityrosine bridge formation and thyroid hormone synthesis are tightly linked and are both dependent on N-glycans

Formation of dityrosine bridges is a ubiquitous process mainly attributed to oxidative stress leading to protein degradation and cellular damages. Here we show that dityrosine formation is involved in a physiological process, thyroid hormone synthesis, and is strictly dependent on structural characteristics, namely N-glycans, presented by the protein acting as the prothyroid hormone. We used two isoforms of the N-terminal thyroid hormone forming domain (NTD) of human thyroglobulin: one without N-glycan (19 kDa isoform) and the other with high mannose type structures (25 kDa isoform). Both isoforms were able to form iodotyrosines after in vitro iodination. However, iodotyrosine coupling to form thyroxine did not occur with the unglycosylated 19 kDa NTD. In contrast, the 25 kDa isoform formed thyroxine. Strikingly, thyroxine synthesis was accompanied by dimerization of the 25 kDa isoform and formation of a dityrosine bridge; none of this was observed with the 19 kDa isoform. Taken as a whole, our results indicate that dimerization through dityrosine bridging accompanies and could have a role in thyroid hormone synthesis.