Comparative aspects of the distribution, metabolism, and excretion of six iodothyronines in the rat

We have studied the kinetics of 3 iodothyronines, 3,3'-diiodothyronine (T2), 3',5'-T2, and 3'-monoiodothyronine (T1), in groups of young adult male rats maintained under normal steady state physiological conditions. We have also performed a comparative analysis of these results, combined with corresponding kinetic indices of T4, T3, and rT3, to obtain a more comprehensive understanding of normal thyroid hormone production, distribution, and metabolism. Tracer doses of 125I-labeled 3,3'-T2, 3',5'-T2, and 3'-T1 were separately injected iv, and blood samples were collected 6-12 times for each iodothyronine in optimized sequential kinetic studies designed to maximize the precision of kinetic parameters. Labeled iodothyronines were separated quantitatively from their metabolites in each plasma sample by Sephadex G-25 column chromatography. Conventional kinetic analysis of the resulting data generated distribution volume, clearance, turnover, and mean residence time indices for each iodothyronine, and concomitant compartmental analysis of the same data provided additional results useful for integration and comparative analysis of the 6 iodothyronines. Kinetic parameters for all but T4 and T3 were similar, suggesting that similar mechanisms are responsible for the transport, metabolism, and distribution of nonhormonal iodothyronines. All but T4 and T3 (and, to a much lesser extent, 3'-T1) were almost completely and irreversibly metabolized, whereas 24-30% of the hormones (and 6% of 3'-T1) were excreted as such in feces only. Three-pool models fitted individual plasma kinetic data sets best in all cases (for all 6 iodothyronines), each with a plasma, a slowly exchanging (slow), and a rapidly exchanging (fast) pool, and kinetic parameters of interest were quantified for each iodothyronine (Ti). Quantitative analysis of an integrated 18-pool model for all 6 Tis revealed several other features of physiological interest. The fractional transport rate of T3 into the fast pool (liver, at least) is about an order of magnitude larger than that for all other Tis, supporting the hypothesis that transport of T3 into fast tissues (e.g. liver cells) is selectively amplified relative to that of the 5 other iodothyronines studied. Simultaneous and direct comparison of the 6 plasma kinetic data sets also supports this result. In addition, composite slow tissue pools, which probably exclude liver and kidney, contained the largest whole body fractions of all Tis (greater than 50%), and these also appear to be major sites of whole body T4 monodeiodinations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of human iodothyronine sulfotransferases

Sulfation is an important pathway of thyroid hormone metabolism that facilitates the degradation of the hormone by the type I iodothyronine deiodinase, but little is known about which human sulfotransferase isoenzymes are involved. We have investigated the sulfation of the prohormone T4, the active hormone T3, and the metabolites rT3 and 3,3'-diiodothyronine (3,3'-T2) by human liver and kidney cytosol as well as by recombinant human SULT1A1 and SULT1A3, previously known as phenol-preferring and monoamine-preferring phenol sulfotransferase, respectively. In all cases, the substrate preference was 3,3'-T2 > rT3 > T3 > T4. The apparent Km values of 3,3'-T2 and T3 [at 50 micromol/L 3'-phosphoadenosine-5'-phosphosulfate (PAPS)] were 1.02 and 54.9 micromol/L for liver cytosol, 0.64 and 27.8 micromol/L for kidney cytosol, 0.14 and 29.1 micromol/L for SULT1A1, and 33 and 112 micromol/L for SULT1A3, respectively. The apparent Km of PAPS (at 0.1 micromol/L 3,3'-T2) was 6.0 micromol/L for liver cytosol, 9.0 micromol/L for kidney cytosol, 0.65 micromol/L for SULT1A1, and 2.7 micromol/L for SULT1A3. The sulfation of 3,3'-T2 was inhibited by the other iodothyronines in a concentration-dependent manner. The inhibition profiles of the 3,3'-T2 sulfotransferase activities of liver and kidney cytosol obtained by addition of 10 micromol/L of the various analogs were better correlated with the inhibition profile of SULT1A1 than with that of SULT1A3. These results indicate similar substrate specificities for iodothyronine sulfation by native human liver and kidney sulfotransferases and recombinant SULT1A1 and SULT1A3. Of the latter, SULT1A1 clearly shows the highest affinity for both iodothyronines and PAPS, but it remains to be established whether it is the prominent isoenzyme for sulfation of thyroid hormone in human liver and kidney.     

Metabolism of 3,3'-diiodothyronine in rat hepatocytes: interaction of sulfation with deiodination

Production of 3,3'-diiodothyronine (3,3'-T2) is an important step in the peripheral metabolism of thyroid hormone in man. The rapid clearance of 3,3'-T2 is accomplished to a large extent in the liver. We have studied in detail the mechanisms of this process using monolayers of freshly isolated rat hepatocytes. After incubation with 3,[3'-125I]T2, chromatographic analysis of the medium revealed two major metabolic routes: outer ring deiodination and sulfation. We recently demonstrated that sulfate conjugation precedes and in effect accelerates deiodination of 3,3'-T2. In media containing different serum concentrations the cellular clearance rate was determined by the nonprotein-bound fraction of 3,3'-T2. At substrate concentrations below 10(-8) M 125I- was the main product observed. At higher concentrations deiodination became saturated, and 3,3'-T2 sulfate (T2S) accumulated in the medium. Saturation of 3,3'-T2 clearance was found to occur only at very high (greater than 10(-6)M) substrate concentrations. The sulfating capacity of the cells exceeded that of deiodination by at least 20-fold. Deiodination was completely inhibited by 10(-4) M propylthiouracil or thiouracil, resulting in the accumulation of T2S while clearance of 3,3'-T2 was little affected. No effect was seen with methimazole. Hepatocytes from 72-h fasted rats showed a significant reduction of deiodination but unimpaired sulfation. Other iodothyronines interfered with 3,3'-T2 metabolism. Deiodination was strongly inhibited by 2 microM T4 and rT3 (80%) but little by T3 (15%), whereas the clearance of 3,3'-T2 was reduced by 27% (T4 and rT3) and 12% (T3). It is concluded that the rapid hepatic clearance of 3,3'-T2 is determined by the sulfate-transferring capacity of the liver cells. Subsequent outer ring deiodination of the intermediate T2S is inhibited by propylthiouracil and by fasting, essentially without an effect on overall 3,3'-T2 clearance.     

Simultaneous turnover studies of thyroxine, 3,5,3' and 3,3',5'-triiodothyronine, 3,5-, 3,3'-, and 3',5'- diiodothyronine, and 3'-monoiodothyronine in chronic renal failure

The present study evaluates the sequential extra-thyroidal monodeiodination of thyroid hormones through tri-, di-, and monoiodothyronines in chronic renal failure (CRF) in man. Simultaneous turnover studies of T4, T3, rT3, 3,5-diiodothyronine (3,5-T2), 3,3'-T2, 3',5'-T2, 3'5'-T2, and 3'-monoiodothyronine (3--T1) were conducted in six patients with CRF (creatinine clearance, 9-18 ml/min) using the single-injection, noncompartmental approach. Serum levels of T4, T3, and 3,5-T2 were reduced to two thirds of control levels (P less than 0.05), whereas serum rT3 and 3,3'-T2 levels were reduced to a minor degree. Serum 3'-5'-T1 was doubled (p less than 0.05). The MCRs of T4, rT3, and 3',5'-T2 were enhanced to 168%, 127%, and 187% of normal (P less than 0.05), respectively, whereas those of T3, 3,5-T2, 3,3'-T2, and 3'-T1 were unaffected. The mean production rates (PRs) of the iodothyronines in CRF were as follows (CRF vs. control values, expressed as nanomoles per day/70 kg): T4, 119 vs. 125; T3, 26 vs. 44 (P less than 0.01); rT3, 49 vs, 48; 3,5-T2, 3.5 vs. 7.2 (P less than 0.001); 3,3'-T2, 25 vs. 35 (P less than 0.01); 3',5'-T2, 25 vs. 14 (P less than 0.01); and 3'-T1, 39 vs. 30. Previous studies have demonstrated reduced phenolic ring (5'-) deiodination of T4 in CRF, which is supported by the present finding of unaltered PR of T4 and reduced PR of T3. In contrast the 5'-deiodination of T3 leading to the formation of 3,5-T2 was found unaffected by CRF, since the conversion rate (CR) of T3 to 3,5-T2 (PR 3,5-T2/PR T3) was unaltered (16% vs. 15% in controls). The tyrosylic ring (5-) deiodination of T4 to rT3 was unaffected in patients with CRF, the CR being 42% vs. 40% in controls, in contrast to an enhanced CR of rT3 to 3',5'-T2 (53% vs. 29%, P less than 0.01), which also is a 5-deiodination step. In conclusion, our data show that CRF profoundly changes the kinetics of all iodothyronines studied. Furthermore, our data are compatible with the existence of more than one 5'-deiodinase as well as more than one 5-deiodinase in man.     

Rapid and selective inner ring deiodination of thyroxine sulfate by rat liver deiodinase

Previous studies have shown that the inner ring deiodination (IRD) of T3 and the outer ring deiodination (ORD) of 3,3'-diiodothyronine are greatly enhanced by sulfate conjugation. This study was undertaken to evaluate the effect of sulfation on T4 and rT3 deiodination. Iodothyronine sulfate conjugates were chemically synthetized. Deiodination was studied by reaction of rat liver microsomes with unlabeled or outer ring 125I-labeled sulfate conjugate at 37 C and pH 7.2 in the presence of 5 mM dithiothreitol. Products were analyzed by HPLC or after hydrolysis by specific RIAs. T4 sulfate (T4S) was rapidly degraded by IRD to rT3S, with an apparent Km of 0.3 microM and a maximum velocity (Vmax) of 530 pmol/min X mg protein. The Vmax to Km ratio of T4S IRD was increased 200-fold compared with that of T4 IRD. However, formation of T3S by ORD of T4S could not be observed. The rT3S formed was rapidly converted by ORD to 3,3'-T2 sulfate, with an apparent Km of 0.06 microM and a Vmax of 516 pmol/min X mg protein. The enzymic mechanism of the IRD of T4S was the same as that of the deiodination of nonsulfated iodothyronines, as shown by the kinetics of stimulation by dithiothreitol or inhibition by propylthiouracil. The IRD of T4S and the ORD of rT3 were equally affected by a number of competitive inhibitors, suggesting a single enzyme for the deiodination of native and sulfated iodothyronines. In conjunction with previous findings on the deiodination of T3S, these results suggest that sulfation leads to a rapid and irreversible inactivation of thyroid hormone.     

Renal handling of thyroxine, 3,5,3'- and 3,3',5'-triiodothyronine, 3,3'- and 3',5'-diiodothyronine in man

The 24-h urinary excretion and renal clearance of thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), and 3',5'-diiodothyronine (3',5'-T2) were measured in 17 healthy subjects. The median urinary excretion was (pmol/24h) T4: 1242, T3: 828, rT3: 12.9, 3,3'-T2: 331, and 3',5'-T2: 5.8. The corresponding renal clearances were in median (ml/min) T4: 31, T3: 133, rT3: 15, 3,3'-T2: 683, and 3',5'-T2: 4.5. The clearances differed mutually (P less than 0.01) as well as from the creatinine clearance (P less than 0.01) which was in median 87 ml/min. Thus, all iodothyronines studied were subject to tubular transport mechanisms besides glomerular filtration. The 3 iodothyronines with 2 iodine atoms in the phenolic ring of the thyronine molecule, T4, rT3 and 3',5'-T2, were mainly tubularly reabsorbed, whereas those with only one iodine atom in the phenolic ring, T3 and 3,3'-T2, were mainly tubularly secreted. It might be hypothesized that the number of iodine atoms in the phenolic ring determines the direction of the tubular transport (presence of 2 iodine atoms is associated with tubular reabsorption, and of one iodine atom with secretion), whereas the rate of tubular transport decreases with decreasing number of iodine atoms in the tyrosylic ring.     

Deiodination of iodothyronine sulfamates by type I iodothyronine deiodinase of rat liver

The substrate behavior of synthetic N-sulfonated iodothyronines (iodothyronine sulfamates, TiNS) for the type I deiodinase was compared with that of the naturally occurring 4'-O-sulfonated iodothyronines (iodothyronine sulfates, TiS), which have been shown to be deiodinated 40-200 times more efficiently than the native iodothyronines. Deiodination was studied in incubations of rat liver microsomes with unlabeled or 3' (5')-125I-labeled T4NS, rT3NS, T3NS, and 3,3'-T2NS at 37 C and pH 7.2 in the presence of 5 mM dithiothreitol. Reaction products were analyzed by RIA or Sephadex LH-20 and HPLC. Kinetic studies were performed under initial reaction rate conditions to determine the apparent Michaelis Menten (Km) constants and maximum velocity values. In contrast to T4S, which is converted only by inner ring deiodination (IRD), T4NS underwent both IRD and outer ring deiodination (ORD), similar to T4, but more rapidly. At 10 nM T4NS substrate, T3NS was the major product observed, while no rT3NS accumulated due to its rapid conversion to 3,3'-T2NS. At least one third of the 3,3'-T2NS was converted by IRD, unlike 3,3'-T2 which is a pure ORD substrate. The type I deiodination efficiencies of T4NS IRD and ORD were 17-fold higher than with T4, mainly due to approximately 32-fold lower apparent Km values. Deiodination of rT3, the preferred type I substrate, was not improved by sulfamation. T3NS and 3,3'-T2NS were deiodinated 4-10 times more efficiently than T3 and 3,3'-T2, respectively, due to 2- to 4-fold decreases in apparent Km values with a concomitant doubling of maximum velocity values. N-Sulfonation stimulates type I deiodination to a similar extent as other side-chain modifications that eliminate the positive charge of the nitrogen (e.g. iodothyroacetic acids). However, the effects are less dramatic than those induced by 4'-sulfation with respect to both efficiency and specificity of the catalytic process.     

Sulfation of thyroid hormone by estrogen sulfotransferase.

Sulfation is one of the pathways by which thyroid hormone is inactivated. Iodothyronine sulfate concentrations are very high in human fetal blood and amniotic fluid, suggesting important production of these conjugates in utero. Human estrogen sulfotransferase (SULT1E1) is expressed among other tissues in the uterus. Here we demonstrate for the first time that SULT1E1 catalyzes the facile sulfation of the prohormone T4, the active hormone T3 and the metabolites rT3 and 3,3'-diiodothyronine (3,3'-T2) with preference for rT3 approximately 3,3'-T2 > T3 approximately T4. Thus, a single enzyme is capable of sulfating two such different hormones as the female sex hormone and thyroid hormone. The potential role of SULT1E1 in fetal thyroid hormone metabolism needs to be considered.     

Characterization of iodothyronine sulfatase activities in human and rat liver and placenta

In conditions associated with high serum iodothyronine sulfate concentrations, e.g. during fetal development, desulfation of these conjugates may be important in the regulation of thyroid hormone homeostasis. However, little is known about which sulfatases are involved in this process. Therefore, we investigated the hydrolysis of iodothyronine sulfates by homogenates of V79 cells expressing the human arylsulfatases A (ARSA), B (ARSB), or C (ARSC; steroid sulfatase), as well as tissue fractions of human and rat liver and placenta. We found that only the microsomal fraction from liver and placenta hydrolyzed iodothyronine sulfates. Among the recombinant enzymes only the endoplasmic reticulum-associated ARSC showed activity toward iodothyronine sulfates; the soluble lysosomal ARSA and ARSB were inactive. Recombinant ARSC as well as human placenta microsomes hydrolyzed iodothyronine sulfates with a substrate preference for 3,3'-diiodothyronine sulfate (3,3'-T(2)S) approximately T(3) sulfate (T(3)S) > rT(3)S approximately T(4)S, whereas human and rat liver microsomes showed a preference for 3,3'-T(2)S > T(3)S > rT(3)S approximately T(4)S. ARSC and the tissue microsomal sulfatases were all characterized by high apparent K(m) values (>50 microM) for 3,3'-T(2)S and T(3)S. Iodothyronine sulfatase activity determined using 3,3'-T(2)S as a substrate was much higher in human liver microsomes than in human placenta microsomes, although ARSC is expressed at higher levels in human placenta than in human liver. The ratio of estrone sulfate to T(2)S hydrolysis in human liver microsomes (0.2) differed largely from that in ARSC homogenate (80) and human placenta microsomes (150). These results suggest that ARSC accounts for the relatively low iodothyronine sulfatase activity of human placenta, and that additional arylsulfatase(s) contributes to the high iodothyronine sulfatase activity in human liver. Further research is needed to identify these iodothyronine sulfatases, and to study the physiological importance of the reversible sulfation of iodothyronines in thyroid hormone metabolism.     

AMNIOTIC FLUID CONCENTRATIONS OF 3,3'',5''-TRI-IODOTHYRONINE (REVERSE T3), 3,3''-DI-IODOTHYRONINE, 3,5,3''-TRI-IODOTHYRONINE (T3) AND THYROXINE (T4) IN NORMAL AND COMPLICATED PREGNANCY

Amniotic fluid concentrations of 3,3',5'-tri-iodothyronine (rT3), 3,3'-Di-iodothyronine (3,3'-T2), 3,5,3'-tri-iodothyronine (T3) and T4 were studied in 384 women during normal and complicated pregnancy. An inverse correlation was observed between decreasing rT3 and increasing 3,3'-T2 concentrations in amniotic fluid with gestational age. The mean rT3 level in normal pregnancy was 2.81 nmol/1 at 12-20 weeks and decreased significantly to 1.06 nmol/1 at 36-42 weeks of gestation. The mean 3,3'-T2 concentration was 49.1 pmol/1 at12-20 weeks increasing to 119 pmol/1 at 36-42 weeks. The mean T4 value of 3.83 nmol/1 at 12-20 weeks was about half that of later periods. The T3 concentration in a random sample of 45 amniotic fluids ranged from less than 28 to 370 pmol/1 (mean 102 pmol/1). The mean rT3, 3,3'-T2 and T4 values measured in patients with intra-uterine malnutrition, gestation diabetes, tocolysis, placental insufficiency and rhesus incompatibility at 31-40 weeks of gestation were not significantly different from those in uncomplicated pregnancy. Significantly decreased rT3 and T4 concentrations were found in toxaemia. From the results obtained in complicated pregnancy it may be concluded that measurements of iodothyronines, especially rT3, in amniotic fluid have insignificant diagnostic value in the recognition of intra-uterine lesions with the probable exception of fetal hypothyroidism. The analysis of the dependence of iodothyronine concentrations on the gestational age showed a maximum of rT3 and T4 levels between 20 and 30 weeks of pregnancy. This marked rise of iodothyronine concentrations in amniotic fluid at mid-gestation may be due to the onsetting maturation of the hypothalamic-pituitary-thyroid control system of the fetus.     

Thyroid hormone transport by the heterodimeric human system L amino acid transporter

Transport of thyroid hormone across the cell membrane is required for thyroid hormone action and metabolism. We have investigated the possible transport of iodothyronines by the human system L amino acid transporter, a protein consisting of the human 4F2 heavy chain and the human LAT1 light chain. Xenopus oocytes were injected with the cRNAs coding for human 4F2 heavy chain and/or human LAT1 light chain, and after 2 d were incubated at 25 C with 0.01-10 microM [(125)I]T(4), [(125)I]T(3), [(125)I]rT(3), or [(125)I]3,3'-diiodothyronine or with 10-100 microM [(3)H]arginine, [(3)H]leucine, [(3)H]phenylalanine, [(3)H]tyrosine, or [(3)H]tryptophan. Injection of human 4F2 heavy chain cRNA alone stimulated the uptake of leucine and arginine due to dimerization of human 4F2 heavy chain with an endogenous Xenopus light chain, but did not affect the uptake of other ligands. Injection of human LAT1 light chain cRNA alone did not stimulate the uptake of any ligand. Coinjection of cRNAs for human 4F2 heavy chain and human LAT1 light chain stimulated the uptake of phenylalanine > tyrosine > leucine > tryptophan (100 microM) and of 3,3'-diiodothyronine > rT(3) approximately T(3) > T(4) (10 nM), which in all cases was Na(+) independent. Saturation analysis provided apparent Michaelis constant (K(m)) values of 7.9 microM for T(4), 0.8 microM for T(3), 12.5 microM for rT(3), 7.9 microM for 3,3'-diiodothyronine, 46 microM for leucine, and 19 microM for tryptophan. Uptake of leucine, tyrosine, and tryptophan (10 microM) was inhibited by the different iodothyronines (10 microM), in particular T(3). Vice versa, uptake of 0.1 microM T(3) was almost completely blocked by coincubation with 100 microM leucine, tryptophan, tyrosine, or phenylalanine. Our results demonstrate stereospecific Na(+)-independent transport of iodothyronines by the human heterodimeric system L amino acid transporter.     

A radioimmunoassay for measurement of 3,3'-diiodothyronine sulfate: Studies in thyroidal and nonthyroidal diseases, pregnancy, and fetal/neonatal life

Data suggesting that (1) sulfation of the phenolic hydroxyl of iodothyronines plays an important role in thyroid hormone metabolism and (2) maternal serum 3,3'-diiodothyronone sulfate (3,3'-T(2)S) may reflect on the status of fetal thyroid function stimulated us to develop a radioimmunoassay (RIA) for measurement of T(2)S. Our T(2)S RIA is highly sensitive, practical, and reproducible. T(4)S, T(3)S, and T(1)S crossreacted 3.1%, 0.81%, and 5.3%, respectively; thyroxine (T(4)), triiodothyronine (T(3)), and reverse (r)T(3), 3,3'-T(2) and 3'-T(1) crossreacted <0.1%. Although rT(3) sulfate (rT(3)S) crossreacted 55% in 3,3'-T(2)S RIA, its serum levels are very low and have little influence on serum T(2)S values reported here. T(2)S was measured in ethanol extracts of serum, amniotic fluid, and urine. Recovery of nonradioactive T(2)S added to serum was 96%. The dose-response curves of inhibition of binding of (125)I-T(2)S to anti-T(2)S by serial dilutions of ethanol extracts of serum or urine were essentially parallel to the standard curve. The detection threshold of the RIA varied between 0.17 and 0.50 nmol/L (or 10 and 30 ng/dL). The coefficient of variation (CV) averaged 9% within an assay and 13% between assays. The serum concentration of T(2)S was [mean +/- SE, nmol/L] 0.86 +/- 0.59 in 36 normal subjects, 2.2 +/- 0.06 in 10 hyperthyroid patients (P <.05), 0.73 +/- 0.10 in 11 hypothyroid patients (not significant [NS]), 6.0 +/- 1.5 in 16 patients with systemic nonthyroidal illness (P <.001), 18 +/- 2.5 in 16 newborn cord blood sera (P <.02), 2.7 +/- 0.32 in 25 pregnant women [15 to 40 weeks gestation, P <.001], 0.94 +/- 0.10 in 10 hypothyroid women receiving T(4) replacement therapy (NS), and 2.0 +/- 0.38 in 11 hypothyroid women treated with T(4) replacement and oral contraceptives (P <.02); serum T(2)S levels in the third trimester of pregnancy were similar to those in the second trimester of pregnancy. T(2)S concentration in amniotic fluid was 12.5 +/- 2.7 nmol/L (n = 7) at 15 to 20 weeks gestation, and it decreased markedly to 3.3 +/- 1.3 nmol/L (n = 3) at 35 to 38 weeks gestation. Urinary excretion of T(2)S in random urine samples of 19 normal subjects was 10.9 +/- 1.3 nmol/g creatinine. (1) T(2)S is a normal component of human serum, urine, and amniotic fluid, and serum T(2)S levels change substantially in several physiologic and pathologic conditions; (2) high serum T(2)S in pregnancy may signify increased transfer of T(2)S from fetal to maternal compartment, estrogen-induced increase in T(2)S production, decreased clearance, or a combination of these factors. The data do not support the notion that fetal thyroid function is the only or the predominant factor responsible for high serum T(2)S in pregnant women.     

Phenolic and tyrosyl ring iodothyronine deiodination by the Caco-2 human colon carcinoma cell line

Thyroid hormone metabolism was studied in the human Caco-2 colon carcinoma cell line, which at confluence exhibits several functions of differentiated enterocytes. Cells were harvested two to 17 days after reaching confluence. Intact cells and homogenates were tested for deiodination of [125I]-labeled substrates. Small amounts of thyroxine (T4) were converted by homogenates to 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), and 1-, with no detectable production of 3,5,3'-triiodothyronine (T3) by homogenates or cells. rT3 was converted to 3,3'-T2 and 1- with an apparent Michaelis constant (Km) for rT3 of 24 nmol/L; 6-n-propyl-2-thiouracil (PTU) had a 50% inhibitory concentration of 30 nmol/L and abolished rT3 5'-deiodination at 1 mmol/L in the presence of 20 mmol/L dithiothreitol (DTT). T3 was deiodinated to 3,3'-T2 and 3'-monoiodothyronine (3'-T1) with an apparent Michaelis constant (Km) for T3 of 5.7 nmol/L; this reaction was not inhibited by 1 mmol/L PTU. Phenolic and tyrosyl ring deiodinating activities were maximal four and six days, respectively, after the cells reached confluence. Homogenates of cells grown in standard medium containing fetal calf serum had fivefold higher rT3 5'-deiodinating activity than cells grown in a serum-free defined culture medium, reflecting a fivefold difference in the apparent Vmax with no difference in the apparent Km for rT3. There was no difference in T3 5-deiodination rates in homogenates of Caco-2 cells grown in the two media until 12 days postconfluence, when cells grown in standard medium had higher activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Handling of iodothyronines by the liver and kidney in patients with chronic liver disease

Possible arterio-venous gradients of T4, T3, rT3 and 3,3'-diiodothyronine (3,3'-T2) across the liver and the kidneys were measured in 9 patients with varying degrees of liver failure undergoing diagnostic catheterization. Plasma iodothyronine levels were measured in peripheral, hepatic and renal veins before and at 10-min intervals until 60 min after iv injection of 400 micrograms of TRH. In 2 patients estimated hepatic plasma flow and effective renal plasma flow were determined as well. In these 2 patients, no significant differences between iodothyronine levels in arterial and peripheral venous plasma were found. T4 and T3 levels were not significantly different between peripheral, renal and hepatic veins. Hepatic vein rT3 and 3,3'-T2 concentrations were 10.7 +/- 8.3% (mean +/- SD, P less than 0.005) and 36 +/- 18% (P less than 0.001) lower than those in the peripheral vein (N = 9). Renal vein rT3 was just (6.2 +/- 7.5%, P less than 0.05) lower than rT3 in peripheral vein, whereas 3,3'-T2 was not different between the two veins. Estimates of hepatic and renal plasma flow were in agreement with values from the literature. On the basis of these data approximate hepatic clearance rates of 110 and 380 1/day for rT3 and 3,3'-T2 and a renal clearance rate of about 35 1/day for rT3 were calculated. Sixty min after TRH, plasma T3 was increased to 147 +/- 56% (P less than 0.05) and 3,3'-T2 in peripheral plasma was increased to 142 +/- 36% (P less than 0.025), whereas plasma T4 and rT3 did not change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of thyroxine, 3,5,3'-triiodothyronine and 3,3'5'-triiodothyronine in euthyroid man

The secretion of iodothyronines from the normal human thyroid gland was assessed by radioimmunoassay analyses of the concentrations of thyroxine (T4), 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (reverse T3, rT3) in thyroid venous and peripheral venous blood. The subjects studied were euthyroid patients undergoing parathyroid surgery. Measurements were carried out both under apparently normal conditions, following peroral T3 pre-treatment, and before and after acute administration of TSH into a thyroid artery. In the control subjects, significant gradients between thyroid venous and peripheral venous concentrations were recorded both for T4, T3 and rT3, suggesting that all three iodothyronines are secreted by the normal human thyroid. T3 pre-treatment seemed to reduce this secretion, and acute administration of TSH promoted rapid, marked, and concomitant increments in the thyroid venous concentrations of all three iodothyronines. Hence, it appears that not only T4 but also T3 and rT3 are secreted by the normal human thyroid gland, and that TSH stimulates the secretion of all three iodothyronines. On the other hand, calculations of the relative secretion rates uielded the relation T4:T3:rT3 as 85:9:1. This indicates that, in euthyroid subjects, most of T3, and almost all of rT3, is produced by extrathyroidal conversion of T4 and not by direct thyroidal secretion.     

Targeted disruption of the type 1 selenodeiodinase gene (Dio1) results in marked changes in thyroid hormone economy in mice

The type 1 deiodinase (D1) is thought to be an important source of T3 in the euthyroid state. To explore the role of the D1 in thyroid hormone economy, a D1-deficient mouse (D1KO) was made by targeted disruption of the Dio1 gene. The general health and reproductive capacity of the D1KO mouse were seemingly unimpaired. In serum, levels of T4 and rT3 were elevated, whereas those of TSH and T3 were unchanged, as were several indices of peripheral thyroid status. It thus appears that the D1 is not essential for the maintenance of a normal serum T3 level in euthyroid mice. However, D1 deficiency resulted in marked changes in the metabolism and excretion of iodothyronines. Fecal excretion of endogenous iodothyronines was greatly increased. Furthermore, when compared with both wild-type and D2-deficient mice, fecal excretion of [125I]iodothyronines was greatly increased in D1KO mice during the 48 h after injection of [125I]T4 or [125I]T3, whereas urinary excretion of [125I]iodide was markedly diminished. From these data it was estimated that a majority of the iodide generated by the D1 was derived from substrates other than T4. Treatment with T3 resulted in a significantly higher serum T3 level and a greater degree of hyperthyroidism in D1KO mice than in wild-type mice. We conclude that, although the D1 is of questionable importance to the wellbeing of the euthyroid mouse, it may play a major role in limiting the detrimental effects of conditions that alter normal thyroid function, including hyperthyroidism and iodine deficiency.     

The effects of Soybean Diet on Thyroid Hormone and Thyrotropin Levels in Aging Rats

OBJECTIVE: To estimate the effect of soybean diet on serum level of thyroid hormone, its metabolites and thyrotropin (TSH) during aging in rats. METHODS: Male Donryu rats were fed laboratory chow containing 40 (Group A) or 10 volume percent (Group B) soybean protein, while controls (Group C) received regular laboratory chow. Groups of 10 animals of each groups were sacrificed by decapitation at the age of 12, 18, 24 and 30 months. Serum total thyroxine (T4), free thyroxine (FT4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine (3,3'-T2) and TSH concentrations were measured by specific radioimmunoassays. RESULTS: In Group A the level of T3 decreased significantly at from the age of 18 months, while in other groups such decrease was found only from the age of 24 months. Such changes were closely resembled by these in the level of 3,3'-T2, while inverse changes were observed in the level of rT3 which was increased in Group A from the age of 18 months and in the other groups from the age of 24 months. Serum T4 and FT4 level was decreased in all groups at the age of 30 months and no changes were observed in the level of TSH. CONCLUSIONS: The findings suggest that the level of T4, FT4 and T3 with its metabolite 3,3'-T2 stepwise decreased with aging, while that of rT3 showed inversely and increase. These changes were influenced by the content of soybean protein in the diet, the most rapid changes being found in the group with the high content of such protein.     

Monodeiodination of triiodothyronine and reverse triiodothyronine during low and high calorie diets

The impact of varying caloric intake on peripheral monodeiodination and plasma disposal of T3, rT3, and the three diiodothyronines (T2) was studied in five normal subjects while they were consuming a low calorie diet (1200 Cal/day) and again while receiving a high calorie diet (3600 Cal/day). Toward the end of each diet period 240 nmol 3,3'-T2 (126 micrograms) and 80 nmol 3',5'-T2 (42 micrograms) were infused for 7 h, and a bolus injection of 137 nmol 3,5-T2 (72 micrograms) was followed by a 12-h infusion of 69 nmol 3,5-T2 (36 micrograms) and 111 nmol rT3 (72 micrograms) on another day. [125I]T3 (30 muCi) was injected on the third day. The T2 and rT3 concentrations were measured by RIA during the 2 days of infusion, and the serum disappearance of [125I]T3 was studied by immunoprecipitation and trichloroacetic acid precipitation of the labeled T3. Four to 5% of the plasma disposal of T3 was accounted for by 3'-monodeiodination, and 36-39% by 5-monodeiodination. Increasing caloric intake resulted in a higher overall plasma disposal rate of T3, but no change in the percentage of T3 metabolized by monodeiodination pathways. In contrast, 5'-monodeiodination accounted for 21% of the total plasma disposal of rT3 during the low calorie diet and 45% during the high calorie intake. This increase in 5'-monodeiodination of rT3 was at the expense of alternative pathways of disposal. A marked increase in the plasma clearance rate of 3,5-T2 was also found during the high calorie diet, indicating that the level of caloric intake affects pathways of metabolism other than outer ring monodeiodination. These studies emphasize the important role played by diet in the regulation of peripheral thyroid hormone metabolism through modulating outer ring monodeiodination, and that overnutrition changes other pathways of iodothyronine metabolism as well.     

Euthyroid hyperthyroxinemia due to a generalized 5'-deiodinase defect

We studied an 11-yr-old girl with asymptomatic hyperthyroxinemia, who remained euthyroid and healthy for 5 yr of follow-up. Besides having elevated serum T4 concentrations, her serum free T4 concentrations were consistently elevated, as measured by three different methods, including equilibrium dialysis and ultrafiltration. Serum total and free T3 concentrations were in the low normal range, and serum 3,5-diiodothyronine (3,5-T2) levels were low, suggesting reduced 5'-deiodination of both T4 and T3. Serum total and free rT3 and total and free 3', 5'-T2 concentrations were all markedly elevated, whereas serum total and free 3,3'-T2 were low, suggesting unaltered 5-deiodination of T4 to rT3 and of rT3 to 3',5'-T2 in combination with reduced 5'-deiodination of rT3 and 3',5'-T2. The girl had a small diffuse goiter, her serum TSH response to TRH was exaggerated, and thyroid radioiodine uptake was elevated, suggesting slightly increased TSH secretion and, consequently, increased thyroid secretion. Both T3 and T4 administration resulted in suppressed basal as well as TRH-stimulated serum TSH concentrations, and radioiodine uptake was suppressed during T3 administration. Our data suggest reduced activity of several (all?) peripheral 5'-deiodination pathways, including possibly also thyrotroph T4 5'-deiodination. Thus, this girl seems to have a previously unrecognized syndrome of generalized 5'-deiodinase deficiency.     

Propranolol and thyroid hormone metabolism.

Propranolol decreases plasma T3 and increases plasma rT3 in a dose-dependent manner due to a decreased production rate of T3 and a decreased metabolic clearance rate of rT3, respectively, caused by inhibition of the conversion of T4 into T3 and of rT3 into 3,3'-T2. This inhibition of 5'-deiodination is not secondary to inhibition of thyroid hormone transport across the plasma membrane. Propranolol and its major metabolite, 4-hydroxypropranolol, are not directly responsible for these effects, but an unidentified metabolite of propranolol might be involved. beta-blockers ameliorate clinical symptoms and signs of thyrotoxicosis independent of the decrease of plasma 13, that is confined to beta-blockers with membrane-stabilizing activity, such as propranolol and alprenolol. The decrease of plasma T3, however, appears responsible for some of the metabolic responses to beta-blockers.