Altered serum levels of thyroxine, triiodothyronines and diiodothyronines in endogenous depression

Serum levels of thyroxine (T4), 3,3',5-triiodothyronine (T3), 3,3',5-triiodothyronine (rT3), 3,5-diiodothyronine (3,5-T2), 3,3'-diiodothyronine (3,3'-T2) and 3',5'-diiodothyronine (3',5'-T2) were studied in 80 patients with endogenous depression before and after electroconvulsive treatment (ECT). Compared to the values found after recovery, the patients when depressed had significant increased serum levels of T4, rT3, 3,3'-T2 and 3',5'-T2. Serum concentrations of T3 and 3,5-T2 were not significantly altered. Similarly the free T4 index (FT4I) was increased, while the free T3 index (FT3I) was unaffected. Previous studies have shown a reduced TSH response to TRH in patients with endogenous depression and that the long-term outcome after ECT is strongly related to changes in the TSH response. However, patients with increased TSH response to TRH (n = 23) had a pattern of serum iodothyronine concentrations similar to those (n = 57) with an unchanged TSH response. A similar pattern was also found in 7 patients with nonendogenous psychosis, in whom the TSH response to TRH was unchanged after recovery. It is concluded that the alterations of the TSH response to TRH found in endogenous depression cannot be explained by changes of FT4I or FT3I.     

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.     

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)     

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.     

Thyronamines are isozyme-specific substrates of deiodinases

3-Iodothyronamine (3-T 1 AM) and thyronamine (T AM) are novel endogenous signaling molecules that exhibit great structural similarity to thyroid hormones but apparently antagonize classical thyroid hormone (T(3)) actions. Their proposed biosynthesis from thyroid hormones would require decarboxylation and more or less extensive deiodination. Deiodinases (Dio1, Dio2, and Dio3) catalyze the removal of iodine from their substrates. Because a role of deiodinases in thyronamine biosynthesis requires their ability to accept thyronamines as substrates, we investigated whether thyronamines are converted by deiodinases. Thyronamines were incubated with isozyme-specific deiodinase preparations. Deiodination products were analyzed using a newly established method applying liquid chromatography and tandem mass spectrometry (LC-MS/MS). Phenolic ring deiodinations of 3,3',5'-triiodothyronamine (rT3AM), 3',5'-diiodothyronamine (3',5'-T2AM), and 3,3'-diiodothyronamine (3,3'-T2AM) as well as tyrosyl ring deiodinations of 3,5,3'-triiodothyronamine (T3AM) and 3,5-diiodothyronamine (3,5-T2AM) were observed with Dio1. These reactions were completely inhibited by the Dio1-specific inhibitor 6n-propyl-2-thiouracil (PTU). Dio2 containing preparations also deiodinated rT(3)AM and 3',5'-T2AM at the phenolic rings but in a PTU-insensitive fashion. All thyronamines with tyrosyl ring iodine atoms were 5(3)-deiodinated by Dio3-containing preparations. In functional competition assays, the newly identified thyronamine substrates inhibited an established iodothyronine deiodination reaction. By contrast, thyronamines that had been excluded as deiodinase substrates in LC-MS/MS experiments failed to show any effect in the competition assays, thus verifying the former results. These data support a role for deiodinases in thyronamine biosynthesis and contribute to confining the biosynthetic pathways for 3-T 1 AM and T 0 AM.     

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.     

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.     

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.     

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.     

Metabolism of triiodothyronine in rat hepatocytes

The metabolism of T3 by isolated rat hepatocytes was analyzed by Sephadex LH-20 chromatography, HPLC, and RIA for T3 sulfate (T3S) and 3,3'-diiodothyronine (3,3'-T2). Type I iodothyronine deiodinase activity was inhibited with propylthiouracil (PTU), and phenol sulfotransferase activity by SO4(2-) depletion or with competitive substrates or inhibitors. Under normal conditions, labeled T3 glucuronide and I- were the main products of [3'-125I]T3 metabolism. Iodide production was decreased by inhibition (PTU) or saturation (greater than 100 nM T3) of type I deiodinase, which was accompanied by the accumulation of T3S and 3,3'-T2S. Inhibition of phenol sulfotransferase resulted in decreased iodide production, which was associated with an accumulation of 3,3'-T2 and 3,3'-T2 glucuronide, independent of PTU. Formation of 3,3'-T2 and its conjugates was only observed at T3 substrate concentrations below 10 nM. Thus, T3 is metabolized in rat liver cells by three quantitatively important pathways: glucuronidation, sulfation, and direct inner ring deiodination. Whereas T3 glucuronide is not further metabolized in the cultures, T3S is rapidly deiodinated by the type I enzyme. As confirmed by incubations with isolated rat liver microsomes, direct inner ring deiodination of T3 is largely mediated by a low Km, PTU-insensitive, type III-like iodothyronine deiodinase, and production of 3,3'-T2 is only observed if its rapid sulfation is prevented.     

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.     

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.     

Cloning and characterization of type III iodothyronine deiodinase from the fish Oreochromis niloticus.

Type III iodothyronine deiodinase (D3) catalyzes the inner ring deiodination (IRD) of T4 and T3 to the inactive metabolites rT3 and 3,3'-diiodothyronine (3,3'-T2), respectively. Here we describe the cloning and characterization of complementary DNA (cDNA) coding for D3 in fish (Oreochromis niloticus, tilapia). This cDNA contains 1478 nucleotides and codes for a protein of 267 amino acids, including a putative selenocysteine (Sec) residue, encoded by a TGA triplet, at position 131. The deduced amino acid sequence shows 57-67% identity with frog, chicken, and mammalian D3, 33-39% identity with frog, fish (Fundulus heteroclitus) and mammalian D2, and 30-35% identity with fish (tilapia), chicken, and mammalian D1. The 3' UTR contains a putative Sec insertion sequence (SECIS) element. Recombinant tilapia D3 (tD3) expressed in COS-1 cells and native tD3 in tilapia brain microsomes show identical catalytic activities, with a strong preference for IRD of T3 (Km approximately 20 nM). IRD of [3,5-125I]T3 by native and recombinant tD3 are equally sensitive to inhibition by substrate analogs (T3 > T4 > rT3) and inhibitors (gold thioglucose > iodoacetate > propylthiouracil). Northern analysis using a tD3 riboprobe shows high expression of a 1.6-kb messenger RNA in gill and brain, although D3 activity is much higher in brain than in gill. The characterization of tD3 cDNA provides new information about the structure-activity relationship of iodothyronine deiodinases and an important tool to study the regulation of thyroid hormone bioactivity in fish.     

Effect of 3,5,3'-triiodothyronine-induced hyperthyroidism on iodothyronine metabolism in the rat: evidence for tissue differences in metabolic responses

We studied the effect of T3-induced hyperthyroidism on the outer ring (5' or 3') monodeiodination of T4 (to T3) and 3',5'-diiodothyronine [3',5'-T2; to 3'-monoiodothyronine (3'-T1)] and on the inner ring (3 or 5) monodeiodination of 3,5-T2 (to 3-T1) by various rat tissues. Weight-matched pairs of male Sprague-Dawley rats were given either saline or T3 (20 micrograms/100 g BW daily) ip for 3 days. The metabolism of the iodothyronines was studied on day 4 in homogenates of the tissues in the presence of 25 mM dithiothreitol. Hyperthyroidism was associated with a significant (P less than 0.05) increase in T4 to T3 monodeiodinating activity in the liver (mean, 95%), kidney (mean, 60%), and heart (mean, 153%), but not in skeletal muscle, small intestine, spleen, testis, cerebral cortex, or cerebellum. The monodeiodinating activity converting 3',5'-T2 to 3'-T1 was greatly increased (P less than 0.05) in the heart (mean, 750%), spleen (mean, 462%), and skeletal muscle (mean, 167%), but not in liver, kidney, small intestine, testis, cerebral cortex, or cerebellum. In the case of liver and kidney, however, there was evidence of an activation of 3',5'-T2 monodeiodinating activity, as suggested by a significant increase in the activity in the absence of added dithiothreitol. The monodeiodination of 3,5-T2 to 3-T1 increased significantly only in the cerebral cortex (mean, 525%) and liver (mean, 69%) and not in any other tissue. The time course of the above-mentioned changes in iodothyronine metabolism was studied in groups of rats (five per group) given T3 (20 micrograms 100 g BW-1 day-1) 6-72 h before death. Significant increases in 3',5'-T2 (to 3'-T1) monodeiodination in the heart and 3,5-T2 (to 3-T1) monodeiodination in the cerebral cortex were evident within 6 h of T3 administration. Changes in T4 to T3 monodeiodinating activity in the kidney and liver, however, did not become statistically significant until 24 and 72 h, respectively. The various effects of T3 on the tissues became maximal between 48 and 72 h after the initiation of T3 treatment. Our data suggest that most tissues, including some that have been considered unresponsive to thyroid hormones, e.g. brain and spleen, demonstrate substantial metabolic changes after T3 administration. The tissue responses are variable in degree; in some instances, they are specific for the substrate and type of tissue.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of triiodothyroacetic acid (TA3) in rat liver. I. Deiodination of TA3 and TA3 sulfate by microsomes

The deiodination of the acetic acid side-chain analogs of T3 as well as 3,3'-diiodothyronine (3,3'-T2) was investigated by incubating 125I-labeled 3,3',5-triiodothyroacetic acid (TA3) and 3,3'-diiodothyroacetic acid (3,3'-TA2) with rat liver microsomes at 37 C and pH 7.2 in the presence of 5 mM dithiothreitol. TA3 sulfate (TA3S) and 3,3'-TA2S were also tested as substrate since sulfation is known to accelerate T3 and 3,3'-T2 conversion. Reaction products were analyzed on Sephadex LH-20 and HPLC. TA3 underwent only inner ring deiodination (IRD), but 3,3'-TA2 was equally converted by IRD and outer ring deiodination (ORD). TA3S was metabolized very rapidly by IRD to 3,3'-TA2S which was only observed transiently due to its rapid deiodination predominantly in the outer ring. Kinetic studies under initial reaction rate conditions yielded apparent Michaelis-Menten (Km) values (micromolar) of 1.8 for TA3, 0.8 for 3,3'-TA2, and 0.004 for TA3S, and 0.02 for 3,3'-TA2S and Vmax values (picomoles per min/mg protein) of 174 for TA3, 49 for 3,3'-TA2, 21 for TA3S, and 63 for 3,3'-TA2S. The Vmax/Km ratios for the IRD of TA3 and TA3S were 16 and 930 times higher, respectively, relative to T3. Deiodinations were sensitive to propylthiouracil inhibition, indicating the involvement of the type I iodothyronine deiodinase. Furthermore, the iodothyroacetic acid derivatives competitively inhibited the ORD of rT3 with apparent inhibition constant (Ki) values (0.45 microM for TA3, 4 nM for TA3S, and 0.04 microM for 3,3'-TA2S) in agreement with corresponding Km values. We conclude that 1) TA3 and 3,3'-TA2 are better substrates than T3 and 3,3'-T2 for the type I deiodinase of rat liver; 2) the IRD of TA3 and ORD of 3,3'-TA2 are markedly enhanced by sulfation similar to the parent iodothyronines; and 3) TA3S in the best known substrate for IRD due to its very high affinity for the type I deiodinase.     

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)     

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.     

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)     

Acute effects of thyroid hormone analogs on sodium currents in neonatal rat myocytes.

We previously reported that T3(3,3',5-triiodo-L-thyronine) acutely increases sodium currents (INa) in neonatal rat myocytes. Here we compare the effects of several thyroid hormone analogs, including T4(3,3',5,5'-tetraiodo-L-thyronine), rT3(3,3',5'-triiodo-L-thyronine), D-T3(3,3',5-triiodo-D-thyronine), 3,5-T2(3,5-diiodo-L-thyronine), DIT (3,5-diiodo-L-tyrosine), MIT (3-monoiodo-L-tyrosine), tetrac (3,3',5,5'-tetraiodo-thyroacetic acid), triac (3, 3',5-triiodo-thyroacetic acid), and tyrosine, on INa in cultured neonatal rat myocytes (n ranged from 9 to 28 for each comparison). T4, T3, 3,5-T2, and DIT (10 n m) all increased current density relative to control to a similar degree: to 1.22+/-0.2, 1.21+/-0.03, 1.16+/-0.02 and 1.16+/-0.03, respectively, P<0.05. In contrast, thyroid hormone analogs with an altered side group of the inner iodophenyl ring, including tetrac, triac, and D-T3, had no effect on INa nor did rT3, MIT or tyrosine. Pretreatment with rT3 inhibited the effects of T4, T3, 3,5-T2, and DIT. Conversely, the dose-dependent inhibitory effect of amiodarone, an iodinated benzofuran derivative that antagonizes thyroid hormone actions, on INa was blocked when myocytes were pretreated with T3(100 n m, n=3), suggesting an interaction of T3 with amiodarone. The enhancement of INa by T3 and 3, 5-T2 could not be blocked by propranolol, suggesting that the effects are not mediated through beta -adrenergic signaling pathways. In conclusion, the present results suggest that the acute effects of thyroid hormone and analogs on cardiac INa are mediated by a non-genomic thyroid hormone receptor with a unique structure-activity relationship.     

Effect of adrenaline on biliary excretion of triiodothyronines in rats mediated by alpha 1-adrenoceptors and related to the inhibition of 5'-monodeiodination in liver

Biliary excretion of thyroxine (T4),3,5,3'-triiodothyronine (T3),3,3,5'-triiodothyronine (rT3) and diiodothyronines (3,3'-T2,3,5-T2 and 3',5'-T2) was estimated with the aid of radioimmunoassay in 3-4 subsequent 2-h samples of bile obtained from pentobarbital anesthetized rats through the tubing inserted in bile duct. The excretion of T3 was significantly decreased during 4-h infusion of 2400 ng/kg/min adrenaline in normal rats or during 6-h infusion of the latter dose in the animals preinjected with 2 micrograms T4. Moreover, the excretion of rT3 was significantly increased after the infusion of 1200 and 2400 ng/kg/min adrenaline. Such increase after 1200 and 2400 ng/kg/min adrenaline was prevented by a single dose of 10 mg/kg phentolamine (alpha 1-2-antagonist) and that after 2400 ng/kg/min adrenaline also by 2.5 mg/kg prazosin (alpha 1-antagonist) injected at the beginning of the infusion, but not by 6 mg/kg yohimbine (alpha 2-antagonist) injected every 60 min during 4-h infusion. In addition, increased rT3 excretion was found during the infusion of alpha 1-agonist methoxamine (1.5 mg/kg/4 h), while no such effect of the infusion of alpha 2-agonist azepexol (10 mg/kg/4 h) was observed. It may be suggested that the effect of adrenaline was mediated predominantly by alpha 1-adrenoceptors and that the observed changes in biliary excretion of T3 and rT3 were related to the inhibition of 5'-monodeiodination in the liver.