Proton nuclear magnetic resonance assignments of thyroid hormone and its analogues

1H NMR data of a series of thyroid hormone analogues, e.g., thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), 3-monoidothyronine (3-T1), 3'-monoiodothyronine (3'-T1), and thyronine (TO) in dimethylsulfoxide (DMSO) have been obtained on a 300 MHz spectrometer. The chemical shift and coupling constant are determined and tabulated for each aromatic proton. The inner tyrosyl ring protons in T4, T3, and 3,5-T2 have downfield chemical shifts with respect to those of the outer phenolic ring protons. Four-bond cross-ring coupling has been observed in all the monoiodinated rings. However, this long-range coupling does not exist in T4, diiodinated on both rings, and T0, containing no iodines on the rings. There is no evidence that at 30 degrees C these iodothyronines have any motional constraint in DMSO solution. In addition to identification of the hormones, the potential use of some characteristic peaks as probes in binding studies is discussed.     

Serum concentrations of 3, 3'-diiodothyronine, 3', 5'-diiodothyronine, and 3, 5-diiodothyronine in altered thyroid states

To investigate the thyroid hormone metabolism in altered states of thyroid function, serum concentrations of 3, 3'-diiodothyronine (3, 3'-T2), 3', 5'-T2 and 3, 5-T2 as well as T4, T3 and rT3 were determined by specific radioimmunoassays in 17 hyperthyroid and 10 hypothyroid patients, before and during the treatment. Serum T4, T3, rT3, 3, 3'-T2 and 3', 5'-T2 concentrations were all higher in the hyperthyroid patients than in age-matched controls and decreased to the normal ranges within 3 to 4 months following treatment with antithyroid drugs. In the hypothyroid patients, these iodothyronine concentrations were lower than in age-matched controls and returned to the normal ranges after 2 to 3 months treatment with T4. In contrast, serum 3, 5-T2 concentrations in hyperthyroid patients (mean +/- SE : 4.0 +/- 0.5 ng/dl) were not significantly different from those in controls (3.9 +/ 0.4 ng/dl), although they tended to decrease in 3 of 6 patients after the antithyroid drug therapy. Serum 3, 5-T2 levels in the hypothyroid patients (3.8 +/- 0.6 ng/dl) were also within the normal range and showed no significant change following the T4 replacement therapy. However, serum 3, 5-T2 as well as 3, 3'T2 concentrations rose significantly with a marked rise in serum T3 following T3 administration, 75 micrograms/day for 7 days, in Graves' patients in euthyroid state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Existence of extrathyroidal conversion inhibitor (IEC) in starved animals and its influence on thyroid hormones deiodination in liver and kidney in vitro

To find out whether an inhibitor of extrathyroidal conversion of iodothyronines is present in sera of starved animals, pig liver and kidney homogenates were incubated with T4, T3 or rT3 and dithiotreitol in the presence of evaporated diethyl ether extracts of sera obtained from fed and starved (1-12 days) rabbits. Sera extracts of short-term (1-4 days) starved rabbits caused a significant inhibition of T4 to T3 conversion (54% on day 3) and T4 to rT3 deiodination (52% on day 2) in liver homogenates. Extracts of sera from long-term (8 and 12 days) starved animals diminished only liver T4 to T3 conversion on day 8 and had no influence on liver T4 to rT3 conversion. 5'-deiodination of rT3 (to 3,3'-T2) in liver was gradually decreased by extracts of sera from animals starved during 2-12 days. Liver rT3-5-deiodination (to 3',5'-T2) was significantly impaired on day 4 and totally depressed by long-term starvation. In vitro T3 to 3,3'-T2 conversion in liver was markedly (59-103%) increased by ether extracts of sera from short-term fasted rabbits and considerably inhibited (62-72%) by long-term fasting. T4 to T3 conversion in kidney was significantly influenced by sera extracts obtained neither from short-term fasted rabbits and considerably inhibited (62-72%) by long-term fasting. T4 to T3 conversion in kidney was significantly influenced by sera extracts obtained neither from short-term nor from long-term fasted rabbits but T4-5-deiodination (to rT3) was reduced by sera extracts of short-term fasted animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of streptozotocin-induced diabetes mellitus on hypothalamic-pituitary-thyroid axis in rats

The effects of streptozotocin-induced diabetes mellitus on the hypothalamic-pituitary-thyroid axis in rats were studied. Streptozotocin (60 mg/kg) was injected ip. Rats were decapitated at two and four weeks after the streptozotocin treatment. Thyrotropin releasing hormone (TRH), thyrotropin (TSH), thyroxine (T4), 3,3',5-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2) and 3',5'-diiodothyronine (3',5'-T2) were measured by means of the specific radioimmunoassay for each. Immunoreactive TRH (ir-TRH) contents in the hypothalamus significantly decreased at four weeks (p less than 0.02). Basal TSH levels in plasma significantly decreased (p less than 0.005, p less than 0.001), and plasma ir-TRH and TSH responses to cold were significantly inhibited after the streptozotocin treatment (p less than 0.001). The plasma TSH response to TRH was decreased, but not significantly. The plasma T4 and T3 levels fell significantly. RT3 did not change throughout the experiment. 3,3'-T2 levels in plasma fell significantly, whereas 3',5'-T2 increased. Blood glucose levels rose significantly after streptozotocin treatment, but insulin treatment led to partial restoration. The findings suggest that streptozotocin-induced diabetes mellitus affects various sites of the hypothalamic-pituitary-thyroid axis in rats.     

pH-dependency of iodothyronine metabolism in isolated perfused rat liver.

1. Isolated livers from fed male rats were perfused for 2 h with T4 (L-thyroxine), T3 (L-3,3',5-tri-iodothyronine) or rT3 (L-3,3',5'-tri-iodothyronine) at different pH values (7.1--7.6) in a fully synthetic medium, whereby normal metabolic functions were maintained without addition of rat blood constituents or albumin. 2. T3 output into the medium and net T3 production reached a maximum at a pH of the medium of 7.2 and significantly decreased with alteration of the pH when livers were perfused with T4 as a substrate. 3. However, the net T4 and T3 uptake by the liver, as well as the hepatic T4 and T3 content after perfusion, were not dependent on the pH of the perfusion when livers were offered T4 or T3 as substrates respectively. 4. Determination of intracellular pH by the analysis of the distribution of the weak acid dimethyloxazolidinedione allows the conclusion that the pH optimum of iodothyronine 5'-deiodinase in the intact perfused liver corresponds to the maximum determined in vitro for the membrane-bound enzyme localized in the endoplasmic reticulum. 5. The rapid 5'-deiodination of rT3 to 3,3'-T2 (L-3,3'-di-iodothyronine), the fast disappearance of 3,3'-T2, and the fact that no net rT3 production from T4 could be detected, supports the hypothesis that in rat liver iodothyronine 5'-deiodinase activity seems to predominate over iodothyronine 5-deiodinase activity. 6. Thus the rat liver can be considered in normal physiological situations as an organ forming T3 from T4 and deiodinating rT3 originating from extrahepatic tissues, whereby the cellular iodothyronine 5'-deiodination rate is controlled by the intracellular pH.     

Increase of biliary excretion of reverse triiodothyronine in rats during the infusion of neurotensin possibly resulting from the inhibition of iodothyronine 5'-monodeiodination.

Male rats weighing about 350 g were inserted polyethylene tubes into the bile duct and femoral vein under pentobarbital anaesthesia. After taking the first (control) 2-h bile sample the control group (n = 24) was infused saline for 4 h and the other group (n = 14) was infused neurotensin in a dose of 27 micrograms per animal per 4 h. The concentration of thyroxine (T4), triiodothyronine (T3) and reverse triiodothyronine (rT3) in the bile was estimated by radioimmunoassay. No significant differences between groups were found in the biliary excretion of T4 and T3, while the excretion of rT3 after the infusion of neurotensin was significantly increased which was not the case in controls. Since neurotensin is known to increase glycemia which effect might be or might not be mediated by glucagon, it may be suggested that these results bring an additional support for the previously reported coincidence between a prevailing effect of gluconeogenetic hormones and inhibition of iodothyronine 5'-deiodination in the liver.     

Bile acids and bile alcohols in a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency: effects of chenodeoxycholic acid treatment

Duodenal bile, urine, plasma, and feces from a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency were analyzed by fast atom bombardment mass spectrometry and gas chromatography-mass spectrometry to investigate the formation and excretion of abnormal bile acids and bile alcohols. The biliary bile salts consisted of glycocholic acid (25%) and of sulfated and glycine conjugated di- and trihydroxycholenoic acids (55%), two C27 bile acids, and eleven sulfated bile alcohols (mainly tetrols, 20%), all having 3 beta,7 alpha-dihydroxy-delta 5 or 3 beta,7 alpha,12 alpha-trihydroxy-delta 5 ring structures. In plasma, sulfated cholenoic acids constituted 65% and unconjugated 3 beta,7 alpha-dihydroxy-5-cholestenoic acid 25% of the total level, 71 micrograms/ml. The urinary excretion of the former was 30.4 mg/day and that of unsaturated bile alcohol sulfates, mainly pentols, 7 mg/day. The predominant bile acid in feces was an unconjugated epimer of 3 beta,7 alpha,12 alpha-trihydroxy-5-cholenoic acid, and small amounts of cholic acid were present. The minimum total excretion was 11.3 mg/day. Treatment with chenodeoxycholic acid resulted in marked clinical improvement and normalized liver function tests. Further studies are needed to define the mechanism of action. Plasma bile acids decreased to 1.6 micrograms/ml and urinary excretion to 3.4 mg/day. Chenodeoxycholic and ursodeoxycholic acids became predominant in all samples. The fecal excretion of unsaturated cholenoic acid sulfates increased to 40 mg/day compared to 89 mg/day of saturated bile acids. The results provide further support for a defective hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency, and indicate that the 3 beta-hydroxy-delta 5 bile acids are formed via 7 alpha-hydroxycholesterol. The formation of glycocholic acid may be due to an incomplete enzyme defect or to transformation of the 3 beta-hydroxy-delta 5 structure by bacterial and hepatic enzymes during an enterohepatic circulation.     

The effect of iodothyronines on the conversion of thyroxine into 3,3''-5-tri-iodothyronine in isolated rat renal tubules.

Isolated rat renal tubules prepared by collagenase digestion were used to study the effects of 3,3',5'-tri-iodothyronine ('reverse T3', rT3) and other iodothyronines on the formation of 3,3',5-tri-iodothyronine (T3) from thyroxine (T4). rT3 inhibited the conversion with a dose response over the concentration range 1.5nM-1.5microM. The inhibition was competitive in nature. Both 3,3'-di-iodothyronine and 3',5'-di-iodothyronine also inhibited the production of T3 and T4 in isolated rat renal tubules, but tetraiodothyroacetic acid and 3,5-di-iodothyronine were found to have no effect. These experiments demonstrate in an intact cell system that some naturally occurring iodothyronines have significant effects on T4 deiodination.     

Sulfation of iodothyronines by recombinant human liver steroid sulfotransferases.

Sulfation is an important pathway in the metabolism of thyroid hormones. Sulfated iodothyronines are elevated in nonthyroidal illnesses and in the normal human fetal circulation. We assayed and characterized COS-1 cell expressed recombinant human liver dehydroepiandrosterone sulfotransferase (DHEA ST or SULT2A1) and estrogen sulfotransferase (EST or SULT1E1) activities for the first time with triiodothyronine (T(3)) as the substrate. Several biochemical properties that included apparent K(m) values, thermal stabilities, and responses to the inhibitors 2, 6-dichloro-4-nitrophenol and NaCl were tested. SULT2A1, a member of the hydroxysteroid sulfotransferase family, used 3,3'-T(2) more readily than T(3) and 3,5-T(2) as substrates, but had the lowest apparent K(m) value for T(3) of any reported human SULT. SULT1E1, a member of the phenol sulfotransferase family, used 3,3'-T(2) and rT(3) more readily than T(3), and also displayed the greatest specificity for T(4) among human SULTs. SULT2A1 may contribute more to iodothyronine sulfation than previously suspected. Potential roles of both steroid sulfotransferases in the enhanced sulfation of nonthyroidal illnesses and fetal development invite further investigation.     

Placental outer and inner ring monodeiodination of thyroxine and triiodothyronines in the rabbit

Both inner- and outer-ring iodothyronines deiodinating activity was found in homogenates of rabbit placentas. The T4 to rT3 and T3 to 3,3'-T2 deiodinating activity was already high on day 10 before delivery but decreased being about 7 times lowered on day 5. Once the T3 to 3,3'-T2 monodeiodination reached a low and a relatively steady level, the outer ring deiodination of T4 begun, reaching a peak value at about day 3 before term and then fell again. The fetal serum thyroid hormones levels were low, showing no significant variability during the period of observation. The results suggested that in the rabbit, representing animals in which the thyroid gland activity begins early in fetal life, there are two distinct phases of the placental monodeiodinating activity. The first is characterized by a high inner-ring deiodinating activity (yielding rT3) and is followed by the second phase with a high outer-ring deiodinating activity (yielding T3) declining just before term.     

rT3 production in normal man, assessed from variations in serum rT3 during short-term rT3 infusion.

The metabolic clearance rate (MCR) of 3,3'5'-triiodothyronine (reverse T3, rT3) was estimated in normal human subjects by a modified noncompartmental method using the integrated increase in serum rT3 following intravenous infusion of 0.10 nmol/min rT3 for 4 hr. The MCR-rT3 was calculated to be 102.8 +/- 17.01/day and the daily rT3 disposal to be 33.0 +/- 9.5 nmol (mean +/- SD, n = 6). The MCR-rT3 compares well to that of previous studies employing tracer kinetic methods. The disposal rate of rT3 estimated in the present study is considerably lower than found in some previous studies. The discrepancy is due to differences in the measured levels of serum rT3 in normal subjects.     

The effect of extrathyroidal conversion inhibitor from food-deprived animals on iodothyronines deiodination by pituitary and cerebral cortical homogenates in vitro

The influence of an inhibitor of iodothyronines' extrathyroidal conversion on T4, T3 and rT3 deiodination by adult pig pituitary and cerebral cortical homogenates has been investigated. The homogenates were incubated with T4, T3 and rT3 in the presence of 5 mM dithiothreitol and evaporated diethyl ether extracts of sera obtained from fed and starved (1-14 days) rabbits. The extracts had no influence either on T4 to T3 or on T4 to rT3 conversion in cerebral cortex. Deiodination of rT3 to 3,3'-T2 in that tissue was significantly inhibited only by the extracts of sera obtained from 4 days starved rabbits. Inner-ring deiodination of both rT3 and T3 was not changed by the extracts got from short-term (1-4 days) fasted animals but was significantly reduced by the extracts from long-term (7-14 days) food-deprived subjects. Pituitary conversion of T4 to T3 was diminished by 35% in the presence of sera extracts gained from 1-9 days fasted rabbits and by about 50% on day 14 of fasting, but only the latter change was statistically significant. Short-term fasting inhibited T4 to rT3 conversion on days 2 and 4. Both deiodinations of rT3 and 5-deiodination of T3 were affected by extracts of sera collected during long-term fasting.     

Abnormal urinary excretion of unsaturated dicarboxylic acids in patients with medium-chain acyl-CoA dehydrogenase deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently described metabolic disorder of fatty acid oxidation in humans. Acute episodes are usually characterized biochemically by the appearance of nonketotic dicarboxylic aciduria. In addition, other abnormal metabolites, such as suberylglycine, n-hexanoylglycine, 3-phenylpropionylglycine, and octanoylcarnitine, are excreted in the urine. Urinary organic acids were determined using dual capillary column gas-liquid chromatography and gas-liquid chromatography/mass spectrometry. In three cases of MCAD deficiency we observed a disproportionate increase in the excretion of unsaturated dicarboxylic acids compared to either fasting control children with expected ketotic dicarboxylic aciduria or patients with nonketotic dicarboxylic aciduria not associated with MCAD deficiency. The most significant increase was in the urinary excretion of cis-4-decendioic acid. Additionally, the urinary excretions of cis-3-octenedioic and cis-5-decenedioic acids were slightly decreased whereas the excretion of cis-5-dodecenedioic acid was increased. These data are consistent with the notion that as a result of MCAD deficiency the metabolic oxidation of unsaturated fatty acids such as linoleate and oleate is inhibited more than saturated fatty acids.     

Alterations in fatty acid composition and trehalose concentration ofSaccharomyces brewing strains in response to heat and ethanol shock

Summary The effects of heat and ethanol shock on fatty acid composition and intracellular trehalose concentration of lager and ale brewing yeasts were examined. Exposure of cells to heat shock at 37°C or 10% (v/v) ethanol for 60 min resulted in a significant increase in the ratio of the total unsaturated to saturated fatty acyl residues and the intracellular trehalose concentration of cells. A similar increase in the amount of unsaturated fatty acids was observed in cells after 24 h of fermentation of 16°P (degree Plato) or 25°P wort, at which time more than 2% (v/v) ethanol was present in the growth medium. These results suggest that unsaturated fatty acids and high concentrations of intracellular trehalose may protect the cells from the inhibitory effects of heat and ethanol shock.     

Measurement of thyroid hormone concentrations in human placenta

Concentrations of thyroxine (T4), 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) in the placenta were measured in 7 patients with abortion, in 9 patients with premature delivery, in 16 normal pregnancies and in 4 pregnant women with Graves' disease. The placentas, obtained at delivery, were homogenized and centrifuged at 800 X g. T4, T3 and rT3 concentrations in the supernatants were extracted with 3 vol. of 99% ethanol and measured by RIAs. In normal pregnancy, placental T4, T3 and rT3 concentrations were 18.8 +/- 5.9 (mean +/- SD), 0.026 +/- 0.012, and 1.70 +/- 0.49 ng/g tissue, respectively. Ratios of rT3/T3 and rT3/T4 in the placenta were about 12 and 2.3 times as high as those in the fetal sera, respectively. There was a significant positive correlation between the placental T4 and the maternal or cord serum T4 concentrations. However, no correlation was found between the placental T3 or rT3 concentrations and the maternal or cord T3 or rT3 concentrations. In 4 patients with Graves' disease, the placental T4 concentration was elevated. These results indicate that the placental T4 concentration is influenced by both the maternal and fetal serum T4, and elevated ratios of rT3/T3 and rT3/T4 in the placenta might be due to the active placental 5-monodeiodination.     

Mechanism of action of iodothyronine-5'-deiodinase.

Production of 3,3'-di-iodothyronine (3,3'-T2) from 3,3',5'-tri-iodothyronine (reverse T3, rT3) as catalysed by rat liver microsomal fraction was measured with a specific radioimmunoassay. The effect of the addition of 2-thiouracil and of varying concentrations of cofactor (dithiothreito) on the kinetic parameters of this reaction were studied. It was found that thiouracil is an uncompetitive inhibitor with respect to substrate and a competitive inhibitor with respect to cofactor. The effect of a decrease in the concentration of cofactor was similar to the effect of addition of thiouracil, i.e. a proportional decrease in Km and V. The results strongly suggest that enzymatic 5'-deiodination of iodothyronines follows a ping-pong mechanisms, which may be envisaged as a transiodination and the subsequent reduction of the iodo-enzyme complex by cofactor. The intermediate is probably a sulfenyl iodide form of the enzyme, which reacts with thiouracil to yield a mixed disulfide.     

Peroxidatic degradation and ether link cleavage of thyroxine in a particulate fraction of human thyroid

The present study was undertaken to investigate degradation of thyroxine (T4) mediated by thyroid peroxidase in man. A particulate fraction (1,000-100,000 x g) of normal human thyroid tissue was prepared and used as crude enzyme. 125I-T4 and unlabeled T4 were incubated with the particulate fraction in buffer containing glucose and glucose oxidase for generation of H2O2. After incubation, iodoamino acids were extracted with ethanol and the products of T4 degradation were analyzed by thin layer chromatography. In this system, T4 was degraded in time-, temperature- and pH-dependent manners, but not in the absence of the H2O2-generating system. The rate of degradation was related to concentration of the particulate fraction. The reaction was inhibited by methimazole, propylthiouracil and catalase. When [3',5'-125I] T4 was used as a tracer, major labeled products of T4 degradation were inorganic iodide and ethanol-unextracted fraction and no detectable labeled 3,5,3'-triiodothyronine (T3) or 3,3',5'-triiodothyronine (rT3) was generated. From a kinetic study by adding various doses of unlabeled T4, the apparent Km value for T4 was 30 microM and the Vmax value was 230 pmol/mg protein/min. When [3,5-125I] T4 was incubated with enzyme preparation, one third of degraded T4 was recovered as diiodotyrosine (DIT) and half of 125I-DIT was degraded in parallel incubation. No formation of radiolabeled DIT was observed in incubation with Na- 125I done in tandem. These findings suggest that thyroid hormones can be metabolized by peroxidase in human thyroid by pathways that include cleavage of ether linkage.     

Effects of ethanol on the lipid composition of bovine vascular smooth muscle cells in culture

Ethanol (50 mM) had no effect on the growth rate or viability of arterial smooth muscle cells over 3.5 days. The cholesterol:phospholipid ratio of the cells was unchanged after 7 days exposure. The major phospholipid components phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were unchanged by ethanol exposure. Sphingomyelin content fell significantly within 12 hr. There were major changes in the fatty acid composition of the phospholipids with a reduction in saturated fatty acids and an increase in unsaturated fatty acids.     

Thyroid hormones and their effects: a new perspective

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