A radioimmunoassay for measurements of 3,3'-diiodothyronine (3,3'-T2) in unextracted serum is described. Antisera to 3,3'-T2 were produced by immunization of rabbits with 3,3'-T2 coupled to human serum albumin. Scatchard plot analysis of the chosen antiserum gave an effective equilibrium constant of 1.1x 10 11 l/mol. The 3,3'-T2 binding capacity in undiluted serum was calculated to be 9.1 mmol/m. Monolabelled ( 125 I)3,3'T2 having a specific activity of 3800 mCi/mg was synthetized from 3-monoiodothyronine. The lowest concentration of standard inducing a consistent fall in per cent bound ( 125 I)3,3'-T2 was 6.0 pmol/l (0.24fmol/tube). Triiodothyronine (T3) cross-reacted significantly in the assay. At 50% inhibition of binding of ( 125 I)3,3'-T2, the cross-reaction was 0.29% (mol/mol), but at the low physiological concentrations of T3 it amounted to approximately 1%. T3 cross-reaction was corrected for in every assay. The interference of 3,3'-T2 binding proteins in serum was precluded by adding 8-anilino-1-napthalenesulphonic acid (ANS). Mean 3,3'-T2 concentrations were 38.9 pmol/l in fourteen normal young females and 48.9 pmol/l in fifteen normal young males. When 3,3'-T2 was measured(1) in extracts of serum, (2) in assays where T3 was added to standards in amounts equivalent to those present in the samples and T3 correction omitted, and (3) using a different 3,3'-T2 antibody, similar levels of 3,3'-T2 were found in normal serum. (Auth.)
Burger, A.; Sakoloff, C.
A specific radioimmunoassay for 3,3'-diiodothyronine (T 2 ) is described which is capable of detecting as little as 1.3 ng/dl. The antiserum recognizes mainly T 2 ; biliary conjugates of T 2 bind slightly to the antibody. The intraassay and interassay coefficients of variation were, respectively, 5.7% and 13.1%. T 2 was detected in the serum of hypothyroid patients treated with triiodothyronine (T 3 ) and in euthyroid subjects treated with reverse triiodothyronine (rT 3 ). These results suggest that both T 3 and rT 3 are deiodinated to T 2 . Serum concentrations of T 2 in normal subjects decreased with age. Between 20 and 40 years the mean concentration was 4.3 +- 2.0 ng/dl (2 SD), between 41 and 60 years it varied from 1.9 to 5.8 ng/dl (3.8 +- 0.3 ng/dl, SE) and in elderly subjects have 60 years concentrations varied from unmeasurable to 4 ng/dl (2.9 +- 0.4 ng/dl, SE). Low serum T 2 concentrations were found in anorexia nervosa (2.5 +- 0.3 ng/dl, SE). In hypothyroidism the serum concentrations were low or unmeasurable. As most of the hypothyroid subjects were elderly their serum T 2 concentrations overlapped with the low values found in the elderly euthyroid subjects. In classical hyperthyroidism serum T 2 concentrations were greatly increased (3.3 to 31 ng/dl (11.8 +- 2.7 ng/dl, SE) but in ''T 3 toxicosis'' the concentrations were only modestly increased
Padron, Alvaro Souto; Neto, Ruy Andrade Louzada; Pantaleão, Thiago Urgal; de Souza dos Santos, Maria Carolina; Araujo, Renata Lopes; de Andrade, Bruno Moulin; da Silva Leandro, Monique; de Castro, João Pedro Saar Werneck; Ferreira, Andrea Claudia Freitas; de Carvalho, Denise Pires
In general, 3,5-diiodothyronine (3,5-T2) increases the resting metabolic rate and oxygen consumption, exerting short-term beneficial metabolic effects on rats subjected to a high-fat diet. Our aim was to evaluate the effects of chronic 3,5-T2 administration on the hypothalamus-pituitary-thyroid axis, body mass gain, adipose tissue mass, and body oxygen consumption in Wistar rats from 3 to 6 months of age. The rats were treated daily with 3,5-T2 (25, 50, or 75 μg/100 g body weight, s.c.) for 90 days between the ages of 3 and 6 months. The administration of 3,5-T2 suppressed thyroid function, reducing not only thyroid iodide uptake but also thyroperoxidase, NADPH oxidase 4 (NOX4), and thyroid type 1 iodothyronine deiodinase (D1 (DIO1)) activities and expression levels, whereas the expression of the TSH receptor and dual oxidase (DUOX) were increased. Serum TSH, 3,3',5-triiodothyronine, and thyroxine were reduced in a 3,5-T2 dose-dependent manner, whereas oxygen consumption increased in these animals, indicating the direct action of 3,5-T2 on this physiological variable. Type 2 deiodinase activity increased in both the hypothalamus and the pituitary, and D1 activities in the liver and kidney were also increased in groups treated with 3,5-T2. Moreover, after 3 months of 3,5-T2 administration, body mass and retroperitoneal fat pad mass were significantly reduced, whereas the heart rate and mass were unchanged. Thus, 3,5-T2 acts as a direct stimulator of energy expenditure and reduces body mass gain; however, TSH suppression may develop secondary to 3,5-T2 administration. © 2014 The authors.
Dietrich, Johannes W.; Müller, Patrick; Schiedat, Fabian; Schlömicher, Markus; Strauch, Justus; Chatzitomaris, Apostolos; Klein, Harald H.; Mügge, Andreas; Köhrle, Josef; Rijntjes, Eddy; Lehmphul, Ina
Background Although hyperthyroidism predisposes to atrial fibrillation, previous trials have suggested decreased triiodothyronine (T3) concentrations to be associated with postoperative atrial fibrillation (POAF). Therapy with thyroid hormones (TH), however, did not reduce the risk of POAF. This study reevaluates the relation between thyroid hormone status, atrial electromechanical function and POAF. Methods Thirty-nine patients with sinus rhythm and no history of atrial fibrillation or thyroid disease undergoing cardiac surgery were prospectively enrolled. Serum concentrations of thyrotropin, free (F) and total (T) thyroxine (T4) and T3, reverse (r)T3, 3-iodothyronamine (3-T1AM) and 3,5-diiodothyronine (3,5-T2) were measured preoperatively, complemented by evaluation of echocardiographic and electrophysiological parameters of cardiac function. Holter-ECG and telemetry were used to screen for POAF for 10 days following cardiac surgery. Results Seven of 17 patients who developed POAF demonstrated nonthyroidal illness syndrome (NTIS; defined as low T3 and/or low T4 syndrome), compared to 2 of 22 (p < 0.05) patients who maintained sinus rhythm. In patients with POAF, serum FT3 concentrations were significantly decreased, but still within their reference ranges. 3,5-T2 concentrations directly correlated with rT3 concentrations and inversely correlated with FT3 concentrations. Furthermore, 3,5-T2 concentrations were significantly elevated in patients with NTIS and in subjects who eventually developed POAF. In multivariable logistic regression FT3, 3,5-T2, total atrial conduction time, left atrial volume index and Fas ligand were independent predictors of POAF. Conclusion This study confirms reduced FT3 concentrations in patients with POAF and is the first to report on elevated 3,5-T2 concentrations in cardiac NTIS. The pathogenesis of NTIS therefore seems to involve more differentiated allostatic mechanisms. PMID:26279999
Smallridge, R.C.; Wartofsky, L.; Green, B.J.; Miller, F.C.; Burman, K.D.
A sensitive, reproducible RIA for the measurement of 3'-L-monoiodothyronine (3'T 1 ) is described. Mean intra- and interassay coefficients of variation were 2.4% and 22.5%, respectively. Cross-reactivity with other iodothyronines was negligible, except for 3,3'-L-diiodothyronine (3,3'T 2 ) which started to demonstrate cross-reactivity when 3,3'T 2 levels were elevated above 35 ng/dl. Fifty percent displacement occurred when 500 pg 3,3'T 2 were added to the 3'T 1 assay. Employing this assay, 11 normal subjects and 7 pregnant women had serum 3'T 1 levels below the limits of detectability of the assay ( 1 , with the mean (+-SD) values being 6.5 +- 3.0 ng/dl. Serum 3'T 1 levels were present in all cord sera measured (7.3 +- 2.3 ng/dl; n = 19), and the highest levels of 3'T 1 observed were in 38- to 40-week gestation amniotic fluid specimens (15.4 +- 8.4 ng/dl; n = 20). Compared to other iodothyronines, it seems that a relatively low proportion of 3'T 1 is bound to circulating proteins, as the mean percentage of dialyzable 3'T 1 in 12 normal subjects was 5.7 +- 1.2%. An oral dose of 3'T 1 (120 μg) given to 2 euthyroid individuals resulted in peak serum levels of 28 ng/dl 2 h after ingestion. After iv administration of 3'5'T 2 to 2 athyreotic patients (1 hypothyroid and the other euthyroid on replacement T 4 ), 3'T 1 levels rose from undetectable levels to 20 ng/dl. It was concluded that 3'T 1 is routinely detectable in the serum of hyperthyroid but not normal individuals, and even higher levels are present in cord sera and amniotic fluid. Moreover, the study demonstrates that in vivo conversion of 3',5'T 2 to 3'T 1 may occur
Grasselli, Elena; Canesi, Laura; Portincasa, Piero; Voci, Adriana; Vergani, Laura; Demori, Ilaria
Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in industrialized countries and is associated with increased risk of cardiovascular, hepatic and metabolic diseases. Molecular mechanisms on the root of the disrupted lipid homeostasis in NAFLD and potential therapeutic strategies can benefit of in vivo and in vitro experimental models of fatty liver. Here, we describe the high fat diet (HFD)-fed rat in vivo model, and two in vitro models, the primary cultured rat fatty hepatocytes or the FaO rat hepatoma fatty cells, mimicking human NAFLD. Liver steatosis was invariably associated with increased number/size of lipid droplets (LDs) and modulation of expression of genes coding for key genes of lipid metabolism such as peroxisome proliferator-activated receptors (Ppars) and perilipins (Plins). In these models, we tested the anti-steatotic effects of 3,5-L-diiodothyronine (T2), a metabolite of thyroid hormones. T2 markedly reduced triglyceride content and LD size acting on mRNA expression of both Ppars and Plins. T2 also stimulated mitochondrial oxidative metabolism of fatty acids. We conclude that in vivo and especially in vitro models of NAFLD are valuable tools to screen a large number of compounds counteracting the deleterious effect of liver steatosis. Because of the high and negative impact of liver steatosis on human health, ongoing experimental studies from our group are unravelling the ultimate translational value of such cellular models of NAFLD.
Burger, A.; Sakoloff, C.
A specific radioimmunoassay for 3,3'-diiodothyronine (T/sub 2/) is described which is capable of detecting as little as 1.3 ng/dl. The antiserum recognizes mainly T/sub 2/; biliary conjugates of T/sub 2/ bind slightly to the antibody. The intraassay and interassay coefficients of variation were, respectively, 5.7% and 13.1%. T/sub 2/ was detected in the serum of hypothyroid patients treated with triiodothyronine (T/sub 3/) and in euthyroid subjects treated with reverse triiodothyronine (rT/sub 3/). These results suggest that both T/sub 3/ and rT/sub 3/ are deiodinated to T/sub 2/. Serum concentrations of T/sub 2/ in normal subjects decreased with age. Between 20 and 40 years the mean concentration was 4.3 +- 2.0 ng/dl (2 SD), between 41 and 60 years it varied from 1.9 to 5.8 ng/dl (3.8 +- 0.3 ng/dl, SE) and in elderly subjects have 60 years concentrations varied from unmeasurable to 4 ng/dl (2.9 +- 0.4 ng/dl, SE). Low serum T/sub 2/ concentrations were found in anorexia nervosa (2.5 +- 0.3 ng/dl, SE). In hypothyroidism the serum concentrations were low or unmeasurable. As most of the hypothyroid subjects were elderly their serum T/sub 2/ concentrations overlapped with the low values found in the elderly euthyroid subjects. In classical hyperthyroidism serum T/sub 2/ concentrations were greatly increased (3.3 to 31 ng/dl (11.8 +- 2.7 ng/dl, SE) but in ''T/sub 3/ toxicosis'' the concentrations were only modestly increased (2.4 to 8.8 ng/dl, 5.2 +- 0.8 ng/dl, SE).
Effect of 3,3',5-triiodothyronine and 3,5-diiodothyronine on progesterone production, cAMP synthesis, and mRNA expression of STAR, CYP11A1, and HSD3B genes in granulosa layer of chicken preovulatory follicles.
Sechman, A; Pawlowska, K; Hrabia, A
In vitro studies were performed to assess whether stimulatory effects of triiodothyronine (T3) on progesterone (P4) production in a granulosa layer (GL) of chicken preovulatory follicles are associated with 3',5'-cyclic adenosine monophosphate (cAMP) synthesis and mRNA expression of STAR protein, CYP11A1, and HSD3B. Effects of 3,5-diiodothyronine (3,5-T2) on steroidogenic function in these follicles were also investigated. The GL of F3 to F1 follicles was incubated in medium supplemented with T3 or 3,5-T2, LH, or forskolin (F), and a combination of each iodothyronine with LH or F. Levels of P4 and cAMP in culture media were determined by RIA. Expression of genes involved in P4 synthesis (ie, STAR protein, CYP11A1, and HSD3B) in the GL of F3 to F1 follicles incubated in medium with T3 or 3,5-T2 and their combination with LH was performed by real-time PCR. Triiodothyronine increased basal and LH- and F-stimulated P4 secretion by preovulatory follicles. The 3,5-T2 elevated P4 synthesis by F3, had no effect on F2 follicles, and diminished P4 production by the GL of F1 follicles. It had no effect on LH-stimulated P4 production; however, it augmented F-stimulated P4 production by F2 and F1 follicles. Although T3 did not affect basal and F-stimulated cAMP synthesis by the GL of preovulatory follicles, it increased LH-stimulated synthesis of this nucleotide. However, 3,5-T2 elevated F-stimulated cAMP synthesis in F3 and F2 follicles; it did not change basal and LH-stimulated cAMP production. Triiodothyronine decreased basal STAR and CYP11A1 mRNAs in F3 follicles, increased them in F1 follicles, and elevated HSD3B mRNA levels in F1 follicles. Triiodothyronine augmented LH-stimulated STAR, CYP11A1, and HSD3B mRNA levels in F2 and CYP11A1 in F1 follicles. However, T3 decreased LH-stimulated STAR and HSD3B mRNA levels in F1 follicles. The 3,5-T2 did not affect basal STAR and CYP11A1 mRNA expression in all investigated follicles; however, it decreased LH-stimulated STAR
Hommel, E; Faber, J; Kirkegaard, C
was 276 pmol/d, whereas the median excretion of glucuronidated and sulfated 3,5-T2 in 7 healthy subjects was 448 and 451 pmol/d, respectively. The median excretion of 154 pmol/d in 9 hypothyroid patients did not differ from that found in controls. In contrast 12 patients with hyperthyroidism had...
Chopra, I.J.; Geola, F.; Solomon, D.H.; Maciel, R.M.B.
An RIA has been developed for 3'5'-diiiodothyronine (3',5'-T 2 ) in unextracted serum. Interference in binding of radioactive 3',5'-T 2 to anti-3',5'-T 2 was minimized by using phosphate buffer and merthiolate. The detection threshold of the RIA was 2.5 ng/dl. Recovery of nonradioactive 3',5'-T 2 averaged 99%. T 4 , T 3 , and rT 3 cross-reacted with anti-3',5'-T 2 antibody 0.0025, 2 concentrations in ng/dl were 2.4 in 53 normal subjects, 4.2 in 7 hypothyroid patients, 14.9 in 34 hyperthyroid patients, 13.5 in 25 patients with hepatic cirrhosis, and 14.3 in 31 newborns' cord blood serum. The values for the latter four groups were significantly different from normal. The serum 3',5'-T 2 concentration of 7.7 ng/dl in eight subjects in the third trimester of pregnancy did not differ from normal when serum T 4 and T 3 were elevated. Oral administration of 300 μg rT 3 to 9 normal subjects led to an increase in serum 3'5'-T 2 concentration of 45% at 1h. Total fasting in 3 obese subjects was associated with an increase in serum 3',5'-T 2 from 8.6 to 16.3 ng/dl at 6 to 8 days; rT 3 increased similarly, while T 3 decreased and T 4 did not change. Administration of dexamethasone to 4 hyperthyroid patients was associated with nearly parallel increases in serum 3',5'-T 2 and rT 3 and a decrease in T 3 . The 3',5'-T 2 concentrations in amniotic fluids were 15.2 ng/dl at 15 to 20 weeks gestation and 5.8 ng/dl at 33 to 40 weeks. Pronase hydrolysates of 9 normal thyroid glands contained 350 μgT 4 and 0.24 μg 3',5'-T 2 /g wet wt. It was estimated that thyroidal secretion contributes 2 in serum of normal man. The data suggest that 3',5'-T 2 is a normal component of human serum and almost all 3',5'-T 2 in serum derives from extrathyroidal sources
Full Text Available 3,5,3'-triiodo-L-thyronine (T3 plays a crucial role in regulating metabolic rate and fuel oxidation; however, the mechanisms by which it affects whole-body energy metabolism are still not completely understood. Skeletal muscle (SKM plays a relevant role in energy metabolism and responds to thyroid state by remodeling the metabolic characteristics and cytoarchitecture of myocytes. These processes are coordinated with changes in mitochondrial content, bioenergetics, substrate oxidation rate, and oxidative phosphorylation efficiency. Recent data indicate that emerging iodothyronines have biological activity. Among these, 3,5-diiodo-L-thyronine (T2 affects energy metabolism, SKM substrate utilization, and mitochondrial functionality. The effects it exerts on SKM mitochondria involve more aspects of mitochondrial bioenergetics; among these, respiratory chain activity, mitochondrial thermogenesis, and lipid-handling are stimulated rapidly. This minireview focuses on signaling and biochemical pathways activated by T3 and T2 in SKM that influence the above processes. These novel aspects of thyroid physiology could reveal new perspectives for understanding the involvement of SKM mitochondria in hypo- and hyperthyroidism.
Assunta eLombardi; Maria eMoreno; Pieter eDe Lange; Susanna eIossa; Rosa Anna eBusiello; Fernando eGoglia
3,5,3'-triiodo-L-thyronine (T3) plays a crucial role in regulating metabolic rate and fuel oxidation; however, the mechanisms by which it affects whole-body energy metabolism are still not completely understood. Skeletal muscle (SKM) plays a relevant role in energy metabolism and responds to thyroid state by remodeling the metabolic characteristics and cytoarchitecture of myocytes. These processes are coordinated with changes in mitochondrial content, bioenergetics, substrate oxidation rate, ...
Lombardi, Assunta; Moreno, Maria; de Lange, Pieter; Iossa, Susanna; Busiello, Rosa A.; Goglia, Fernando
3,5,3′-Triiodo-L-thyronine (T3) plays a crucial role in regulating metabolic rate and fuel oxidation; however, the mechanisms by which it affects whole-body energy metabolism are still not completely understood. Skeletal muscle (SKM) plays a relevant role in energy metabolism and responds to thyroid state by remodeling the metabolic characteristics and cytoarchitecture of myocytes. These processes are coordinated with changes in mitochondrial content, bioenergetics, substrate oxidation rate, ...
Perrild, H; Skovsted, L; Korsgaard Christensen, L [Department of Internal Medicine and Endocrinology, Herlev Hospital, DK-2730 Herlev, Denmark
Alkaline Sephadex G-25 columns were used to separate labelled 3,5,3',5'-thyroxine, 3,5,3'-triiodothyronine, 3,3',5'-triiodothyronine and 3,3'-diiodothyronine from the serum binding proteins followed by a quantitative elution of each hormone by coupling to its respective antibody. It is shown that although these antibodies (diluted 1:1500-1:100 000) in our radioimmunoassays are highly specific they show a high degree of non-specific binding when they are used in the concentrations necessary to get a maximal recovery of the hormones in column separating experiments.
Moreno, Maria; Giacco, Antonia; Di Munno, Celia; Goglia, Fernando
A growing number of researchers are focusing their attention on the possibility that thyroid hormone metabolites, particularly 3,5-diiodothyronine (T2), may actively regulate energy metabolism at the cellular, rather than the nuclear, level. Due to their biochemical features, mitochondria have been the focus of research on the thermogenic effects of thyroid hormones. Indeed, mitochondrial activities have been shown to be regulated both directly and indirectly by T2-specific pathways. Herein, we describe the effects of T2 on energy metabolism. Copyright © 2017 Elsevier B.V. All rights reserved.
Pillai, M.R.A.; Nagvekar, U.H.; Desai, C.N.; Mani, R.S.
A method standardized for the preparation of high specific activity labelled triiodothyronine (T 3 ) is discussed. Iodine-125 labelled T 3 with a specific activity of 3 mCi μg was prepared by iodinating 3,5-diiodothyronine (T 2 ) and purifying it over Sephadex G-25 gel. Radochemical purity and stability evaluations were done by paper chromatography. Specific activity of the labelled T 3 prepared was estimated by the self-displacement method. The use of this high specific activity labelled T 3 in radioimmunoassay increased the sensitivity considerably. The advantage of this procedure is that the specific activity of labelled T 3 formed is independent of reaction yield and labelled T 3 yield. (author)
Huang, Biao; Yu, Huixin; Bao, Jiandong; Zhang, Manda; Green, William L; Wu, Sing-Yung
Using compound W (a 3,3'-diiodothyronine sulfate [T 2 S] immuno-crossreactive material)-specific polyclonal antibodies and homogeneous time-resolved fluorescence immunoassay assay techniques (AlphaLISA) to establish an indirect competitive compound W (ICW) quantitative detection method. Photosensitive particles (donor beads) coated with compound W or T 2 S and rabbit anti-W antibody were incubated with biotinylated goat anti-rabbit antibody. This constitutes a detection system with streptavidin-coated acceptor particle. We have optimized the test conditions and evaluated the detection performance. The sensitivity of the method was 5 pg/mL, and the detection range was 5 to 10 000 pg/mL. The intra-assay coefficient of variation averages W levels in extracts of maternal serum samples. This may have clinical application to screen congenital hypothyroidism in utero.
Daniel F Vatner
Full Text Available Thyroid hormone mimetics are alluring potential therapies for diseases like dyslipidemia, nonalcoholic fatty liver disease (NAFLD, and insulin resistance. Though diiodothyronines are thought inactive, pharmacologic treatment with 3,5- Diiodo-L-Thyronine (T2 reportedly reduces hepatic lipid content and improves glucose tolerance in fat-fed male rats. To test this, male Sprague Dawley rats fed a safflower-oil based high-fat diet were treated with T2 (0.25 mg/kg-d or vehicle. Neither 10 nor 30 days of T2 treatment had an effect on weight, adiposity, plasma fatty acids, or hepatic steatosis. Insulin action was quantified in vivo by a hyperinsulinemic-euglycemic clamp. T2 did not alter fasting plasma glucose or insulin concentration. Basal endogenous glucose production (EGP rate was unchanged. During the clamp, there was no difference in insulin stimulated whole body glucose disposal. Insulin suppressed EGP by 60% ± 10 in T2-treated rats as compared with 47% ± 4 suppression in the vehicle group (p = 0.32. This was associated with an improvement in hepatic insulin signaling; insulin stimulated Akt phosphorylation was ~2.5 fold greater in the T2-treated group as compared with the vehicle-treated group (p = 0.003. There was no change in expression of genes thought to mediate the effect of T2 on hepatic metabolism, including genes that regulate hepatic lipid oxidation (ppara, carnitine palmitoyltransferase 1a, genes that regulate hepatic fatty acid synthesis (srebp1c, acetyl coa carboxylase, fatty acid synthase, and genes involved in glycolysis and gluconeogenesis (L-pyruvate kinase, glucose 6 phosphatase. Therefore, in contrast with previous reports, in Sprague Dawley rats fed an unsaturated fat diet, T2 administration failed to improve NAFLD or whole body insulin sensitivity. Though there was a modest improvement in hepatic insulin signaling, this was not associated with significant differences in hepatic insulin action. Further study will be
Wojtczak, A; Luft, J R; Cody, V
The crystal structure of human transthyretin (TTR) complexed with milrinone (2-methyl-5-cyano-3,4'-bipyridin-6(1H)-one), a positive inotropic cardiac agent, has been refined to R = 17.4% for 8-1.9-A resolution data. This report provides the first detailed description of protein interactions for an inotropic bipyridine agent which is an effective thyroid hormone binding competitor to transthyretin. Milrinone is bound along the 2-fold axis in the binding site with its substituted pyridone ring located deep within the channel of the two identical binding domains of the TTR tetramer. In this orientation the 5-cyano group occupies the same site as the 3'-iodine in the TTR complex with 3,3'-diiodothyronine (Wojtczak, A., Luft, J., and Cody, V. (1992) J. Biol. Chem. 267, 353-357), which is 3.5 A deeper in the channel than thyroxine (Blake, C. C. F., and Oately, S. J., (1977) Nature 268, 115-120). These structural results confirm computer modeling studies of milrinone structural homology with thyroxine and its TTR binding interactions and explain the effectiveness of milrinone competition for thyroxine binding to TTR. To understand the weaker binding affinity of the parent inotropic drug, amrinone (5-amino-3,4'-bipyridin-6(1H)-one), modeling studies of its TTR binding were carried out which indicate that the 5-amino group cannot participate in strong interactions with TTR and the lack of the 2-methyl further weakens amrinone binding.
Inhibitory effects of unlabeled iodothyronines on the metabolism of thyroxine (T 4 ), 3,3',5-triiodothyronine (T 3 ) and 3,3',5'-triiodothyronine (reverse T 3 , rT 3 ) were investigated in continuously cultured monkey hepatocarcinoma cells which showed a rapid metabolism of the thyroid hormones. Nonphenolic ring deiodination of [3',5'- 125 I]-T 4 and [3'- 125 I]-T 3 was strongly inhibited by excess T 3 , 3,5-diiodothyronine (3,5-T 2 ) and T 4 , whereas rT 3 was the least effective inhibitor. Phenolic ring deiodination of [3',5'- 125 I]-rT 3 was strongly affected by excess unlabeled rT 3 . However, the inhibitory effect of T 4 , T 3 and 3,5-T 3 was much weaker than that of rT 3 . It was concluded that rT 3 is apparently the most effective inhibitor of phenolic ring deiodination but the least effective inhibitor of nonphenolic ring deiodination. (author)
To introduce radioimmunoassay, 3, 3', 5'-triiodothyronine (reverse-T 3 ) was coupled to bovine serum albumin by the carbodiimide technique and rabbits were immunized with the conjugates obtained. The immunizations were performed by multiple site intradermal injections at places in which cornynebacterium parvum was previously injected to enhance immunologic reaction. After 3 months the rabbits raised antisera to reverse-T 3 of a high titer and specificity. To obtain labelled 125 I-reverse T 3 , 3,3'-diiodothyronine was used. Iodination was performed by the chloramine T technique and the iodination mixture was subjected to gel filtration on Sephadex G-25 (fine) column. The purified monolabelled 125 I-reverse T 3 had a specific activity of 3,000 milli Curie/mg. The reverse T 3 radioimmunoassay of a high sensitivity (ca 2 pg/tube) was introduced in the clinical studies and facilitated direct determination of reverse T 3 in sera without the need of plasma extractions. The interference of serum proteins (TBG) was avoided by adding 8-anilino-1-naphtalene sulfonic acid to serum samples. Separation of free from antibody bound antigens was achieved by polyethylene glycol precipitation or immunoprecipitation. (author)
Kruh, J.; Tichonicky, L.
1) Injection of triiodothyronine to rats stimulates protein kinase activity in liver chromatin nonhistone proteins. A significant increase was found after two daily injections. A 4-fold increase was observed with the purified enzyme after eight daily injections of the hormone. No variations were observed in cytosol protein kinase activity. Electrophoretic pattern, effect of heat denaturation, effect of p-hydroxymercuribenzoate seem to indicate that the enzyme present in treated rats is not identical to the enzyme in control animals, which suggests that thyroid hormone has induced nuclear protein kinase. Diiodothyronine, 3, 3', 5'-triiodothyronine have no effect on protein kinase. 2) Chromatin non-histone proteins isolated from rats injected with triiodothyronine incorporated more 32 P when incubated with [γ- 32 P]ATP than the chromatin proteins from untreated rats. Thyroidectomy reduced the in vitro 32 P incorporation. It is suggested that some of the biological activity of thyroid hormone could be mediated through its effect on chromatin non-histone proteins. (orig.) [de
Boado, R.J.; Ulloa, E.R.; Zaninovich, A.A.
Wistar rats were treated with 7.8 or 260 nmols T4/100 g BW, 1.5 or 260 nmols T3/100 g BW, or saline as control. Twenty minutes later 1 μg TRH/100 g BW was injected iv. Heparinized blood samples were drawn at times 0 and 30 minutes (10 min post-TRH) for determination of plasma TSH, T4 and T3 by RIA. Other group of rats were administered with 150 μCi of 3',5'- 125 I-T4 prepared by iodination of 3,5-diiodothyronine. Thirty minutes later the hypophyses were removed, and chromatographed. Other group of animals were treated with 5 mg of iopanoic acid (IOP)/100 g BW. Thereafter, rats were injected iv with 260 nmols T4 or T3/100 g BW and the TRH-test performed as described above. In the control group there was a 11-fold increase in plasma TSH at 10 minutes post-TRH. In rats treated with 260 nmols T4 the post-TRH increment in plasma TSH was 5+-1-fold (p 125 I-T3 in the hypophyses 30 minutes after 125 I-T4 administration. The present data indicate that T4 is capable of depressing the release of TSH in response to TRH stimulation in normal rats. (M.E.L.) [es
Sorimachi, K [Dokkyo Univ. Tochigi (Japan). School of Medicine
Inhibitory effects of unlabeled iodothyronines on the metabolism of thyroxine (T/sub 4/), 3,3',5-triiodothyronine (T/sub 3/) and 3,3',5'-triiodothyronine (reverse T/sub 3/, rT/sub 3/) were investigated in continuously cultured monkey hepatocarcinoma cells which showed a rapid metabolism of the thyroid hormones. Nonphenolic ring deiodination of (3',5'-/sup 125/I)-T/sub 4/ and (3'-/sup 125/I)-T/sub 3/ was strongly inhibited by excess T/sub 3/, 3,5-diiodothyronine (3,5-T/sub 2/) and T/sub 4/, whereas rT/sub 3/ was the least effective inhibitor. Phenolic ring deiodination of (3',5'-/sup 125/I)-rT/sub 3/ was strongly affected by excess unlabeled rT/sub 3/. However, the inhibitory effect of T/sub 4/, T/sub 3/ and 3,5-T/sub 3/ was much weaker than that of rT/sub 3/. It was concluded that rT/sub 3/ is apparently the most effective inhibitor of phenolic ring deiodination but the least effective inhibitor of nonphenolic ring deiodination.
Lombardi, Assunta; De Matteis, Rita; Moreno, Maria; Napolitano, Laura; Busiello, Rosa Anna; Senese, Rosalba; de Lange, Pieter; Lanni, Antonia; Goglia, Fernando
Iodothyronines such as triiodothyronine (T(3)) and 3,5-diiodothyronine (T(2)) influence energy expenditure and lipid metabolism. Skeletal muscle contributes significantly to energy homeostasis, and the above iodothyronines are known to act on this tissue. However, little is known about the cellular/molecular events underlying the effects of T(3) and T(2) on skeletal muscle lipid handling. Since FAT/CD36 is involved in the utilization of free fatty acids by skeletal muscle, specifically in their import into that tissue and presumably their oxidation at the mitochondrial level, we hypothesized that related changes in lipid handling and in FAT/CD36 expression and subcellular redistribution would occur due to hypothyroidism and to T(3) or T(2) administration to hypothyroid rats. In gastrocnemius muscles isolated from hypothyroid rats, FAT/CD36 was upregulated (mRNA levels and total tissue, sarcolemmal, and mitochondrial protein levels). Administration of either T(3) or T(2) to hypothyroid rats resulted in 1) little or no change in FAT/CD36 mRNA level, 2) a decreased total FAT/CD36 protein level, and 3) further increases in FAT/CD36 protein level in sarcolemma and mitochondria. Thus, the main effect of each iodothyronine seemed to be exerted at the level of FAT/CD36 cellular distribution. The effect of further increases in FAT/CD36 protein level in sarcolemma and mitochondria was already evident at 1 h after iodothyronine administration. Each iodothyronine increased the mitochondrial fatty acid oxidation rate. However, the mechanisms underlying their rapid effects seem to differ; T(2) and T(3) each induce FAT/CD36 translocation to mitochondria, but only T(2) induces increases in carnitine palmitoyl transferase system activity and in the mitochondrial substrate oxidation rate.
Chopra, I.J.; Santini, F.; Hurd, R.E.; Chua Teco, G.N.
A highly sensitive, specific, and reproducible RIA has been developed to measure T 4 sulfate (T 4 S) in ethanol extracts of serum. rT 3 sulfate (rT 3 S) cross-reacted 7.1%, and T 3 S cross-reacted 0.59% in the RIA; T 4 , T 3 , rT 3 and 3,3'-diiodothyronine cross-reacted 0.004% or less. The recovery of nonradioactive T 4 S added to serum averaged 95%. The detection threshold of the RIA was 18 pmol/L. The coefficient of variation averaged 6.9% within an assay and 12% between assays. T 4 S was bound by T 4 -binding globulin and albumin in serum. The free fraction of T 4 S in four normal sera averaged 0.06% compared to a value of 0.03% for T 4 (P 4 S was (mean ± SE) 19 ± 1.2 pmol/L in normal subjects, 33 ± 10 in hyperthyroid patients with Graves disease, 42 ± 15 in hypothyroid patients, 34 ± 6.9 in patients with systematic nonthyroidal illnesses, 21 ± 4.3 in pregnant women at 15-40 weeks gestation, and 245 ± 26 in cord blood sera of newborns; the value in the newborn was significantly different from normal (P 4 S. The T 4 S content of the thyroid gland was less than 1/4000th that of T 4 . We conclude that (1) T 4 S is a normal component of human serum, and its levels are markedly increased in newborn serum and amniotic fluid; and (2) the sulfation pathway plays an important role in the metabolism of T 4 in man. 28 refs., 4 figs., 2 tabs
Chopra, I.J.; Santini, F.; Hurd, R.E.; Chua Teco, G.N. (Univ. of California Center for the Health Sciences, Los Angeles (United States))
A highly sensitive, specific, and reproducible RIA has been developed to measure T[sub 4] sulfate (T[sub 4]S) in ethanol extracts of serum. rT[sub 3] sulfate (rT[sub 3]S) cross-reacted 7.1%, and T[sub 3]S cross-reacted 0.59% in the RIA; T[sub 4], T[sub 3], rT[sub 3] and 3,3[prime]-diiodothyronine cross-reacted 0.004% or less. The recovery of nonradioactive T[sub 4]S added to serum averaged 95%. The detection threshold of the RIA was 18 pmol/L. The coefficient of variation averaged 6.9% within an assay and 12% between assays. T[sub 4]S was bound by T[sub 4]-binding globulin and albumin in serum. The free fraction of T[sub 4]S in four normal sera averaged 0.06% compared to a value of 0.03% for T[sub 4] (P < 0.001). The serum concentration of T[sub 4]S was (mean [+-] SE) 19 [+-] 1.2 pmol/L in normal subjects, 33 [+-] 10 in hyperthyroid patients with Graves disease, 42 [+-] 15 in hypothyroid patients, 34 [+-] 6.9 in patients with systematic nonthyroidal illnesses, 21 [+-] 4.3 in pregnant women at 15-40 weeks gestation, and 245 [+-] 26 in cord blood sera of newborns; the value in the newborn was significantly different from normal (P < 0.001). Administration of sodium ipodate (Oragrafin; 3 g, orally) to hyperthyroid patients was associated with a transient increase in serum T[sub 4]S. The T[sub 4]S content of the thyroid gland was less than 1/4000th that of T[sub 4]. We conclude that (1) T[sub 4]S is a normal component of human serum, and its levels are markedly increased in newborn serum and amniotic fluid; and (2) the sulfation pathway plays an important role in the metabolism of T[sub 4] in man. 28 refs., 4 figs., 2 tabs.
Chopra, I J; Huang, T S; Hurd, R E; Solomon, D H
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