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Introduction Reliability in the use of placentome (including placenta, umbilical cord, and cord blood) biomarkers requires an understanding of their distributions. Here we aim to develop a simple and proper placenta sampling scheme, and to evaluate the placental distributions of biomarkers. Methods We developed a continuous cooling chain protocol off delivery room and cryo-subsampling method for placenta sampling. The levels of thyroid hormones (THs), elements, persistent organic pollutants (POPs), monoamines, and vitamin E were measured using UPLC-Q-TOF-MS, HPLC-ICP-MS, HPLC-EcD, and HRGC-HRMS, respectively. The distributions of biomarkers were assessed. Results In human placentome, l -thyroxine (T4), Cd, Se, Zn, Cu, Fe, Ca, K, Mg, α-tocopherol, β-tocopherol, and β-tocotrienol levels were higher in placenta than in umbilical cord, while Pb and Mn were concentrated in human cord. In porcine placentome, T4, 3,3′,5′-triiodo- l -thyronine (rT3), 3,3′-diiodo- l -thyronine, Cd, Pb, Zn, K, and Al levels were higher in the cord. The intraclass correlation coefficient (ICC) was l -thyronine, rT3, α-tocopherol, and 7 elements in human basal plate, indicating low reliability. rT3, Cd, Zn, Mn, and Cu were significantly concentrated in the central region in human placenta, while higher levels of As, Cd, Cr, and Al were found in the periphery region in porcine placenta. Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) showed moderate reliability (ICC: 0.40–0.98) except PCB-81, -126, and BDE-208, while polychlorinated dibenzo-p-doixins/furans (PCDD/Fs) showed poor reliability (ICC: 0.07–0.31). Discussion These results highlight the complexity of placenta sampling. This study provides a novel and simple sampling approach in investigating placental exposomics.

Thyroid hormones are important regulatory hormones, acting on nearly every cell in the body. The two main thyroid hormones are l-thyroxine (tetraiodo-l-thyronine, T4) and 3,3′,5-triiodo-l-thyronine (T3), which are produced in the thyroid gland and secreted into the blood stream. Other important thyroid hormone metabolites are 3,3′-diiodo-l-thyronine (T2) and l-thyronine (T0), which may show increased levels in circulation due to dietary iodine deficiency or other medical disorders. Owing to their central role in cellular functions, sensitive and specific detection methods for thyroid hormones are needed. In this work, native mass spectrometry (MS) was used to quantitate thyroid hormone binding to the anti-T4 antibody Fab fragment. First, the binding affinity for T2 was determined via direct ligand titration experiments. Then, the affinities for the other ligands were determined by competition experiments using T2 as the "low-affinity" reference ligand. The highest affinity was measured for T3, followed by T4, T2, and T0 (Kd = 29, 3.4, and 260 nM and 130 μM, respectively). Thus, it is evident that the number and positions of the iodine substituents within the thyronine rings are important for the ligand binding affinity of anti-T4 Fab. Surprisingly, structurally related tetrahalogen bisphenols were also able to bind to anti-T4 Fab with nanomolar affinities.

Thyroid hormones have been proposed as anti obesity drugs due to their effects on basal metabolism and the ability to increase energy expenditure. However, their clinical use has been strongly curbed by the concomitant onset of thyrotoxicosis. In this setting, several studies have been undertaken to assess the role of 3,5 diiodo- L-thyronine (T2), an endogenous metabolite of thyroid hormone derived from the enzymatic deiodination of triodothyronine T3. The metabolic effects of T2 are similar to those induced by T3. However, these effects appear to involve different and not welldefined mechanisms that make this molecule clinically useful as potential drug in the treatment of pathological conditions such as obesity and hepatic steatosis. The main pharmacological target of T2 appears to be the mitochondria. Therefore, the administration of T2 to obese subjects might improve the mitochondrial performance, which is generally recognized to be reduced in these subjects who must oxidize greater quantities of substrates. In this context, it can be hypothesized that T2, by acting mainly on mitochondrial function and oxidative stress, might be able to prevent and revert the tissue damages and hepatic steatosis induced by a hyperlipidic diet and a concomitant reduction in the circulating levels LDL and triglycerides as well. This review the discuss the mechanisms of action of T2 and the possible, future clinical uses of T2 analogs for the treatment lipid dysmetabolism related to obesity and overweight.

3,5-diiodo-thyronine (T2), an endogenous metabolite of thyroid hormones, exerts beneficial metabolic effects. When administered to overweight rats receiving a high fat diet (HFD), it significantly reduces body fat accumulation, which is a risk factor for the development of an inflammatory state and of related metabolic diseases. In the present study, we focused our attention on T2 actions aimed at improving the adverse effects of long-lasting HFD such as the adipocyte inflammatory response. For this purpose, three groups of rats were used throughout: i) receiving a standard diet for 14 weeks; ii) receiving a HFD for 14 weeks, and iii) receiving a HFD for 14 weeks with a simultaneous daily injection of T2 for the last 4 weeks. The results showed that T2 administration ameliorated the expression profiles of pro- and anti-inflammatory cytokines, reduced macrophage infiltration in white adipose tissue, influenced their polarization and reduced lymphocytes recruitment. Moreover, T2 improved the expression of hypoxia markers, all altered in HFD rats, and reduced angiogenesis by decreasing the pro-angiogenic miR126 expression. Additionally, T2 reduced the oxidative damage of DNA, known to be associated to the inflammatory status. This study demonstrates that T2 is able to counteract some adverse effects caused by a long-lasting HFD and to produce beneficial effects on inflammation. Irisin and SIRT1 pathway may represent a mechanism underlying the above described effects.

The effects of thyroid hormone metabolites (THMs) other than T3, rT3 and T4 are largely unknown, partially due to the lack of adequate methods. For adequate analysis, internal standards for all THMs are essential, but unfortunately not commercially available. Reported approaches for the synthesis of T0, 3,5-T2, TA3 and TA4 lack sensitivity and/or are not adaptable for 13C6-labeled analogues. In this paper, we describe the synthesis of four 13C6-labeled THMs, T0-13C6, 3,5-T2-13C6, TA3-13C6, TA4-13C6. Starting with 13C6-bromo-benzene, a short and versatile synthesis route was developed in which the formation of the diphenyl ether by a Chan-Lam coupling reaction was fundamental.

Background: Thyroid hormones (THs) are critical for brain development. Whether low-moderate fluoride exposure affects thyroid function and what the impact is on children's intelligence remain elusive. Objectives: We conducted a cross-sectional study to examine the associations between low-moderate fluoride exposure and thyroid function in relation to children's intelligence. Methods: We recruited 571 resident children, aged 7–13 years, randomly from endemic and non-endemic fluorosis areas in Tianjin, China. We measured fluoride concentrations in drinking water and urine using the national standardized ion selective electrode method. Thyroid function was evaluated through the measurements of basal THs [(total triiodothyronine (TT3), total thyronine (TT4), free triiodothyronine (FT3), free thyronine (FT4)] and thyroid-stimulating hormone (TSH) levels in serum. Multivariable linear and logistical regression models were used to assess associations among fluoride exposure, thyroid function and IQ scores. Results: In adjusted models, every 1 mg/L increment of water fluoride was associated with 0.13 uIU/mL increase in TSH. Every 1 mg/L increment of urinary fluoride was associated with 0.09 ug/dL decrease in TT4, 0.009 ng/dL decrease in FT4 and 0.11 uIU/mL increase in TSH. Fluoride exposure was inversely related to IQ scores (B = −1.587; 95% CI: −2.607, −0.568 for water fluoride and B = −1.214; 95% CI: −1.987, −0.442 for urinary fluoride). Higher TT3, FT3 were related to the increased odds of children having high normal intelligence (OR = 3.407, 95% CI: 1.044, 11.120 for TT3; OR = 3.277, 95% CI: 1.621, 6.623 for FT3). We detected a significant modification effect by TSH on the association between urinary fluoride and IQ scores, without mediation by THs. Conclusions: Our study suggests low-moderate fluoride exposure is associated with alterations in childhood thyroid function that may modify the association between fluoride and intelligence. Keywords: Low-moderate fluoride exposure, Thyroid function, IQ scores, School-age children

Background Type 2 diabetes has an underlying pathology with thyroid dysfunction. However, few studies have investigated the association between thyroid hormones and diabetic peripheral neuropathy. Our aim was to evaluate the relationship between thyroid hormones and electrophysiological properties of peripheral nerves in type 2 diabetes. Patients and Methods The medical records of 308 patients with type 2 diabetes were enrolled in this study. Subjects stratified by sex were divided into subgroups based on the diagnosis of nerve conduction study. The nerve conduction parameters were separately described with the spectrum of thyroid hormones. Multivariate regression models to analyze the potential links between thyroid hormones and nerve conduction parameters. Results The serum free triiodine thyronine levels between normal and abnormal nerve conduction groups were statistically different in total (4.55±0.65 vs 4.37±0.63, P Conclusions Serum free triiodine thyronine level is associated with nerve conduction in diabetes. Low free triiodine thyronine may be a potential risk for diabetic peripheral neuropathy.

Thyroid hormones (THs), which mainly consist of 3, 3', 5-triiodo-l-thyronine (T3) and L-thyroxine (T4), play a critical role in regulating biological processes such as growth and metabolism in various animal species. Thus, accurate measurement of T3 and T4, especially physiologically active free (protein-unbound) forms, in serum/plasma is needed for the evaluation of TH homeostasis. However, such high-precision determination of free THs is lacking for non-human species. The present study aimed to develop a highly sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of six free THs in serum/plasma, which is applicable to not only humans but also non-human species. Two different physical separation steps, ultrafiltration (UF) and equilibrium dialysis (ED), were examined to obtain the free TH fraction. Several experimental conditions were carefully optimized and validated for UF or ED using the commercially available bovine serum. As a result, UF at 1100 × g and 37 °C for 30 min with a 30 kDa ultrafiltration device (Centrifree YM-30, Millipore) yielded excellent precision (CV:10%). The optimized ED step also yielded high precision (CV:10%) and the measurement values were approximately equal to those of UF, but at least 16 h were required to reach equilibrium. Thus, UF combined with LC-MS/MS was finally chosen, in terms of the time needed for the measurement. Acceptable accuracy (recovery: 70%-110%) and intra- and inter-day precision (CV:10% and12%, respectively) were obtained, when triplicate analyses in three different days were conducted using the bovine serum. The developed analytical method was successfully applied to the determination of free THs in serum/plasma samples of humans, cats, and dogs. Furthermore, comparison with free T4 concentrations measured by a common immunoassay method evidently indicated that the ultrafiltration-LC-MS/MS method developed in this study can increase the specificity and accuracy of TH measurement.

The transplacental passage of thyroid hormones (THs) is of great significance since the maternal THs are vitally important in ensuring the normal fetal development. In this paper, we determined the concentrations of seven THs, viz. L-thyroxine (T4), 3,3′,5-triiodo- l -thyronine (T3), 3,3′,5′-triiodo- l -thyronine (rT3), 3,3′-diiodo- l -thyronine (T2), 3,5-diiodo- l -thyronine (rT2), 3-iodo- l -thyronine (T1) and 3-iodothyronamine (T1AM), in placenta using isotope dilution liquid chromatography quadrupole time-of-flight mass spectrometry. We optimized the method using isotopically labeled quantification standards (13C6-T4, 13C6-T3, 13C6-rT3 and 13C6-T2) and recovery standard (13C12-T4) in combination with solid-liquid extraction, liquid–liquid extraction and solid phase extraction. The linearity was obtained in the range of 0.5–150 pg uL−1 with R2 values >0.99. The method detection limits (MDLs) ranged from 0.01 ng g−1 to 0.2 ng g−1, while the method quantification limits (MQLs) were between 0.04 ng g−1 and 0.7 ng g−1. The spike-recoveries for THs (except for T1 and T1AM) were in the range of 81.0%–112%, with a coefficient of variation (CV) of 0.5–6.2%. The intra-day CVs and inter-day CVs were 0.5%–10.3% and 1.19%–8.88%, respectively. Concentrations of the THs were 22.9–35.0 ng g−1 T4, 0.32–0.46 ng g−1 T3, 2.86–3.69 ng g−1 rT3, 0.16–0.26 ng g−1 T2, and

Both 3,5-diiodo-L-thyronine (3,5-T2) and 3,5,3'-triiodo-L-tyronine (T3) affect energy metabolism having mitochondria as a major target. However, the underlying mechanisms are poorly understood. Here, using a model of chemically induced hypothyroidism in male Wistar rats, we investigated the effect of administration of either 3,5-T2 or T3 on liver oxidative capacity through their influence on mitochondrial processes including: proton-leak across the mitochondrial inner membrane; complex I-, complex II- and glycerol-3-phosphate-linked respiratory pathways; respiratory complex abundance and activities as well as individual complex aggregation into supercomplexes. Background/Aims: Both 3,5-diiodo-L-thyronine (3,5-T2) and 3,5,3'-triiodo-L-tyronine (T3) affect energy metabolism having mitochondria as a major target. However, the underlying mechanisms are poorly understood. Here, using a model of chemically induced hypothyroidism in male Wistar rats, we investigated the effect of administration of either 3,5-T2 or T3 on liver oxidative capacity through their influence on mitochondrial processes including: proton-leak across the mitochondrial inner membrane; complex I-, complex II- and glycerol-3-phosphate-linked respiratory pathways; respiratory complex abundance and activities as well as individual complex aggregation into supercomplexes. Methods: Hypothyroidism was induced by propylthiouracil and iopanoic acid; 3,5-T2 and T3 were intraperitoneally administered at 25 and 15 μg/100 g BW for 1 week, respectively. Resulting alterations in mitochondrial function were studied by combining respirometry, Blue Native-PAGE followed by in-gel activity, and Western blot analyses. Results: Administration of 3,5-T2 and T3 to hypothyroid (hypo) rats enhanced mitochondrial respiration rate with only T3 effectively stimulating proton-leak (450% vs. Hypo). T3 significantly enhanced complex I (+145% vs. Hypo), complex II (+66% vs. Hypo), and glycerol-3 phosphate dehydrogenase (G3PDH)-linked oxygen consumptions (about 6- fold those obtained in Hypo), while 3,5-T2 administration selectively restored Euthyroid values of complex II- and increased G3PDH- linked respiratory pathways (+165% vs. Hypo). The mitochondrial abundance of all respiratory complexes and of G3PDH was increased by T3 administration whereas 3,5-T2 only increased complex V and G3PDH abundance. 3,5-T2 enhanced complex I and complex II in gel activities with less intensity than did T3, and T3 also enhanced the activity of all other respiratory complexes tested. In addition, only T3 enhanced individual respiratory component complex assembly into supercomplexes. Conclusions: The reported data highlight novel molecular mechanisms underlying the effect elicited by iodothyronine administration to hypothyroid rats on mitochondrial processes related to alteration in oxidative capacity in the liver. The differential effects elicited by the two iodothyronines indicate that 3,5-T2, by influencing the kinetic properties of specific mitochondrial respiratory pathways, would promote a rapid response of the organelle, while T3, by enhancing the abundance of respiratory chain component and favoring the organization of respiratory chain complex in supercomplexes, would induce a slower and prolonged response of the organelle.

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.

Introduction: Free thyroxine (T4), free triiodothyronine thyroid (T3) and thyroid stimulating hormone (TSH) are recognized as regulators of lipid synthesis, mobilization and degradation. Objective: To find a relationship between dyslipidemia and thyroid disease in an adult population. Methods: This was a cross-sectional study of 819 individuals. TSH, free T4, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) were measured. Results: The general population showed normal TC, normal LDL-C, low HDL-C, and elevated TG. The prevalence of dyslipidemia was as follows: elevated TC 31.9%, HDL-C

OBJECTIVE: To find out the relationship of thyroid hormone profile of females with outcomes after intra cytoplasmic sperm injection (ICSI). METHOD: It was a cross sectional study conducted in Islamabad Clinic Serving Infertile Couple from June 2013 till August 2015. T3 (triiodo thyronine), T4 (thyroxine) and TSH (thyroid stimulating hormone) of 168 consented females was estimated after they underwent the first step of treatment protocol (ovarian down regulation) for ICSI. Pregnant group had ß hCG result more than 25 IU/mL while the rest were included in the non-pregnant group. Both groups were compared by using independent sample t-test. Pearson correlation coefficient was used to associate T3 and T4 with other pregnancy variables with their significance. RESULTS: Non pregnant women had significantly higher mean values for T3 and T4 as compared to pregnant women (p

The conversion of white adipose cells into beige adipose cells is known as browning, a process affecting energy metabolism. It has been shown that 3,5 diiodo-l-thyronine (T2), an endogenous metabolite of thyroid hormones, stimulates energy expenditure and a reduction in fat mass. In light of the above, the purpose of this study was to test whether in an animal model of fat accumulation, T2 has the potential to activate a browning process and to explore the underlying mechanism. Three groups of rats were used: (i) receiving a standard diet for 14 weeks, (ii) receiving a high-fat diet (HFD) for 14 weeks, and (iii) receiving a high fat diet for 10 weeks and being subsequently treated for four weeks with an HFD together with the administration of T2. We showed that T2 was able to induce a browning in the white adipose tissue of T2-treated rats. We also showed that some miRNA (miR133a and miR196a) and MAP kinase 6 were involved in this process. These results indicate that, among others, the browning may be another cellular/molecular mechanism by which T2 exerts its beneficial effects of contrast to overweight and of reduction of fat mass in rats subjected to HFD.

3,5-diiodo-L-thyronine (3,5-T2) is an endogenous derivative of thyroid hormone with potential metabolic effects. It has been detected in human blood by immunological methods, but a reliable assay based on mass spectrometry (MS), which is now regarded as the gold standard in clinical chemistry, is not available yet. Therefore, we aimed at developing a novel ad-hoc optimized method to quantitate 3,5-T2 and its isomers by MS in human serum. Serum samples were obtained from 28 healthy subjects. Two ml of serum were deproteinized with acetonitrile and then subjected to an optimized solid phase extraction-based procedure. To lower background noise, the samples were furtherly cleaned by hexane washing and acetonitrile precipitation of residual proteins. 3,5-T2 and its isomers 3,3′-T2 and 3′,5′-T2 were then analyzed by HPLC coupled to tandem MS. Accuracy and precision for T2 assay were 88–104% and 95–97%, respectively. Recovery and matrix effect averaged 78% and +8%, respectively. 3,5-T2 was detected in all samples and its concentration averaged (mean ± SEM) 41 ± 5 pg/ml, i.e., 78 ± 9 pmol/l. In the same samples the concentration of 3,3′-T2 averaged 133±15 pg/ml, i.e., 253±29 pmol/l, while 3′,5′-T2 was not detected. 3,5-T2 concentration was significantly related to 3,3′-T2 concentration (r = 0.540, P < 0.01), while no significant correlation was observed with either T3 or T4 in a subset of patients in which these hormones were assayed. In conclusion, our method is able to quantify 3,5-T2 and 3,3′-T2 in human serum. Their concentrations lie in the subnanomolar range, and a significant correlation was detected between these two metabolites in healthy individuals.

When administered to rats receiving a high-fat diet (HFD), 3,5-diiodo-L-thyronine (3,5-T2) [at a dose of 25 &mu, g/100 g body weight (BW)] is known to increase energy expenditure and to prevent HFD-induced adiposity. Here, we investigated which cellular and molecular processes in visceral white adipose tissue (VAT) contributed to the beneficial effect of 3,5-T2 over time (between 1 day and 4 weeks following administration). 3,5-T2 programmed the adipocyte for lipolysis by rapidly inducing hormone sensitive lipase (HSL) phosphorylation at the protein kinase A-responsive site Ser563, accompanied with glycerol release at the 1-week time-point, contributing to the partial normalization of adipocyte volume with respect to control (N) animals. After two weeks, when the adipocyte volumes of HFD-3,5-T2 rats were completely normalized to those of the controls (N), 3,5-T2 consistently induced HSL phosphorylation at Ser563, indicative of a combined effect of 3,5-T2-induced adipose lipolysis and increasing non-adipose oxidative metabolism. VAT proteome analysis after 4 weeks of treatment revealed that 3,5-T2 significantly altered the proteomic profile of HFD rats and produced a marked pro-angiogenic action. This was associated with a reduced representation of proteins involved in lipid storage or related to response to oxidative stress, and a normalization of the levels of those involved in lipogenesis-associated mitochondrial function. In conclusion, the prevention of VAT mass-gain by 3,5-T2 occurred through different molecular pathways that, together with the previously reported stimulation of resting metabolism and liver fatty acid oxidation, are associated with an anti adipogenic/lipogenic potential and positively impact on tissue health.

It is a common notion that thyroid status is linked to energy expenditure and body weight, and thyroid hormones (THs) have always deserved attention for their potential use as pharmacological agents to counteract obesity and lipid metabolism-related disorders. Besides 3,5,3′,5′-tetraiodo- l -thyronine (thyroxine or T4) and 3,3′,5,-triiodo- l -thyronine (T3), other iodothyronines can be produced in the thyroid or in peripheral tissues through the process of stepwise deiodination. Among these TH metabolites, 3,5-diodo- l -thyronine (T2) is of particular interest for its effects on energy metabolism. After a brief overview of TH production and actions at the cellular level, this chapter will focus on the mechanisms underlying the lipid-lowering effects of the iodothyronines T3 and T2, with particular regard to the regulation of lipid metabolism in the liver.

2,4,6-tribromophenol (TBP) is generally used as a brominated flame retardant but is produced in the degradation of tetrabromobisphenol-A. Although TBP is frequently detected in the environment and in various biota, including fish species, we still know little about its toxicity and environmental health risk. Here we investigated the bioconcentration and effects of TBP on the thyroid endocrine system by using zebrafish as a model. Zebrafish embryos (2 h post-fertilization, hpf) were exposed to five concentrations of TBP (0, 0.3, 1, 10, and 100 μg/L) until 144 hpf. According to our chemical analysis, TBP underwent bioconcentration in zebrafish larvae. However, acute exposure to TBP did not affect the hatching of embryos or their risk of malformation, nor the growth and survival of larvae, indicating low developmental toxicity of TBP. The whole-body thyroxine (T4) contents were significantly increased in zebrafish larvae after exposure to TBP, indicating thyroid endocrine disruption occurred. Gene transcription levels in the hypothalamic-pituitary-thyroid (HPT) axis were also examined in larvae; these results revealed that the transcription of corticotrophin-releasing hormone (crh), thyrotropin-releasing hormone (trh), and thyroid-stimulating hormone (tshβ) were all significantly downregulated by exposure to TBP. Likewise, genes encoding thyronine deiodinases (dio1, dio2, and dio3a/b) and thyroid hormone receptors (trα and trβ) also had their transcription downregulated in zebrafish. Further, the gene transcription and protein expression of binding and transport protein transthyretin (TTR) were significantly increased after TBP exposure. Taken together, our results suggest the bioavailability of and potential thyroid endocrine disruption by TBP in fish.

Studies involving human genetic mutations and mutant mouse models have provided irrevocable evidence for a key role for thyroid hormones (THs) in the regulation of skeletal growth. While T3 binds to TH receptors with higher affinity than T4, T4 occupied TH receptors have also been reported in the nucleus under euthyroid conditions raising the possibility that T4 bound nuclear receptors may be biologically relevant in thyroid syndromes with elevated free T4 and reduced T3 levels. We, therefore, evaluated the direct effects of T4, T3, and their metabolites (rT3 and T2) in stimulating osteoblast differentiation using MC3T3-E1 preosteoblasts which do not produce detectable levels of deiodinases. Under serum-free conditions, a 24-h treatment of MC3T3-E1 cells with THs and their metabolites caused a dose-dependent increase in the expression of osteoblast differentiation markers, osterix, and osteocalcin. Circulating concentrations of T3 (~1 ng/ml) and T4 (~30 ng/ml) showed similar potency in stimulating osteoblast differentiation marker expression, while rT3 and T2 were less potent than T3 and T4. Moreover, T3 and T4 treatments elevated the IGF-1 mRNA level suggesting the involvement of IGF-1 signaling in the TH regulation of osteoblast differentiation. We conclude that an elevated T4 level in the absence of T3 may exert stimulatory effects on osteoblast differentiation. The establishment of cell-specific effects of T4 on osteoblasts may provide a strategy to generate T4 mimics that exert skeletal specific effects without the confounding T3 effects on other tissues.

This paper describes a novel analytical methodology for the simultaneous determination of absolute and total concentrations of 11 native thyroid hormones and associated metabolites, viz. thyroxine (T4), 3,3', 5-triiodothyronine (T3), 3,3', 5'-triiodothyronine (rT3), 3,5-diiodothyronine (3,5-T2), 3,3'- diiodothyronine (3,3'-T2), 3-iodothyronine (T1), thyronine (T0), 3-iodothyronamine (T1AM), tetraiodothyroacetic acid (Tetrac), triiodothyroacetic acid (Triac), and diiodothyroacetic acid (Diac), in 50-μL of plasma or serum. The method was optimized using four isotopic labeled surrogate and internal standards in combination with solid-phase extraction and LC-MS/MS. The methodology was further evaluated using amphibian plasma and serum with matrix-matched calibration applied for quantification. Method detection limits are 3.5 pg T4, 1.5 pg T3, 2.9 pg rT3, 1.7 pg 3,3'-T2, 2.3 pg 3,5-T2, and between 0.3 and 7.5 pg for the remaining six metabolites in 50 μL aliquots of blood sera or plasma. Accuracies and repeatabilities for all analytes were between 88 and 103 % and 1.31 and 17.2 %, respectively. Finally, we applied the method on adult frog (Xenopus laevis) plasma and tadpole (Rana (Lithobates) catesbeiana) serum. We observed up to seven different thyroid hormones and associated metabolites in tadpole serum. This method will enable researchers to improve the assessment of thyroid homeostasis and endocrine disruption in animals and humans. Graphical Abstract Quantification of 11 thyroid hormones and metabolites from 50 μL plasma or serum using protein denaturation in combination with solid-phase extraction followed by LC-MS/MS.

Peroxynitrite is a highly reactive oxidant effecting cell signaling and cell death. Here we report a fluorescent protein probe to selectively detect peroxynitrite. A novel unnatural amino acid, thyronine (Thy), was genetically encoded in E. coli and mammalian cells by evolving an orthogonal tRNA(Pyl)/ThyRS pair. Incorporation of Thy into the chromophore of sfGFP or cpsGFP afforded a virtually non-fluorescent reporter. Upon treatment with peroxynitrite, Thy was converted into tyrosine via O-dearylation, regenerating GFP fluorescence in a time- and concentration-dependent manner. Genetically encoded thyronine may also be valuable for other redox applications.

3,5-diiodo-l-thyronine (T2) is an endogenous derivative of thyroid hormone that has been suggested to regulate energy expenditure, resting metabolic rate and oxygen consumption with a mechanism that involves the activation of mitochondrial function. In this study, we focused on the cardiac effects of T2, which have been poorly investigated so far, by using both in vitro and ex vivo models. As a comparison, the response to T3 and T4 was also determined. Rat cardiomyoblasts (H9c2 cells) were used to determine T2, T3, and T4 uptake by high-performance liquid chromatography–tandem mass spectrometry. In the same experimental model, MTT test, crystal violet staining, and glucose consumption were investigated, using T2 concentrations ranging from 0.1 to 10 µM. To assess cardiac functional effects, isolated working rat hearts were perfused with T2, T3, or T4 in Krebs-Ringer buffer, and the hemodynamic variables were recorded. T2 was taken up by cardiomyoblasts, and in cell lysate T2 levels increased slowly over time, reaching higher concentrations than in the incubation medium. T2 significantly decreased MTT staining at 0.5–10 µM concentration (P

Bisphenol AF (BPAF) is extensively used as a raw material in industry, resulting in its widespread distribution in the aqueous environment. However, the effect of BPAF on the hypothalamic-pituitary-thyroidal (HPT) axis remains unknown. For elucidating the disruptive effects of BPAF on thyroid function and expression of the representative genes along the HPT axis in zebrafish (Danio rerio) embryos, whole-body total 3,3′,5-triiodothyronine (TT3), total 3,5,3′,5′-tetraiodothyronine (TT4), free 3,3′,5-triiodothyronine (FT3) and free 3,5,3′,5′-tetraiodothyronine (FT4) levels were examined following 168 h post-fertilization exposure to different BPAF concentrations (0, 5, 50 and 500 μg/L). The results showed that whole-body TT3, TT4, FT3 and FT4 contents decreased significantly with the BPAF treatment, indicating an endocrine disruption of thyroid. The expression of thyroid-stimulating hormone-β and thyroglobulin genes increased after exposing to 50 μg/L BPAF in seven-day-old larvae. The expressions of thyronine deiodinases type 1, type 2 and transthyretin mRNAs were also significantly up-regulated, which were possibly associated with a deterioration of thyroid function. However, slc5a5 gene transcription was significantly down-regulated at 50 μg/L and 500 μg/L BPAF exposure. Furthermore, trα and trβ genes were down-regulated transcriptionally after BPAF exposure. It demonstrates that BPAF exposure triggered thyroid endocrine toxicity by altering the whole-body contents of thyroid hormones and changing the transcription of the genes involved in the HPT axis in zebrafish larvae.

BackgroundThere are a few papers reporting the association between thyroid hormones, cholesterol and lipoprotein in euthyroid cardiovascular patients.ObjectiveOur objective was to investigate the relationship between thyroid hormones, cholesterol and lipoprotein in euthyroid cardiovascular patients.MethodsThe study population consisted of 73 consecutive euthyroid cardiovascular patients. The average age of the patients was 57±16years, and 52% were male. The serum levels of free 3,3′,5′-triiodo-l-thyronine (FT3), free thyroxine (3,5,3′,5′-tetraiodo-l-thyronine; FT4), thyroxine stimulating hormone (TSH), apolipoprotein, fatty acids and cholesterol were assessed.ResultsWe found a strong, significant positive correlation between the serum levels of FT4 and low-density lipoprotein cholesterol (LDL-C) (r=0.442, p

3,3′,5-triiodo-L-thyronine (T3) improves hepatic lipid accumulation by increasing lipid catabolism but it also increases lipogenesis, which at first glance appears contradictory. Recent studies have shown that 3,5-diiodothyronine (T2), a natural thyroid hormone derivative, also has the capacity to stimulate hepatic lipid catabolism, however, little is known about its possible effects on lipogenic gene expression. Because genes classically involved in hepatic lipogenesis such as SPOT14, acetyl-CoA-carboxylase (ACC), and fatty acid synthase (FAS) contain thyroid hormone response elements (TREs), we studied their transcriptional regulation, focusing on TRE-mediated effects of T3 compared to T2 in rats receiving high-fat diet (HFD) for 1 week. HFD rats showed a marked lipid accumulation in the liver, which was significantly reduced upon simultaneous administration of either T3 or T2 with the diet. When administered to HFD rats, T2, in contrast with T3, markedly downregulated the expression of the above-mentioned genes. T2 downregulated expression of the transcription factors carbohydrate-response element-binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) involved in activation of transcription of these genes, which explains the suppressed expression of their target genes involved in lipogenesis. T3, however, did not repress expression of the TRE-containing ChREBP gene but repressed SREBP-1c expression. Despite suppression of SREBP-1c expression by T3 (which can be explained by the presence of nTRE in its promoter), the target genes were not suppressed, but normalized to HFD reference levels or even upregulated (ACC), partly due to the presence of TREs on the promoters of these genes and partly to the lack of suppression of ChREBP. Thus, T2 and T3 probably act by different molecular mechanisms to achieve inhibition of hepatic lipid accumulation.

Novel mechanisms associated with therapeutically safe thyroid hormone (TH) therapy are emerging. We have shown that oral triiodo-L-thyronine (T3) offers safe cardioprotection in coronary ligation myocardial infarction (MI), ligation ischemia-reperfusion injury, diabetic cardiomyopathy, etc. via restoration of gene expression. However, safe therapeutic effects following atherosclerosis-driven MI and role of long noncoding RNAs (lncRNAs) is unknown. We employed a mouse model of scavenger receptor B1 knockout with hypomorphic apolipoprotein E. Young adult heterozygote littermates served as controls and all mice received high fat (HF) diet for one month. Along with HF diet, a cohort of homozygotes (HypoE) received therapeutic dose of T3 (5.5 μg/kg/d) in drinking water ad libitum. In HypoE mice, Paigen HF diet induced interstitial fibrotic MI with severe hypertrophic (Heart wt./Body wt., HW/BW: control:4.6±0.14; HypoE:12.9±0.75; p2-fold; p

The effects of halogen derivatives of thyronine (tetraiodotironine and triiodothyronine) and fluorescein (Rose Bengal, phloxine B, erythrosin, eosin Y, and fluorescein) on the dipole potential of membranes composed of diphytanoylphosphocholine, diphytanoylphosphoserine, and diphytanoylphosphoethanolamine were investigated. A quantitative description of the modifying action of the agents was presented as characteristic parameters of the Langmuir adsorption isotherm: the maximum changes in the dipole potential of the membrane at an infinitely high concentration of modifiers and the desorption constant, characterizing their inverse affinities to the lipid phase. It was shown that the iodine-containing hormones led to a less significant reduction in the dipole potential of phospholipid membranes compared to the xanthene dyes, Rose Bengal, phloxine B, and erythrosin. The latter were characterized by the highest affinity for the lipid membranes compared to tetraiodotironine and triiodothyronine. It was found that the effect of iodine-containing hormones and xanthene dyes on the membrane dipole potential was caused by their uncharged and charged forms, respectively.

Background Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have great potential as a cell source for therapeutic applications such as regenerative medicine, disease modeling, drug screening, and toxicity testing. This potential is limited, however, by the immature state of the cardiomyocytes acquired using current protocols. Tri-iodo- l -thyronine (T3) is a growth hormone that is essential for optimal heart growth. In this study, we investigated the effect of T3 on hiPSC-CM maturation. Methods and results A one-week treatment with T3 increased cardiomyocyte size, anisotropy, and sarcomere length. T3 treatment was associated with reduced cell cycle activity, manifest as reduced DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor p21. Contractile force analyses were performed on individual cardiomyocytes using arrays of microposts, revealing an almost two-fold higher force per-beat after T3 treatment and also an enhancement in contractile kinetics. This improvement in force generation was accompanied by an increase in rates of calcium release and reuptake, along with a significant increase in sarcoendoplasmic reticulum ATPase expression. Finally, although mitochondrial genomes were not numerically increased, extracellular flux analysis showed a significant increase in maximal mitochondrial respiratory capacity and respiratory reserve capability after T3 treatment. Conclusions Using a broad spectrum of morphological, molecular, and functional parameters, we conclude that T3 is a driver for hiPSC-CM maturation. T3 treatment may enhance the utility of hiPSC-CMs for therapy, disease modeling, or drug/toxicity screens.

Experimental studies have highlighted that the administration of 3,5-diiodo-L-thyronine (T2) to rats fed diets rich in lipids induces a decrease of cholesterol and triglycerides plasma levels and body weight (BW) without inducing liver steatosis. On the basis of these observations we carried out some experimental in vivo studies to assess the effects of multiple high doses of T2 on the pituitary thyroid axis of rats fed diet rich in lipids. Fifteen male Wistar rats were divided into three groups of five animals each. The first group (N group) received standard diet, the second group was fed with a high fat diet (HFD group), while the third group (HFDT2 group) was additionally given T2 intraperitoneally at a dose level of 70 µg/100 g of BW three times a week up to four weeks. At the end of the treatment, blood sample from each animal was collected, centrifuged and the serum was stored at -20°C. The serum concentrations of thyroidstimulating hormone (TSH), triiodothyronine, thyroxine, adrenocorticotropic hormone, triglycerides, cholesterol, glucose, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase were then determined. In addition, liver of rats was examined by histology in order to assess the presence and degree of steatosis. The administration of T2 to rats fed with a high fat diet suppressed TSH secretion (P=0.013) while no steatosis was observed in the liver of these animals. Our data show that multiple administrations of high doses of T2 to rats fed diets rich in lipid inhibit TSH secretion and prevent the onset of liver steatosis in these animals.