Determination of 3-iodothyronamine (3-T1AM) in mouse liver using liquid chromatography-tandem mass spectrometry.

3-iodothyronamine (3-T1AM) has been suggested as a novel chemical messenger and potent trace amine-associated receptor 1 ligand in the CNS that occurs naturally as endogenous metabolite of the thyroid hormones. Discrepancies and variations in 3-T1AM plasma and tissue concentrations have nonetheless caused controversy regarding the existence and biological role of 3-T1AM. These discussions are at least partially based on potential analytical artefacts caused by differential decay kinetics of 3-T1AM and the widely used deuterated quantification standard D4-T1AM. Here, we report a novel LC-MS/MS method for the quantification of 3-T1AM in biological specimens using stable isotope dilution with 13C6-T1AM, a new internal standard that showed pharmacodynamic properties comparable to endogenous 3-T1AM. The method detection limit (MDL) and method quantification limit (MQL) of 3-T1AM were 0.04 and 0.09 ng/g, respectively. The spike-recoveries of 3-T1AM were between 85.4% and 94.3%, with a coefficient of variation of 3.7-5.8%. The intra-day and inter-day variations of 3-T1AM were 8.45-11.2% and 3.58-5.73%, respectively. Endogenous 3-T1AM liver values in C57BL/6J mice were 2.20 ± 0.49 pmol/g with a detection frequency of 50%. Higher liver 3-T1AM values were found when C57BL/6J mice were treated with N-acetyl-3-iodothyronamine or O-acetyl-3-iodothyronamine. Overall, our new stable isotope dilution LC-MS/MS method improves both the sensitivity and selectivity compared with existing methods. The concomitant possibility to quantify additional thyroid hormones such as thyroxine, 3,5,3'-triiodo-L-thyronine, 3,3',5'-triiodo-L-thyronine, 3,3'-diiodo-L-thyronine, and 3,5-diiodo-L-thyronine further adds to the value of our novel method in exploring the natural occurrence and fate of 3-T1AM in biological tissues and fluids.

Electrochemical sensing of the thyroid hormone thyronamine (T0AM) via molecular imprinted polymers (MIPs).

Recent studies have shown that besides the well-known T3 (triiodothyronine) and T4 (thyroxine) there might be other important thyroid hormones, in particular T0AM (thyronamine) and T1AM (3-iodothyronamine). The absence of a large number of studies showing their precise importance might be explained by the limited number of analytical methodologies available. This work aims to show an electroanalytical alternative making use of electropolymerized molecularly imprinted polymer (MIPs). The MIPs' polymerization is performed on the surface of screen-printed carbon electrodes (SPCEs), using 4-aminobenzoic acid (4-ABA) as the building and functional monomer and the analyte T0AM as the template. The step-by-step construction of the SPCE-MIP sensor was studied by cyclic voltammetry (CV) and by electrochemical impedance spectroscopy (EIS). After optimization, by means of square-wave voltammetry, the SPCE-MIP showed suitable selectivity (in comparison with other thyroid hormones and catechol amines), repeatability (intra-day of 3.9%), a linear range up to 10 µmol L-1 (0.23 × 103 µg dL-1) with an r2 of 0.998 and a limit of detection (LOD) and quantification (LOQ) of 0.081 and 0.27 µmol L-1 (1.9 and 6.2 µg dL-1), respectively.

Recovery of 3-Iodothyronamine and Derivatives in Biological Matrixes: Problems and Pitfalls.

BACKGROUND: Difficulties have been reported in quantitating 3-iodothyronamine (T1AM) in blood or serum, and tentatively attributed to problems in extraction or other pre-analytical steps. For this reason, even cell culture experiments have often be performed with unphysiological protein-free media. The aim of this study was to evaluate the recovery of exogenous T1AM added to a standard cell culture medium, namely Dulbecco's minimum essential medium (DMEM) supplemented with fetal bovine serum (FBS), and to other biological matrixes. METHODS: Cell culture media (Krebs-Ringer buffer, DMEM, FBS, DMEM + FBS, used either in the absence or in the presence of NG108-15 cells) and other biological matrixes (rat brain and liver homogenates, human plasma, and blood) were spiked with T1AM and/or deuterated T1AM (d4-T1AM) and incubated for times ranging from 0 to 240 minutes. Samples were then extracted using a liquid/liquid method and analyzed using liquid chromatography coupled to mass spectrometry in order to assay T1AM and its metabolites, namely 3-iodothyroacetic acid (TA1), thyronamine, thyroacetic acid, N-acetyl-T1AM, and T1AM esters. RESULTS: In FBS-containing buffers, T1AM decreased exponentially over time, with a half-life of 6-17 minutes, depending on FBS content, and after 60 minutes, it averaged 0-10% of the baseline. T1AM metabolites were not detected, except for minimum amounts of TA1. Notably, d4-T1AM decreased over time at a much lower rate, reaching 50-70% of the baseline at 60 minutes. These effects were completely abolished by protein denaturation and partly reduced by semicarbazide. In the presence of cells, T1AM concentration decreased virtually to 0 within 60 minutes, but TA1 accumulated in the incubation medium, with quantitative recovery. Spontaneous decrease in T1AM concentration with isotopic difference was confirmed in rat organ homogenates and human blood. CONCLUSIONS: These results suggest binding and sequestration of T1AM and/or its aldehyde derivative by blood and tissue proteins, with significant isotope effects. These issues might account for the technical problems complicating the analytical assays of endogenous T1AM.

Influence of iodothyronine conjugates of bovine serum albumin and horseradish peroxidase on enzyme immunosorbent assay of thyroid hormones.

Enzyme-linked immunosorbent assays (ELISA's) reported for thyroxine (T4) and 3,5,3'-triiodothyronine (T3), involved coupling of the haptens through (i) carboxylic group to carrier protein for producing antibodies and (ii) amino group to detection labels. To improve the titer and specificity of antibodies, immunogens were prepared by coupling of carboxyl group to bovine serum albumin (BSA) either directly or through adipic acid dihydrazide (ADH), after protecting amino group through acetylation of T4 and T3. Direct coupling resulted in the incorporation of 40-50 moles of T4 and T3 per BSA molecule and helped in improving immunogenic response and specificity, especially of T4. High epitope density of immunogens evoked better antibody response, since attachement of ADH as spacer, introduced 18-27 moles of haptens into carrier protein and had less effect on antibody development, with T3 being exception. Detection labels were prepared by coupling horseradish peroxidase (HRP) to amino group of thyroid hormones directly and after preparing their methyl esters, which provided sensitive displacement curves in combination with the antibodies developed against N-acetylated-T4 and T3. Unlike methyl esters, T4-HRP and T3-HRP showed higher sensitivity and seemed to be related to the affinity of the labels for binding the antibody.

In vivo molecular imaging of [125I]-labeled 3-iodothyronamine: a hibernation-inducing agent.

The present investigation was carried out with the objective of studying in vivo imaging of 3-iodothyronamine (T(1)AM) compound in mice. A simple and efficient synthesis of [(125)I]-T(1)AM was established, and a molecular imaging study was performed using micro-SPECT/CT at 1h post-injection of [(125)I]-T(1)AM. Imaging studies revealed the activity in the gastrointestinal tract and liver, indicating that [(125)I]-T(1)AM was distributed primarily in the liver, and excreted into the gastrointestinal tract through a bile duct.

Detection of 3-iodothyronamine in human patients: a preliminary study.

CONTEXT AND OBJECTIVE: The primary purpose of this study was to detect and quantify 3-iodothyronamine (T(1)AM), an endogenous biogenic amine related to thyroid hormone, in human blood. DESIGN: T(1)AM, total T(3), and total T(4) were assayed in serum by a novel HPLC tandem mass spectrometry assay, which has already been validated in animal investigations, and the results were related to standard clinical and laboratory variables. SETTING AND PATIENTS: The series included one healthy volunteer, 24 patients admitted to a cardiological ward, and 17 ambulatory patients suspected of thyroid disease, who underwent blood sampling at admission for routine diagnostic purposes. Seven patients were affected by type 2 diabetes, and six patients showed echocardiographic evidence of impaired left ventricular function. INTERVENTIONS: No intervention or any patient selection was performed. MAIN OUTCOME MEASURES: serum T(1)AM, total and free T(3) and T(4), routine chemistry, routine hematology, and echocardiographic parameters were measured. RESULTS: T(1)AM was detected in all samples, and its concentration averaged 0.219 ± 0.012 pmol/ml. The T(1)AM concentration was significantly correlated to total T(4) (r = 0.654, P < 0.001), total T(3) (r = 0.705, P < 0.001), glycated hemoglobin (r = 0.508, P = 0.013), brain natriuretic peptide (r = 0.543, P = 0.016), and γ-glutamyl transpeptidase (r = 0.675, P < 0.001). In diabetic vs. nondiabetic patients T(1)AM concentration was significantly increased (0.232 ± 0.014 vs. 0.203 ± 0.006 pmol/ml, P = 0.044), whereas no significant difference was observed in patients with cardiac dysfunction. CONCLUSIONS: T(1)AM is an endogenous messenger that can be assayed in human blood. Our results are consistent with the hypothesis that circulating T(1)AM is produced from thyroid hormones and encourage further investigations on the potential role of T(1)AM in insulin resistance and heart failure.

Thyronamines--past, present, and future.

Thyronamines (TAMs) are a newly identified class of endogenous signaling compounds. Their structure is identical to that of thyroid hormone and deiodinated thyroid hormone derivatives, except that TAMs do not possess a carboxylate group. Despite some initial publications dating back to the 1950s, TAMs did not develop into an independent area of research until 2004, when they were rediscovered as potential ligands to a class of G protein-coupled receptors called trace-amine associated receptors. Since this discovery, two representatives of TAMs, namely 3-iodothyronamine (3-T(1)AM) and thyronamine (T(0)AM), have been detected in vivo. Intraperitoneal or central injection of 3-T(1)AM or T(0)AM into mice, rats, or Djungarian hamsters caused various prompt effects, such as metabolic depression, hypothermia, negative chronotropy, negative inotropy, hyperglycemia, reduction of the respiratory quotient, ketonuria, and reduction of fat mass. Although their physiological function remains elusive, 3-T(1)AM and T(0)AM have already revealed promising therapeutic potential because they represent the only endogenous compounds inducing hypothermia as a prophylactic or acute treatment of stroke and might thus be expected to cause fewer side effects than synthetic compounds. This review article summarizes the still somewhat scattered data on TAMs obtained both recently and more than 20 yr ago to yield a complete and updated picture of the current state of TAM research.

Tissue distribution and cardiac metabolism of 3-iodothyronamine.

3-iodothyronamine (T1AM) is a novel relative of thyroid hormone, able to interact with specific G protein-coupled receptors, known as trace amine-associated receptors. Significant functional effects are produced by exogenous T1AM, including a negative inotropic and chronotropic effect in cardiac preparations. This work was aimed at estimating endogenous T1AM concentration in different tissues and determining its cardiac metabolism. A novel HPLC tandem mass spectrometry assay was developed, allowing detection of T1AM, thyronamine, 3-iodothyroacetic acid, and thyroacetic acid. T1AM was detected in rat serum, at the concentration of 0.3±0.03 pmol/ml, and in all tested organs (heart, liver, kidney, skeletal muscle, stomach, lung, and brain), at concentrations significantly higher than the serum concentration, ranging from 5.6±1.5 pmol/g in lung to 92.9±28.5 pmol/g in liver. T1AM was also identified for the first time in human blood. In H9c2 cardiomyocytes and isolated perfused rat hearts, significant Na+-dependent uptake of exogenous T1AM was observed, and at the steady state total cellular or tissue T1AM concentration exceeded extracellular concentration by more than 20-fold. In both preparations T1AM underwent oxidative deamination to 3-iodothyroacetic acid. T1AM deamination was inhibited by iproniazid but not pargyline or semicarbazide, suggesting the involvement of both monoamine oxidase and semicarbazide-sensitive amine oxidase. Thyronamine and thyroacetic acid were not detected in heart. Finally, evidence of T1AM production was observed in cardiomyocytes exposed to exogenous thyroid hormone, although the activity of this pathway was very low.

An online solid-phase extraction-liquid chromatography-tandem mass spectrometry method to study the presence of thyronamines in plasma and tissue and their putative conversion from 13C6-thyroxine.

Thyronamines are exciting new players at the crossroads of thyroidology and metabolism. Here, we report the development of a method to measure 3-iodothyronamine (T(1)AM) and thyronamine (T(0)AM) in plasma and tissue samples. The detection limit of the method was 0.25 nmol/l in plasma and 0.30 pmol/g in tissue both for T(1)AM and for T(0)AM. Using this method, we were able to demonstrate T(1)AM and T(0)AM in plasma and liver from rats treated with synthetic thyronamines. Although we demonstrated the in vivo conversion of (13)C(6)-thyroxine ((13)C(6)-T(4)) to (13)C(6)-3,5,3'-triiodothyronine, we did not detect (13)C(6)-T(1)AM in plasma or brain samples of rats treated with (13)C(6)-T(4). Surprisingly, our method did not detect any endogenous T(1)AM or T(0)AM in plasma from vehicle-treated rats, nor in human plasma or thyroid tissue. Although we are cautious to draw general conclusions from these negative findings and in spite of the fact that insufficient sensitivity of the method related to extractability and stability of T(0)AM cannot be completely excluded at this point, our findings raise questions on the biosynthetic pathways and concentrations of endogenous T(1)AM and T(0)AM.

In-line coupling of a reversed-phase column to cope with limited chemoselectivity of a quinine carbamate-based anion-exchange type chiral stationary phase.

A chiral stationary phase based on tert-butylcarbamoyl quinine has shown remarkable enantiomer separation capability for the thyroid hormone thyroxine (T(4)) and its structural analogue triiodothyronine (T(3)) employing hydroorganic buffered mobile phases (typical RP conditions). To overcome the problem of a somehow limited chemoselectivity for the critical peak pair between adjacent L-thyroxine (L-T(4)) and D-thyroxine (D-T(4)) peaks on the chiral anion-exchanger CSP when all four compounds need to be analysed simultaneously like in impurity profiling of L-T(4 )drug products, an RP column (Gemini C18) was serially coupled with the chiral anion-exchanger column to add a hydrophobic selectivity increment and to improve thereby the critical resolution between L-T(3) and D-T(4). Various parameters such as organic modifier content, pH, buffer concentration and type, type of achiral column as well as sequence of achiral and chiral column have been investigated with individual and tandem columns. With the optimized conditions and use of the tandem column a significantly improved separation, as compared to the chiral anion-exchanger column alone, with a critical resolution as high as 3.7 and an almost equal band spacing of the four components of the test mixture could be obtained. The sequence of the columns (achiral-chiral or chiral-achiral) had no significant effect on the separation performance.

Differentiation of monoiodothyronines using electrospray ionization tandem mass spectrometry.

In this work two monoiodothyronines, 3-T1 and 3'-T1, have been analyzed using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed based on our data obtained by ESI-MS/MS. MS2 spectra in either negative or positive ion mode can be used to differentiate 3-T1 and 3'-T1. Based on the relative abundance of fragment ions in MS2 spectra in the negative ion mode, quantification of the 3-T1 and 3'-T1 isomers in mixtures is achieved without prior separation. Solid-phase extraction in combination with ESI-MS/MS provides a practicable procedure that can be used to determine the molar ratio of 3-T1 and 3'-T1 in human serum with an error less than 3%. The detection limits for 3-T1 and 3'-T1 were 0.5 and 0.7 pg/microL, respectively.

Development of a validated HPLC method for the determination of iodotyrosines and iodothyronines in pharmaceuticals and biological samples using solid phase extraction.

Identification, separation and quantitation of iodoaminoacids, is essential for the biological research and the clinical diagnosis of thyroid gland disease. Under this aspect a reversed-phase high-performance liquid chromatographic method was developed for the determination of thyroid gland hormones and some of their primary metabolites, 3,3',5,5'-tetra-iodo-L-thyronine (L-thyroxine), 3,3',5-tri-iodo-L-thyronine, 3,5-di-iodo-L-thyronine, L-thyronine, 3,5-di-iodo-L-tyrosine, 3-iodo-L-tyrosine and l-tyrosine. Analysis was performed on an Inertsil C(18) column with photodiode-array detection, using a 25 min gradient scale program of a binary mobile phase consisted of 0.1% aqueous solution of trifluoroacetic acid at pH 3 as solvent A and acetonitrile as solvent B, at a flow rate of 1 mL/min. Quantitation was performed using were obtained using theophylline as internal standard. The method was applied to commercial pharmaceuticals and biological samples (serum, urine and tissue). Drug-free urine and serum samples were spiked with known concentrations of the analytes standards and pretreated by solid phase extraction to remove matrix interferences. C(18) cartridges were used, yielding recoveries ranging from 87.1% to 107.6% for serum samples and from 92.1% to 98.7% for urine samples. With regard to total-T(4) concentrations in serum samples, results are cross-validated with RIA and found to agree well.

5'-deiodinase activity and circulating thyronines in lactating cows.

To investigate the correlation between lactation and thyroid hormone metabolism, the authors studied concentrations of total and free thyroxine (T4 and fT4), triiodothyronine (T3 and fT3), and reverse triiodothyronine (rT3) in plasma and milk, as well as liver and mammary gland 5'-deiodinase (5'D) activity in dry, early, middle, and late lactating dairy cows. Cows in early lactation show lower plasma levels of T4 and rT3 than dry, middle, and late lactating animals, whereas T3 shows the lowest plasma levels in the dry period; free T4 and T3 show a similar pattern. In early lactation there is a clear decrease in liver 5'D associated with a notable increase in mammary 5'D. Concentrations of T4 and T3 in milk drop significantly in the first few days after delivery, whereas rT3 increases up to the fourth month. The findings suggest a relationship between the hypothyroid status of lactating cows and the rearrangement of organ-specific 5'-deiodinase activity related to the maintenance of the udder's function.

Identification of a nuclear protein from rat developing brain as heterodimerization partner with thyroid hormone receptor-beta.

Thyroid hormone receptors (TR) are ligand-activated transcription factors that modulate the expression of certain target genes in a developmental and tissue-specific manner. These specificities are determined by the tissue distribution of the TR isoforms alpha1 and beta1, the structure of the thyroid hormone response element (TRE) bound by the receptor, and heterodimerization partners. Among these, retinoid X receptors (RXR) have been recognized as the principal partners for TR. The present work reports the identification of a novel nuclear protein from 19-day-old embryonic rat brain that displays a distinct interaction pattern with TR isoforms at the level of the TRE of two genes known to be differentially expressed and regulated by thyroid hormone (T3): the ubiquitous malic enzyme and the brain-specific myelin basic protein. Electrophoretic gel mobility shift assays demonstrate that only TRbeta1 forms a specific complex with the rat brain nuclear factor on the myelin basic protein-TRE, but not on the malic enzyme-TRE. Thus, the interaction is selectively determined by both the receptor isoform and the structure of the TRE. The expression of this brain nuclear factor is restricted to the perinatal period, when myelination is sensitive to T3. Gel supershift assays with RXR-specific antibodies indicate that this factor is not one of the known RXR isoforms. However, it is most likely a new member of the RXR subfamily because it could be supershifted with an antibody raised against the highly conserved DNA-binding domain of RXRs.

Identification and quantitation of iodotyrosines and iodothyronines in proteins using high-performance liquid chromatography by photodiode-array ultraviolet-visible detection.

We describe a new method for the separation, identification and quantitation of iodotyrosines and iodothyronines [3-monoiodo-L-tyrosine (MIT), 3,5-diiodo-L-tyrosine (DIT), L-thyronine (T0), 3,5-diiodo-L-thyronine (T2), 3,5,3'-triiodo-L-thyronine (T3), reverse 3,3',5'-triiodo-L-thyronine (rT3) and 3,3',5,5'-tetraiodo-L-thyronine (T4)]. Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed on a Nucleosil C8 column with photodiode-array UV-Vis detection. A clearly defined elution profile was obtained of each iodoamino acid (iodotyrosines and iodothyronines) using a linear gradient from 20 to 80% phase B (90% acetonitrile, 10% water, 0.1% TFA), phase A (water, 0.1% TFA, pH 2.0) eluted over 40 min. Iodoamino acid composition was determined, taking into account retention times and spectral characteristics. Thyroid protein samples were digested enzymatically and the complex mixture of IAA was then injected onto the RP-HPLC system. A photodiode-array detector with a dynamic range in the UV-Vis region was used in the HPLC system to monitor the absorbance at different wavelengths continuously, collecting data which were compared with standard samples. Each IAA was quantitated using linear calibration curves obtained at 280 nm. This method allowed identification and quantitation of iodoamino acids from diverse sources in the range 2-500 ng, avoiding the need to radiolabel samples. The technique was tested with in vitro iodinated and non-iodinated human thyroglobulin and the recoveries ranged from 84 to 91%.

Computer-assisted molecular modeling of benzodiazepine and thyromimetic inhibitors of the HepG2 iodothyronine membrane transporter.

T3 cellular uptake is inhibited in the presence of benzodiazepines (BZs). The structure-activity relationship of BZ inhibition correlates strongly with halogen substitution of the nonfused phenyl ring and indicates that this ring is required for activity. A structure-activity series of thyromimetic (TH) inhibitors of the HepG2 iodothyronine transporter further point out the critical importance of the amino group of the alanine side chain, its L-stereo configuration, and the size of the substituents of the inner and outer phenyl rings. A third series of compounds, reported to interact at related sites, were inactive as HepG2 iodothyronine transport inhibitors, and therefore the potent inhibitors were restricted to the BZ and TH compounds. Using both of these BZ and TH structure-activity series along with computer-assisted molecular modeling techniques, we determined which chemical structural components were important at the transporter interaction site. By superimposing structures from active chemicals, excluding residues from poor inhibitors, and incorporating molecular electropotential data, we developed a five-point model of BZ conformational similarity to the endogenous transporter ligand, L-T3: the alkyl substitution at the N1 of the BZ ring seems to simulate the alanine side chain of T3, and the electro-negative halogen and oxygen atoms of substituents at R3/R7/R2'/R4' of BZ form a pyramidal pharmacophore that seems to correspond with the 3-l/5-l/3'-l/4'-OH substituents of T3, respectively. These points, suggesting a tilted cross-bow formation, may be sites for ligand interaction with the iodothyronine transporter.

Comparison of high-performance liquid chromatographic detection methods for thyronine and tyrosine residues in toxicological studies of the thyroid.

Four high-performance liquid chromatographic methods for the detection of thyroid hormones (iodinated thyronines) and precursors (iodinated tyrosines) have been developed and evaluated. Two methods consist of direct determination of the parent compounds with detection at ultraviolet wavelength (230 nm) and with electrochemical detection. The two other methods consist of a pre-column derivatization (with fluorenylmethyl chloroformate and dabsyl chloride) prior to high-performance liquid chromatographic analysis. The various methods were evaluated based on their practical use and sensitivity. The method with direct ultraviolet detection turned out to be the most practical method. With this method analyses of thyroid homogenates have been performed from rats from a toxicological experiment.

Resolution and binding site determination of D,L-thyronine by high-performance liquid chromatography using immobilized albumin as chiral stationary phase. Determination of the optical purity of thyroxine in tablets.

Human and bovine serum albumin bound to silica or aminopropyl silica were used as chiral stationary phases (CSPs). D,L-Thyronine, D,L-tryptophan, N-benzoyl-D,L-phenylalanine, D,L-warfarin and D,L-benzoin could be resolved on these CSPs using a mobile phase of 0.05 M phosphate buffer, pH 7.0. The capacity factor of D-thyronine was higher than that of L-thyronine. The resolution of D,L-thyronine was completely lost by the presence of bilirubin in the mobile phase, but only little affected by caprylate. By contrast, the resolution of D,L-tryptophan was not affected by bilirubin, but lost by the presence of caprylate. These results are consistent with binding of D-thyronine to the bilirubin binding site and L-tryptophan to the caprylate binding site in albumin, respectively, and suggests that such "displacement chromatography" can be used for the determination of binding sites. The optical purity of D-thyroxine in tablets was determined indirectly after de-iodination by catalytic hydrogenation.