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.

In the brain of mice, 3-iodothyronamine (T1AM) is converted into 3-iodothyroacetic acid (TA1) and it is included within the signaling network connecting thyroid hormone metabolites with histamine.

3-iodothyronamine (T1AM) and its oxidative product, 3-iodotyhyroacetic acid (TTA1A), are known to stimulate learning and induce hyperalgesia in mice. We investigated whether i)TA1 may be generated in vivo from T1AM, ii) T1AM shares with TA1 the ability to activate the histaminergic system. Tandem mass spectrometry was used to measure TA1 and T1AM levels in i) the brain of mice following intracerebroventricular (i.c.v.) injection of T1AM (11µgkg(-1)), with or without pretreatment with clorgyline, (2.5mgkg(-1) i.p.), a monoamine oxidase inhibitor; ii) the medium of organotypic hippocampal slices exposed to T1AM (50nM). In addition, learning and pain threshold were evaluated by the light-dark box task and the hot plate test, respectively, in mice pre-treated subcutaneously with pyrilamine (10mgkg(-1)) or zolantidine (5mgkg(-1)), 20min before i.c.v. injection of T1AM (1.32 and 11µgkg(-1)). T1AM-induced hyperalgesia (1.32 and 11µgkg(-1)) was also evaluated in histidine decarboxylase (HDC(-/-)) mice. T1AM and TA1 brain levels increased in parallel in mice injected with T1AM with the TA1/T1AM averaging 1.7%. Clorgyline pre-treatment reduced the increase in both T1AM and TA1. TA1 was the main T1AM metabolite detected in the hippocampal preparations. Pretreatment with pyrilamine or zolantidine prevented the pro-learning effect of 1.32 and 4µgkg(-1) T1AM while hyperalgesia was conserved at the dose of 11µgkg(-1) T1AM. T1AM failed to induce hyperalgesia in HDC(-/-) mice at all the doses. In conclusion, TA1 generated from T1AM, but also T1AM, appears to act by modulating the histaminergic system.

Distribution of exogenous [125I]-3-iodothyronamine in mouse in vivo: relationship with trace amine-associated receptors.

3-Iodothyronamine (T1AM) is a novel chemical messenger, structurally related to thyroid hormone, able to interact with G protein-coupled receptors known as trace amine-associated receptors (TAARs). Little is known about the physiological role of T1AM. In this prospective, we synthesized [125I]-T1AM and explored its distribution in mouse after injecting in the tail vein at a physiological concentration (0.3 nM). The expression of the nine TAAR subtypes was evaluated by quantitative real-time PCR. [125I]-T1AM was taken up by each organ. A significant increase in tissue vs blood concentration occurred in gallbladder, stomach, intestine, liver, and kidney. Tissue radioactivity decreased exponentially over time, consistent with biliary and urinary excretion, and after 24 h, 75% of the residual radioactivity was detected in liver, muscle, and adipose tissue. TAARs were expressed only at trace amounts in most of the tissues, the exceptions being TAAR1 in stomach and testis and TAAR8 in intestine, spleen, and testis. Thus, while T1AM has a systemic distribution, TAARs are only expressed in certain tissues suggesting that other high-affinity molecular targets besides TAARs exist.

3-Iodothyronamine: a modulator of the hypothalamus-pancreas-thyroid axes in mice.

BACKGROUND AND PURPOSE Preclinical pharmacology of 3-iodothyronamine (T1AM), an endogenous derivative of thyroid hormones, indicates that it is a rapid modulator of rodent metabolism and behaviour. Since T1AM undergoes rapid enzymatic degradation, particularly by MAO, we hypothesized that the effects of T1AM might be altered by inhibition of MAO. EXPERIMENTAL APPROACH We investigated the effects of injecting T1AM (i.c.v.) on (i) feeding behaviour, hyperglycaemia and plasma levels of thyroid hormones and (ii) T1AM systemic bioavailability, in overnight fasted mice, under control conditions and after pretreatment with the MAO inhibitor clorgyline. T1AM (1.3, 6.6, 13, 20 and 26 µg·kg(-1) ) or vehicle were injected i.c.v. in fasted male mice not pretreated or pretreated i.p. with clorgyline (2.5 mg·kg(-1) ). Glycaemia was measured by a glucorefractometer, plasma triiodothyronine (fT3) by a chemiluminescent immunometric assay, c-fos activation immunohistochemically and plasma T1AM by HPLC coupled to tandem-MS. KEY RESULTS T1AM, 1.3 µg·kg(-1) , produced a hypophagic effect (-24% vs. control) and reduced c-fos activation. This dose showed systemic bioavailability (0.12% of injected dose), raised plasma glucose levels and reduced peripheral insulin sensitivity (-33% vs. control) and plasma fT3 levels. These effects were not linearly related to the dose injected. Clorgyline pretreatment strongly increased the systemic bioavailability of T1AM and prevented the hyperglycaemia and reduction in fT3 induced by T1AM. CONCLUSIONS AND IMPLICATIONS T1AM induces central and peripheral effects including hyperglycaemia and a reduction in plasma fT3 levels in fasted mice. These effects critically depend on the concentration of T1AM or its metabolites in target organs.

The importance of employing homologous serum free of thyronines in radioimmunoassays to assess circulating thyroid hormones in rainbow trout

A pesar de ser abundante, la información relacionada con los valores de tironinas circulantes en la trucha arcoiris, es confusa. El presente estudio demuestra la importancia de mantener especies sero-homólgoas durante la medición de tironinas con RIE para eviatar falsos resultados. La utilización de suero homólogo (trucha) libre de tironinas en la curva estándar permite la estandarización de radioinmunoensayos exactos y específcos para cuantificar los niveles circulates de T3 y T4 en 10 microl de suero de trucha arcoiris. El límite de detección fue de 50 pg para ambas tironinas. Los coeficientes de variación inter e intra ensayo fueron menores al 10 por ciento. Los valores promedio para T3 y T4 + - DEM en la población de truchas estudiadas (n=34) fueron 23.6 + - 1.2 y 12.0 + - 2.3 ng/ml respectivamente. Se obsevó una correlación positiva y significativa (p < 0.005) entre el peso corporal y T3 confirmando la participación activa de esta tironina en el proceso de crecimiento.

The disposition of SK&F L-94901, a selective thyromimetic in rat, dog and cynomolgus monkey.

SK&F L-94901 is a novel thyromimetic, structurally related to thyroxine. The absorption, distribution, excretion and metabolism of radiochemically labelled [14C]-SK&F L-94901 has been investigated in the rat, dog and cynomolgus monkey. Oral absorption from solution was low or moderate in all three species. The compound was widely distributed and rapidly excreted, although traces of radioactivity were still evident in some tissues at 7 days post-dose, particularly in the kidney where radioactivity was located in an area approximating to the corticomedullary junction. Elimination of [14C]-SK&F L-94901 was both metabolic, mediated by the liver, and renal. The major metabolic routes of elimination were via oxidative deamination to lactate and acetate derivatives.

Fecal and urinary excretion of six iodothyronines in the rat.

Fecal and urinary excretion rates of six iodothyronines were assessed in the rat maintained under normal steady state physiological conditions, to gain a more comprehensive understanding of the mechanisms of control of normal thyroid hormone economy and metabolism. Groups of young adult male rats were injected with trace doses of T4, T3, rT3, 3,3'-diiodothyronine (T2), 3',5'-T2, or 3'-monoiodothyronine, each labeled with 125I, and feces and urine were collected separately for up to 10 days. Pooled fecal pellets were homogenized in saline, extracted in ethanol, evaporated under vacuum, and reconstituted in NaOH. Fecal extracts and urine were chromatographed on Sephadex G25 columns under conditions providing quantitative separations of components of interest. A new technique was also developed, based on a model of the in vitro extraction and measurement process, to correct chromatographic results for possible variable recoveries and possible artifactious degradation of radioactively labeled components. No iodothyronines or their conjugates were excreted in urine; all radioactivity was in the form of iodide. In feces, about 30% of the [125I]T3 injected was excreted as T3; and 24% of the [125I]T4 injected was excreted as T4, plus 4% as T3. Together, these results imply that about 24% of endogenous T4 production is excreted as T4 and 76% is irreversibly metabolized; and for T3, about 30% of endogenous T3 production is excreted as T3 and 70% is degraded. For the nonhormonal iodothyronines, about 6% of injected monoiodothyronine, 3% of injected 3',5'-T2, 2% of injected 3,3'-T2, and less than 1% of injected rT3 were excreted in feces as such, indicating that these substances are nearly completely deiodinated in vivo. Very little (1-7%) iodide was excreted as such in feces, which also were devoid of measurable conjugates. An open question is whether the substantial wastage of thyroid hormones in feces represents poor hormone economy in the usually accepted sense or a functional property of overall thyroid hormone regulation.