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.

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 < 0.05). Crystal violet staining confirmed a reduction of cell viability only upon treatment with 10 µM T2, while equimolar T3 and T4 did not share this effect. Glucose consumption was also significantly affected as indicated by glucose uptake being increased by 24 or 35% in cells exposed to 0.1 or 1.0 µM T2 (P < 0.05 in both cases). On the contrary, T3 did not affect glucose consumption which, in turn, was significantly reduced by 1 and 10 µM T4 (−24 and −41% vs control, respectively, P < 0.05 and P < 0.01). In the isolated perfused rat heart, 10 µM T2 produced a slight and transient reduction in cardiac output, while T3 and T4 did not produce any hemodynamic effect. Our findings indicate that T2 is taken up by cardiomyoblasts, and at 0.1–1.0 µM concentration it can modulate cardiac energy metabolism by increasing glucose consumption. Some evidence of toxicity and a transient impairment of contractile performance are observed only at 10 µM concentration. These effects appear to be specific for T2, since they are not reproduced by T3 or T4.

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.

Background3-Iodothyronamine (T1AM) is an endogenous messenger chemically related to thyroid hormone. Recent results indicate significant transcriptional effects of chronic T1AM administration involving the protein family of sirtuins, which regulate important metabolic pathways and tumor progression. Therefore, the aim of this work was to compare the effect of exogenous T1AM and 3,5,3′-triiodo-L-thyronine (T3) chronic treatment on mammalian sirtuin expression in hepatocellular carcinoma cells (HepG2) and in primary rat hepatocytes at micromolar concentrations.Materials and methodsSirtuin (SIRT) activity and expression were determined using a colorimetric assay and Western blot analysis, respectively, in cells treated for 24 h with 1–20 μM T1AM or T3. In addition, cell viability was evaluated by the MTTtest upon 24 h of treatment with 0.1–20 μM T1AM or T3.ResultsIn HepG2, T1AM significantly reduced SIRT 1 (20 μM) and SIRT4 (10–20 μM) protein expression, while T3 strongly decreased the expression of SIRT1 (20 μM) and SIRT2 (any tested concentration). In primary rat hepatocytes, T3 decreased SIRT2 expression and cellular nicotinamide adenine dinucleotide (NAD) concentration, while on sirtuin activity it showed opposite effects, depending on the evaluated cell fraction. The extent of MTT staining was moderately but significantly reduced by T1AM, particularly in HepG2 cells, whereas T3 reduced cell viability only in the tumor cell line.ConclusionsT1AM and T3 downregulated the expression of sirtuins, mainly SIRT1, in hepatocytes, albeit in different ways. Differences in mechanisms are only observational, and further investigations are required to highlight the potential role of T1AM and T3 in modulating sirtuin expression and, therefore, in regulating cell cycle or tumorigenesis.

T1AM, a derivative of thyroid hormones, and its major catabolite, TA1, produce effects on memory acquisition in rodents. In the present study, we compared the effects of exogenous T1AM and TA1 on protein belonging to signal transduction pathways, assuming that TA1 may strengthen T1AM’s effects in brain tissue. A hybrid line of cancer cells of mouse neuroblastoma and rat glioma (NG 108-15), as well as a human glioblastoma cell line (U-87 MG) were used. We first characterized the in vitro model by analyzing gene expression of proteins involved in the glutamatergic cascade and cellular uptake of T1AM and TA1. Then, cell viability, glucose consumption, and protein expression were assessed. Both cell lines expressed receptors implicated in glutamatergic pathway, namely Nmdar1, Glur2, and EphB2, but only U-87 MG cells expressed TAAR1. At pharmacological concentrations, T1AM was taken up and catabolized to TA1 and resulted in more cytotoxicity compared to TA1. The major effect, highlighted in both cell lines, albeit on different proteins involved in the glutamatergic signaling, was an increase in phosphorylation, exerted by T1AM but not reproduced by TA1. These findings indicate that, in our in vitro models, T1AM can affect proteins involved in the glutamatergic and other signaling pathways, but these effects are not strengthened by TA1.