Mass spectrometry allows a nice insight into the interconversion reactions of the thyroid hormones, working on different types of sample. T1AM is an endogenous compound with chemical features similar to thyroid hormone. That is mainly due to its structure, which is based on the same carbon skeleton of thyroxine. In principle, T1AM could be produced from T4 by enzymatic decarboxylation and deiodination. At the molecular level it is a potent agonist of the TAAR1, and an agonist ligand against the &alpha-2A adrenergic receptor. When administered pharmacologically it induced hypometabolic state opposite to that induced by excess of thyroid hormone. In isolated working rat heart and rat cardiomyocytes, T1AM produced a reversible dose-dependent negative inotropic effect. In the present study we investigate the catabolism and uptake of exogenous T1AM in rat cardiac tissues, biological fluids and cardiomyoblast cells, using HPLC-ESI-MS-MS. Ex vivo assay: isolated working rat hearts were subjected to perfusion with T1AM alone or in presence of different monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) inhibitors; circulating buffer and tissue homogenates were also analyzed. In vivo assay: after administration of T1AM, rat biological fluids were investigated. In vitro assay: cultured rat cardiomyoblasts (H9C2) were treated with T1AM alone or in presence of different MAO and SSAO inhibitors, using perfusion buffer as medium for the assay. At different incubation time, medium and cell lysate were collected and submitted to analysis. Analytical method: reverse phase HPLC coupled to ESI-MS-MS was performed, in positive and negative MRM ion mode, acquiring 3-4 transitions for T1AM and some of its catabolites. T1AM and its oxidative deamination product, 3-iodothyroacetic acid (TA1), were evaluated in cell lysate, in cell growth medium, and in circulating perfusion buffer . Similar studies involving also heart tissue homogenates and biological fluids upon treatment are in progress. Preliminary data indicated that T1AM is metabolized to TA1 both in working rat heart and in H9C2 cells. In particular TA1 showed a significant increase in cell lysate reaching its maximum in 40 min and only a moderate increment in cell medium. The pretreatment of H9C2 cells with different amounts of iproniazide significantly inhibited T1AM conversion to TA1. The same mechanism was found in circulating perfusion buffer and iproniazide acted as an inhibitor as well. The reduction of T1AM cellular uptake in presence of MAO and SSAO inhibitors constitutes the evidence for the involvement of these enzymes in T1AM catabolism. In conclusions, HPLC-ESI-MS-MS proved to be a first choice technique to elucidate the mechanism involved in T1AM catabolism in rat heart. Further investigations are in progress to clarify T1AM metabolism products and their presence in rat tissue homogenates and biological fluids.
Evaluation of 3-iodothyronamine (T1AM) in cell preparation, tissue homogenates and biological fluids by HPLC-ESI-MS-MS
SABA, ALESSANDRO;CHIELLINI, GRAZIA;GHELARDONI, SANDRA;ZUCCHI, RICCARDO
2009-01-01
Abstract
Mass spectrometry allows a nice insight into the interconversion reactions of the thyroid hormones, working on different types of sample. T1AM is an endogenous compound with chemical features similar to thyroid hormone. That is mainly due to its structure, which is based on the same carbon skeleton of thyroxine. In principle, T1AM could be produced from T4 by enzymatic decarboxylation and deiodination. At the molecular level it is a potent agonist of the TAAR1, and an agonist ligand against the &alpha-2A adrenergic receptor. When administered pharmacologically it induced hypometabolic state opposite to that induced by excess of thyroid hormone. In isolated working rat heart and rat cardiomyocytes, T1AM produced a reversible dose-dependent negative inotropic effect. In the present study we investigate the catabolism and uptake of exogenous T1AM in rat cardiac tissues, biological fluids and cardiomyoblast cells, using HPLC-ESI-MS-MS. Ex vivo assay: isolated working rat hearts were subjected to perfusion with T1AM alone or in presence of different monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) inhibitors; circulating buffer and tissue homogenates were also analyzed. In vivo assay: after administration of T1AM, rat biological fluids were investigated. In vitro assay: cultured rat cardiomyoblasts (H9C2) were treated with T1AM alone or in presence of different MAO and SSAO inhibitors, using perfusion buffer as medium for the assay. At different incubation time, medium and cell lysate were collected and submitted to analysis. Analytical method: reverse phase HPLC coupled to ESI-MS-MS was performed, in positive and negative MRM ion mode, acquiring 3-4 transitions for T1AM and some of its catabolites. T1AM and its oxidative deamination product, 3-iodothyroacetic acid (TA1), were evaluated in cell lysate, in cell growth medium, and in circulating perfusion buffer . Similar studies involving also heart tissue homogenates and biological fluids upon treatment are in progress. Preliminary data indicated that T1AM is metabolized to TA1 both in working rat heart and in H9C2 cells. In particular TA1 showed a significant increase in cell lysate reaching its maximum in 40 min and only a moderate increment in cell medium. The pretreatment of H9C2 cells with different amounts of iproniazide significantly inhibited T1AM conversion to TA1. The same mechanism was found in circulating perfusion buffer and iproniazide acted as an inhibitor as well. The reduction of T1AM cellular uptake in presence of MAO and SSAO inhibitors constitutes the evidence for the involvement of these enzymes in T1AM catabolism. In conclusions, HPLC-ESI-MS-MS proved to be a first choice technique to elucidate the mechanism involved in T1AM catabolism in rat heart. Further investigations are in progress to clarify T1AM metabolism products and their presence in rat tissue homogenates and biological fluids.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
