New insights into the thyroid endocrine system by HPLC-ESI-MS-MS

MS allows an estimate of THs and their metabolites in different tissues. For the first time endogenous T1AM was detected. We have recently reported the discovery of 3-iodothyronamine (T1AM), a novel endogenous compound, which was supposed to derive from thyroid hormone (TH) through decarboxylation and deiodination. When administrated in rodents it showed significant functional effects, such as reducing body temperature and cardiac contractility. However, a quantitative and comprehensive analysis of T1AM distribution and metabolism has not been performed in any tissue. In the present study we describe novel liquid chromatography tandem mass spectrometry methods that allowed the simultaneous evaluation of endogenous Thyroxine (T4), 3,5,3’-triiodothyronine (T3), and T1AM, as well as its putative metabolites, such as thyronamine (T0AM), 3-iodothyroacetic acid (TA1) and thyroacetic acid (TA0) and their distribution in different tissues. Tissue sample preparation: tissues were washed with saline, weighted, finely minced, homogenized in PBS, and after centrifugation, the supernatant was collected. MS analysis: reverse phase HPLC coupled to ESI/MS/MS was performed, in positive and negative MRM ion mode, acquiring 3-4 transitions for each compound. Method 1: positive ion mode, to get the best sensitivity on T1AM, T3 and T4. Method 2 used for the contemporaneous detection of T0AM, T1AM, TA0, TA1, T3, and T4. T0AM and T1AM were detected in positive ion mode, while TA0, TA1, T3, and T4 were detected in negative ion mode. Both MS methods made use of the same HPLC run, based on a mobile phase gradient: Solvent A: Water; Solvent B: Metanol/Acetonitrile 1:4. In homogenates obtained from different rat tissues (heart, liver, kidney, skeletal muscle, stomach, lung, and brain) T1AM was detected, at concentrations ranging from 0.3 pmol/ml in serum to over 90 pmol/g in liver. While the accuracy of our assay is still limited, these results confirm that T1AM is an endogenous compound with a widespread distribution. Its serum concentration was approximately on the same order of magnitude than serum T3 and about two orders of magnitude lower than serum T4, but tissue concentrations were close to or greater than tissue T3 and T4 levels. The values which we obtained are in good agreement with the total tissue content which has been determined by radio-immunological techniques. T0AM detection in serum and in the tissues showing the highest T1AM content suggests T1AM to T0AM convertion. Other potential catabolites were not observed, but we cannot exclude that their tissue concentrations may be lower that the dection limit of our assay