Deiodinases (5′-D1, 5′-D2, 5-D3) control the systemic and local bioavailability of thyroid hormones by removing iodine from their substrates. Thyronamine (T0AM) and 3-iodothyronamine (3-T1AM) are possible novel metabolites of classical thyroid hormones which have been demonstrated to occur endogenously and to display unique effects such as reducing body temperature in mice and activating the plasma membrane bound G-Protein coupled receptor TAAR1 (Scanlan et al., 2004). As the pathways of thyronamine biosynthesis are still unknown, we reasoned whether deiodinases might be involved.
In preliminary experiments using classical 125I release assays the HepG1 cell line was found to express a specific 5′-D1 activity of 1.2+0.29 pmol iodide released×mg−1×min−1 but not to exhibit 5′-D2 or 5-D3 activity at all. Thus, HepG2 cells were used to study the ability of 5′-D1 to accept thyronamines as substrates. Cells were homogenized in HEPES buffer containing sucrose, EDTA and DTT. Homogenates were incubated for 2 h at 37 °C in the absence or presence of 1 mM PTU in 100 mM sodium phosphate buffer at pH=6.8 containing 1 mM EDTA, 20 mM DTT and various concentrations of the following substrates: thyronamine (T0AM), 3-iodothyronamine (3-T1AM), 3,5-diiodothyronamine (3,5-T2AM), 3,5,3′-triiodothyronamine (3,5,3′-T3AM), 3,5,3′,5′-tetraiodothyronamine (T4AM) as well as rT3 and 3′,5′-diiodothyronine (3′,5′-T2) as positive controls. Deiodination products were analysed using a newly established selected reaction monitoring (SRM) based liquid chromatography tandem mass spectrometry (LC-MS/MS) method.
5′-D1 from HepG2 cells did not deiodinate any of the thyronamines at the substrate concentrations tested (50 nM to 20 μM). Thus, a role of 5′-D1 in thyronamine deiodination is rather unlikely. The ability of 5′-D2 and 5-D3 to accept thyronamines as substrates still remains to be tested.
Supported by Graduate College 1208, DFG