Metabolism of reverse triiodothyronine by isolated rat hepatocytes.

Reverse triiodothyronine (rT3) is metabolized predominantly by outer ring deiodination to 3,3'-diiodothyronine (3,3'-T2) in the liver. Metabolism of rT3 and 3,3'-T2 by isolated rat hepatocytes was analyzed by Sephadex LH-20 chromatography, high performance liquid chromatography, and radioimmunoassay, with closely agreeing results. Deiodinase activity was inhibited with propylthiouracil (PTU) and sulfotransferase activity by sulfate depletion or addition of salicylamide or dichloronitrophenol. Normally, little 3,3'-T2 production from rT3 was observed, and 125I- was the main product of both 3,[3'-125I]T2 and [3',5'-125I]rT3. PTU inhibited rT3 metabolism but did not affect 3,3'-T2 clearance as explained by accumulation of 3,3'-T2 sulfate. Inhibition of sulfation did not affect rT3 clearance but 3,3'-T2 metabolism was greatly diminished. The decrease in I- formation from rT3 was compensated by an increased recovery of 3,3'-T2 up to 70% of rT3 metabolized. In conclusion, significant production of 3,3'-T2 from rT3 by rat hepatocytes is only observed if further sulfation is inhibited.

Metabolism of triiodothyronine in rat hepatocytes.

The metabolism of T3 by isolated rat hepatocytes was analyzed by Sephadex LH-20 chromatography, HPLC, and RIA for T3 sulfate (T3S) and 3,3'-diiodothyronine (3,3'-T2). Type I iodothyronine deiodinase activity was inhibited with propylthiouracil (PTU), and phenol sulfotransferase activity by SO4(2-) depletion or with competitive substrates or inhibitors. Under normal conditions, labeled T3 glucuronide and I- were the main products of [3'-125I]T3 metabolism. Iodide production was decreased by inhibition (PTU) or saturation (greater than 100 nM T3) of type I deiodinase, which was accompanied by the accumulation of T3S and 3,3'-T2S. Inhibition of phenol sulfotransferase resulted in decreased iodide production, which was associated with an accumulation of 3,3'-T2 and 3,3'-T2 glucuronide, independent of PTU. Formation of 3,3'-T2 and its conjugates was only observed at T3 substrate concentrations below 10 nM. Thus, T3 is metabolized in rat liver cells by three quantitatively important pathways: glucuronidation, sulfation, and direct inner ring deiodination. Whereas T3 glucuronide is not further metabolized in the cultures, T3S is rapidly deiodinated by the type I enzyme. As confirmed by incubations with isolated rat liver microsomes, direct inner ring deiodination of T3 is largely mediated by a low Km, PTU-insensitive, type III-like iodothyronine deiodinase, and production of 3,3'-T2 is only observed if its rapid sulfation is prevented.

Development of a radioimmunoassay for triiodothyronine sulfate.

This paper is the first description of a radioimmunoassay (RIA) for triiodothyronine sulfate (T3S). Rabbits were immunized against T3S coupled to bovine serum albumin using carbodiimide. All animals produced antibodies to T3S but also even higher titers of T3 antibodies. Ka values for binding of T3 and T3S to these antisera varied between 2 x 10(10) and 8 x 10(10) M-1. One of the antisera (#8193) was selected for use in the T3S RIA because of a high titer of T3S antibodies (final dilution 1:50,000), a high sensitivity to T3S (less than 2.5 fmol/tube), and a low crossreactivity by T3 (0.4%). This RIA provides a tool for the study of the importance of sulfation as a metabolic pathway for T3.