Establishment of a non-isotopic activity assay for thyroid hormone transporters

Iodine determination depending on the Sandell-Kolthoff-reaction (S-K) is a frequently used method for measuring urinary iodine secretion as a marker for iodine status in population studies. In brief, the method relies on the photometric detection of the redox-reaction between yellowish Ce(IV) and As(III) to colorless Ce(III) and As(V). Iodine, as a catalyst for this reaction, speeds up the destaining process in a concentration-dependent manner. The S-K-reaction follows a straightforward protocol with limited needs regarding instrumentation or costly chemicals. Recently, we have developed a battery of thyroid axis-related in-vitro assays, using this method to determine activities of iodine- releasing enzymatic activities (e.g. deiodinase activities). This method may complement or even replace the classical approaches utilizing radioactive tracer molecules [1].

We have now expanded this set of detectable activities towards thyroid hormone transporters, e.g. MCT8. With a cell line (MDCK-1), stably expressing the human thyroid hormone transporter hMCT8, we are able to detect significant uptake of the preferred substrate T3, compared to the MOCK-transfected cells. The concentration- and time-dependent T3 transport was decreased by adding the inhibitor Bromosulphthaleine (BSP). Titrating BSP concentration allowed calculating an IC50-value of ˜75µM, which was verified by a parallel experiment using the classical radioactive 125I-T3 uptake assay (IC50˜80µM).

The novel uptake assay can be performed in microtiter plate format, thereby being well-suited for systematic screening approaches to identify endocrine disruptive substances or specific uptake modulators or inhibitors. The readout of the assay depends solely on the substrate-bound iodine, thereby rendering this method independent from the availability of specially synthesized (radio-)tracer molecules, and providing a cost effective and flexible novel screening method.

Supported by the Deutsche Forschungsgemeinschaft (RE 3038/1 – 1)

[1] Renko, K., Hoefig, C. S., Hiller, F., Schomburg, L. & Köhrle, J. Identification of iopanoic acid as substrate of type 1 deiodinase by a novel nonradioactive iodide-release assay. Endocrinology 153, 2506 – 2513 (2012).