Project

Tech Resource for Sandell-Kolthoff-based Assays

Goal: The resource (www.sandell-kolthoff.de or www.sandell-kolthoff.com) is meant to give easy access to and interaction upon these type of thyroid-axis-related assays, which might be, in some cases, an alternative to radioactive setups. Goal of this project is to establish and curate the platform on the long run.

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Kostja Renko
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Watch your Cerium!
Just recently I ran into some issues with the Ce-Solutions from Honeywell/Fluka. Background reaction was severely increased in two recent batches....as there is no (known) specification for this product towards its apllication for the Sandell-Kolthoff-Rct I can not fully recommmend its use anymore (as I did on Sandell-Kolthoff.com).
Don't get me wrong, it doesn't completely mess up the measurement but reduces the dynamic range between singnal (+iodide) and background.
Cheers
Kostja
 
Grata pela informação.
Abs,
 
Roopa Jayarama-Naidu
added 2 research items
Thyroid hormones (TH) are actively taken up into target cells via TH-transmembrane transporters (THTT). Their activity and expression patterns define a layer of endocrine regulation that is poorly understood. Therefore, THTT are potential targets for interfering agents (endocrine disruptors, ED) as well as for pharmacological interventions. Inactivating mutations have been identified as the underlying cause of heritable diseases (MCT8-associated Allan-Herndon-Dudley Syndrome) and might also define a class of subclinical TH-insensitivity. As a basic tool to solve questions regarding THTT substrate specificity, activation or inactivation by compounds and functional changes from mutations, uptake assays with radiolabeled tracers are standard. Due to the need for radioactive isotopes, this technique is limited to screening of labelled substrates and disadvantaguous regarding handling, setup and regulatory issues. To overcome these hurdles, we developed an uptake-assay protocol utilizing nonradioactive ligands. In brief, uptake of non-radioactive iodine-containing substrate molecules was monitored via Sandell-Kolthoff-reaction. The novel assay was designed to the common microtiter plate layout. As a prove-of-principle, we measured TH-uptake by MCT8-transfected MDCK1-cells. Titrations with bromosulphthalein as an example for inhibitor screening setups and a side-by-side comparison with the radioactive method prove this assay to be reliable, sensitive and convenient. Furthermore, the method was applicable on primary murine astrocytes, which enables high-throughput screening (HTS) studies on in-vitro model systems with physiological transporter regulation. Due to its design, it is applicable for HTS of modulatory compounds, but it is also a safe, inexpensive and an easily accessible method for functional testing of THTT in basic science.
Kostja Renko
added 3 research items
Enzymatic 5'- and 5-deiodination are key reactions for local and systemic activation and inactivation of iodothyronines and thyronamines. Expression of the three deiodinase (DIO) isoenzymes is regulated by a number of parameters, including thyroid status, genotype, micronutrient availability, and disease-related signaling. In addition, DIO are potential targets of pharmacological as well as environmentally derived substances, which might affect their enzymatic activity (endocrine disruptors). With the classical DIO activity assay, testing depends on the availability of radioactively labeled substrates (e.g. (125)I-rT(3)) to monitor the release of radioactive iodide. Recently, liquid chromatography-tandem mass spectrometry was described as an alternative method apparently resolving this limitation. However, it has a high demand in technical equipment and analytical routine and is limited in sample number by considerable measuring time. We therefore combined the classical deiodination assay with an easily accessible photometric method taking advantage of the Sandell-Kolthoff reaction for measuring iodide release. In brief, iodine works as a catalyst within this redox reaction between Ce(4+) and As(3+) leading to an acceleration of destaining. Furthermore, the protocol was adapted to minimize handling effort and time consumption. Because this method is not dependent on radioactivity, it expands the substrate spectrum of the classical method. Suitability of this assay was tested with tissue samples from animal experiments (hepatic Dio1 activity in hypo- and hyperthyroid mice) and established DIO inhibitors. As a new but not unexpected finding, the alleged inhibitor iopanoic acid turned out to be a DIO substrate. This finding was confirmed by liquid chromatography-tandem mass spectrometry, and its potential clinical impact requires further studies.
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. & Kohrle, J. Identification of iopanoic acid as substrate of type 1 deiodinase by a novel nonradioactive iodide-release assay. Endocrinology 153, 2506 – 2513 (2012).
Kostja Renko
added 2 research items
Deiodinases (DIO1, 2 and 3) are key enzymes in thyroid hormone (TH) activation and inactivation with impact on energy metabolism, development, cell differentiation and a number of other physiological processes. The three DIO isoenzymes thus constitute sensitive rate-limiting components within the TH axis, prone to dysregulation by endocrine disruptive compounds or disease state. In animal models and cell culture experiments, they serve as readout for local TH-status and disarrangement of the hormonal axis. Furthermore, some human diseases are characterized by apparent deiodinase dysregulation, e.g. the low T3 syndrome in critical illness. Consequently, these enzymes are targets of interest for the development of pharmacological compounds with modulatory activities. Until now, the portfolio of inhibitors for these enzymes is limited. In the clinics, the DIO1-specific inhibitor propylthiouracil (PTU) is in use for treatment of severe hyperthyroidism. Other well-known inhibitors, e.g. iopanoic acid (IA) or aurothioglucose (ATG), are non-selective and block all three isoenzymes. Furthermore, DIO3 was shown to be a potential oncogenic gene, which is strongly expressed in some tumors and might, in consequence, protect tumor tissue form differentiation by TH. With respect to its role in tumorigenesis, specific inhibitors of DIO3 as a potential target for anticancer drugs would be highly desirable. To this end, a flexible and convenient assay for high-throughput screening (HTS) is needed. We recently described a non-radioactive screening assay, utilizing the classical Sandell-Kolthoff-reaction as readout for iodide release from the substrate molecules. While we used murine liver as enzyme source, the assay was limited to murine Dio1 activity testing. Now, we describe the use of recombinant proteins as enzyme sources within the assay, expanding its suitability from murine Dio1 to human DIO1, DIO2 and DIO3. As prove-of-concept, deiodination reactions catalyzed by these recombinant enzymes were monitored with various non-radioactive substrates and confirmed by LC-MS/MS. The contrast agent and known DIO-inhibitor IA was characterized as readily accepted substrate by DIO2 and Dio3. In a screening approach using established endocrine disrupting compounds, the natural food ingredient genistein was identified as a further DIO1-specific inhibitor while xanthohumol turned out to potently block the activity of all three isoenzymes.
Iodotyrosine deiodinase (DEHAL1) is a crucial enzyme in iodine homeostasis. Unbound mono- and diiodotyrosines are indispensable byproducts of thyroid hormone biosynthesis. Their iodine needs to be recovered to avoid iodine deficiency, as observed in genetic defects in DEHAL1. Despite its importance, the enzyme is rarely studied. The deiodination process can be monitored by radioactive tracers or via techniques involving mass spectrometry (MS). However, isotope-labeled molecules are expensive, not always commercially available, and their use is legally restricted, while MS requires sophisticated, costly and sensitive instrumentation. To circumvent these difficulties, we adapted the non-radioactive iodothyronine deiodinase (DIO) assay to determine DEHAL1 activity by a colorimetric readout, based on the Sandell-Kolthoff-reaction. DEHAL1 was recombinantly expressed and used to optimize the assay in microtiter format. We applied the setup to scenarios of alternative substrate screening or search for compounds potentially acting as endocrine disrupting compounds, without identifying novel readily accepted substrates or inhibitors yet. Next, the assay was adapted to ex vivo material, and activity was reliably determined from rodent kidney and other tissues. Analyzing two mouse models of hyperthyroidism, we observed a decreased renal Dehal1 activity and mRNA expression. Our results show that this non-radioactive DEHAL1 assay is suited to screen for potential endocrine disrupters and to monitor endogenous Dehal1 expression. We harmonized the assay protocols to enable DIO and DEHAL1 activity measurements from the same samples. Hereby, a more complete view on iodide metabolism by these predominant deiodinating activities can be obtained from a given sample by a similar process flow.
Kostja Renko
added a project goal
The resource (www.sandell-kolthoff.de or www.sandell-kolthoff.com) is meant to give easy access to and interaction upon these type of thyroid-axis-related assays, which might be, in some cases, an alternative to radioactive setups. Goal of this project is to establish and curate the platform on the long run.