Name: Colorimetric Assessment of Deiodinase 1 Activity in Human Liver Microsomes Using the Sandell-Kolthoff Reaction (Video) | JoVE Journal
Uploaded: 2026-04-10T13:05:00.000+00:00
Duration: 8 min
Description: Learn a colorimetric assay for Deiodinase 1 activity in human liver microsomes using the Sandell-Kolthoff reaction. This non-radioactive method enables high-throughput screening of chemicals that disrupt thyroid hormone homeostasis.

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5 minutes

The authors want to thank Dr. Andreas Weber for his work during the initial establishment of the DIO1-SK assay.

<ol><li>Kabir, E. R., Rahman, M. S., Rahman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+on+endocrine+disruptors+and+their+possible+impacts+on+human+health%5BTitle%5D)+AND+%22Environ+Toxicol+Pharmacol%22%5BJournal%5D)">A review on endocrine disruptors and their possible impacts on human health.</a> Environ Toxicol Pharmacol. 40 (1), 241-258 (2015).</li>
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<li>França, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+type+1+iodothyronine+deiodinase+%28DIO1%29+mutations+cause+abnormal+thyroid+hormone+metabolism%5BTitle%5D)+AND+%22Thyroid%22%5BJournal%5D)">Human type 1 iodothyronine deiodinase (DIO1) mutations cause abnormal thyroid hormone metabolism.</a> Thyroid. 31 (2), 202-207 (2020).</li>
<li>Weber, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+the+predictivity+of+the+DIO1-SK+assay+to+investigate+DIO1+inhibition+in+human+liver+microsomes%5BTitle%5D)+AND+%22Appl+Vitro+Toxicol%22%5BJournal%5D)">Assessment of the predictivity of the DIO1-SK assay to investigate DIO1 inhibition in human liver microsomes.</a> Appl Vitro Toxicol. 9 (2), 44-59 (2023).</li>
<li>Weber, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+approach+method+to+study+thyroid+hormone+disruption%3A+optimization+and+standardization+of+an+assay+to+assess+the+inhibition+of+DIO1+enzyme+in+human+liver+microsomes%5BTitle%5D)+AND+%22Appl+Vitro+Toxicol%22%5BJournal%5D)">A new approach method to study thyroid hormone disruption: optimization and standardization of an assay to assess the inhibition of DIO1 enzyme in human liver microsomes.</a> Appl Vitro Toxicol. 8 (3), 67-82 (2022).</li>
<li>Renko, K., Hoefig, C. S., Hiller, F., Schomburg, L., Köhrle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+iopanoic+acid+as+substrate+of+type+1+deiodinase+by+a+novel+nonradioactive+iodide-release+assay%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">Identification of iopanoic acid as substrate of type 1 deiodinase by a novel nonradioactive iodide-release assay.</a> Endocrinology. 153 (5), 2506-2513 (2012).</li>
<li>Renko, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+nonradioactive+screening+method+identifies+genistein+and+xanthohumol+as+potent+inhibitors+of+iodothyronine+deiodinases%5BTitle%5D)+AND+%22Thyroid%22%5BJournal%5D)">An improved nonradioactive screening method identifies genistein and xanthohumol as potent inhibitors of iodothyronine deiodinases.</a> Thyroid. 25 (8), 962-968 (2015).</li>
<li>Olker, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Screening+the+ToxCast+phase+1%2C+phase+2%2C+and+e1k+chemical+libraries+for+inhibitors+of+iodothyronine+deiodinases%5BTitle%5D)+AND+%22Toxicol+Sci%22%5BJournal%5D)">Screening the ToxCast phase 1, phase 2, and e1k chemical libraries for inhibitors of iodothyronine deiodinases.</a> Toxicol Sci. 168 (2), 430-442 (2019).</li>
<li>Sane, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+characterization+of+highly+potent+and+isoenzyme-selective+inhibitors+of+deiodinase+type+I+via+a+nonradioactive+high-throughput+screening+method%5BTitle%5D)+AND+%22Thyroid%22%5BJournal%5D)">Identification and characterization of highly potent and isoenzyme-selective inhibitors of deiodinase type I via a nonradioactive high-throughput screening method.</a> Thyroid. 35 (5), 576-589 (2025).</li>
<li>Visser, T. J., Van Overmeeren, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Binding+of+radioiodinated+propylthiouracil+to+rat+liver+microsomal+fractions%3A+stimulation+by+substrates+for+iodothyronine+5%E2%80%B2-deiodinase%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">Binding of radioiodinated propylthiouracil to rat liver microsomal fractions: stimulation by substrates for iodothyronine 5′-deiodinase.</a> Biochem J. 183 (1), 167-169 (1979).</li>
<li>Hindrichs, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+and+fast+online+SPE-LC-MS%2FMS+method+to+quantify+thyroid+hormone+metabolites+in+rat+plasma%5BTitle%5D)+AND+%22Chem+Res+Toxicol%22%5BJournal%5D)">A novel and fast online SPE-LC-MS/MS method to quantify thyroid hormone metabolites in rat plasma.</a> Chem Res Toxicol. 37 (1), 33-41 (2024).</li>
<li>Kolthoff, I. M., Sandell, E. B., Lingane, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modified+persulfate-arsenite+method+for+manganese%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">A modified persulfate-arsenite method for manganese.</a> Anal Chem. 8 (1), 73-73 (1936).</li>
<li>Stefanidis, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+the+robustness+and+accuracy+of+the+deiodinase+1+Sandell%E2%80%93Kolthoff+assay%3A+a+comparative+analysis%5BTitle%5D)+AND+%22Appl+Vitro+Toxicol%22%5BJournal%5D)">Assessment of the robustness and accuracy of the deiodinase 1 Sandell–Kolthoff assay: a comparative analysis.</a> Appl Vitro Toxicol. 10 (4), 80-90 (2024).</li>
<li>Satya, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guidance+document+on+standardised+test+guidelines+for+evaluating+chemicals+for+endocrine+disruption%5BTitle%5D)+AND+%22OECD+Environ+Health+Saf+Publ%22%5BJournal%5D)">Guidance document on standardised test guidelines for evaluating chemicals for endocrine disruption.</a> OECD Environ Health Saf Publ. 150, (2012).</li>
<li>Joseph, O., Eberle, M., Lieberman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolites+in+urine+that+interfere+with+the+Sandell%E2%80%93Kolthoff+assay+for+urinary+iodine%5BTitle%5D)+AND+%22Biol+Trace+Elem+Res%22%5BJournal%5D)">Metabolites in urine that interfere with the Sandell–Kolthoff assay for urinary iodine.</a> Biol Trace Elem Res. 202 (2), 466-472 (2024).</li>
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</ol>

The authors K.S., N.H, D.F.-W., and R.L. are employees of BASF SE, a chemical company, which may use the DIO1 assay to develop and register commercial products in the future.

The DIO1-SK assay provides a non-radioactive, cost-effective, and scalable alternative to traditional methods for assessing DIO1 activity, such as radiolabeled assays or mass spectrometry. Its prevalidation for human liver microsomes and strong concordance with SPE-LC-MS/MS measurements of thyroid hormone levels confirm its reliability and regulatory relevance, as reflected by its inclusion in the OECD test guideline workplan. The simplicity of the Sandell-Kolthoff (SK) reaction makes the assay accessible to laboratories without advanced instrumentation.
In the present protocol, several practical aspects were specified in greater detail compared with previously published DIO1-SK procedures to further support robustness and reproducibility in a routine 96-well format. These clarifications focus on the rationale for the control concept (solvent, negative and positive controls), the decision to use freshly prepared ion-exchange resin plates rather than re-using previously cast plates, and SK reaction conditions that improve handling robustness in multi-well plate measurements, while keeping the biological interpretation consistent with prior standardization work6,7.
Despite its strengths, several critical steps must be addressed to accurately quantify the iodide released during the DIO1-mediated conversion of rT3 to T2. This is achieved via the Sandell-Kolthoff reaction, which requires precise timing and consistent reagent handling to ensure reproducibility. The SK reaction is a rapid catalytic process in which iodide accelerates the reduction of yellow cerium (IV) to colorless cerium (III) by arsenite; the rate of color change is directly proportional to the iodide concentration. Because the reaction proceeds quickly once the arsenic solution is added, it is essential that the measurement is started without delay. In a 96-well plate format, slow or inconsistent addition of the arsenic solution can introduce significant variability between wells. Therefore, the use of a multichannel pipette or an automated dispenser is strongly recommended to ensure uniform and rapid reagent addition across all wells.
To maximize comparability across wells and between plates, the reagent addition order in the SK reaction should be kept consistent with the described workflow: Cerium is present as the chromogenic substrate, and the reaction is initiated by arsenite addition, which defines the start time and therefore requires rapid, synchronized dispensing. Alternative reagent addition orders were not evaluated in this setup; thus, changing the order is not recommended when reproducing the protocol. In addition, the cerium concentration can influence the practical performance characteristics of the SK readout (e.g., signal window and robustness). While 25 mM cerium was defined as a validated standard condition during earlier assay standardization, higher concentrations such as 40 mM were shown to increase signal window and z&#8242;-factor without affecting IC50 values or biological interpretation, as already described in previous work6. In this protocol, 40 mM cerium is used to ensure complete and rapid iodide complexation under the assay conditions and to improve robustness and signal stability in routine plate-based measurements. The acceptance criteria (e.g., z&#8242;-factor, IC50 values of the reference inhibitor) are based on historical performance observed in prior validation studies and is intended as a routine assay performance check.
The use of human liver microsomes introduces biological variability, making the standardization of microsome batch-specific iodide release activity essential. Careful preparation of ion exchange resin-filled filter plates is necessary to avoid uneven resin distribution across the filter plate and to ensure complete separation of iodide from potentially interfering substances. With respect to the resin workflow, freshly casting ion exchange resin-filled filter plates was chosen to minimize the potential risk of additional variability arising from resin saturation, iodide or protein carryover, and potential loss of binding efficiency upon reuse. Re-using resin and filter plates is technically feasible, however might harbour some risks that were not systematically evaluated in this study. It may introduce batch-to-batch drift if resin binding capacity is reduced or if residual matrix components remain in the resin bed after prior runs. For laboratories considering recycling, a pragmatic recommendation is to validate resin performance after reuse (e.g., by monitoring background and standard curve behavior and ensuring consistent iodide capture) and to discontinue reuse if increased background, reduced dynamic range, or inconsistent separation is observed. It is recommended to utilize resin-filled filter plates for single use, as this protocol has been specifically developed based on that methodology.
Additionally, the solubility of each test item must be determined in an appropriate vehicle, as the maximal concentration tested is limited by solubility (typically up to 1 mM with 1% DMSO). Because all test compounds are applied in DMSO as the solvent, the solvent control defines the baseline DIO1 activity under assay-relevant conditions and serves as the most appropriate reference condition for normalization and comparison. In contrast, 1-thio-&#946;-D-glucose sodium salt was included as a negative control because it is structurally similar to the positive control aurothioglucose (ATG) but chemically inert with respect to iodothyronine deiodination and does not interfere with microsomal redox systems or DIO1 catalytic activity. Its inclusion helps distinguish true DIO1-specific effects from nonspecific effects related to compound addition, handling, or assay matrix interactions. A no-treatment/no-vehicle control was not used as the primary reference condition because it would not reflect the actual assay environment in which DMSO is present in all compound-treated wells; therefore, the solvent control is the relevant baseline for assay interpretation.
Troubleshooting focuses on identifying interferences in the SK reaction. Iodine-containing, chelating, or strongly oxidizing substances may artificially affect the colorimetric readout14,17,18. To address this, the protocol includes interference testing without microsomes. Furthermore, the use of dithiothreitol (DTT) as a cofactor can also lead to non-enzymatic iodide release from certain test substances. In such cases, orthogonal methods like SPE-LC-MS/MS are recommended to confirm DIO1 inhibition. Nonspecific interference via general denaturing effects (e.g., surfactants) can generate false positives, so a general strategy to identify frequent hitters, using in silico tools or integrated testing approaches, can be valuable.
The assay has several limitations. It is susceptible to interference from test substances that react with assay components or release iodide independently of DIO1 activity. The assay does not distinguish between specific inhibition of DIO1 and unspecific interference with enzyme function or structural integrity. To improve specificity, inhibition of additional microsomal enzymes can be analyzed in parallel, and comparing inhibitory potencies across different enzyme assays may provide further evidence.
A further strategy for identifying possible artifacts is to include integrity or viability tests, as commonly performed in cell-based, in vitro assays, to define the maximum test concentration. While a direct integrity test is lacking for the DIO1-SK assay, its use within an in vitro test battery allows integration of cytotoxicity data from other cell-based assays to inform concentration selection. If such cross-assay information is not available, the upper concentration in the DIO1-SK assay is limited by solubility, which may not reflect relevant in vivo exposures. However, toxicokinetic approaches can be used to better define and interpret in vitro test concentrations.
The DIO1-SK assay is particularly valuable in toxicological screening and mechanistic studies of endocrine disruption. It addresses a key event in thyroid hormone homeostasis and can be integrated into in vitro test batteries. Its robustness, reproducibility, and adaptability to different laboratory settings and throughput needs make it a valuable tool for chemical safety assessment and regulatory validation. The assay provides a robust in vitro measure of DIO1 inhibition under standardized conditions and supports classification of test substances relative to a well&#8209;characterized reference inhibitor (6&#8209;PTU). However, in vivo data on DIO1 inhibition are scarce, and direct correlation of in vitro DIO1 inhibition outcomes with in vivo effects is therefore not currently applicable for estimating in vivo predictivity. Accordingly, assay results should be interpreted as mechanistic evidence for interference with one key event in thyroid hormone metabolism and integrated with additional lines of evidence (e.g., other thyroid MoA assays and orthogonal measurements where needed). Importantly, the DIO1&#8211;SK assay has been evaluated in multiple validation-oriented studies (optimization/standardization, predictivity assessment, and robustness/accuracy analysis)6,7,15, and as the method progresses toward OECD implementation, the present article provides a level of step-by-step procedural detail intended to facilitate transfer and consistent implementation across additional laboratories.

Endocrine disruptors (EDs) are chemicals that interfere with hormonal regulation and consequently cause adverse effects in humans1,2. Deiodinases (DIO) are membrane-associated enzymes, with selenocysteine as a key residue in their active sites. This enzyme family comprises three distinct isoforms (DIO1, DIO2, and DIO3), which collectively modulate circulating thyroid hormone (TH) concentrations and govern their tissue-specific availability within cells3. Thyroxine (T4), which serves as a prohormone, and triiodothyronine (T3), the active form, play vital roles in regulating energy expenditure, growth, developmental processes, and thermoregulation in the body4. The DIO isoforms differ in the characteristics of their deiodination site specificity, kinetic properties, and substrate preferences3. DIO1 is the main deiodinase isoform in adult liver and exhibits strong substrate affinity for reverse triiodothyronine (rT3) and sulfated iodothyronines, while DIO2 preferentially utilizes T4 and rT3, and DIO3 primarily acts on T4 and T3. Using human liver microsomes and rT3 as substrate allows selective detection of DIO1 activity, minimizing interference from other isoforms. While the precise consequences of substance-induced DIO1 inhibition remain to be fully elucidated, evidence from human gene mutations affecting DIO1 clearly demonstrates that DIO1 can disrupt the thyroid hormone system5. As a result, there is a critical need for robust and reproducible methods to investigate DIO1 inhibition.
The goal of the presented method is to quantify DIO1 activity in vitro using a non-radioactive, colorimetric approach based on the Sandell-Kolthoff (SK) reaction. DIO1 catalyzes the conversion of reverse rT3 to T2, a process that results in the release of iodide ions. The SK reaction enables sensitive detection of the released iodide, allowing determination of iodide release activity (IRA) as a direct measure of DIO1 activity. This assay enables the detection of inhibitory effects of test substances on DIO1, supporting mechanistic toxicology, screening, or regulatory decision-making. It is particularly relevant for investigating DIO1 inhibition as a molecular initiating event of adverse outcome pathways leading to thyroid disruption, as outlined by international validation efforts such as those coordinated by EURL ECVAM6,7. The method was originally developed in 20128,9 and further optimized6,7 in response to the growing need for robust, reproducible, and scalable in vitro assays that can be integrated into test batteries for endocrine screening, especially in the context of thyroid hormone metabolism. Recently, the application of analog SK-based setups for medium-10 to high-throughput screening11 was published, demonstrating the broad applicability of the SK-based approach.
Even though the DIO1-SK assay indirectly measures DIO1 activity by quantifying IRA rather than TH products, it offers several advantages over alternative techniques. Unlike methods that rely on radiolabeled substrates12 or mass spectrometry-based quantification of depleted or formed thyroid hormones13, the SK reaction provides a simple, scalable, and cost-effective readout by measuring iodide release at 415 nm14. While LC-MS/MS methods with solid-phase extraction (SPE-LC-MS/MS) offer high specificity and sensitivity for quantifying rT3 and T2, they require expensive instrumentation and are less suitable for higher-throughput screening. In contrast, the DIO1-SK assay can be implemented in standard laboratory settings and has been prevalidated for use with human liver microsomes, demonstrating high reproducibility and predictivity6,7. In a comparative study, results from the SK reaction, specifically iodide release, were directly compared with quantification of thyroid hormone metabolites (rT3 and T2) using SPE-LC-MS/MS15. The findings revealed a strong concordance between the two approaches across a range of test substances and inhibition potencies. For all substances tested, changes in iodide release activity (IRA) measured by the SK reaction closely mirrored alterations in rT3 and T2 levels determined by mass spectrometry. This further supported the validity of the SK readout as a surrogate for DIO1 activity. These comparative analyses confirm that the SK assay provides a robust and reliable alternative to more complex analytical techniques, and that iodide release can be measured with confidence to assess DIO1 inhibition in vitro.
However, the SK method has certain limitations that must be considered: It is susceptible to interference from substances that either contain free iodide as an impurity, release iodide non-enzymatically, or react with assay components such as dithiothreitol (DTT). DTT is included as a reducing cofactor to maintain deiodinase catalytic competence by supporting regeneration of the active-site selenocysteine in its reduced state during turnover. Because DTT is a strong reductant, certain test substances (or impurities) may undergo redox reactions that can influence the colorimetric readout and, in some cases, lead to DTT-dependent assay artifacts unrelated to enzymatic iodide release. Such interferences can lead to false-negative results for DIO1 inhibition, as the measured iodide may not originate from the enzymatic conversion of rT3. Thus, while the SK assay is suitable for routine screening and most test substances, LC-MS/MS could be used for confirmation whenever assay interference is suspected or for substances with complex chemical properties.
The DIO1-SK assay is currently being readied for inclusion as an OECD test guideline in an international PEPPER (Public-private platform for the validation of endocrine disruptors characterization methods) project, which highlights its growing importance for regulatory applications and the international harmonization of endocrine disruptor testing strategies. The assay may be integrated into comprehensive in vitro test batteries, supporting both chemical safety assessment and mechanistic investigations into endocrine disruption16. When combined with assays addressing additional key events along the thyroid adverse outcome pathway, it contributes to a more complete understanding of thyroid hormone disruption mechanisms.
In addition, the protocol&#8217;s scalability and high reproducibility make it well-suited for medium- to high-throughput screening. This is essential for evaluating large numbers of substances during drug development and in academic settings, as recently demonstrated11. Researchers interested in DIO1 inhibition, whether for screening, mechanistic toxicology, or regulatory decision-making, will benefit from a robust, accessible, and versatile assay. The validation of this method will make it a standard tool for research on thyroid hormone system disruption and for evaluating chemical safety.

<table><tbody><tr><td>1-Thio-&amp;beta;-D-glucose sodium salt</td><td>Sigma-Aldrich</td><td>T6375-1G</td><td>CAS 10593-29-0</td></tr><tr><td>3,3&amp;rsquo;,5&amp;rsquo;-triiodothyronine (rT3)&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td>sc-209692</td><td>CAS 5817-39-0</td></tr><tr><td>6-Propyl-2-thiouracil (6PTU)</td><td>Supelco</td><td>P3755-10G</td><td>CAS 51-52-5</td></tr><tr><td>Acetic acid</td><td>Thermo Fisher</td><td>9526-33</td><td>CAS 64-19-7</td></tr><tr><td>Analytical balance</td><td>NA</td><td>NA</td><td>capable of accurately weighing up to 30 g with 0.1 mg readability</td></tr><tr><td>Arsenic sodium oxide (NaAsO2)</td><td>Thermo Fisher</td><td>041533.AP</td><td>CAS 7784-46-5</td></tr><tr><td>Assay plates (96-well format)</td><td>Merck</td><td>Z707902-108EA</td><td>e.g. tissue culture plates, 96-well plate, flat bottom, polystyrene, 0.34 cm2, sterile, 108/cs, TPP</td></tr><tr><td>Aurothioglucose (ATG)</td><td>Sigma-Aldrich</td><td>A0606-5MG</td><td>CAS 12192-57-3</td></tr><tr><td>Centrifuge Tubes 15 and 50&amp;nbsp;mL</td><td>Merck</td><td>Z707724-800EA, Z707716-320EA</td><td>e.g. TPP centrifuge tubes, volume 15 and 50 mL, polypropylene,&amp;nbsp;</td></tr><tr><td>Centrifuge with swing-out rotor for microtiter plates</td><td>NA</td><td>NA</td><td>Should be high enough to fit a 96-deep well plate with 96-well filter plate on top (at least about 6 cm high)</td></tr><tr><td>Cerium (IV) ammonium sulphate (Ce(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;4&lt;/sub&gt;(SO&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>22269-100G-F</td><td>CAS 10378-47-9</td></tr><tr><td>CO2 Incubator</td><td>NA</td><td>NA</td><td>capable of keeping temperatures of 37&amp;deg;C, 5% CO2 and &amp;ge;90% humidity</td></tr><tr><td>Deep well plates (96-well format)</td><td>Supelco</td><td>575653-U</td><td>e.g. SPE 96-Deep Square Well Collection Plate, well volume 2 mL, polypropylene</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>AppliChem</td><td>A3672,0250</td><td>CAS 67-68-5</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>D9779-1G</td><td>CAS 3483-12-3</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>APO456787707-500G</td><td>CAS 6381-92-6</td></tr><tr><td>Filter plates (96-well format)</td><td>Merck</td><td>WHA77002810</td><td>e.g. Micro-Plates, 96-well, clear polystyrene, 800 &amp;micro;L, DNA Binding, Whatman</td></tr><tr><td>Gas-tight plate sealers</td><td>Thermo Fisher</td><td>232698</td><td>e.g. Sealing tape, polyester, sterile, Sealing tape, polyester, sterile, Nunc</td></tr><tr><td>HEPES</td><td>Pan Biotech</td><td>P05-01100</td><td>CAS 7365-45-9</td></tr><tr><td>Human liver microsomes</td><td>Biovit</td><td>X008070</td><td>e.g. INVITROCYP 150-Donor Pooled Human Liver Liver Microsomes</td></tr><tr><td>Iodide (IC standard)</td><td>Supelco</td><td>41271-100ML</td><td>e.g. Iodide standard for IC, 1000 mg/L in water, Sigma-Aldrich</td></tr><tr><td>Ion exchange resin like Dowex 50WX2</td><td>Roth</td><td>334.4</td><td>CAS 12612-37-2; Mesh 100-200</td></tr><tr><td>Microcentrifuge tubes 1.5&amp;nbsp;mL</td><td>Merck</td><td>EP0030120086-1PAK</td><td>e.g. Eppendorf&amp;reg; Safe-Lock microcentrifuge tubes, volume 1.5 mL, natural</td></tr><tr><td>Multichannel dispenser capable of delivering 50 to 300&amp;nbsp;&amp;micro;L per step</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Multichannel pipette capable of delivering 10 to 100&amp;nbsp;&amp;micro;L per step</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>pH meter with electrode and calibration buffers</td><td>NA</td><td>NA</td><td>capable of reading +/- 0.1 pH units</td></tr><tr><td>Photometer for absorbance measurement</td><td>Tecan Trading AG</td><td>NA</td><td>e.g. Sunrise Absorbance Reader, INSTSUN-3</td></tr><tr><td>Pipets capable of delivering 1 to 10&amp;nbsp;&amp;micro;L</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Pipets capable of delivering 10 to 100&amp;nbsp;&amp;micro;L</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Pipets capable of delivering 100 to 1000&amp;nbsp;&amp;micro;L&amp;nbsp;</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Pipets for higher volumes</td><td>NA</td><td>NA</td><td>serological pipettes, e.g. 10, 25, 50 mL</td></tr><tr><td>Plate shaker</td><td>Thermo Fisher</td><td>NA</td><td>e.g. Thermo Scientific H+P MONOSHAKE VORTEXER microtiter plate, directly controlled</td></tr><tr><td>Repeater pipette</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S9888-25G</td><td>CAS: 7647-14-5, e.g. sodium chloride, ACS reagent, &amp;ge;99.0 %</td></tr><tr><td>Statistics software</td><td>NA</td><td>NA</td><td>Able to perform regression analysis that reflect assay characteristics and able to calculate inhibitory concentrations e.g. GraphPad Prims 8, GraphPad</td></tr><tr><td>Sulfuric acid (H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)&amp;nbsp;</td><td>Supelco</td><td>1007311000</td><td>CAS 7664-93-9</td></tr><tr><td>Volumetric flask</td><td>NA</td><td>NA</td><td>certified with defined volume</td></tr></tbody></table>

colorimetric assessment of deiodinase 1 activity in human liver microsomes using the sandell-kolthoff reaction

Colorimetric Assessment of Deiodinase 1 Activity in Human Liver Microsomes Using the Sandell-Kolthoff Reaction

This method enables the identification of test substances that inhibit DIO1 activity and may thus interfere with thyroid hormone homeostasis. The protocol presented describes a non-radioactive, colorimetric method for assessing the effects of test substances on the enzymatic activity of Deiodinase 1 (DIO1) in human liver microsomes. The assay is based on the Sandell-Kolthoff (SK) reaction, which quantifies iodide released during the deiodination of reverse triiodothyronine (rT3) to diiodothyronine (T2). The yellow cerium (IV) is reduced to colorless cerium (III) in the presence of iodide, allowing photometric detection at 415 nm. In this standardized setup, the cerium (IV) concentration is set to 40 mM to improve signal stability and robustness under the described conditions. To ensure assay reliability and reproducibility, a comprehensive control setup is included: 6-propyl-2-thiouracil (reference item), aurothioglucose (positive control), 1-thio-&#946;-D-glucose sodium salt (negative control), and 1% dimethyl sulfoxide (solvent control). The protocol includes a range-finding assay to determine appropriate test concentrations, standardization of microsome batch-specific iodide release activity and structured chemical interference testing in the absence of microsomes, with an optional follow-up assessment without dithiothreitol (DTT) to identify DTT-dependent artifacts. The DIO1-SK assay is suitable for medium- to high-throughput screening and mechanistic toxicology studies. Its robustness, scalability, and applicability to human microsomes make it a valuable tool for investigating endocrine disruption. This protocol supports regulatory validation efforts.

This protocol describes a colorimetric method to reliably assess the influence of chemicals on Deiodinase 1 (DIO1) activity in human liver microsomes using the Sandell-Kolthoff reaction. It includes predefined acceptance criteria and statistical performance metrics and supports ongoing efforts toward OECD test guideline inclusion.

This method enables the identification of test substances that inhibit DIO1 activity and may thus interfere with thyroid hormone homeostasis. The protocol ...

The assay quantifies DIO1 inhibition using human liver microsomes. It was validated and optimized for robust and standardized handling. This protocol is used in mechanistic toxicology and screening to identify chemicals inhibiting DIO1 and disrupting thyroid hormone homeostasis.This protocol measures iodide release from RT3 through deiodination via the Sandell-Kolthoff reaction, a practical surrogate readout for DIO1 activity. To begin, obtain an appropriate rectangular transparent container. Add about 250 grams of ion-exchange resin to the container.Wash the resin with 10%acetic acid and let the resin suspension rest for 10 minutes. Then, slightly tilt the container to aspirate the supernatant. Repeat the wash at least five times in total, or until no more color leaks into the solvent.After the last wash, add 10%acetic acid, so it makes up approximately 50%of the total volume. Tilt the container so the resin moves to one side. Stabilize the container in the tilted position, and wait about 10 minutes for the phases to separate.Place a 96 well filter plate on top of a 96 deep well plate. Add 100 microliters of 10%acetic acid into each well of the 96 well filter plate. Cut the tips used for resin casting at a height of about one centimeter to widen the opening at the end.Use the cut tip to dispense 300 microliters of ion-exchange resin into each well of the 96 well filter plate. Centrifuge the plate assembly with a swing out rotor for microtiter plates at 100 to 200G for one minute to elute the acetic acid into a 96 deep well plate. Check whether the resin is distributed evenly across the plate.Seal the plate with an impermeable sheet of plastic. Store the plate at four degrees Celsius for a maximum of six months. Prepare iodide dilutions from the iodide source in deionized water.Add 50 microliters of pure deionized water as a control to a 96 well plate in triplicate. Add 50 microliters of the prepared iodide dilutions to the same plate in triplicate. Add 50 microliters of 40 millimolar cerium solution to each well.Start the reaction by adding 50 microliters of 25 millimolar arsenite solution. Immediately after adding arsenite, measure the absorbance with a plate reader at 415 nanometers. Then measure the absorbance every minute for 21 minutes.Prepare the reference item, positive control, negative control, and test item dilution series at the concentration shown. Prepare the substrate mix and a microsome suspension. Place the tubes on ice.Add 10 microliters of the reference item dilutions, controls, and test item dilutions to the wells according to the plate layout. Add 40 microliters of microsome suspension to each well followed by 50 microliters of freshly prepared substrate mix. Seal the plate with an impermeable sheet of plastic.Place it on a shaker in an incubator at 37 degrees Celsius and 850 revolutions per minute for two hours. Then place the plate on ice to stop the reaction and leave it there until measurement. Place the ion-exchange resin filled 96 well filter plate on top of a 96 deep well plate.Add 150 microliters of 10%acetic acid to each well of the filter plate to wet the resin. Centrifuge the plate assembly in a centrifuge with a swing out rotor for microtiter plates at 100 to 200G for one minute to elute the acetic acid into the 96 deep well plate. Replace the used 96 deep well plate with a fresh plate.Retrieve the incubated 96 well plate from ice. Add 133 microliters of 10%acetic acid to each well. Shake the plate briefly to mix the samples.Transfer 175 microliters of each sample to the ion-exchange resin filled 96 well filter plate, maintaining the original plate layout. Centrifuge the plate assembly with a swing out rotor for microtiter plates at 100 to 200G for one minute to pass the samples through the resin into the 96 deep well plate. Then remove the ion-exchange resin filled 96 well filter plate.Transfer 50 microliters of the undiluted sample from the 96 deep well plate into a new 96 well plate. Add 50 microliters of 40 millimolar cerium solution to all wells and start the reaction by adding 50 microliters of 25 millimolar arsenite solution to each well. Immediately after adding arsenite, measure the absorbance with a plate reader at 415 nanometers.Then measure the absorbance every minute for 21 minutes. The background corrected Sandell-Kolthoff signal increased with increasing microsomal protein. The signal varied across post ion-exchange dilutions.The one to two dilution yielded the highest signal, while maintaining a sigmoidal response. Comparison of inhibition curves identified five micrograms of microsomal protein per well as the lowest amount producing a sigmoidal response. The reference inhibitor showed a concentration-dependent decrease in iodide release activity, with a sigmoidal inhibition profile.Tannic acid showed a comparable inhibitory profile with high maximum inhibition. And dibutyl phthalate showed no inhibition across the tested concentration range. These results showed that the assay clearly distinguished inhibitory from non-inhibitory concentration response profiles under standardized conditions.Maintaining strict uniform handling is a key challenge. A slight variation to timing and handling can significantly affect protocol efficiency. Future studies can adapt the standardized workflow to other sources of deiodinating enzymes, enabling assays for DIO2, DIO3, or dehalogenases.

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Learn a colorimetric assay for Deiodinase 1 activity in human liver microsomes using the Sandell-Kolthoff reaction. This non-radioactive method enables high-throughput screening of chemicals that disrupt thyroid hormone homeostasis.

Colorimetric Assessment of Deiodinase 1 Activity in Human Liver Microsomes Using the Sandell-Kolthoff Reaction

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