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-β-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.