Assessment of the Robustness and Accuracy of the Deiodinase 1 Sandell–Kolthoff Assay: A Comparative Analysis

Introduction: This scientific publication presents the third prevalidation study of the deiodinase 1 Sandell–Kolthoff (DIO1-SK) assay, investigating its robustness and accuracy. The study compares the released iodide as the original read-out of the assay with concentrations of the thyroid hormones reversed triiodothyronine and 3,3′-diiodo-L-thyronine as the substrate and the product of the enzymatic reaction, respectively. Moreover, it is examining the impact of iodine-releasing substances, evaluating the alkaline phosphatase (ALP) assay as a specificity test, and investigating the temporal robustness of the assay. Methods: The study utilized a comparative analysis between the SK method and solid-phase-extraction liquid chromatography MS/MC to evaluate the concordance between the released iodide and thyroid hormone levels. The effects of iodine-releasing substances on the SK reaction were examined, and the ALP assay was used to assess specificity and microsome integrity. The temporal robustness of the DIO1-SK assay was investigated by varying incubation times. Results and Discussion: The comparative analysis revealed a strong concordance between released iodide and thyroid hormone levels, validating the read-out of the SK assay. Iodide-releasing substances were found to interfere with the SK reaction, specifically impacting the iodide release activity and should be tested for their potential to react with dithiothreitol. The ALP assay showed clear limitations in assessing test system integrity and substance specificity. The DIO1-SK assay demonstrated robustness to different incubation times, indicating its stability and reliability. Conclusion: The findings of this study characterize the robustness of the DIO1-SK assay, its limitations, and potential areas for improvement. Considering the identified limitations with iodine-containing substances, the DIO1-SK assay serves as a reliable tool to investigate the influence of substances on DIO1 activity and thus is an assay addressing one key event of impacting the thyroid hormone homeostasis, which can lead to endocrine disruption.