Optimization of an in vitro assay methodology for competitive binding of thyroidogenic xenobiotics with thyroxine on human transthyretin and albumin

Katie L. Hill, Timo Hamers, Jorke H. Kamstra, William G. Willmore, Robert J. Letcher*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Thyroid hormones (THs) are involved in the regulation of many physiological processes in vertebrates. Competition for TH binding sites on serum transport proteins can interfere with delivery of THs to target tissues, and this is a potential mechanism of action of exogenous thyroidogenic substances. To date, detailed accounts of in vitro methods for competitive binding with THs on TH transport proteins (human or wildlife) are sparse. In the limited number of published studies on in vitro radio-labelled TH-TH transport protein interactions, method descriptions were brief and with insufficient details for successful replication. Furthermore, upon review of these methodologies, we identified several opportunities for optimization. The present study addresses the methodological deficiencies and describes, in detail, a fully optimized and validated competitive T4 radio-ligand binding assay with human transthyretin (TTR) and albumin (ALB). • Significant improvements were made over previous methods, including better maintenance of protein stability and enhanced measurement of competition between different ligands.• Sample size was reduced to allow use of small pre-packed size exclusion chromatography columns, which eliminates the rinsing step during the separation procedure.• The assay was parameterized for use with T4 and human TTR and ALB.

Original languageEnglish
Pages (from-to)404-412
Number of pages9
JournalMethodsX
Volume4
DOIs
Publication statusPublished - 20 Oct 2017

Funding

This study was financially supported by the Chemicals Management Plan (CMP; Environment and Climate Change Canada) (to R.J.L.), a Discovery Grant from the Natural and Engineering Science Research Council (NSERC) of Canada (to R.J.L. and W.G.W.), and the NSERC CREATE Program (to R.J.L. and W.G.W.). We thank Environment and Climate Change Canada staff at the National Wildlife Research Centre France Maisonneuve and Lewis Gauthier, for their practical assistance in the lab.

FundersFunder number
Chemicals Management Plan
Natural and Engineering Science Research Council
Environment and Climate Change Canada

    Keywords

    • Albumin
    • Competitive protein binding assay
    • Exogenous ligands
    • Human
    • Thyroid hormones
    • Transport proteins
    • Transthyretin

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