An optimized radioimmunoassay for quantification of total serum thyroxine (T4) in fetal, neonatal, and pregnant rats

Identifying xenobiotics that interrupt the thyroid axis has significant public health implications, given that thyroid hormones are required for brain development. As such, some developmental and reproductive toxicology (DART) studies now require or recommend serum total thyroxine (T4) measurements in pregnant, lactating, and developing rats. However, serum T4 concentrations are normally low in the fetus and pup which makes quantification difficult. These challenges can be circumvented by technologies like mass spectrometry, but these approaches are expensive and not always widely available. To demonstrate the feasibility of measuring T4 using a commercially available assay, we examine technical replicates of rat serum samples measured both by liquid chromatography mass spectrometry (LC/MS/MS) and radioimmunoassay (RIA). These samples were obtained from rats on gestational day 20 (dams and fetuses) or postnatal day 5 (pups), following maternal exposure to the goitrogen propylthiouracil (0–3 ppm) to incrementally decrease T4. We show that with assay modification, it is possible to measure serum T4 using low sample volumes (25–50 μL) by an RIA, including in the GD20 fetus exposed to propylthiouracil. This proof-of-concept study demonstrates the technical feasibility of measuring serum T4 in DART studies.

Impact/Purpose

Measuring serum thyroxine (T4) in rats is an important endpoint in some standardized developmental and reproductive toxicology studies. However, measuring this hormone is not necessarily an easy task, as it is normally in low concentration during development. These and other technical challenges have recently led to discussions about whether this experiment is feasible for chemical registrants. In this work we show that it is possible to measure serum T4 in developing rats using a commercially purchased radioimmunoassay that was modified to improve its limit of detection. We then compare the same serum samples ran with mass-spectrometry, a highly sensitive detection technology. Our results show that it is possible to measure serum T4 in rats with a relatively inexpensive kit, and the results are comparable to those obtained by mass spectrometry - a more expensive and technically difficult methodology. In conclusion, this work demonstrates that serum T4 measurements are a feasible experiment in toxicology studies, and this endpoint can be used to identify serum T4 reductions resultant from exposure to endocrine disrupting chemicals. 

Citation

O'Shaughnessy, Katherine, M. Hotchkiss, A. Buckalew, A. Murr, M. Gilbert, AND T. Stoker. An optimized radioimmunoassay for quantification of total serum thyroxine (T4) in fetal, neonatal, and pregnant rats. Elsevier Science Ltd, New York, NY, 100:107303, (2023). [DOI: 10.1016/j.ntt.2023.107303]

DOI: An optimized radioimmunoassay for quantification of total serum thyroxine (T4) in fetal, neonatal, and pregnant rats