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. The technical limitations quantifying serum thyroxine (T4) in developing rats by commercially available methods. In euthyroid (control) fetuses on GD20, serum T4 concentrations are expected to be only ∼3 ng/mL (O'Shaughnessy et al., 2018a), which is below the quantitation limits of many commercial T4 assays previously used in published toxicology studies. This includes the T4 RIA commercial kits from ICN Biomedicals (Orangeburg, NY) (Bansal and Zoeller, 2019) and Siemen's Coat-A-Count® Kit (Malvern PA) (Louis et al., 2017, Crofton et al., 2007). Given these historically utilized assays, it would be difficult or impossible to quantify serum T4 in control GD20 rats. The sensitivity of mass-spectrometry allows quantification of serum T4 in the GD20 fetus and postnatal rat pup following a goitrogen exposure (Hassan et al., 2017; Ko et al., 2021; O'Shaughnessy et al., 2018a; O'Shaughnessy et al., 2018b; O'Shaughnessy et al., 2019) (see dark purple box and line), but this technology may not be available in certain laboratories. Additionally, the mass-spectrometry methods require a solid phase extraction step, which is time consuming and expensive. The modified In