Studies of Propylthiouracil Using a Newly Developed Radioimmunoassay*

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Abstract
A RIA for the antithyroid drug 6-propyl-2-thiouracil (PTU) has been developed. Antibodies to PTU were generated in rabbits after synthesizing 6-propionic acid-2-thiouracil and conjugating this derivative to either bovine serum albumin or porcine thyroglobulin. [35S]JPTU was used as a tracer. At a final antibody dilution of 1:400, the assay could detect PTU in amounts as low as 2.5 ng. Cross-reaction studies suggested that the thiol group of PTU was necessary for immunoactivity. The known circulating, urinary, and intrathyroidal metabolites of PTU had negligible cross-reactivity in the assay. It was observed that serum samples containing PTU lost immunoactivity over the course of several weeks. Gel filtration chromatography of [35S]PTU incubated in fresh human serum revealed that [35S]PTU radioactivity eluted in two peaks (peaks I and II). Peak I eluted with serum protein, was not immunoactive, and grew progressively larger with increasing incubation time in serum (15 min to 8 h). Peak II corresponded to free PTU. Polyacrylamide gel electrophoresis of the PTU-serum complex (peak I) in the presence of sodium dodecyl sulfate resulted in complete conversion to free PTU, suggesting that the drugprotein interaction was a noncovalent process. This was supported further by the observation that 0.1% sodium dodecyl sulfate restored full immunoactivity to PTU stored in serum. Similar effects were obtained with 5 mM dithiothreitol, indicating that the interaction may involve sulfhydryl groups. After a single oral 150-mg dose of PTU, PTU immunoactivity appeared in the serum within 5 min. The half-time of PTU disappearance was significantly shorter in hyperthyroid patients than in controls (75 vs 90 min; P < 0.01). Peak PTU levels (∼4 µg/ml) were similar in both groups; there was no correlation of either of these parameters with serum T4, T.3, or body surface area.