Preparation, purification, and stability of high specific activity 125I-labeled thyronines.

A modified chloramine-T method is described for the preparation of several radioiodothyronines by an exchange reaction, which results in low specific activity preparations, or by an addition reaction, which yields radioiodothyronines with specific activities up to 7 mCi/microgram. Purification by paper chromatography is shown to be more convenient than by LH-20 chromatography and provides better resolution among the various thyronines. Radioiodothyronines with only a single iodine atom in the outer (3,5,3'-triiodothyronine and 3,3'-diiodothyronine) are stable for several months when stored in organic solvents. The least stable radioiodothyronines are those with two 125I atoms in the outer ring (3,3',5'-triiodothyronine (rT3) produced from 3-iodothyronine and thyroxine (T4) from 3,5-diiodothyronine). The stability of rT3 and T4 stored in human plasma at 4 C is much greater than when stored in buffer at the same pH. The use of high specific activity [125I]rT3 has permitted the development of a radioimmunoassay with a sensitivity of 1 pg rT3/ml incubation volume.

Thyroid hormone analogues. Synthesis of 3'-substituted 3,5-diiodo-L-thyronines and quantitative structure-activity studies of in vitro and in vivo thyromimetic activities in rat liver and heart.

Twenty-nine novel 3'-substituted derivatives of the thyroid hormone 3,3',5-triiodo-L-thyronine (T3) have been synthesized by using established methods and by a new route involving manipulation of a 3'-formyl intermediate. In vitro hormone receptor binding (to intact nuclei) and in vivo thyromimetic activity (induction of mitochondrial 3-phosphoglycerate oxidoreductase, GPDH) were measured in rat liver and heart for these new analogues and for the 18 previously reported 3'-substituted 3,5-diiodo-L-thyronines. Analysis of the binding data using theoretical conformational and quantitative structure-affinity methods implies that the 3'-substituent recognition site on the thyroid hormone receptor is hydrophobic and limited in depth to the length of the natural iodo substituent, but has sufficient width to accommodate a phenyl or cyclohexyl group. Receptor binding is reduced by approximately 10-fold in 3'-acyl derivatives which form strong intramolecular acceptor hydrogen bonds with the adjacent 4'-hydroxyl. The compounds studied showed no differences in their relative affinities for heart and liver nuclei, suggesting that receptors in these tissues are similar. However, the relationships between thyromimetic activity (induction of GPDH) and nuclear binding showed some tissue differences. A high correlation between activity and binding is observed for full agonists in the heart, but an equally significant correlation for the liver data is only seen when 3'-substituent bulk (molar refractivity) is included in the analysis. These results suggest the possibility that differential tissue penetration or access to receptors may occur in vivo.

Establishment of the sulfated 3,3'-diiodothyronine radioimmunoassay and its application in pregnant women.

Sulfation of iodothyronines is a major alternate pathway of thyroid hormone metabolism during fetal development. Sulfated 3,3'-diiodothyronine (T2S) is a low end metabolite of this pathway. Its clinical implications in the prenatal evaluation of fetal thyroid disorders are now being intensively investigated. A highly sensitive and reproducible radioimmunoassay (RIA) for T2S has been established in our laboratory. The detection threshold of the RIA approximated 5 pg T2S. The dose-response curve of T2S was essentially linear between 5-200 pg. An essentially parallel correlation of the dose-response curves for inhibition of binding of radiolabeled T2S and T2S antiserum was found between serial dilutions of serum extracts and the standards. The average intra- and inter-assay coefficients of variation were 7% and 15%, respectively. By applying the T2S RIA, we found that serum titers of T2S in pregnant women increased proportionately to the gestational age (first trimester vs. second trimester vs. third trimester: 30.1 +/- 1.4 vs. 41.6 +/- 1.9 vs. 98.0 +/- 3.9 ng/dL; p < 0.001 each). A high concentration of T2S was detected in cord and maternal serum at birth as compared to the results for non-pregnant women (165.1 +/- 10.3 vs. 113.7 +/- 5.6 vs. 7.5 +/- 0.9 ng/dL). The T2S levels decreased remarkably in maternal circulation 10 days after partuition. Four pregnant women who had two blood samplings four or more weeks apart during the third trimester showed invariable increases of serum T2S titers at later sampling times. Additionally, in the case of a pregnant woman who received two doses of T4 injections (200 micrograms/wk) intraamniotically for a previous Cretin birth, the maternal serum levels of T2S increased promptly from 47 ng/dL [corrected] to 96 ng/dL. Our findings imply that the established T2S RIA is clinically applicable, provide further evidence that the coincident increase of serum T2S titers in pregnant women may reflect ontogenesis of fetal thyroid hormone maturation, and provide a clue to the fetal thyroid status in the prenatal stage. However, more knowledge is still needed regarding the transfer and transformation of sulfated iodothyronine(s) from the fetal compartment to maternal circulation.