RADIOIMMUNOASSAY FOR MEASUREMENT OF 3,5-DIIODOTHYRONINE

A highly specific, simple and reproducible RIA [radioimmunoassay] was developed for measurement of 3,5-L-diiodothyronine (3,5T2) in unextracted serum. The assay employs an inner ring-labeled [125I]3,5T2 as the tracer and a 3,5T2-binding antibody produced by immunization of rabbits with 3,5-T2 human serum albumin conjugate. The interference in binding of radioactive 3,5T2 to anti-3,5T2 by serum proteins was minimized using 0.15 M Tris buffer (pH 8.2) and sodium salicylate. The detection threshold of the RIA was 0.5 ng/100 ml. Recovery of nonradioactive 3,5T2 added to serum averaged 96%. Compared to 3,5T2, 3-monoiodothyronine (3T1), 3,5,3''-triiodothyroacetic acid, and purified T3 [triiodothyroxine] inhibited the binding of radioactive 3,5T2 to the 3,5T2 antibody only to the extent of 0.6, 0.28% and 0.03%, respectively. T4 [thyroxine], r[reverse]T3, 3,3''T2, 3'',5''T2, and KI cross-reacted less than 0.001% with 3,5T2-binding sites on the antibody. Serum 3,5T2 concentrations were (mean .+-. SD) 7.3 .+-. 3.5 ng/100 ml in 30 normal subjects, 8.5 .+-. 3.6 ng/100 ml in 16 hyperthyroid patients, 9.5 .+-. 5.0 ng/100 ml in 16 hypothyroid patients, 13 .+-. 6.1 ng/100 ml in 6 pregnant subjects, 2.5 .+-. 1.7 ng/100 ml in 6 patients with hepatic cirrhosis and 5.1 .+-. 2.8 ng/100 ml in 14 newborn cord sera. The values in the latter 3 groups differed significantly from that in normal subjects. 3,5T2 concentrations in 14 amniotic fluids obtained at 33-40 wk of gestation averaged 0.9-0.7 ng/100 ml. This value was significantly lower than that in normal human serum. Pronase hydrolysates of 8 autopsy specimens of normal thyroid glands contained (mean .+-. SE) 332 .+-. 51 .mu.g T4 and 0.16 .+-. 0.06 .mu.g 3,5T2/g wet wt. Thus, the content of 3,5T2 in thyroglobulin is about 0.07% that of T4 on a molar basis, suggesting that thyroidal secretion of 3,5T2 contributes minimally to 3,5T2 measured in the serum of normal man. 3,5T2 is a normal component of human serum, amniotic fluid and thyroid gland; serum 3,5T2 is reduced in the low T3 syndrome in clinically euthyroid patients with nonthyroid illnesses and in the newborn; serum 3,5T2 is not a good indicator of the state of thyroid function.