A Radioimmunoassay for Measurement of 3,3′, 5′-Triiodothyronine (Reverse T3)

Abstract

A highly specific antiserum to 3,3′,5′-triiodothyronine (reverse T₃, rT₃) was prepared by immunization of rabbits with D,L-rT₃-human serum albumin conjugate. Of the various thyroid hormone derivatives tested, only 3,3′-diiodothyronine (3,3′-T₂) cross-reacted significantly (10%) with rT₃-binding sites on the antiserum, while thyroxine (T₄) and triiodothyronine (T₃) cross-reacted by less than 0.1%. The antiserum was used in a simple, sensitive, precise, and reproducible radioimmunoassay (RIA) for measurement of rT₃ in ethanolic extracts of serum. The dose-response curves of inhibition of the binding of [¹²⁵I]rT₃ to antibody obtained by serial dilutions of serum extracts were essentially parallel to the standard assay curve. Recovery of nonradioactive rT₃ added to serum before extraction averaged 93%. Serum rT₃ concentrations were found to be (mean±SD) 41±10 ng/100 ml in 27 normal subjects, 103±49 ng/100 ml in 22 hyperthyroid patients, 19±9 ng/100 ml in 12 hypothyroid patients, and 54±7 ng/100 ml in five subjects with elevated serum thyroxine-binding globulin: the values in each of the latter three groups of individuals were significantly different from normal. Reverse T₃ was detected regularly in normal or supranormal concentrations in serum of 12 hypothyroid patients rendered euthyroid or mildly hyperthyroid by treatment with synthetic T₄. It is suggested that serum rT₃ values noted here should be taken to reflect the relative changes in serum rT₃ rather than its absolute values in health and thyroid disease. True serum rT₃ may be somewhat different because: (a) D.L-rT₃ employed in the standard curve and L-rT₃ present in human serum may react differently with anti-D,L-rT₂. (b) Even though 3,3′-T₂, which cross-reacted 10% in rT₃ RIA, has been considered unlikely to be present in human serum, it may circulate in low levels. (c) Cross-reaction of T₄ in rT₃ RIA of 0.06% although small, could contribute to RIA estimates of rT₂; the effect of T₄ would be particularly important in case of serum of hyperthyroid patients. Thus, serum rT₃ concentration in hyperthyroid patients averaged 89±48 μg/100 ml after correction for cross-reaction effects of T₄: this value was about 14% lower than that before correction (see above). Serum rT₃ concentration in cord sera of seven newborns averaged 136±19 ng/100 ml; it was clearly elevated and within the range of values seen in hyperthyroid patients. This was the case when the mean T₄ concentration in the newborn cord sera was moderately higher than normal and about one-half that in hyperthyroid patients, whereas serum T₃ was markedly below the normal adult level. A Pronase hydrolysate of thyroglobulin prepared from pooled normal thyroid glands contained 0.042, 3.0, and 0.16 μg/mg protein of rT₃, T₄, and T₃, respectively. The various data suggest that: (a) rT₃ is a normal component of human serum and thyroglobulin: (b) peripheral metabolism of T₄ is an important source of the rT₃ present in serum: (c) peripheral conversion of T₄ to T₃ and rT₃ may not necessarily be a random process.