A Radioimmunoassay for 3,3′-L-Diiodothyronine (3,3′T₂)

Abstract

The present report describes the development of a radioimmunoassay for 3,3′-L-diiodothyronine (3,3′-T2) which may be performed on unextracted serum. Utilizing a specific antiserum to 3,3′-L-T2-bovine serum albumin conjugates developed in rabbits, cross-reactivity was less than 0.5% with 3,5,3′-triiodothyronine (T3) and 3,3′,5′-triiodothyronine (reverse T3) and less than 0.01% with thyroxine (T4). Intra-assay variation averaged 2.9% and inter-assay variation was 7.8% and 18.5% when serum samples with 3,3′T2 concentrations of 8 ng/dl and 12 ng/dl, respectively, were analyzed. Assay sensitivity was considered to be 6 ng/dl by statistical criteria. Although routine assays were performed on unextracted samples with 8-anilino-l-naphthalene sulfonic acid (ANS) used to inhibit 3,3′T2 binding to serum proteins, comparable values were obtained if samples were extracted with ethanol prior to analysis. Exposure of serum samples to acid hydrolysis, a procedure which theoretically might inhibit binding of iodothyronines as a glucuronide or sulfate, did not appear to alter measurable values of 3,3′T2. The mean (±SE) serum 3,3′T2 concentration in 18 euthyroid subjects was 17 ± 1 ng/dl. Thyrotoxic patients generally had elevated 3,3′T2 levels (29 ± 2 ng/dl; n = 5) and hypothyroid individuals tended to have decreased 3,3′T2 concentrations (range <6–22 ng/dl; n = 11). In 8 nonnal thyroid glands the concentration of 3,3′T2 was 0.40 ± .03 /µg/g of tissue. Thyrotropin-releasing hormone (TRH) administration in 7 euthyroid subjects did not evoke an increase in serum 3,3′T2 levels when measured 90 min after injection. In contrast, serum 3,3′T2 concentrations did increase 90 min following TRH administration in two sheep, although it could not be discerned whether this rise in circulating 3,3′T2 was secondary to thyroidal secretion or to peripheral conversion. 6–22> In summary, these data suggest that 3,3′T2 circulatesin the serum of normal individuals and tends to parallel serum concentrations of T3, T4 and rT3 in various states of thyroid function.