A new unextracted-sample radioimmunoassay method for hepatic endogenous nuclear L-tri-iodothyronine content. Validity of its use in determining nuclear receptor binding characteristics

Abstract

Endogenous l-tri-iodothyronine content in an hepatic nuclear extract was measured by a new unextracted-sample radioimmunoassay method using 8-anilinonaphthalene-1-sulphonic acid to inhibit the l-[(125)I]tri-iodothyronine binding to the nuclear l-tri-iodothyronine receptor within the extract. For this method, the lower sensitivity limit was 3.125 pg/tube, the recovery of added l-tri-iodothyronine was 90-120%, and the between-assay coefficient of variation was 10%. The amount of endogenous l-tri-iodothyronine was 10-40 pg/0.2 ml of hepatic nuclear extract from euthyroid rats, compared with less than 3.125 pg/0.2 ml from thyroidectomized rats. The results obtained by this new method were compared with a Sephadex G-25 column extracted-sample radioimmunoassay method and showed a good agreement. The values for the endogenous l-tri-iodothyronine content were utilized to correct for the l-tri-iodothyronine concentration within the binding assay mixture in order to accurately determine by Scatchard analysis the binding characteristics of the nuclear l-tri-iodothyronine receptor. The validity of the correction for endogeneous l-tri-iodothyronine was demonstrated by using a nuclear extract from a thyroidectomized rat which was preincubated with a small known amount of l-tri-iodothyronine before determining the nuclear l-tri-iodothyronine receptor binding characteristics. When the Scatchard plots were corrected for the preincubated dose, the results obtained were similar to true values, but they were falsely lower when not corrected. It is concluded that the necessity and validity of using endogenous l-tri-iodothyronine corrections in the Scatchard analytical computations of the nuclear l-tri-iodothyronine receptor binding characteristics has been demonstrated, being particularly more important for affinity constant than maximum binding capacity.