A competitive ligand binding assay for measurement of thyroid hormone-binding inhibitor in serum and tissues

Abstract

A competitive ligand-binding assay (CLBA) is described for measurement of an inhibitor(s) of serum binding of T4 in ether extracts of serum and in homogenates and extracts of tissues. The CLBA is based on the effect of thyroid hormone binding inhibitor (THBI) on partition of a constant amount of radiolabeled ligand [(125I]T4) between fixed amounts of serum and an anti-T4 antibody. The method is convenient, rapid, sensitive, and reproducible. The coefficient of variation averaged 8.9% within an assay and 12.8% between assays. Several fatty acids, e.g. arachidonic acid, lauric acid, linolenic acid, and linoleic acid, had potent THBI activity in the CLBA; arachidonic acid was more potent than the other fatty acids. Since oleic acid cross-reacted substantially with T4-binding sites on anti-T4, its THBI activity was examined by an equilibrium dialysis method; it was about 77% as potent as arachidonic acid. Arachidic, myristic, palmitic, and stearic acids, choleserol, various phospholipids and triglycerides (triolein and tripalmitin) had little or no THBI activity in the CLBA. THBI activity was detected in the sera of 50% (60% when serum T4 was low and 42% when it was normal) of 34 patients with nonthyroid illnesses (NTI) when studied by CLBA and in 59% (67% when serum T4 was low and 53% when it was normal) of patients when determined by the inhibitory ratio (normalized dialysis ratio/normalized binding ratio). THBI values obtained by the CLBA correlated significantly (r = 0.58; P less than 0.001) with those obtained by the inhibitory ratio method. The dose-response curve of an ether extract of pooled sera of hospitalized patients was parallel to that of arachidonic acid in the CLBA. Among various rat tissues, the small intestine had the most THBI activity in both homogenates and ether extracts of homogenates. Ether (2 vol) extracted about 63% of the THBI activity in small intestine homogenate at pH 5.2. THBI activity was demonstrable in all particulate fractions (especially mitochondria and endoplasmic reticulum) of small intestine homogenate; cytosol contained little or no THBI activity. THBI activity changed little after treatment of small intestine homogenate with trypsin or protease inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)