Chromatographic Differences Between Circulating and Pituitary Thyrotropins12

Abstract

To explore the possibility that circulating immunoreactive human thyrotropin (hTSH) may be heterogeneous, sera were obtained from 3 untreated patients with primary hypothyroidism before and after the administration of synthetic thyrotropin-releasing hormone (TRH), and were examined by both gel chromatography on Sephadex G-100 and anion exchange chromatography on DEAE-Sephadex A-50. For comparison, purified unlabeled pituitary hTSH was examined under identical conditions. ¹²⁵I-pituitary hTSH was cochromatographed in all experiments as an internal standard. Unlabeled pituitary hTSH and serum hTSH were measured in all chromatographic fractions by radioimmunoassay. Unlabeled pituitary hTSH eluted as an essentially symmetrical single component during gel chromatography but appeared to contain multiple components during anion exchange chromatography. In comparison to a series of reference proteins of known molecular size, the molecular radii of ¹²⁵I-pituitary hTSH and unlabeled pituitary hTSH were estimated to be 28.4 Å and 28.8 Å, respectively. Immunoreactive serum hTSH eluted asymmetrically during gel and anion exchange chromatography, appeared to consist of more than one component in both chromatographic systems, and contained at least one component of greater apparent size and lesser net negative charge than purified unlabeled pituitary hTSH. TRH increased the concentrations of serum hTSH chromatographic components proportionally. The gel chromatographic components of serum hTSH were not immunochemically distinguished from each other or from purified pituitary hTSH. These observations suggest that circulating immunoreactive hTSH displays both charge and apparent size heterogeneity in primary hypothyroidism.