W. Klootwijk

Effect of Starvation and Subsequent Refeeding on Thyroid Function and Release of Hypothalamic Thyrotropin-Releasing Hormone

Effects of starvation on thyroid function were studied in 5- to 6-week-old (R x U) F1 rats. Starvation lowered plasma TSH in female, but not in male rats. Plasma T4 and T3 levels decreased, whereas the dialysable T4 fraction increased during starvation. Free T4 (FT4) levels decreased rapidly in females, but only after prolonged fasting in male rats. Glucose decreased, and free fatty acid levels increased during starvation. Peripheral TRH levels did not change during food deprivation. Since effects of starvation were most apparent in young female rats, such rats were used to study hypothalamic TRH release during starvation and subsequent refeeding. Basal in vitro hypothalamic TRH secretion was less in starved rats than in control or refed animals. In vitro hypothalamic TRH release in medium with 56 mM KCl increased 3-fold compared to basal release, and in these depolarization conditions TRH release was similar between hypothalami from control, starved and refed rats. In rats starved for 2 days, TRH level in hypophysial portal blood was lower than that of controls. Thus, diminished thyroid function during starvation may at least in part be caused by a reduced hypothalamic TRH release.

A new radioimmunoassay of thyrotropin-releasing hormone

Several radioimmunoassays for the measurement of thyrotropin-releasing hormone (pGlu-His-ProNHI, TRH) have been described. Antisera have been produced mainly by immunization of rabbits with TRH-protein conjugates in which coupling was effected via the imidazole of the histidyl moiety of TRH. For this purpose bis-diazotized benzidine has been most widely used [ 11. In another procedure TRH is reacted with p-diazoniumphenylacetic acid and the derivative is coupled to protein with the aid of a carbodiimide [2,3] . An entirely different approach has been the introduction of an amide bond between pGlu-His-Pro-OH and NH2 -groups of a protein carrier resulting in the formation of a TRH-like structure, i.e., pGlu-His-Pro-NH-protein [4]. We here report the attachment of TRH to hemocyanin (HC) using the bifunctional reagent 1 ,S-difluoro2,4-dinitrobenzene (DFDNB) [5]. Administration of the conjugate to rabbits elicited the production of antisera, which were used in the radioimmunoassay of the hypothalamic hormone.

Approaches to a markedly increased sensitivity of the radioimmunoassay for thyrotropin-releasing hormone by derivatization

Antisera to thyrotropin-releasing hormone (pGlu-His-Pro-NH2, TRH) have previously been produced in rabbits by immunization with a conjugate having TRH linked to a carrier protein by means of dinitrophenylene (Dnp) moiety. Studies on the specificity of the antisera obtained suggested that the sensitivity of the radioimmunoassay for TRH may be increased substantially by prior conversion of the hormone in to dinitrophenylene derivatives. To test this possibility, several TRH-Dno derivatives were prepared by reaction of TRH with equimolar amounts of 1,5-difluoro-2,4-dinitrobenzene yielding Nim-(5-fluoro-2,4-dinitrophenyl)TRH. This intermediate was reacted with ammonia, histamine, tyramine or N alpha-acetyl-lysine methyl ester (N alpha Ac-Lys-OMe) to yield the respective unsubstituted and N-substituted Nim-(5-amino-2,4-dinitrophenyl)TRH derivatives: TRH-Dnp-NH2, TRH-Dnp-histamine, TRH-Dnp-tyramine and TRH-Dnp-N alpha Ac-Lys-OMe. Nim-(2,4-Dinitrophenyl)TRH was prepared similarly by reaction of TRH with 1-fluoro-2,4-dinitrobenzene. The products were isolated by means of high-performance liquid chromatography (HPLC) and were found to be pure by HPLC and thin-layer chromatography using several solvent systems. TRH-Dnp-histamine and TRH-Dnp-tyramine were labelled with 125I using the chloramine-T method. The labelled products were purified to homogeneity by ion-exchange chromatography on SP-Sephadex and adsorption chromatography on Sephadex LH-20, respectively, and were found by HPLC to be pure.

Degradation of thyrotropin releasing hormone and a related compound by rat liver and kidney homogenate.

The inactivation of the hypothalamic hormone by rat liver and kidney homogenates was studied, using specific radioimmunoassays for the measurement of thyrotropin releasing hormone, pGlu-His-Pro-NH2 (TRH), and for an analogous peptide, pGlu-His-Pro-OH(TRH-OH), which has been proposed as a major metabolite of TRH [NAIR et al., 1971]. The inactivation of TRH and the free acid was found to be rapid. Heat lability and saturation kinetics suggest the involvement of enzymatic processes. In liver homogenate, TRH-OH production from TRH was observed. The accumulation of TRH-OH was substantial in experiments employing near-saturation concentrations of TRH. The liver and kidney are ascribed as major sites for breakdown of TRH in vivo.

Preparation of radioiodine labelled thiouracil derivatives

Abstract 2-Thiouracil and 6-propyl-2-thiouracil are radioiodinated with high yield uding the chloramine-T method at low pH to prevent oxidation of the sulfur. The radioiodine-labelled derivatives are purified by adsorption chromatography on Sephadex G-10. The identity and purity of the products obtained are demonstrated by both normal- and reversed-phase high pressure liquid chromatography. The specific activity of the tracers amounts to approximately 1750 Ci mmol −1 . Conditions for storage are described as well. These radioactive compounds may be used in the investigation of the mechanism of action of thyroid peroxidase as well as of iodothyronine deiodinases.