Inhibition of degradation and measurement of immunoreactive thyrotropin-releasing hormone in rat blood and plasma.

Abstract

A radioimmunoassay (RIA) for thyrotropin-releasing hormone (TRH) is described. The cross-reactivity of the antiserum was tested with 26 analogs of TRH, 5 amino acids, and LH-releasing hormone. Use of this RIA revealed that inactivation of TRH by rat blood was prevented if the blood was frozen and thawed prior to its incubation with TRH. This procedure did not interfere with the binding of TRH to its antibody. The degradation of TRH by blood or plasma was inhibited by 2,3-dimercaptopropanol (BAL) or benzamidine, but these compounds nonspecifically inhibited the binding of [125I]TRH to anti-TRH. At 37 C, 50% of the synthetic TRH added to rat blood was degraded within seconds, whereas at 1C, 60-65% was recovered after 90 min. When blood was frozen and thawed prior to its incubation with TRH at 1C, essentially all of the hormone was recovered after a 90-min incubation period. In contrast, incubation of TRH with frozen and thawed blood at 37 C resulted in a rapid loss of TRH. BAL (10 mM) or bensamidine (100 mM) afforded complete protection for TRH for at least 30 min at 1 C. At 37 C, protection was incomplete. Exposure of rats to cold (2C) resulted in a significant increase in serum TSH levels, but TRH was undetectable (less than3 pg) in 100 mul of blood regardless of whether the blood contained BAL (10 mM) or benzamidine (100 mM), or was frozen quickly and thawed before RIA. However, when 5-8 ml of trunk blood was extracted with methanol, 8-11 pg/ml of TRH was found, and the TRH levels were slightly but significantly elevated after cold exposure.