Histidyl-proline diketopiperazine cyclo (His-Pro): Measurement by radioimmunoassay in human blood in normal subjects and in patients with hyper- and hypothyroidism

An extraction procedure for cyclo (His-Pro) in human blood has been developed, employing sequential extraction with 90% methanol and 0.3 M perchloric acid. Chromatographic elution profiles of immunoreactive cyclo (His-Pro) in blood extracts measured by radioimmunoassay corresponded precisely to those of {3H} cyclo (His-Pro) following separation with Sephadex G-25, DEAE-cellulose, and SP-Sephadex columns, and also HPLC. Concentrations of cyclo (His-Pro) in normal human subjects (5073 ± 730 fmoles/ml) were approximately 50 times greater than respective TRH concentrations measured in identical samples. Cyclo (His-Pro) concentrations in hypothyroid patients (12373 ± 802 fmoles/ml) were significantly higher than those in normal and those in hyperthyroid patients (5949 ± 566 fmoles/ml). TRH concentrations, by contrast, were similar in all 3 groups. Pyroglutamateaminopeptidase activity which catalyzes the conversion of TRH to cyclo (His-Pro), was identified also in peripheral plasma. The plasma enzyme activity in normal subjects was 76 ± 27 fmoles cyclo (His-Pro) formed/min/ml plasma. This activity was not statistically increased in hypothyroid patients, so that the increased concentrations of cyclo (His-Pro) in hypothyroidism could not be attributed to the enhanced conversion of TRH in the peripheral blood.