A FAST PROTEIN LIQUID-CHROMATOGRAPHY (FPLC) METHOD FOR STUDY OF THYROROPIN-RELEASING HORMONE (TRH) AND ITS METABOLITE HISTIDYL-PROLINE DIKETOPIPERAZINE (CHP) IN HUMAN BLOOD - DEGRADATION IN LIVER AND PANCREATIC DISEASES

We have developed a convenient method combining fast protein liquid ch romatography (FPLC) with sensitive radioimmunoassay (RIA) for thyrotro pin-releasing hormone (TRH) to separate and identify TRH and its metab olite histidyl-proline diketopiperazine (CHP) and applied this to stud y inactivation of TRH by blood extracts from patients with liver cirrh osis (LC) and acute edematous pancreatitis (AP). Blood samples spiked with TRH and CHP were extracted by cold methanol and injected on a rev erse-phase FPLC column. A linear gradient was applied for separation. Subsequent analyses of fractions by RIA for TRH revealed that only fra ctions 9-10 contained TRH. Separation by retention time (9.9 +/- 0.8 m in for TRH, 10.5 +/- 0.6 min for CHP, mean +/- SEM) was highly reprodu cible. For degradation studies, pooled sera from patients with LC and AP were incubated with TRH and CHP for 60 min. Inactivation of TRH was less rapid in the presence of blood extract from LC patients than tha t from normal subjects or AP patients. CHP was more stable than TRH. T hese data suggest that activity of TRH-degrading enzymes is reduced in liver disease, whereas it does not appear to be altered in AP. Degrad ation of CHP does not closely reflect metabolic processing of its majo r precursor. This rapid and sensitive method may be applicable for fur ther investigations on the metabolism of TRH in organic fluids.