URINARY-EXCRETION OF THE TRH-LIKE PEPTIDE PYROGLUTAMYL-GLUTAMYL-PROLINEAMIDE IN RATS

TRH-like immunoreactivity (TRH-LI) was estimated in methanolic extract s of rat tissues and blood by RIA using antiserum 4319, which binds mo st peptides with the structure pClu-X-ProNH(2), or antiserum 8880, whi ch is specific for TRH (pGlu-His-ProNH(2)). TRH-LI (determined with an tiserum 4319) and TRH (determined with antiserum 8880) contents were 8 and 8 ng/g in brain, 216 and 222 ng/g in hypothalamus, 6.5 and 6 ng/g in pancreas, 163 and 116 ng/g in male pituitary, 105 and 77 ng/g in f emale pituitary, 1 and 0.1 ng/g in salivary gland, 61 and 42 ng/g in t hyroid, 12 and 3 ng/g in adrenal, 3 and 0.3 ng/g in prostate, and 11 a nd 0.8 ng/g in ovary respectively. Blood TRH-LI (antiserum 4319) and T RH (antiserum 8880) levels were 31 and 18 pg/ml in male rats, and 23 a nd 10 pg/ml in female rats respectively. Unextracted serum obtained fr om blood kept for at least Ih at room temperature no longer contained authentic TRH but still contained TRH-LI (males 20.3+/-3.1, females 15 .9+/-3.0 pg/ml; means+/-S.E.M.). Isocratic reverse-phase HPLC showed t hat TRH-LI in serum is largely pGlu-Glu-ProNH(2) (<EEP-NH2), a peptide previously found in prostate and anterior pituitary. In urine, TRH-LI (antiserum 4319) and TRH (antiserum 8880) levels were 3.21+/-0.35 and 0.32+/-0.04 ng/ml in male rats and 3.75+/-0.22 and 0.37+/-0.04 ng/ml in female rats respectively (means+/-S.E.M.). Anion-exchange chromatog raphy on QAE-Sephadex showed that urine of normally fed rats contains both basic/neutral TRH-LI (b/nTRH-LI) and acidic TRH-LI (aTRH-LI) in a ratio of approximate to 40:60, and further analysis by HPLC indicated that aTRH-LI represents <EEP-NH2. Analysis of food extracts and urine from fasted rats demonstrated that b/nTRH-LI is derived from food par ticles spilled by the rats during urine collection, while aTRH-LI is e ndogenously produced. While urinary aTRH-LI levels were higher in fema le than in male rats (2.99+/-0.41 vs 2.04+/-0.20 ng/ml), the daily uri nary excretion was similar in both sexes (females 15.6+/-1.4, males 19 .5+/-2.0 ng/day). Intravenously injected <EEP-NH2 disappeared from ser um with a half-life of approximate to 1h, and was recovered unchanged and quantitatively in urine. In contrast, when <EEP-NH2 was administer ed with food, only approximate to 0.5% was recovered in urine. The uri nary clearance rate of serum TRH-LI amounted to 0.52+/-0.10 ml/min in males and 0.34+/-0.05 ml/min in females. In view of the presence of <E EP-NH2 in the anterior pituitary gland, and the regulation of its cont ent in parallel with gonadotrophins, we examined the possibility that serum <EEP-NH2 is of pituitary of origin and correlates with gonadotro phin secretion. However, treatments that alter pituitary <EEP-NH2 cont ent and gonadotrophin release had no effect on serum TRH-LI or urinary aTRH-LI. In conclusion, the TRH-like peptide <EEP-NH2 is present in r at serum and is excreted into the urine. Moreover, <EEP-NH2 in serum a nd urine is not derived from rat food and is probably not of pituitary origin.