Urinary excretion of the TRH-like peptide pyroglutamyl-glutamyl-prolineamide in rats
Authors: Klootwijk W , de Boer R D H , Sleddens-Linkels E , Cockle S M , de Herder W W , Bauer K , Visser T J , de Greef W J 
Published: December 2004

Abstract
TRH-like immunoreactivity (TRH-LI) was estimated in methanolic extracts of rat tissues and blood by RIA using antiserum 4319, which binds most peptides with the structure pGlu-X-ProNH2, or antiserum 8880, which is specific for TRH (pGlu-His-ProNH2). TRH-LI (determined with antiserum 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 female pituitary, 1 and 0·1 ng/g in salivary gland, 61 and 42 ng/g in thyroid, 12 and 3 ng/g in adrenal, 3 and 0·3 ng/g in prostate, and 11 and 0·8 ng/g in ovary respectively. Blood TRH-LI (antiserum 4319) and TRH (antiserum 8880) levels were 31 and 18 pg/ml in male rats, and 23 and 10 pg/ml in female rats respectively. Unextracted serum obtained from blood kept for at least 1 h 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 that TRH-LI in serum is largely pGlu-Glu-ProNH2 (<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 chromatography 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 ≈ 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 particles spilled by the rats during urine collection, while aTRH-LI is endogenously produced. While urinary aTRH-LI levels were higher in female than in male rats (2·99 ± 0·41 vs 2·04 ± 0·20 ng/ml), the daily urinary excretion was similar in both sexes (females 15·6 ± 1·4, males 19·5 ± 2·0 ng/day). Intravenously injected <EEP-NH2 disappeared from serum with a half-life of ≈ 1 h, and was recovered unchanged and quantitatively in urine. In contrast, when <EEP-NH2 was administered with food, only ≈ 0·5% was recovered in urine. The urinary 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 <EEP-NH2 in the anterior pituitary gland, and the regulation of its content in parallel with gonadotrophins, we examined the possibility that serum <EEP-NH2 is of pituitary origin and correlates with gonadotrophin secretion. However, treatments that alter pituitary <EEP-NH2 content 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 rat serum and is excreted into the urine. Moreover, <EEP-NH2 in serum and urine is not derived from rat food and is probably not of pituitary origin.
Journal of Endocrinology (1997) 153, 411–421