title: Urinary excretion of the TRH-like peptide pyroglutamyl-glutamyl-prolineamide in rats.
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/n TRH-LI) and acidic TRH-LI (aTRH-LI) in a ratio of approximately 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/n TRH-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 approximately 1 h, and was recovered unchanged and quantitatively in urine. In contrast, when < EEP-NH2 was administered with food, only approximately 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.
authors.affiliation: Departments of Internal Medicine and Clinical Endocrinology, Erasmus University, Rotterdam, The Netherlands.
authors.forename: W
authors.initials: W
authors.lastname: Klootwijk
authors.forename: R D
authors.initials: RD
authors.lastname: de Boer
authors.forename: E
authors.initials: E
authors.lastname: Sleddens-Linkels
authors.forename: S M
authors.initials: SM
authors.lastname: Cockle
authors.forename: W W
authors.initials: WW
authors.lastname: de Herder
authors.forename: K
authors.initials: K
authors.lastname: Bauer
authors.forename: T J
authors.initials: TJ
authors.lastname: Visser
authors.forename: W J
authors.initials: WJ
authors.lastname: de Greef
mesh_terms: D000311:Adrenal Glands / Q000737:chemistry; D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005260:Female; D006207:Half-Life; D007031:Hypothalamus / Q000737:chemistry; D008297:Male; D008657:Metabolic Clearance Rate; D010053:Ovary / Q000737:chemistry; D010179:Pancreas / Q000737:chemistry; D010902:Pituitary Gland / Q000737:chemistry; D011467:Prostate / Q000737:chemistry; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D051381:Rats; D017208:Rats, Wistar; D012469:Salivary Glands / Q000737:chemistry; D013961:Thyroid Gland / Q000737:chemistry; D013973:Thyrotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000032:analysis / Q000493:pharmacokinetics / Q000652:urine*
chemical_list: D013973:Thyrotropin-Releasing Hormone; C059697:pyroglutamyl-glutamyl-proline amide; D011761:Pyrrolidonecarboxylic Acid