Radioimmunoassay for TA-0910, a new stable thyrotropin releasing hormone analogue and its metabolite, TA-0910 acid-type, in human plasma and urine.

Radioimmunoassay (RIA) was investigated for the determination of TA-0910 and its main metabolite, TA-0910 acid-type, in human plasma and urine. TA-0910 is a new metabolically stable analogue of thyrotropin releasing hormone (TRH). Antiserum was raised in the rabbit against the 1-fluoro-2,4-dinitrophenyl derivative of TA-0910 or TA-0910 acid-type conjugated to keyhole limpet hemocyanin (KLH). The radioligand was prepared by iodination with 125I of the histidine imidazole ring of TA-0910 or TA-0910 acid-type. Cross-reactivities of anti-TA-0910 or TA-0910 acid-type antiserum for TA-0910, its metabolite and related compounds were low. The calibration range was 0.02-5 ng ml-1 using 100 microliters human plasma or urine. Inter-day variations of TA-0910 and TA-0910 acid-type assay in plasma were 3.5-15.5 and 1.8-9.4%, respectively. The variations of the assay in urine were the same as those in plasma. The recovery of TA-0910 and TA-0910 acid-type spiked in plasma or urine samples was approximately 100%. Furthermore, this method was applied to the determination of TA-0910 and TA-0910 acid-type in human plasma and urine samples, for the evaluation of the pharmacokinetics of TA-0910 in humans. From the results it was demonstrated that he developed RIA was useful for the determination of TA-0910 and TA-0910 acid-type in human plasma and urine, and was applicable to pharmacokinetic studies in humans.

Renal clearance of the thyrotropin-releasing hormone-like peptide pyroglutamyl-glutamyl-prolineamide in humans.

TRH-like peptides have been identified that differ from TRH (pGlu-His-ProNH2) in the middle amino acid. We have estimated TRH-like immunoreactivity (TRH-LI) in human serum and urine by RIA with TRH-specific antiserum 8880 or with antiserum 4319, which binds most peptides with the structure pGlu-X-ProNH2. TRH was undetectable in serum (< 25 pg/mL), but TRH-LI was detected with antiserum 4319 in serum of 27 normal subjects, 21 control patients, and 12 patients with carcinoid tumors (range 17-45, 5-79, and 18-16,600 pg/mL, respectively). Because serum was kept for at least 2 h at room temperature, which causes degradation of TRH, pGlu-Phe-ProNH2, and pGlu-Tyr-ProNH2, serum TRH-LI is not caused by these peptides. On high-performance liquid chromatography, serum TRH-LI coeluted with pGlu-Glu-ProNH2 (< EEP-NH2), a peptide produced in, among others, the prostate. Urine of normals and control patients also contained TRH-LI (range 1.14-4.97 and 0.24-5.51 ng/mL, respectively), with similar levels in males and females. TRH represented only 2% of urinary TRH-LI, and anion-exchange chromatography and high-performance liquid chromatography revealed that most TRH-LI in urine was < EEP-NH2. In patients with carcinoid tumors, increased urinary TRH-LI levels were noted (range 1.35-962.4 ng/mL). Urinary TRH-LI correlated positively with urinary creatinine, and the urinary clearance rate of TRH-LI was similar to the glomerular filtration rate. In addition, serum TRH-LI was increased in 17 hemodialysis patients (43-373 pg/mL). This suggests that serum < EEP-NH2 is cleared by glomerular filtration with little tubular resorption. The possible role of the prostate as a source of urinary TRH-LI was evaluated in 11 men with prostate cancer, showing a 25% decrease in urinary TRH-LI excretion after prostatectomy (0.19 +/- 0.02 vs. 0.15 +/- 0.01 ng/mumol creatinine, mean +/- SEM). However, TRH-LI was similar in spontaneously voided urine and in urine obtained through a nephrostomy cannula from 16 patients with unilateral urinary tract obstruction (0.15 +/- 0.01 vs. 0.14 +/- 0.01 ng/mumol creatinine). These data indicate that: 1) TRH-LI in human serum represents largely < EEP-NH2, which is cleared by renal excretion; 2) part of urinary < EEP-NH2 is derived from prostatic secretion into the blood and not directly into urine; and 3) urinary < EEP-NH2 can be used as marker for carcinoid tumors.

Urinary excretion of the TRH-like peptide pyroglutamyl-glutamyl-prolineamide in rats.

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.

Pharmacokinetics of the new thyrotropin releasing hormone analogue montirelin hydrate. 3rd communication: identification of metabolites in rat urine.

The metabolism of montirelin hydrate (CAS 90243-66-6, NS-3) was studied in rats after intravenous administration of 14C-labeled or unlabeled NS-3. 1. Four radioactive metabolites (M-1 to M-4) were found in the urine after administration of 14C-NS-3. M-3 (major metabolite) and M-2 showed the same Rf values as (-)-N-[[(3R,6R)-6-methyl-5-oxo-3-thiomorpholinyl]carbonyl]-L-histi dyl-L- proline (CNK-6004) and (+)-N-[[(3R,6R)-6-methyl-5-oxo-3- thiomorpholinyl]carbonyl]-L-histidine (CNK-6001), respectively. 2. M-3 and M-2 were purified from the urine after administration of unlabeled NS-3, and their chemical structures were identified by mass spectrometry, 1H-nuclear magnetic resonance spectroscopy, thin-layer chromatography and high performance liquid chromatography. Consequently, M-3 was identified as CNK-6004 formed by deamidation at a prolinamide moiety of NS-3, and M-2 as CNK-6001 formed by deprolination of CNK-6004.

Intra- and extravascular turnover of thyrotrophin-releasing hormone in normal man.

In 14 normal subjects constant TRH infusions for determination of plasma clearance rate (PCR) and half-life of disappearance (t 1/2) of TRH were carried out with simultaneous determination of half-life of disappearance of TRH in serum in vitro (t 1/2p). PCR, t 1/2 and t 1/2p were 1532 +/- 423 ml/min, 6.6 +/- 1.5 min and 16.8 +/- 9.4 min respectively (mean +/- S.D.) and displayed only minor fluctuations when determined repeatedly in the same subjects (coefficients of variation within individuals were 15.1, 10.6 and 7.5% respectively). Simultaneous determination of PCR, t 1/2 and t 1/2p enabled calculation of the half-life of disappearance of TRH in the extravascular tissue compartment (t 1/2t). Values of t 1/2t (6.3 +/- 1.4 min) correlated to t 1/2p (r = 0.95). Activities of TRH-degrading enzymes in tissues and in serum were independent of sex, phase of female menstrual cycle, time of day and of the concentrations of TRH used. The methods employed for this investigation offer the possibility of examining the degradation of TRH and TRH analogues both in the serum and in the extravascular compartment during various conditions.

Thyrotropin-releasing hormone immunoreactivity in human blood, urine, spinal fluid, amniotic fluid, saliva, and gastric juice.

TRH immunoreactivity levels were measured in human blood, urine, saliva, spinal fluid, amniotic fluid and gastric juice. Urinary TRH excretion during a 48-h period was measured in 11 healthy persons. Blood and urinary TRH immunoreactivity were measured at 2 and 5 h, respectively, after administration of 40 mg of TRH. All the samples were prepurified by SP-Sephadex-C-25 cation-exchange chromatography and subjected to reverse-phase high-pressure liquid chromatography (HPLC). TRH immunoreactivity levels were then measured by our TRH radioimmunoassay. The TRH immunoreactivity (TRH-ir) levels found in urine were 14.6 +/- 2.5 pmol/l; in blood 7.5 +/- 2.0 pmol/l; in spinal fluid 2.8 +/- 1.4 pmol/l, and in gastric juice 23.2 +/- 7.1 pmol/l. In all of the amniotic fluid and saliva samples, in almost one half of the blood and spinal fluid samples, and in almost one third of the gastric juice samples, TRH-ir was below the detectable limit. In blood and urine samples taken after oral administration of TRH, TRH-ir was eluted at the same time as synthetic TRH. The recovery of synthetic TRH added to the samples ranged from 36 to 99%. In all of the biological fluid samples, endogenous TRH-ir was eluted at the same time in HPLC, at 15-18 min, as was synthetic TRH which had been added to the samples. Urine was found to contain two TRH immunoreactive peaks, the second of which was eluted at the same time as synthetic TRH. No diurnal variation in urinary TRH excretion or TRH-ir levels was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased urinary levels of thyrotropin-releasing hormone immunoreactivity in acute pancreatitis.

Thyrotropin-releasing hormone (TRH) has been shown to be present and have actions in the human gastrointestinal tract. We have studied urine TRH immunoreactivity (TRH-ir) levels in healthy subjects and patients with acute pancreatitis, gallstones, ulcerative colitis, or acute gastritis. The urine samples were prepurified by SP-Sephadex-C-25 cation exchange chromatography, subjected to reverse-phase high-pressure liquid chromatography, and assayed in our TRH radioimmunoassay. The mean urine TRH immunoreactivity values of healthy subjects were 4.42 +/- 1 ng/l (x +/- SEM); of patients with acute pancreatitis on the 1st day of hospitalization, 23 +/- 7 ng/l; on the 2nd day, 7 +/- 1 ng/l; and on the 3rd day 9 +/- 2 ng/l. Only the urine TRH levels of the pancreatitis patients on day 1 differed significantly (p less than 0.05) from the levels of the healthy subjects. Circulating TRH appears to be derived mostly from the pancreas, where the islets during acute pancreatitis are affected, and TRH is released into circulation and urine.

Urine TRH immunoreactivity in hypothyroid and hyperthyroid patients.

Urine samples from 8 healthy subjects, from 16 patients with primary hypothyroidism and 8 patients with Graves' hyperthyroidism were pre-purified in SP-Sephadex-C-25 cation-exchange-chromatography, subjected to reverse phase high-pressure liquid chromatography (HPLC) with 0.01 M ammonium acetate pH 4 as a polar and propanol as a non-polar solvent with a 1%/min gradient and assayed in our TRH radioimmunoassay. Urine TRH-immunoreactivity levels were measured before and after 3 months of treatment with thyroxine or methimazole. The urine TRH-levels in healthy subjects were 5.5 +/- 1.4 ng/1 (mean +/- SEM, n = 8). In the hypothyroid patients, the urine TRH levels were 50.6 +/- 40 ng/1 before and 71.7 +/- 45.3 ng/1 after 3 months of treatment with thyroxine. These values did not significantly differ from those in healthy subjects. The large variations were due to highly elevated values in 3 patients. In 2 hypothyroid patients with initially high urine TRH values, 67 and 657 ng/1, urine TRH was measured 5 and 18 months later and was found to have decreased to 5 and 11 ng/1. In the hyperthyroid patients, urine TRH levels were 10.3 +/- 3.9 ng/1 before and 8.9 +/- 3.3 ng/1 after the treatment with methimazole and did not differ significantly from the levels in healthy subjects. After 3 months of treatment, the hyper- and the hypothyroid patients were euthyroid. Our results show, that, except in 2 hypothyroid patients, there does not appear to be any relationship between urine TRH levels and serum TSH or thyroid hormone levels in hypothyroid and hyperthyroid patients.

High pressure liquid chromatography purification of human urinary samples for thyrotropin-releasing hormone radioimmunoassay.

Urine samples from healthy adult subjects and patients who had received TRH orally were prepurified in SP-Sephadex-C-25 cation exchange chromatography, subjected to reverse phase high pressure liquid chromatography (HPLC) starting with 0.01 M ammonium acetate, pH 4, followed by 1%/min gradient of acetonitrile or isopropanol and assayed in a TRH RIA. Two TRH immunoreactive peaks (A and B) were detected by HPLC with an RP-8 column, peak A eluting at 4-10 min and B at 12-14 min. Serial dilutions of peak B produced a line parallel with synthetic TRH by RIA. Synthetic TRH added to urinary samples and urinary TRH immunoreactivity from TRH-treated patients were eluted at 12-14 min. These results suggest that peak B represents endogenous urinary TRH. Urinary TRH levels of eight normal human males and four females were 5.0 +/- 3.4 pg/ml and 5.2 +/- 1.2 pg/ml (mean +/- SD). These values are many times lower than those presented in previous studies.

Evidence for the presence of the putative TRH metabolite, deamido-TRH, in human urine.

Using a specific radioimmunoassay (RIA) for deamido TRH (TRH-OH), TRH-OH immunoreactivity (IR) was detected in the urine of 54% of a group of normal subjects and patients with thyroid disease. Human urine TRH-OH IR eluted in the position of synthetic TRH-OH on Sephadex G-10 gel filtration (GF). In contrast, urine TRH IR, as determined by a specific TRH RIA, did not elute with synthetic TRH. HU-TRH was prepared for studies on its crossreactivity in the TRH-OH RIA. HU-TRH is the term given to a previously described substance(s) which is obtained from human urine by immunoabsorption to anti-TRH antiserum, is reactive in the TRH RIA, and shows non-identity with synthetic TRH on GF. HU-TRH was not reactive in the TRH-OH RIA. These studies suggest that the putative TRH metabolite, deamido TRH, is present in human urine and that HU-TRH is neither TRH-OH or as closely related structurally to TRH-OH as it is to TRH.

TRH-like immunoreactivity in urine, serum and extrahypothalamic brain: non-identity with synthetic pyroglu-hist-pro-NH2 (TRH).

TRH-like immunoreactive substances obtained from several areas of rat brain and from human serum and urine were chromatographically separated by TLC and the resulting immunoreactive 'elution profiles' compared with that obtained for pyroglu-hist-pro-NH2 (TRH). For hypothalamus and septal-preoptic samples TRH was present, but represented less than 100% of the immunoreactive substances. For cortex, amygdala, brain stem, serum and urine, no TRH was detectable in the immunoreactive substances from those samples. The implications of these findings in relation to 'TRH' distribution studies and validation of small peptide RIAs are discussed.

Diurnal variation of the human urinary TRH excretion measured by radioimmunoassay.

Urine TRH was estimated by the radioimmunoassay which was accomplished according to the method of Bassiri and Utiger. Minimum detectable dose of TRH was 25 pg and recovery of TRH ranged from 72% to 112% in our laboratory. Intraassay coefficients of variation were 5.4% to 14.0% and interassay variations were 10.6% to 15.0%. Of the TRH analogues tested, only two (Ser-His-Pro-NH2, Thr-His-Pro-NH2) had potent reactivity to anti-TRH serum in large dose of 100 ng/tube. Urine samples were kept at -20 degrees C after adjusted to pH 3.0 because the inactivation of TRH in urine was markedly dependent on temperature and pH value. Using this radioimmunoassay, diurnal variation of the urinary TRH excretion at regular intervals in normal subjects was observed. Peak TRH excretion occurred around early morning, while minimum of the excretion was observed around noon. Total urinary TRH excretion of 24 hours was 817-1579 ng (M+/-SE: 1241+/-89 ng) in normal subjects. In patients with chronic renal failure, urinary excretions of TRH was obviously lower than those of normal subjects.

Radioimmunoassay of thyrotropin releasing hormone in plasma and urine.

A sensitive and specific radioimmunoassay has been developed capable of measuring thyrotropin releasing hormone (TRH) in extracted human plasma and urine. All of three TRH analogues tested had little cross-reactivity to antibody. Luteinizing hormone releasing hormone, lysine vasopressin, rat growth hormone and bovine albumin were without effect, but rat hypothalamic extract produced a displacement curve which was parallel to that obtained with the synthetic TRH. Sensitivity of the radioimmunoassay was 4 pg per tube with intraassay coefficient of variation of 6.2-9.7%. Synthetic TRH could be quantitatively extracted by methanol when added to human plasma in concentration of 25, 50 and 100 pg/ml. TRH immunoreactivity was rapidly reduced in plasma at 20 degrees C than at 0 degrees C, but addition of peptidase inhibitors, FOY-007 and BAL, prevented the inactivation of TRH for 3 hr at 0 degrees C. The TRH in urine was more stable at 0 degrees C than 20 degrees C, and recovered 75 +/- 4.6% hr after being added. The plasma levels of TRH were 19 pg/ml or less in normal adults and no sex difference was observed. The rate of disappearance of TRH administered i.v. from the blood could be represented as half-times of 4-12 min. Between 5.3-12.3% of the injected dose was excreted into urine within 1 hr as an immunoreactive TRH. These results indicate the usefulness of TRH radioimmunoassay for clinical investigation.

Clearance and identification of thyrotrophin releasing hormone in human urine after intravenous injections.

The urine clearance of TRH after intravenous injection in man has been measured by radioimmunoassay. Between 4.4 percent and 10.7 percent of the dose was excreted within 90 min, the majority within 30 min. The TRH excreted was immunochemically and chromatographically indistinguishable from synthetic TRH and was inactivated by plasma enzymes with the same kinetic characteristics. The immunoreactive TRH-like material in basal urine samples was not TRH however: chromatographically and enzymatically it behaved differently from the synthetic tripeptide.