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Active collagen oligopeptides (ACOP) are bioactive collagen-derived peptides detected by a recently-established ELISA. To facilitate studies of the function and metabolism of these products, this study aims to determine which of these peptides is recognized by a novel anti-ACOP antibody used in this ELISA. We then investigate the effect of collagen peptide (CP) ingestion and exercise on urinary ACOP concentrations in a cohort of university student athletes using colorimetric, LC-MS/MS, and ELISA. We observed that the antibody showed strong cross-reactivity to Pro-Hyp and Gly-Pro-Hyp and weak cross-reactivity to commercial CP. CP ingestion increased the urinary level of ACOP over time, which correlated highly with urinary levels of peptide forms of Hyp and Pro-Hyp. Physical activity significantly decreased the urinary ACOP level. This study demonstrates changes in urinary ACOP following oral CP intake and physical activity using ELISA with the novel anti-ACOP antibody. Thus, ACOP may be useful as a new biomarker for collagen metabolism.

Objective: 18 F-labeled prostate-specific membrane antigen (PSMA) ligand, [ 18 F]PSMA-1007, has the benefit of a higher synthetic yield and minimal excretion in the urine. High detection efficacy was reported in biochemical recurrence (BCR) of prostate cancer after radical prostatectomy. Thus, we evaluated the preliminary diagnostic utility of [ 18 F]PSMA-1007 PET in patients with prostate cancer, focusing on the BCR which is not detected on conventional imaging., Methods: We enrolled a total of 28 patients (age 51-79 years) with BCR of prostate cancer. BCR was defined as a continuous increase in PSA after radical prostatectomy or radiation therapy without any apparent recurrent lesions on conventional diagnostic imaging (CT and bone scintigraphy). PSMA-PET scanning was performed approximately 60 min after intravenous injection of [ 18 F]PSMA-1007 (259 ± 37 MBq). PSMA-PET images were evaluated for lesion detection as well as its relation to PSA values and location., Results: Abnormal uptake, which was suspected to be recurrence or metastasis, was detected in 92.9% (26/28) of patients with BCR. The SUVmax was 8.4 ± 6.4 in local recurrence, 11.5 ± 11.8 in pelvic lymph nodes (LN), and 4.1 ± 1.6 in bone metastasis. The detection rates were 66.7% in the PSA group-1 (0.1-0.5 ng/mL), 85.7% in the PSA group-2 (0.5-1.0 ng/mL), and 100% in the PSA group-3 (above 1.0 ng/mL). Among the PET-positive BCR patients (n = 26), local recurrence was detected in 57.7% (15/26), pelvic LN in 42.3% (11/26), and bone metastasis in 15.4% (4/26). In 53% (8/15) of BCR patients who were suspected of local recurrence, focal uptake was detected adjacent to the bladder on [ 18 F]PSMA-1007 PET. This suggested the significant advantage of having minimal physiological urine excretion., Conclusions: [ 18 F]PSMA-1007 PET showed a high detection rate in recurrent and metastatic lesions. In patients with BCR, its high detection led to suitable treatment strategies, such as salvage radiation therapy or surgical removal of recurrent lymph nodes., Trial Registration: (UMIN Clinical Trials Registry) UMIN000037697.

The purpose of this study was to investigate the mutual effect of systemic inflammatory response syndrome (SIRS) accompanied with fibrinolysis, endotoxemia, and coagulation in severe cases of antipsychotic poisoning. A total of 199 patients were examined, of which 71 were men and 128 were women. The age of the patients was from 22 to 63 years, (45.3 ± 6.1 years on average). According to the results of the course of therapy, the patients were divided into two groups. In the blood plasma, the content of C-reactive protein, fibrinogen and its proteolysis products (oligopeptides, D-dimers), interleukin-6 were determined. In the first 1 to 3 days, in group 1, the level of interleukin-6 decreased and approached the normal level (P ≤ .05). The opposite trend continued throughout the observation of patients from group 2-their levels of interleukin-6 increased day by day (P ≤ .05). The concentration of D-dimer already in 1 day after admission to intensive care in patients from group 2 exceeded the norm by 14 times (P ≤ .05). The level of D-dimer correlated with the level of oligopeptides in blood plasma upon admission, as well as for 3 and 5 days after admission to intensive care: 0.36, 0.76 at P ≤ .05, 0.94 at P ≤ .01, respectively. Similar correlations were obtained for the content of oligopeptides in urine and the level of D-dimer: 0.55, 0.85 at P ≤ .05, 0.93 at P ≤ .01. In this regard, the most pronounced correlation is that between the SIRS score, plasma D-dimer level, and the plasma level of the D-dimer derivatives, oligopeptides., (© 2020 Wiley Periodicals LLC.)

We report the proof-of-concept of a bioaffinity format designed for the early detection of growth hormone secretagogue receptor (GHS-R1a) antagonists in urine samples. We exploit here their atypical behavior in competitive experiments with labeled ghrelin (GHR), namely, the strong promoting effect on the GHR/GHS-R1a interaction at low molar ratios GHR/antagonist. The antagonists potentiate the GHR/GHS-R1a interaction, and they display the same effect on the interaction of GHS-R1a with other agonists listed as doping agents. The developed assay allows the estimation of affinity constants of ligand/receptor and antagonist/receptor binding and is amenable to optical, electrochemical, and mass-sensitive detection. The estimated affinity constants for GHR/GHS-R1a and antagonist/GHS-R1a in the absence of G proteins are in good agreement with recently reported data.

Background: A biomarker, by which we can predict alterations of renal function in normoalbuminuric diabetic patients, is not available. Here, we report that endogenous anti-fibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) represents a potential biomarker to predict alterations in eGFR in normoalbuminuric diabetic patients., Methods: We analyzed 21 normoalbuminuric diabetic patients with eGFR ≥ 30 ml/min/1.73 m 2 and measured AcSDKP levels in first morning void urine. We divided patients into two groups based on the median values: low or high urinary AcSDKP groups (uAcSDKP/Cr low or uAcSDKP/Cr high ). At baseline, no significant differences in sex, age, HbA1c, BMI, serum creatinine levels, etc., were observed between the two groups., Results: During ~ 4 years, the alteration in eGFR [ΔeGFR op (ΔeGFR observational periods)] was significantly stable in uAcSDKP/Cr high group compared with uAcSDKP/Cr low group over time (P = 0.003, χ 2  = 8.58). We also evaluated urine kidney injury molecule-1 (uKim-1) levels and found that ΔeGFR op was also stable in low uKim-1 group compared with high uKim-1 group over time (P = 0.004, χ 2  = 8.38). Patients who fulfilled the criteria for both uAcSDKP/Cr high and uKim-1 low exhibited stable ΔeGFR op (P < 0.001, χ 2  = 30.4) when compared to the remaining patients. Plasma AcSDKP (P = 0.015, χ 2  = 5.94) and urine β2-microglobulin (P = 0.038, χ 2  = 4.31) also display weak but significant predictor of ΔeGFR op as well., Conclusion: AcSDKP represents a potentially useful biomarker to predict alterations in the renal function of patients with diabetes presenting normoalbuminuria.

Background: Rapid urinary trypsinogen-2 dipstick test and levels of urinary trypsinogen-2 and trypsinogen activation peptide (TAP) concentration have been reported as prognostic markers for the diagnosis of acute pancreatitis., Aim: To reconfirm the validity of all these markers in the diagnosis of acute pancreatitis by undertaking a multi-center study in Japan., Methods: Patients with acute abdominal pain were recruited from 17 medical institutions in Japan from April 2009 to December 2012. Urinary and serum samples were collected twice, at enrollment and on the following day for measuring target markers. The diagnosis and severity assessment of acute pancreatitis were assessed based on prognostic factors and computed tomography (CT) Grade of the Japanese Ministry of Health, Labour, and Welfare criteria., Results: A total of 94 patients were enrolled during the study period. The trypsinogen-2 dipstick test was positive in 57 of 78 patients with acute pancreatitis (sensitivity, 73.1%) and in 6 of 16 patients with abdominal pain but without any evidence of acute pancreatitis (specificity, 62.5%). The area under the curve (AUC) score of urinary trypsinogen-2 according to prognostic factors was 0.704, which was highest in all parameter. The AUC scores of urinary trypsinogen-2 and TAP according to CT Grade were 0.701 and 0.692, respectively, which shows higher than other pancreatic enzymes. The levels of urinary trypsinogen-2 and TAP were significantly higher in patients with extended extra-pancreatic inflammation as evaluated by CT Grade., Conclusion: We reconfirmed urinary trypsinogen-2 dipstick test is useful as a marker for the diagnosis of acute pancreatitis. Urinary trypsinogen-2 and TAP may be considered as useful markers to determine extra-pancreatic inflammation in acute pancreatitis., Competing Interests: Conflict-of-interest statement: The qualitative and quantitative analyses of urinary trypsinogen-2 were performed free of charge by Unitika Ltd. (Osaka, Japan). Mayumi T received research funding. The other authors declare no conflict of interest.

Zebrafish (Danio rerio) water tank (ZWT) approach was investigated as an alternative model for metabolism studies based on six different experiments with four model compounds. Sibutramine was applied for the multivariate optimization of ZWT conditions, also for the comparison of the metabolism among ZWT, humans and mice, beyond for the role of CYP2B6 in ZWT. After the optimization, 18 fish and 168 hours of experiments is the minimum requirement for a relevant panel of biotransformation products. A comparison among the species resulted in the observation of the same hydroxylated metabolites, with differences in metabolites concentration ratio. However, the ZWT allowed tuning of the conditions to obtain a specific metabolic profile, depending on the need. In addition, by utilizing CYP2B6 inhibition, a relevant ZWT pathway for the demethylation of drugs was determined. The stereospecificity of the ZWT metabolism was investigated using selegiline and no racemization or inversion transformations were observed. Moreover, the investigation of metabolism of cannabimimetics was performed using JWH-073 and the metabolites observed are the same described for humans, except for the hydroxylation at the indol group, which was explained by the absence of CYP2C9 orthologs in zebrafish. Finally, hexarelin was used as a model to evaluate studies by ZWT for drugs with low stability. As a result, hexarelin displays a very fast metabolization in ZWT conditions and all the metabolites described for human were observed in ZWT. Therefore, the appropriate conditions, merits, and relevant limitations to conduct ZWT experiments for the investigation of drug metabolism are described., (© 2018 John Wiley & Sons, Ltd.)

With improved performances, miniature mass spectrometers are becoming suitable for more practical applications. At the same time, the coupling of an approximate ionization source is essential in terms of minimizing sample preparation and broadening the range of samples that could be analyzed. In this study, an atmospheric pressure laserspray ionization (AP-LSI) source was coupled with our home developed miniature ion trap mass spectrometer. The whole system is compact in size, and biological samples could be directly analyzed with minimum sample preparation. Direct detections of peptides, proteins, drugs in whole blood, and urine could be achieved with high sensitivity. The analyses of tissue sections were demonstrated, and different regions in a tissue section could be differentiated based on their lipid profiles. Results suggest that the coupling of AP-LSI with miniature mass spectrometer is a powerful technique, which could potentially benefit target molecule analysis in biological and medical applications.

Purpose: Radiopharmaceuticals containing the motive tripeptide arginyl-glycyl-asparatic acid (RGD) are known to target ανβ3 integrins during tumor angiogenesis. A more generic kit radiolabeling procedure accommodating Ga-68 from different generators was developed for NOTA-RGD and evaluated for its versatile use and safety in subsequent in vivo applications. The [ 68 Ga]NOTA-RGD kit was further verified for its expected biodistribution and pharmacokinetics in nonhuman primates and its clinical sensitivity to detect solitary pulmonary nodules (SPN) in cancer patients., Procedures: Single vial kits containing 28-56 nmol of NOTA-cyclo-Arg-Gly-Asp-d-Tyr-Lys (NOTA-RGD) and sodium acetate trihydrate buffer were formulated. Versatility of the NOTA-RGD radiolabeling performance and adaption to a TiO 2 - and a SnO 2 -based generator type, characterization and long-term storage stability of the kits were carried out. The blood clearance and urine recovery kinetics as well as the image-guided biodistribution of [ 68 Ga]NOTA-RGD was studied in a vervet monkey model. [ 68 Ga]NOTA-RGD kits were further tested clinically to target solitary pulmonary nodules., Results: The kits could be successfully formulated warranting integrity over 3-4 months with a good [ 68 Ga]NOTA-RGD radiolabeling performance (radiochemical purity >95 %, decay corrected yield 76-94 %, specific activity of 8.8-37.9 GBq/μmol) The kits met all quality requirements to be further tested in vivo. [ 68 Ga]NOTA-RGD cleared rapidly from blood and was majorly excreted via the renal route. The liver, spleen, heart and intestines showed initial uptake with steadily declining tissue activity concentration over time. In addition, the [ 68 Ga]NOTA-RGD kit allowed for delineation of SPN from non-malignant lung tissue in humans., Conclusions: A more versatile radiolabeling procedure using kit-formulated NOTA-RGD and different generator types was achieved. The uncompromised in vivo behavior and efficient targeting of SPN warrants further investigations on the clinical relevance of [ 68 Ga]NOTA-RGD derivatives to implement initial guidelines and management of patients, with regard to integrin targeted imaging.

Growth hormone releasing peptides (GHRPs) could be widely used by cheating athletes because they produce growth hormone (GH) secretion, so may generate an ergogenic effect in the body. Knowledge of the essential amino acids needed in GHRP structure for interaction with the target biological receptor GHSR1a, the absorption through different administration routes, and the maintenance of pharmacological activity of potential biotransformation products may help in the fight against their abuse in sport. Several GHRPs and truncated analogues with the common core Ala-Trp-(D-Phe)-Lys have been studied with a radio-competitive assay for the GHSR1a receptor against the radioactive natural ligand ghrelin. Relevant chemical modifications influencing the activity for positions 1, 2, 3, and 7 based on the structure aa-aa-aa-Ala-Trp-(D-Phe)-Lys have been obtained. To test in vivo the applicability of the activities observed, the receptor assay activity in samples from excretion studies performed after nasal administration of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin was confirmed. Overall results obtained allow to infer structure-activity information for those GHRPs and to detect GHSR1a binding (intact GHRPs plus active metabolites) in excreted urines. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Background: We describe a pharmacodynamic study of the dose-effect relationship of perindopril arginine at 10, 14, and 20mg with in vivo angiotensin-converting enzyme (ACE) activity, assessed by urine and plasma AcSDKP levels, as well as the effect on plasma active renin concentrations and blood pressure., Methods: This randomized, double-blind, four-period, crossover study involved single-dose administration of perindopril arginine (10, 14, and 20mg or placebo) to 32 healthy male normotensive mildly sodium-depleted volunteers. Blood and urine were collected over 48h for AcSDKP, ACE activity, and plasma active renin measurements., Results: There were dose-related increases in urinary AcSDKP excretion and plasma AcSDKP concentration after administration of perindopril, with significant between-period differences (estimate of the median difference in urinary excretion over 48h of AcSDKP, 49.1 [95% CI: 15.3-82.0] nmol for 14 versus 10mg, and 73.2 [95% CI: 44.9-106.3] nmol for 20 versus 14mg). Consequently, a dose-dependent increase in plasma active renin concentration was observed. Even though each dose of perindopril 10 to 20mg was associated with a significant 24-h ambulatory blood pressure reduction versus placebo, no dose-dependency was detected in these normotensive subjects., Conclusions: Administration of perindopril arginine 10, 14, or 20mg to mildly sodium-depleted healthy volunteers is associated with a dose-dependent inhibition of in vivo ACE activity with significant between-dose differences. This effect was associated with a dose-dependent increase in plasma active renin concentration, indicating a dose-dependent blockade of the renin angiotensin system., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Currently liquid chromatography - mass spectrometry (LC-MS) analysis after solid-phase extraction (SPE) on weak cation-exchange cartridges is a method of choice for anti-doping analysis of small bioactive peptides such as growth hormone releasing peptides (GHRPs), desmoporessin, LHRH, and TB-500 short fragment. Dilution of urine samples with phosphate buffer for pH adjustment and SPE on weak cation exchange microelution plates was tested as a means to increase throughput of this analysis. Dilution using 200 mM phosphate buffer provides good buffering capacity without affecting the peptides recoveries. SPE on microelution plates was performed on Waters Positive Pressure-96 Processor with subsequent evaporation of eluates in nitrogen flow. Though the use of smaller sample volume decreases the pre-concentration factor and increases the limits of detection of 5 out of 17 detected peptides, the recovery, linearity, and reproducibility of the microelution extraction were comparable with cartridge SPE. The effectiveness of protocols was confirmed by analysis of urine samples containing ipamorelin, and GHRP-6 and its metabolites. SPE after urine sample dilution with buffer can be used for faster sample preparation. The use of microelution plates decreases consumption of solvents and allows processing of up to 96 samples simultaneously. Cartridge SPE with manual рН adjustment remains the best option for confirmation. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Collagen tripeptide (CTP) is a collagen hydrolysate containing a high concentration of tripeptides with a Gly-X-Y sequence, such as Gly-Pro-Hyp. To test the effects of this preparation, we compared the absorption of peptides in humans after ingestion of a tripeptide fraction of CTP (CTP-100), a CTP preparation containing ca. 50% Gly-X-Y tripeptides (CTP-50), and a collagen peptide that did not contain tripeptides (CP). The postprandial levels of Gly-Pro-Hyp and Pro-Hyp in the plasma increased in those subjects who ingested CTP-100 and CTP-50, and were higher with greater Gly-Pro-Hyp ingestion. This demonstrated that collagen hydrolysates were efficiently absorbed when the collagen was ingested in the tripeptide form. Gly-Pro-Hyp and Pro-Hyp were also found in the urine after ingestion of CTP-100 or CTP-50. Similar to the results for the plasma concentration, the urinary excretion of Gly-Pro-Hyp and Pro-Hyp was also dependent on the amount of Gly-Pro-Hyp ingested. This indicates that ingested Gly-Pro-Hyp and generated Pro-Hyp were relatively stable in the body and were transported to the urine in the peptide form. The concentration of Hyp-Gly in the plasma was low after the ingestion of CP and CTP-100 but higher after the ingestion of CTP-50. Overall, our results suggest that tripeptides derived from collagen are absorbed efficiently by the body.

Growth hormone releasing peptides (GHRPs) stimulate secretion of endogenous growth hormone and are listed on the World Anti-Doping Agency (WADA) Prohibited List. To develop an effective method for GHRPs anti-doping control we have investigated metabolites of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin in urine after nasal administration. Each compound was administrated to one volunteer. Samples were collected for 2 days after administration, processed by solid-phase extraction on weak cation exchange cartridges and analyzed by means of nano-liquid chromatography - high resolution mass spectrometry. Six metabolites of GHRP-1 were identified. GHRP-1 in the parent form was not detected. GHRP-1 (2-4) free acid was detected in urine up to 27 h. GHRP-2, GHRP-2 free acid and GHRP-2 (1-3) free acid were detected in urine up to 47 h after administration. GHRP-6 was mostly excreted unchanged and detected in urine 23 h after administration, its metabolites were detectable for 12 h only. Hexarelin and Ipamorelin metabolized intensively and were excreted as a set of parent compounds with metabolites. Hexarelin (1-3) free acid and Ipamorelin (1-4) free acid were detected in urine samples after complete withdrawal of parent substances. GHRPs and their most prominent metabolites were included into routine ultra-pressure liquid chromatography-tandem mass spectrometry procedure. The method was fully validated, calibration curves of targeted analytes were obtained and excretion curves of GHRPs and their metabolites were plotted. Our results confirm that the detection window after GHRPs administration depends on individual metabolism, drug preparation form and the way of administration., (Copyright © 2015 John Wiley & Sons, Ltd.)

Background: Due to opiorphin's analgesic and antidepressant functions, its illicit use is rumored in some racing jurisdictions. Opiorphin is very difficult to detect due to its hydrophilic nature and rapid degradation in plasma and urine samples. METHODOLOGY & RESULTS: We have developed a sensitive, reliable method for opiorphin detection and confirmation in equine samples, using EDTA to inhibit analyte degradation between the time of collection and analysis. Opiorphin was extracted by weak cation exchange followed by analysis using HILIC-MS/MS. The method was validated and the LOD was determined to be 50 pg/ml in equine plasma and urine., Conclusion: The method has good selectivity and precision and is the first reported method for the detection of opiorphin in equine plasma and urine.

Atazanavir sulfate, an azapeptide inhibitor of HIV protease, has been associated with urolithiasis. A 60-year-old man with atazanavir-induced urinary sediment crystals verified by infrared spectroscopic analysis is described. He had been receiving highly active antiretroviral therapy (HAART) for HIV infection and also had a history of urinary lithiasis and been undergoing urinalysis once every month. Needle-shaped crystals were seen in his urine sediment and infrared spectroscopic analysis revealed that these were atazanavir crystals. Because the presence of the crystals in urine do not always reveal an abnormality in the urinary test strip analysis, the urinary sediment needed to be observed microscopically in order to prevent future urolithiasis and renal failure in this HIV patient receiving atazanavir., (© 2013 S. Karger AG, Basel.)

The pharmacokinetics, mass balance, and metabolite profiles of faldaprevir, a selective peptide-mimetic hepatitis C virus NS3/NS4 protease inhibitor, were assessed at steady state in 7 healthy male subjects. Subjects received oral doses of 480 mg faldaprevir on day 1, followed by 240 mg faldaprevir on days 2 to 8 and 10 to 15. [14C]faldaprevir (240 mg containing 100 μCi) was administered on day 9. Blood, urine, feces, and saliva samples were collected at intervals throughout the study. Metabolite profiling was performed using radiochromatography, and metabolite identification was conducted using liquid chromatography-tandem mass spectrometry. The overall recovery of radioactivity was high (98.8%), with the majority recovered from feces (98.7%). There was minimal radioactivity in urine (0.113%) and saliva. Circulating radioactivity was predominantly confined to plasma with minimal partitioning into red blood cells. The terminal half-life of radioactivity in plasma was approximately 23 h with no evidence of any long-lasting metabolites. Faldaprevir was the predominant circulating form, accounting for 98 to 100% of plasma radioactivity from each subject. Faldaprevir was the only drug-related component detected in urine. Faldaprevir was also the major drug-related component in feces, representing 49.8% of the radioactive dose. The majority of the remainder of radioactivity in feces (41% of the dose) was accounted for in almost equal quantities by 2 hydroxylated metabolites. The most common adverse events were nausea, diarrhea, and constipation, all of which were related to study drug. In conclusion, faldaprevir is predominantly excreted in feces with negligible urinary excretion.

We have reported previously that Ac-SDKP (N-acetyl-seryl-aspartyl-lysyl-proline) reduces fibrosis and inflammation (in macrophages and mast cells). However, it is not known whether Ac-SDKP decreases collagen cross-linking and lymphocyte infiltration; lymphocytes modulate both collagen cross-linking and ECM (extracellular matrix) formation in hypertension. Thus we hypothesized that (i) in AngII (angiotensin II)-induced hypertension, Ac-SDKP prevents increases in cross-linked and total collagen by down-regulating LOX (lysyl oxidase), the enzyme responsible for cross-linking, and (ii) these effects are associated with decreased pro-fibrotic cytokine TGFβ (transforming growth factor β) and the pro-inflammatory transcription factor NF-κB (nuclear factor κB) and CD4+/CD8+ lymphocyte infiltration. We induced hypertension in rats by infusing AngII either alone or combined with Ac-SDKP for 3 weeks. Whereas Ac-SDKP failed to lower BP (blood pressure) or LV (left ventricular) hypertrophy, it did prevent AngII-induced increases in (i) cross-linked and total collagen, (ii) LOX mRNA expression and LOXL1 (LOX-like 1) protein, (iii) TGFβ expression, (iv) nuclear translocation of NF-κB, (v) CD4+/CD8+ lymphocyte infiltration, and (vi) CD68+ macrophages infiltration. In addition, we found a positive correlation between CD4+ infiltration and LOXL1 expression. In conclusion, the effect of Ac-SDKP on collagen cross-linking and total collagen may be due to reduced TGFβ1, LOXL1, and lymphocyte and macrophage infiltration, and its effect on inflammation could be due to lower NF-κB.

Objectives: Atazanavir has been associated with kidney stones and renal failure. We measured urine and plasma concentrations of recent protease inhibitors (PIs) and searched for PI crystals in the urine of asymptomatic patients., Methods: A cross-sectional analysis of HIV-infected patients taking ritonavir-boosted atazanavir 300 mg/day (ATV300/r), unboosted atazanavir 400 mg/day (ATV400), ritonavir-boosted darunavir at either 800 mg/day (DRV800/r) or 1200 mg/day (DRV1200/r) or ritonavir-boosted lopinavir 800 mg/day was performed. Plasma and urine were collected and PI levels measured using HPLC. Crystals were detected and identified in urine using polarized microscopy., Results: PI levels were measured in 266 patients, 142 of whom were assessed for urinary crystals. Their mean age was 46 years. The mean duration of HIV infection was 10.5 years and the mean duration of the current PI-containing regimen was 22.5 months. The mean CD4 cell count was 494 cells/mm(3); 74% showed controlled HIV replication. Median urinary PI levels were 22.3, 14.3, 26.9 and 29.7 mg/L for ATV300/r, ATV400, DRV800/r and DRV1200/r, respectively, significantly higher than plasma levels, which were all <5 mg/L (P < 0.001). In contrast, median urinary lopinavir concentrrations did not significantly differ from plasma concentrations (4.2 and 6.4 mg/L, respectively; P = 0.7) and were significantly lower than those of other PIs (P < 0.001). Atazanavir crystals were found in 7/78 patients receiving ATV300/r (8.9%; 95% CI = 2.6%-15.2%) and darunavir crystals were found in 4/51 patients receiving darunavir (7.8%; 95% CI = 0.4%-15.2%). Longer exposure to atazanavir was the only risk factor associated with the presence of atazanavir crystalluria (P = 0.04)., Conclusions: Unlike lopinavir, atazanavir and darunavir reached high concentrations in urine. Urinary crystals were found in a few patients receiving ritonavir-boosted atazanavir or darunavir and may favour nephrolithiasis.

Aim: To undertake a meta-analysis on the value of urinary trypsinogen activation peptide (uTAP) in predicting severity of acute pancreatitis on admission., Methods: Major databases including Medline, Embase, Science Citation Index Expanded and the Cochrane Central Register of Controlled Trials in the Cochrane Library were searched to identify all relevant studies from January 1990 to January 2013. Pooled sensitivity, specificity and the diagnostic odds ratios (DORs) with 95%CI were calculated for each study and were compared to other systems/biomarkers if mentioned within the same study. Summary receiver-operating curves were conducted and the area under the curve (AUC) was evaluated., Results: In total, six studies of uTAP with a cut-off value of 35 nmol/L were included in this meta-analysis. Overall, the pooled sensitivity and specificity of uTAP for predicting severity of acute pancreatitis, at time of admission, was 71% and 75%, respectively (AUC = 0.83, DOR = 8.67, 95%CI: 3.70-20.33). When uTAP was compared with plasma C-reactive protein, the pooled sensitivity, specificity, AUC and DOR were 0.64 vs 0.67, 0.77 vs 0.75, 0.82 vs 0.79 and 6.27 vs 6.32, respectively. Similarly, the pooled sensitivity, specificity, AUC and DOR of uTAP vs Acute Physiology and Chronic Health Evaluation II within the first 48 h of admission were found to be 0.64 vs 0.69, 0.77 vs 0.61, 0.82 vs 0.73 and 6.27 vs 4.61, respectively., Conclusion: uTAP has the potential to act as a stratification marker on admission for differentiating disease severity of acute pancreatitis.

In the present study, a screening assay was developed comprising 11 prohibited peptides (<1.5 kDa) that are sufficiently purified from urine using weak cation exchange with subsequent determination of all substances by means of nanoUHPLC separation coupled to high resolution tandem mass spectrometry. These peptides included Gonadorelin (LH-RH), Desmopressin and 9 growth hormone releasing peptides (GHRP-1, -2, -4, -5, -6, Hexarelin, Alexamorelin, Ipamorelin and a GHRP-2 metabolite); however, the procedure is expandable to further target analytes or metabolites. The method was validated with a main focus on qualitative result interpretation considering the parameters specificity, linearity (0-500 pg/mL), recovery (45-95%), precision (<20% at 100 pg/mL), limits of detection (2-10 pg/mL), robustnesss and ion suppression. The proof-of-principle was shown by analysing excretion study urine samples for LHRH, Desmopressin and GHRP-2., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Rationale: Precise assessment of renal glomerular filtration rate (GFR) is essential for the early detection of chronic kidney disease. AcSDKP-NH(2), an analogue of the endogenous tetrapeptide AcSDKP, is not degraded in vivo and is freely filtered by the kidney and eliminated in urine; for that reason this analogue is an ideal candidate marker for the assessment of GRF after administration to humans. Proof-of-concept demonstration and lack of toxicity in animals have allowed an ongoing clinical study in which AcSDKP-NH(2) was administered intravenously at a dose of 100 µg and compared with currently available GFR markers. The use of the AcSDKP analogue in clinical practice requires that this novel marker be associated with an analytical method that combines specificity, robustness and high accuracy. We have developed a liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay and compared it with an existing enzyme immunoassay (EIA) for AcSDKP-NH(2)., Methods: Human urine and plasma samples from the clinical study were analyzed by EIA and LC/MS/MS. Before LC/MS/MS assessment, AcSDKP-NH(2) was extracted using mixed-mode cation-exchange solid-phase extraction cartridges. Chromatographic separation was performed by hydrophilic interaction liquid chromatography (HILIC), before analysis with an electrospray ionization triple quadrupole mass spectrometer., Results: Mass spectrometry, through the use of an internal standard, tailored sample preparation and chromatographic separation, has better intra- and inter-assay precision (accuracies between 95 and 101% with CVs <8% for LC/MS/MS vs. accuracies between 90 and 115% with CVs <18% for EIA) and allows greater steadiness in intra-subject concentrations during the infusion (4.4% for LC/MS/MS vs. 8.6% for EIA). Moreover, the LC/MS/MS assay circumvents matrix effects observed in certain instances for the EIA and which may reduce its accuracy., Conclusions: Although the EIA can provide sufficient information in most subjects, the LC/MS/MS assay associated with this new marker should be the reference method., (Copyright © 2011 John Wiley & Sons, Ltd.)

Growth hormone secretagogues (GHS) constitute a new GH deficiency treatment increasing exponentially in number and improved potency and bioavailability over the last decade. The growth hormone releasing activity makes these compounds attractive for the artificial improvement of the human sports skills, now that recombinant human growth hormone (rhGH) administration is effectively detected. The GHS family is extremely diverse both in number and chemical heterogeneity and keeps growing continuously. In this paper, a general screening test is proposed. To develop a universal method, the single common property of growth hormone secretagogues has been targeted: their capacity to bind to the GHS receptor 1a (GHS-R1a). Pretreated urine samples have been tested in a competition assay where eventually the GHS presence detached a radiolabelled ligand from the receptor in a dose-dependent manner. Blank urine samples were processed to determine potential age, gender and exercise effects, and to define a threshold beyond which a specimen is considered positive. Samples from a growth hormone releasing peptide 2 (GHRP-2) excretion study corroborated the screening assay applicability with a detection window of approximately 4.5 h, and results were confirmed by comparison with a dedicated LC-MS quantification of the intact compound.

Degarelix is a novel competitive gonadotropin-releasing hormone receptor blocker (antagonist). In this study, the nonclinical metabolism and excretion of degarelix was investigated in Sprague-Dawley rat, beagle dog, and cynomolgus monkey. Degarelix was found to be stable when incubated in microsomes and cryopreserved hepatocytes from animal liver tissue. Absorption, distribution, metabolism, and excretion studies in male rat, dog, and monkey showed that after a subcutaneous dose of tritium-labeled degarelix, the peptide was rapidly absorbed with C(max) in plasma of 1 to 2 h. The predominant route of excretion was via the kidneys and the bile. In rat and dog, most of the degarelix dose was eliminated within 48 h via urine and feces in equal amounts (40-50% in each matrix), whereas in monkey the major route of excretion was fecal (50%) and renal (22%). In plasma and urine samples from all three species, mainly intact degarelix was detected. In bile and feces samples from rats and dogs, the same truncated peptides of the parent decapeptide were detected. The major metabolites identified represented the N-terminal tetrapeptide, the pentapeptide, and the heptapeptide. From the animal studies, it could be concluded that degarelix is subject to common peptidic degradation in the liver and bile and is fully excreted via metabolic and biliary (as metabolites and parent compound) and urinary (mainly as parent compound) pathways. Systemic exposure to metabolic products seems to be low.

Atazanavir (ATV) is an antiretroviral drug of the protease inhibitor class. Multiple adverse effects of ATV have been reported in clinical practice, such as jaundice, nausea, abdominal pain, and headache. The exact mechanisms of ATV-related adverse effects are unknown. It is generally accepted that a predominant pathway of drug-induced toxicity is through the generation of reactive metabolites. Our current study was designed to explore reactive metabolites of ATV. We used a metabolomic approach to profile ATV metabolism in mice and human liver microsomes. We identified 5 known and 13 novel ATV metabolites. Three potential reactive metabolites were detected and characterized for the first time: one aromatic aldehyde, one α-hydroxyaldehyde, and one hydrazine. These potential reactive metabolites were primarily generated by CYP3A. Our results provide a clue for studies on ATV-related adverse effects from the aspect of metabolic activation. Further studies are suggested to illustrate the impact of these potential reactive metabolites on ATV-related adverse effects.

Atazanavir inhibits UDP-glucuronyl-transferase-1A1 (UGT1A1), which metabolizes raltegravir, but the magnitude of steady-state inhibition and role of the UGT1A1 genotype are unknown. Sufficient inhibition could lead to reduced-dose and -cost raltegravir regimens. Nineteen healthy volunteers, age 24 to 51 years, took raltegravir 400 mg twice daily (arm A) and 400 mg plus atazanavir 400 mg once daily (arm B), separated by ≥3 days, in a crossover design. After 1 week on each regimen, raltegravir and raltegravir-glucuronide plasma and urine concentrations were measured by liquid chromatography-tandem mass spectrometry in multiple samples obtained over 12 h (arm A) or 24 h (arm B) and analyzed by noncompartmental methods. UGT1A1 promoter variants were detected with a commercially available kit and published primers. The primary outcome was the ratio of plasma raltegravir C(tau), or concentration at the end of the dosing interval, for arm B (24 h) versus arm A (12 h). The arm B-to-arm A geometric mean ratios (95% confidence interval, P value) for plasma raltegravir C(tau), area under the concentration-time curve from 0 to 12 h (AUC(0-12)), and raltegravir-glucuronide/raltegravir AUC(0-12) were 0.38 (0.22 to 0.65, 0.001), 1.32 (0.62 to 2.81, 0.45), and 0.47 (0.38 to 0.59, <0.001), respectively. Nine volunteers were heterozygous and one was homozygous for a UGT1A1 reduction-of-function allele, but these were not associated with metabolite formation. Although atazanavir significantly reduced the formation of the glucuronide metabolite, its steady-state boosting of plasma raltegravir did not render the C(tau) with a once-daily raltegravir dose of 400 mg similar to the C(tau) with the standard twice-daily dose. UGT1A1 promoter variants did not significantly influence this interaction.

Administration of exogenous 22 kDa recombinant human growth hormone (rhGH) suppresses the non-22 kDa pituitary growth hormone (GH) secretion by negative feedback; then, the elevated 22 kDa GH to non-22 kDa GH ratio (Rec/Pit ratio) can be utilized to detect doping with rhGH (isoform differential immunoassay). The influence of intravenous administration of growth hormone releasing peptide GHRP-2 on the isoform differential immunoassay for detecting rhGH doping has been investigated.In this study, a reference population (n=100) was used, with 0.04 mg/kg rhGH subcutaneous administration (n=5), 100 μg of GHRP-2 intravenous administration (n=10) and 0.04 mg/kg rhGH combined with 100 μg of GHRP-2 (n=10) in Japanese male subjects. The results indicated that the low dose (0.04 mg/kg) of rhGH led to significantly increased Rec/Pit ratio compared with the Japanese reference limit (P < 0.001). Because GHRP-2 dose led to increases in concentrations of both recombinant GH (recGH) and pituitary GH (pit GH), no significant change in the Rec/Pit ratio was observed (P > 0.05). In a combined administration study, after GHRP-2 dose the Rec/Pit ratios decreased to 39.9-43.9% compared with the elevated ratio caused by the rhGH dose.The results indicated that GHRP-2 administration cannot only be detected by the isoform differential immunoassay but also masks rhGH doping. The analysis of GHRP-2 was found to be suitable for compensating for the disadvantages of the isoform differential immunoassay because GHRP-2 and its metabolite (AA-3) in urine could be detected during the periods of masking of the Rec/Pit ratio by means of liquid chromatography/tandem mass spectrometry., (Copyright (c) 2010 John Wiley & Sons, Ltd.)

In this article, an approach has been developed for the analysis of some small peptides with similar pI values by CE-ESI-MS based on the online concentration strategy of dynamic pH junction. The factors affected on the separation, detection and online enrichment, such as BGE, injection pressure, sheath flow liquid and separation voltage have been investigated in detail. Under the optimum conditions, i.e. using 0.5 mol/L formic acid (pH 2.15) as the BGE, preparing the sample in 50 mM ammonium acetate solution (pH 7.5), 50 mbar of injection pressure for 300 s, using 7.5 mM of acetic acid in methanol-water (80% v/v) solution as the sheath flow liquid and 20 kV as the separation voltage, four peptides with similar pI values, such as L-Ala-L-Ala (pI = 5.57), L-Leu-D-Leu (pI = 5.52), Gly-D-Phe (pI = 5.52) and Gly-Gly-L-Leu (pI = 5.52) achieved baseline separation within 18.3 min with detection limits in the range of 0.2-2.0 nmol/L. RSDs of peak migration time and peak area were in the range of 1.45-3.57 and 4.93-6.32%, respectively. This method has been applied to the analysis of the four peptides in the spiked urine sample with satisfactory results.

Background/aims: The aim of the study was to establish the value of urinary trypsinogen-2 in predicting the severity of acute pancreatitis (AP) and to compare it with the accuracy of the urinary trypsinogen activation peptide (TAP) and the computed tomography severity index (CTSI)., Methodology: The study population consisted of 187 consecutive patients with AP, of whom 38 had severe disease. The predictive values of urinary trypsinogen-2, TAP and CTSI were assessed within 24 h of the onset of symptoms., Results: The mean values of predictive markers in the mild and severe pancreatitis groups were: urinary trypsinogen-2, 59/90 and 25/13 (p < 0.001); urinary TAP, 13.2 +/- 3.3nmol/l and 66.2 +/- 19.3 nmol/l (p < 0.001); and computed tomography severity index, 1.42 +/- 1.1 and 5.31 +/- 2.6 (p < 0.001). The sensitivity, specificity, positive predictive value, negative predictive value, and positive and negative likelihood ratios were calculated for the urinary trypsinogen-2 (65.7%, 66.4%, 33.3%, 88.4%, 1.9, and 0.51), for TAP (greater than 35 nmol/l: 63.2%, 65.8%, 32.0%, 87.5%, 1.9, and 0.58) and for CTSI (greater than 3: 47.4%, 95.3%, 69.2%, 87.7%, 9.0 and 0.55). To differentiate between severe and mild AP, urinary trypsinogen-2 (AUC 0.724) was slightly better than TAP (AUC 0.722), and they were both clearly better than CTSI (AUC 0.597) (p < 0.05). Urinary trypsinogen-2 had significantly lower cost (p < 0.001) than TAP and computed tomography., Conclusion: Urinary trypsinogen-2 was superior to CTSI and was as good as or even better than urinary TAP in the early prediction of severity in AP. This suggests that this simple and quick method deserves routine clinical application.

GHRP-2 (pralmorelin, D-Ala-D-(beta-naphthyl)-Ala-Ala-Trp-D-Phe-Lys-NH(2)), which belongs to a class of growth hormone secretagogue (GHS), is intravenously used to diagnose growth hormone (GH) deficiency. Because it may be misused in expectation of a growth-promoting effect by athletes, the illicit use of GHS by athletes has been prohibited by the World Anti-Doping Agency (WADA). Therefore, the mass spectrometric identification of urinary GHRP-2 and its metabolite D-Ala-D-(beta-naphthyl)-Ala-Ala-OH (AA-3) was studied using liquid chromatography/electrospray ionization tandem mass spectrometry for doping control purposes. The method consists of solid-phase extraction using stable-isotope-labeled GHRP-2 as an internal standard and subsequent ultra-performance liquid chromatography/tandem mass spectrometry, and the two target peptides were determined at urinary concentrations of 0.5-10 ng/mL. The recoveries ranged from 84 to 101%, and the assay precisions were calculated as 1.6-3.8% (intra-day) and 1.9-4.3% (inter-day). Intravenous administration of GHRP-2 in ten male volunteers was studied to demonstrate the applicability of the method. In all ten cases, unchanged GHRP-2 and its specific metabolite AA-3 were detected in urine., (Copyright 2010 John Wiley & Sons, Ltd.)

Asp-Phe-Leu-Ala-Glu (DE5) is a frequent sequence of many toxic middle molecular peptides that accumulate in uremic patients. To eliminate these peptides by hemoperfusion, three adsorbents (CP1-Zn(2+), CP2-Zn(2+), and CP3-Zn(2+)) were designed on the basis of coordination and hydrophobic interactions. Adsorption experiments indicated that CP2-Zn(2+) had the highest affinity for DE5 among these three adsorbents. Also, the adsorption capacity of CP2-Zn(2+) in DE5 and DE5-containing peptides was about 2-6 times higher than that of peptides without the DE5 sequence. Linear polymers bearing the same functional groups of the adsorbents were used as models to study the adsorption mechanism via isothermal titration calorimetry (ITC) and computer-aided analyses. The results indicated that coordination and hydrophobic interactions played the most important roles in their affinity. When two carboxyl moieties on Asp and Glu residues coordinated to CP2-Zn(2+), the hydrophobic interaction took place by the aggregation of the hydrophobic amino acid residues with phenyl group on CP2-Zn(2+). The optimal collaboration of these interactions led to the tight binding and selective adsorption of DE5-containing peptides onto CP2-Zn(2+). These results may provide new insight into the design of affinity adsorbents for peptides containing DE5-like sequences.

The (99m)Tc complex of NC100692 is being evaluated as a diagnostic agent for the detection of angiogenesis. In the present study, human urine samples from a clinical Phase I study were analysed using reversed-phase liquid chromatography coupled with an ion-trap mass spectrometer (LC-MS) in order to estimate the amount of intact NC100692 and any metabolites excreted in urine following intravenous injection of 150microg (99m)Tc-NC100692. Only intact NC100692 was observed in these urine samples, no metabolites were detected, in contrast to data earlier reported for rat urine where two metabolites and no NC100692 were observed. Within 4-8h after injection, 30+/-6% of the injected NC100692 was excreted in the urine, with the majority (23+/-5%) recovered within the first hour post-injection. There was good agreement between the calculated urinary recoveries of NC100692 and total radioactivity in urine samples voided approximately 1h post-injection. NC100692 constituting 98+/-24% of the total urinary (99m)Tc recovery indicating that the (99m)Tc excreted during this period was most likely excreted as (99m)Tc-NC100692. The ratio of NC100692 to (99m)Tc decreased in the urine samples as a function of time following injection for all subjects; this change is most likely due to reduced accuracy in the results at low levels of NC100692.

In the early 1970s, a peptide fraction with insulin potentiating activity was purified from human urine but the identity and origins of the active constituent remained unknown. Here we identify the active component and characterize its origins. The active peptide was identified as an alpha amidated tetrapeptide with the sequence GHTD-amide. The peptide was synthesized and tested for stimulation of glycogen synthesis and insulin potentiation by insulin tolerance testing in insulin-deficient rats, which confirmed GHTD-amide as the active peptide. Tissue localization using a peptide-specific anti-serum and epifluorescent and confocal microscopy showed decoration of pancreatic islets but not other tissues. Confocal microscopy revealed co-localization with insulin and immunogold and electron microscopy showed localization to dense core secretory granules. Consistent with these observations GHTD-amide was found in media conditioned by MIN6 islet beta cells. Sequence database searching found no annotated protein in the human proteome encoding a potential precursor for GHTD-amide. We conclude that the insulin potentiating activity originally described in human urine is attributable to the tetrapeptide GHTD-amide. GHTD-amide is a novel peptide produced by pancreatic beta cells and no precursor protein is present in the annotated human proteome. Stimulation of glycogen synthesis and co-localization with insulin in beta cells suggest that GHTD-amide may play a role in glucose homeostasis by enhancing insulin action and glucose storage in tissues.

To study the endogenous intoxication syndrome in patients with food toxic infections is essential in revealing the pathogenetic mechanisms underlying this disease. For this, the authors measured the level of low and average molecular weight, as well as their protein component--oligopeptides in plasma, red blood cells, and urine in the course of the disease. There were increased levels of the study parameters, which depended on the stage and degree of a pathological process. The determination of the level of low and average molecular weight and oligopeptides in plasma, red blood cells, and urine may serve as a marker of the intoxication syndrome; the level of the study parameters may be used as additional criteria for the severity of the process, the prediction of disease development and comorbidity, and as a criterion for recovery completeness.

BPPs have been identified in the venom of the Bothrops jararaca snake, or deduced from precursor proteins expressed either in the venom gland or in the brain of the snake. Their potentiating activity on bradykinin (Bk) is assumed to occur through a somatic angiotensin-converting enzyme (sACE) inhibitory mechanism. We have demonstrated that synthetic BPPs show remarkable functional differences, despite their high amino acid sequence similarities. Recently, we demonstrated that BPP-10c, after i.p. administration, was found in its intact form and in the form of a unique metabolite (des-Pro(10) BPP-10c) in mouse urine. Given this finding, we selected a number of BPPs with different structure-activities - BPP-5a (

Context: The incidence of post-ERCP pancreatitis is 1-22%. It continues to be a difficult problem for endoscopist and patient. Uncovering an agent that may be used to prevent its occurrence is critical., Objective: The aim of our study was to investigate the role of etanercept in the prevention of post-ERCP pancreatitis., Design: Endoscopic retrograde pancreatography (ERP)-induced injury was performed in dogs using a previously established endoscopic model of post-ERCP pancreatitis., Animals: Eight study dogs underwent ERP: 4 were pre-treated with etanercept one day before the procedure and 4 were untreated. In addition, three control dogs not undergoing ERP were also studied., Main Outcome Measures: Serum levels of amylase, lipase, and TNF-alpha, as well as the ratio of urinary trypsinogen activation peptide (TAP) and urinary creatinine, were measured before and after ERP. Necropsy was performed on post-operative day 5. All pancreatic specimens were graded by two blinded pathologists according to a validated scoring system., Results: Eight study dogs developed mild to moderate clinical pancreatitis with hyperamylasemia (11,538+/-4,065 U/L vs. 701+/-157 U/L; post-ERP peak levels vs. baseline values: P<0.001) and hyperlipasemia (3,637+/-2,333 U/L vs. 246+/-125 U/L; P=0.003). Mean total injury score was significantly elevated in study dogs compared to control dogs (6.16+/-1.85 vs. 1.06+/-0.49; P=0.001). There were escalating total injury scores concordant with more elaborate methods of endoscopically-induced injury although the trend did not reach the statistical significance (P=0.223). When comparing untreated to etanercept-treated dogs, there were no significant differences in serum amylase levels (P=0.903), serum lipase levels (P=0.771), TAP/creatinine urinary ratio (P=0.912), and pancreatic injury score (P=0.324)., Conclusion: Etanercept is ineffective in prevention of mild to moderate post-ERCP pancreatitis in canines. ERP-induced pancreatic injury can be used as a reliable animal model for studies investigating therapy and prevention of post-ERCP pancreatitis.

Two days after castration, urinary free felinine plus N-acetylfelinine decreased 24% in male cats, but, by day 5, the concentration had not decreased to that routinely found in males that have been castrated for several months. In a second experiment, three groups of castrated adult male cats received different subcutaneous injections: control (carrier), testosterone, testosterone plus estradiol. A fourth group of intact adult female cats received a testosterone injection. Urine was collected and analysed for free felinine, N-acetylfelinine and 3-methylbutanolglutathione. Baseline blood testosterone and estradiol concentrations were low during the pre-period, but increased sharply after hormone injections. The concentration of all three urinary metabolites increased as a result of testosterone injections with estradiol not modulating the effect. The effect of testosterone was not gender dependent. The concentration of free felinine, N-acetylfelinine and 3-methylbutanolglutathione in the urine remained low in the placebo control group throughout the study. The relative molar contribution of free felinine to the total amount of felinine containing compounds increased due to testosterone treatment, while the contribution of 3-methylbutanolglutathione and N-acetylfelinine decreased. Testosterone increases free felinine, N-acetylfelinine and 3-methylbutanolglutathione excretion in castrated adult male and intact female cats, whereas estradiol does not modulate this effect.

Autism is a complex neurodevelopmental disorder with unknown etiology. One hypothesis regarding etiology in autism is the "opioid peptide excess" theory that postulates that excessive amounts of exogenous opioid-like peptides derived from dietary proteins are detectable in urine and that these compounds may be pathophysiologically important in autism. A selective LC-MS/MS method was developed to analyze gliadinomorphin, beta-casomorphin, deltorphin 1, and deltorphin 2 in urine. The method is based on on-line SPE extraction of the neuropeptides from urine, column switching, and subsequent HPLC analysis. A limit of detection of 0.25 ng/mL was achieved for all analytes. Analyte recovery rates from urine ranged between 78% and 94%, with relative standard deviations of 0.2-6.8%. The method was used to screen 69 urine samples from children with and without autism spectrum disorders for the occurrence of neuropeptides. The target neuropeptides were not detected above the detection limit in either sample set.

The purpose of this study was to compare the clinical utility of the serum concentration of feline trypsin-like immunoreactivity (fTLI), the plasma and urine concentrations of trypsinogen-activation peptide (TAP), and the ratio of the urine TAP and creatinine concentrations (TAP:Cr) in the diagnosis of feline acute pancreatitis. We used 13 healthy cats and 10 cats with a diagnosis of acute pancreatitis. The mean serum fTLI and plasma TAP concentrations were significantly higher in the cats with acute pancreatitis than in the healthy cats (P < 0.05); the mean urine TAP concentrations and the median urine TAP:Cr ratios were not significantly different. Among the cats examined in this study, there was no benefit of plasma TAP over serum fTLI in the evaluation of suspected acute pancreatitis.

Domestic cats spray urine with species-specific odor for territorial marking. Felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid), a putative pheromone precursor, is excreted in cat urine. Here, we report that cauxin, a carboxylesterase excreted as a major urinary component, regulates felinine production. In vitro enzyme assays indicated that cauxin hydrolyzed the felinine precursor 3-methylbutanol-cysteinylglycine to felinine and glycine. Cauxin and felinine were excreted age dependently after 3 months of age. The age-dependent increases in cauxin and felinine excretion were significantly correlated. In mature cats, cauxin and felinine levels were sex-dependently correlated and were higher in males than in females. In headspace gas of cat urine, 3-mercapto-3-methyl-1-butanol, 3-mercapto-3-methylbutyl formate, 3-methyl-3-methylthio-1-butanol, and 3-methyl-3-(2-methyldisulfanyl)-1-butanol were identified as candidates for felinine derivatives. These findings demonstrate that cauxin-dependent felinine production is a cat-specific metabolic pathway, and they provide information for the biosynthetic mechanisms of species-specific molecules in mammals.

Objective: The purpose of this study was to assess patients' and physicians' compliance with ACE inhibitor treatment, by measuring an endogenous biomarker of ACE inhibition, urinary N-acetyl-Ser-Asp-Lys-Pro (AcSDKP), in the Noninsulin-Dependent Diabetes, Hypertension, Microalbuminuria, Proteinuria, Cardiovascular Events, and Ramipril (DIABHYCAR) trial, which compared ramipril (1.25 mg o.d.) with placebo in 4,912 patients with type 2 diabetes and microalbuminuria/proteinuria., Research Design and Methods: The urine AcSDKP-to-creatinine ratio was measured blind to treatment in all participants who completed follow-up and provided spot urine samples (n = 1,871)., Results: The median urinary AcSDKP-to-creatinine ratio was six times higher for ramipril than for placebo. Urinary AcSDKP-to-creatinine ratios displayed a bimodal distribution in both groups, with a very large intergroup overlap. Based on cluster analysis, we defined truly adherent ramipril patients as those with a ratio > or =4 nmol/mmol and truly adherent placebo patients as those with a ratio < 4 nmol/mmol. After excluding patients withdrawing prematurely from the study or known to have used a nonstudy ACE inhibitor, 27.3% of the 597 ramipril patients had ratios <4, indicating poor compliance, and 9.7% of the 621 placebo patients had ratios > or =4, indicating intake of a nonstudy ACE inhibitor. Correcting for compliance by using AcSDKP-guided analysis affected surrogate outcome results (decrease in systolic blood pressure and urinary albumin excretion) only slightly., Conclusions: The systematic use of spot urinary AcSDKP determination facilitated the detection of defects in compliance with ACE inhibitor treatment in both patients and physicians. Urinary AcSDKP measurement could be a useful biomarker for assessing compliance with ACE inhibition in the routine care of diabetic patients.

Context: All work on human trypsinogen activation peptide (TAP) in acute pancreatitis has been carried out with the same assay. Despite the extensive use of this original TAP assay, there is no characterization of the TAP-like immunoreactivity measured., Objective: The aims of this study were to develop an additional TAP assay and to attempt to characterize the TAP-like immunoreactivity found in the urine of patients with acute pancreatitis., Methods: Antibodies against the human TAP were prepared using the whole octapeptide APFD4K, conjugated at its N-terminal end. Characterization of the immunoreactivity measured with these assays was performed using gel filtration of human pancreatic juice before and after activation of trypsinogen with enterokinase., Results: After activation of the pancreatic juice, there was a large initial increase in immunoreactive TAP and a decrease 6-24 hours later. Using our antiserum, we found low levels of immunoreactive TAP in urine from patients with acute pancreatitis, although many of these samples contained high levels of immunoreactive TAP when tested with the commercially available TAP kit (Biotrin). The pentapeptide D4K, used as a standard in the Biotrin kit, showed much lower immunoreactivity than the synthetic octapeptide APFD4K in our assay. The octapeptide, however, reacted similarly to D4K in the Biotrin kit assay., Conclusion: n Our antibody prepared against the synthetic octapeptide APFD4K is directed against the N-terminal part of the octapeptide and does not recognize the pentapeptide D4K. Immunoreactive TAP in urine in acute pancreatitis is mainly composed of the C-terminal pentapeptide, D4K.

Objective: Our objective was to define the pharmacodynamic profile of the new dual neutral endopeptidase (NEP)/angiotensin-converting enzyme (ACE) inhibitor AVE7688., Methods: We compared the effects of single oral doses of AVE7688 (5 and 25 mg) with those of 10 mg ramipril (R10), a selective ACE inhibitor, in a placebo-controlled crossover study in sodium-depleted normotensive subjects. We also compared the effects of 25 mg AVE7688 with those of a renin-angiotensin system (RAS) blockade induced by a high dose of an angiotensin II receptor antagonist (300 mg irbesartan) and a dual blockade of the RAS (150 mg irbesartan plus 10 mg ramipril) in sodium-replete normotensive subjects by use of the same study design. The in vivo inhibition of ACE and NEP was monitored by measuring the urinary excretion of N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) and atrial natriuretic peptide (ANP), respectively. The intensity of RAS blockade was assessed by the increase in plasma active renin concentration., Results: The 24-hour urine AcSDKP cumulative excretion increased significantly more after 25 mg AVE7688 (919 nmol [95% confidence interval (CI), 803-1052 nmol], P < .05) than after 5 mg AVE7688 (706 nmol [95% CI, 612-813 nmol]) or 10 mg ramipril (511 nmol [95% CI, 440-593 nmol]). The 25-mg dose of AVE7866 significantly and transiently (4 to 8 hours after drug intake) increased urinary ANP (2.02 +/- 1.05 ng/h, P < .05), whereas 5 mg AVE7688 (1.14 +/- 0.77 ng/h) and 10 mg ramipril (0.93 +/- 0.65 ng/h) had no effect compared with placebo (0.80 +/- 0.37 ng/h). In the low-salt panel the rise in plasma active renin concentration achieved 24 hours after dosing by 25 mg AVE7688 (247 pg/mL [95% CI, 157-389 pg/mL], P < .05) was significantly higher than that achieved by 5 mg AVE7688 (129 pg/mL [95% CI, 75-221 pg/mL]) or 10 mg ramipril (113 pg/mL [95% CI, 67-193 pg/mL]), which did not differ. In the high-salt panel group the effects of 25 mg AVE7688 on renin release did not significantly differ from those after administration of the combination of 150 mg irbesartan plus 10 mg ramipril or 300 mg irbesartan alone. All of these active drugs similarly decreased blood pressure compared with placebo., Conclusion: AVE7688 at a dose of 25 mg has a favorable pharmacodynamic profile compared with other RAS blockers. These results support further clinical studies of its long-term effects in essential or resistant hypertension, chronic proteinuric nephropathy, and chronic heart failure.

Aim: To assess the usefulness of urinary trypsinogen-2 test strip, urinary trypsinogen activation peptide (TAP), and serum and urine concentrations of the activation peptide of carboxypeptidase B (CAPAP) in the diagnosis of acute pancreatitis., Methods: Patients with acute abdominal pain and hospitalized within 24 h after the onset of symptoms were prospectively studied. Urinary trypsinogen-2 was considered positive when a clear blue line was observed (detection limit 50 microg/L). Urinary TAP was measured using a quantitative solid-phase ELISA, and serum and urinary CAPAP by a radioimmunoassay method., Results: Acute abdominal pain was due to acute pancreatitis in 50 patients and turned out to be extrapancreatic in origin in 22 patients. Patients with acute pancreatitis showed significantly higher median levels of serum and urinary CAPAP levels, as well as amylase and lipase than extrapancreatic controls. Median TAP levels were similar in both groups. The urinary trypsinogen-2 test strip was positive in 68% of patients with acute pancreatitis and 13.6% in extrapancreatic controls (P<0.01). Urinary CAPAP was the most reliable test for the diagnosis of acute pancreatitis (sensitivity 66.7%, specificity 95.5%, positive and negative predictive values 96.6% and 56.7%, respectively), with a 14.6 positive likelihood ratio for a cut-off value of 2.32 nmol/L., Conclusion: In patients with acute abdominal pain, hospitalized within 24 h of symptom onset, CAPAP in serum and urine was a reliable diagnostic marker of acute pancreatitis. Urinary trypsinogen-2 test strip showed a clinical value similar to amylase and lipase. Urinary TAP was not a useful screening test for the diagnosis of acute pancreatitis.

Acute pancreatitis is a common digestive disease of which the severity may vary from mild, edematous to severe, necrotizing disease. An improved outcome in the severe form of the disease is based on early identification of disease severity and subsequent focused management of these high-risk patients. However, the ability of clinicians to predict, upon presentation, which patient will have mild or severe acute pancreatitis is not accurate. Prospective systems using clinical criteria have been used to determine severity in patients with acute pancreatitis, such as the Ranson's prognostic signs, Glasgow score, and the acute physiology and chronic health evaluation II score (APACHE II). Their application in clinical practise has been limited by the time delay of at least 48 h to judge all parameters in the former two and by being cumbersome and time-consuming in the latter. Contrast-enhanced computed tomography is presently the most accurate non-invasive single method to evaluate the severity of acute pancreatitis. It cannot, however, be performed to all patients with acute pancreatitis. Therefore, considerable interest has grown in the development of reliable biochemical markers that reflect the severity of acute pancreatitis. In this article we critically appraise current and new severity markers of acute pancreatitis in their ability to distinguish between mild and severe disease and their clinical utility.

Aim: To evaluate functional activity of the kidneys in patients with nonspecific pulmonary diseases (NSPD)., Material and Methods: Routine renal function tests, registration of iron-induced urine chemiluminescence (CL), estimation of middle-molecular oligopeptides in blood serum and urine were made in 22 patients with pneumonia, 18 patients with chronic non-obstructive bronchitis and 16 ones with chronic obstructive pulmonary disease., Results: Urine CL was subnormal in all the patients. This CL intensity correlated with functional renal characteristics, negatively correlated with endotoxicosis severity. In a favourable course of the disease, CL intensity increased, renal function and content of middle-molecular oligopeptides in biological substrates normalized. In patients with lingering and chronic inflammation CL remained low, renal dysfunction and endotoxicosis persisted., Conclusion: Registration of iron-induced urine CL can be used for control over NSPD patients condition and efficacy of their treatment.

Serum amylase is most commonly used as a biochemical marker of acute pancreatitis (AP). But it lacks specificity. The serum lipase level is more accurate and a better marker. Serum elastase -1 level is specific and remains elevated longer, but its radioimmunoassay is not routinely used. Recently, it can be rapidly measured by latex turbidometric immunoassay with automatic analyzer. Biochemically, only CRP test is available and useful to assess severity, but its sensitivity is unacceptably low in the early course of the disease. Urinary trypsinogen activation peptide (TAP) or trypsinogen-2 is an earlier marker. Increasing knowledge of the inflammatory process in AP has led to possibly useful biochemical indicators of severity, such as cytokines, nonpancreatic synovial type group II PLA2 or granulocyte elastase.