Comparison of independent and combined effects of the neurotensin receptor agonist, JMV-449, and incretin mimetics on pancreatic islet function, glucose homeostasis and appetite control.

BACKGROUND: Neurotensin receptor activation augments the biosctivity of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). JMV-449, a C-terminal neurotensin-like fragment with a reduced peptide bond, represents a neurotensin receptor agonist. METHODS: The present study assessed the actions of JMV-449 on pancreatic beta-cells alone, and in combination with GIP and GLP-1. Further studies examined the impact of JMV-449 and incretin mimetics on glucose homeostasis and appetite control in mice. RESULTS: JMV-449 was resistant to plasma enzyme degradation and induced noticeable dose-dependent insulin-releasing actions in BRIN-BD11 beta-cells. In combination with either GIP or GLP-1, JMV-449 augmented (P < 0.05) the insulinotropic actions of both hormones, as well as enhancing (P < 0.001) insulin secretory activity of both incretin peptides. JMV-449 also increased beta-cell proliferation and induced significant benefits on beta-cell survival in response to cytokine-induced apoptosis. JMV-449 (25 nmol/kg) inhibited (P < 0.05-P < 0.001) food intake in overnight fasted lean mice, and enhanced (P < 0.01) the appetite supressing effects of an enzymatically stable GLP-1 mimetic. When injected co-jointly with glucose, JMV-449 evoked glucose lowering actions, but more interestingly significantly augmented (P < 0.05) the glucose lowering effects of established long-acting GIP and GLP-1 receptor mimetics. In terms of glucose-induced insulin secretion, only GIP receptor signalling was associated with increases in insulin concentrations, and this was not enhanced by JMV-449. CONCLUSION: JMV-449 is a neurotensin receptor agonist that positively augments key aspects of the biological action profile of GIP and GLP-1. GENERAL SIGNIFICANCE: These observations emphasise the, yet untapped, therapeutic potential of combined neurotensin and incretin receptor signalling for diabetes.

Effects of an enzymatically stable C-terminal hexapseudopeptide fragment peptide of xenin-25, ψ-xenin-6, on pancreatic islet function and metabolism.

Xenin-25 undergoes rapid enzyme metabolism following secretion. Early studies demonstrated bioactivity of a C-terminal hexapeptide fragment of xenin-25, namely xenin-6, which were enhanced through introduction of a reduced N-terminal peptide bond, to yield Ψ-xenin-6. The present study was undertaken to define the biological actions and potential antidiabetic properties of Ψ-xenin-6. In vitro enzymatic stability, insulin and glucagon secretory activity, as well as effects on beta-cell survival were determined. Studies in mice were used to assess the impact of Ψ-xenin-6 on glucose homeostasis and satiety. Ψ-xenin-6 was resistant to murine plasma degradation. In BRIN-BD11 cells and isolated murine islets, Ψ-xenin-6 significantly stimulated insulin secretion, and prominently enhanced the insulinotropic actions of GIP. Xenin-6 and Ψ-xenin-6 had no impact on glucagon secretion, although xenin-6 partially reversed the glucagonotropic action of GIP. Further in vitro investigations revealed that, similar to GLP-1, Ψ-xenin-6 significantly augmented proliferation of human and rodent clonal beta-cells, whilst also fully protecting against cytokine-induced beta-cell cytotoxicity, with greater potency than xenin-25 and xenin-6. When administered to mice in combination with glucose, Ψ-xenin-6 significantly reduced glucose levels and enhanced glucose-induced insulin release, with a duration of biological action beyond 8 h. Ψ-xenin-6 also significantly enhanced the glucose-lowering action of GIP in vivo. In overnight fasted mice, Ψ-xenin-6 exhibited satiety actions at both 25 and 250 nmol/kg. These data demonstrates that Ψ-xenin-6 is a metabolically stable C-terminal fragment analogue of xenin-25, with a metabolic action profile that merits further study as a potential antidiabetic compound.

Analysis and behavior of colistin during anaerobic fermentation.

A new analytical method for the determination of colistin in fermenter samples was developed followed by a study on the behavior of this substance during anaerobic fermentation. Analysis of colistin A and B was carried out by liquid chromatography-tandem mass spectrometry. Separation of the analytes was performed on a Security Guard column (4×3mm). Fourteen fermentation tests in batch as well as in continuous reactors were carried out. After 44days of anaerobic digestion of cattle manure, initially spiked with 500mg/kg of colistin sulfate, a considerable decrease of the colistin concentration to less than 1mg/kg could be observed. Furthermore, the daily production of biogas and methane was measured. A correlation between gas production and colistin concentration could not be determined. However, an increase of 10% of the cumulative methane production was observed in those fermenters spiked with an initial bolus of 500mg/kg colistin.

Cytotoxic drug residues in urine of dogs receiving anticancer chemotherapy.

BACKGROUND: The presence of cytotoxic drug residues in urine of dogs may represent an exposure risk for pet owners and other people as well as a potential environmental contaminant. However, studies on cytotoxic drug residues in excretions of clinical patients are lacking in veterinary oncology. HYPOTHESIS: Variable concentrations of cytotoxic residues are present in urine samples, depending on sampling time and substance. ANIMALS: Client-owned dogs with lymphoma or mast cell tumors treated with standard chemotherapy protocols. METHODS: Urine samples were collected before, directly after, and on days after administration of chemotherapy. Measurement of vincristine, vinblastine, cyclophosphamide, and doxorubicin residues in canine urine was performed by a quantitative liquid chromatography tandem mass spectrometry (LC/MS/MS) method. RESULTS: Median cyclophosphamide residue concentration was 398.2 microg/L directly after treatment (d0) and was below the level of detection on days 1-3 (d1, d2, d3). Median vincristine residue concentration was 53.8 microg/L directly after treatment and was 20.2, 11.4, and 6.6 microg/L on days 1, 2, and 3. Median vinblastine residues were 144.9 (d0), 70.8 (d1), 35.6 (d2), and 18.7 microg/L (d3) with low concentrations detectable for 7 days after treatment. Median urine doxorubicin concentrations were 354.0 (d0), 165.6 (d1), 156.9 (d2), and 158.2 microg/L (d3). Low concentrations of doxorubicin were measurable up to 21 days after administration. CONCLUSIONS AND CLINICAL IMPORTANCE: Variable concentrations of chemotherapeutics were measured in urine samples, depending on sampling time point and drug. Findings may inform current chemoprotection guidelines and help minimize exposure risks.

Drug residues in serum of dogs receiving anticancer chemotherapy.

BACKGROUND: The presence of drug residues in blood samples can represent an occupational hazard. However, studies on cytotoxic drug residues in serum of dogs are lacking in veterinary oncology. OBJECTIVE: To evaluate possible occupational hazards associated with handling of blood samples from dogs receiving oncolytic drugs 7 days after treatment. ANIMALS: Twenty-seven client-owned dogs treated for lymphoma or mast cell tumors with vincristine, vinblastine, cyclophosphamide, or doxorubicin. METHODS: Prospective, observational study. Serum samples were either taken 7 days after administration of vincristine, cyclophosphamide, doxorubicin (lymphoma), and vinblastine (mast cell tumor), or 1-2 days after the last concurrent oral administration of cyclophosphamide (mast cell tumor). Additionally, serum was collected within 5 minutes of treatment. Measurement of drug residues in serum was performed by liquid chromatography tandem mass spectrometry (LC/MS/MS). RESULTS: In 33 samples collected within 5 minute of treatment, the median serum concentrations were vincristine: 37 microg/L (range: 11-87 microg/L), vinblastine: 13 microg/L (range: 13-35 microg/L), cyclophosphamide: 2,484 microg/L (range: 1,209-2,778 microg/L), doxorubicin: 404 microg/L (range: 234-528 microg/L). In 81 serum samples collected 7 days after treatment vinblastine (7 microg/L) was detected in 1 sample, and cyclophosphamide (7 and 9 microg/L) in 2 samples collected 1-2 days after oral administration of cyclophosphamide. Medications were not detected in any of the other samples. CONCLUSIONS AND CLINICAL IMPORTANCE: Handling of blood samples from dogs receiving oncolytic chemotherapy 7 days after treatment with vincristine, vinblastine, cyclophosphamide, and doxorubicin should not present a health hazard.

Determination of phoxim residues in eggs by using high-performance liquid chromatography diode array detection after treatment of stocked housing facilities for the poultry red mite (Dermanyssus gallinae).

The poultry red mite Dermanyssus gallinae is the most important ectoparasite of poultry in several European countries. Phoxim is a well-known antiparasitic agent in wide use. Initial studies indicated that this compound could successfully be applied to eliminate D. gallinae in egg-laying birds and in henhouses by treating the cages and the equipment with it. In order to investigate whether phoxim residues are present in eggs from laying hens, we developed a selective and sensitive high-performance liquid chromatography method employing a simple water/acetonitrile gradient system. The amount of phoxim was determined by UV detection at 281 nm, and the presence of the residue was confirmed by diode array detection. The eggs were homogenized for sample pretreatment and extracted with acetonitrile and partitioned with n-hexane. The acetonitrile extract was further purified with silica gel column chromatography. Recovery rates (performed at the 5-120 microg kg(-1) level) were in the range of 86.0-92.1% with relative standard deviations between 3.1% and 16.3%. Based on a signal to noise ratio of 3, the limit of detection of the assay was approximately 2 microg kg(-1). The day-to-day variation in the concentration of phoxim in four contaminated eggs (5.7-51.6 microg kg(-1)) was generally less than 20%. The decision limit (CCalpha) and the detection capability (CCbeta) were 62.0 and 68.7 microg kg(-1), respectively. The applicability of the method was demonstrated in eggs from three clinical trials and from a field study. In these investigations, all animals were kept in conventional battery cages. No sample was found containing more than the maximum residue level of 60 microg kg(-1) for phoxim in eggs as given in Annex I of Council Regulation (EEC) No. 2377/90.

[Acrylamide, antiparasitic agents, dioxins and more: how dangerous are contaminants and residues in food?]. / Acrylamid, Antiparasitika, Dioxine und mehr: Wie gefährlich sind Rückstände und Kontaminanten in Lebensmitteln?

Within the European Union, food safety and consumer protection are topics of highest priority. The evaluation of the safety of contaminants and residues in food is usually based on the determination of the acceptable daily intake (ADI), which in turn is the basis for maximum residue levels (MRLs). As this procedure depends on animal testing, a safety factor of 100 is usually applied to provide a margin of safety. This paper discusses the occurrence of relevant contaminants (e.g. dioxins, acrylamide) and residues (e.g. antibiotics, antiparasitic agents) in food and provides a brief assessment of the risks resulting from the consumption of food containing these substances.

Metabolism and potency of xenin and of its reduced hexapseudopeptide psi fragment in the dog.

Xenin is a 25 amino acid peptide hormone, secreted into the circulation by specific endocrine cells in the duodenal mucosa. Plasma concentrations are elevated after sham feeding and feeding. In the present study the metabolism of xenin and of a C-terminal fragment was investigated. Xenin, its C-terminal hexapeptide, and a pseudopeptide analog psi (CH2NH) hexapeptide in which a psi reduced bond is introduced in the biologically important dibasic motif of the C-terminus were infused or injected intravenously in 14 anaesthetized dogs. Plasma disappearance time, metabolic clearance rate, the generation of metabolites, and biological effects on exocrine pancreatic secretion were determined employing radioimmunoassay, high pressure liquid chromatography, mass spectrometry (MALDI-MS), and sequence analysis. Half time after steady state infusion of xenin was 3.1 min(-1), that of psi xenin 6 was 6.2(-1) (p<0.01) Plasma concentrations of psi xenin 6 were significantly elevated (p<0.01), pancreatic secretion of volume was augmented by a factor of 50, and output of protein by a factor of 30 compared to unmodified xenin 6. MALDI-MS and sequencing after infusion of xenin revealed a C-terminal octapeptide fragment as primary metabolite. Introduction of a reduced pseudobond in the dibasic motif of xenin dramatically enhances biological potency. This indicates that such a reduced pseudopeptide may be useful in the treatment of bowel paralysis.

[Effects of animal housing facilities on residues and contaminants in food]. / Einfluss der Tierhaltung auf Rückstände und Kontaminanten in Lebensmitteln.

There are--especially in the case of food of animal origin--only a few well-controlled studies available comparing organically and conventionally produced food with a focus on residues and contaminants. The differences found seemed to be much lower than expected, and the amounts of residues and contaminants were mostly below regulatory maximum residue levels. In the case of organically and conventionally produced milk there have been slight but usually not significant differences reported. One important observation is, that conventionally produced milk contains aflatoxin M1 more frequently than biologically produced milk. The investigation of eggs from different housing systems for laying hens showed in the case of dioxins the highest concentrations in free range eggs. In contrast, propoxur, an insecticide used against the poultry red mite, was found in eggs from battery cages in higher amounts than in those from enriched cages and in eggs obtained from an aviary system. Further research in this field is highly recommended, but there should be more sophisticated evaluation of the data sets from national and international monitoring programs.

Phase III of the migrating motor complex: associated with endogenous xenin plasma peaks and induced by exogenous xenin.

Xenin, a recently discovered peptide produced by specific endocrine cells of the duodenal mucosa, has shown exocrine, endocrine and motility effects in the gastroenteropancreatic system in animal experiments. The aim of the present investigation was to study the role of xenin in the regulation of duodenojejunal motility of humans. Twenty-nine healthy volunteers from the hospital staff gave informed consent to participate in this investigation. In 20 volunteers, we determined plasma concentrations of immunoreactive xenin at 15 min intervals over a mean time period of 8 h fasting and recorded the interdigestive motor activity of the duodenojejunum. In a double-blind randomized crossover study on other nine subjects, synthetic xenin in a dose of 4 pmol kg-1 min-1 or placebo was infused for 10 min intravenously in the interdigestive period and postprandially after a liquid meal. Duodenojejunal motility was recorded simultaneously. Predefined interdigestive xenin plasma peaks were found to be significantly associated with the phases III of the migrating motor complex. In the interdigestive period, xenin induced a premature phase III activity in each volunteer; this was followed by a second phase III in five out of nine subjects. In the postprandial state, xenin significantly increased contraction frequency and the percentage of aborally propagated contractions. These findings suggest a role of the peptide hormone xenin in modulating interdigestive and postprandial duodenojejunal motility in humans.

Localization of xenin-immunoreactive cells in the duodenal mucosa of humans and various mammals.

Xenin is a 25-amino-acid peptide extractable from mammalian tissue. This peptide is biologically active. It stimulates exocrine pancreatic secretion and intestinal motility and inhibits gastric secretion of acid and food intake. Xenin circulates in the human plasma after meals. In this study, the cellular origin of xenin in the gastro-entero-pancreatic system of humans, Rhesus monkeys, and dogs was investigated by immunohistochemistry and immunoelectron microscopy. Sequence-specific antibodies against xenin detected specific endocrine cells in the duodenal and jejunal mucosa of all three species. These xenin-immunoreactive cells were distinct from enterochromaffin, somatostatin, motilin, cholecystokinin, neurotensin, and secretin cells, and comprised 8.8% of the chromogranin A-positive cells in the dog duodenum and 4.6% of the chromogranin A-positive cells in human duodenum. In all three species, co-localization of xenin was found with a subpopulation of gastric inhibitory polypeptide (GIP)-immunoreactive cells. Immunoelectron microscopy in the canine duodenal mucosa demonstrated accumulation of gold particles in round, homogeneous, and osmiophilic secretory granules with a closely adhering membrane of 187 +/- 19 nm diameter (mean +/- SEM). This cell type was found to be identical to the previously described canine GIP cell. Immunocytochemical expression of the peptide xenin in a subpopulation of chromogranin A-positive cells as well as the localization of xenin immunoreactivity in ultrastructurally characterized secretory granules permitted the identification of a novel endocrine cell type as the cellular source of circulating xenin.

[Substances with pharmacological effects including hormonally active substances in the environment: identification of tetracyclines in soil fertilized with animal slurry]. / Stoffe mit pharmakologischer Wirkung einschliesslich hormonell aktiver Substanzen in der Umwelt: Nachweis von Tetracyclinen in güllegedüngten Böden.

Many drugs used in human medicine are detectable in surface waters from the low to the very low microgram/L concentration range. In drinking waters only some of these substances were detected, the concentrations are usually an order of magnitude below the concentrations found in surface waters. A risk assessment of long time effects caused by a permanent intake of these low concentrations of drug residues cannot be done at this time. Hormonally active substances in surface waters may present an ecotoxicological risk, there are many investigations currently under way to assess this problem. Our investigations show for the first time that residues of the commonly used veterinary drugs tetracycline and chlortetracycline can be detected in the surface of soil (0-40 cm) fertilized with animal slurry. The maximum concentrations found were 32.3 micrograms/kg and 26.4 micrograms/kg respectively. Leaching of these compounds into seeping water sampled at a depth of 80-140 cm could not be detected with the methods employed. The significance of the detected antibiotic residues in soil samples for the quality of food of animal origin or any ecotoxicological consequences needs further investigations. The knowledge about the concentrations of veterinary drug residues resulting from animal husbandry in the environment is the first step for such a risk assessment.

Enhancing of anti-viral activity against HIV-1 by stimulation of CD8+ T cells with thymic peptides.

HIV-1 can be neutralized by soluble factors produced and secreted by activated CD8+ T cells. Production of such anti-viral CD8 factors (including chemokines) can be induced with IL-2 or phytohaemagglutinin (PHA). In addition to PHA or IL-2, we have co-stimulated CD8+ T cells with PHA/IL-2 and a mixture of thymic peptides (TP) of molecular weights below 10 kD. For the activation, CD8+ T cells were purified from peripheral blood mononuclear cells of HIV-1- individuals and any resultant anti-viral activity was monitored using an HIV-1 neutralization assay. Using HIV-1 isolates highly resistant to chemokine inhibition we detected significantly higher levels of HIV-1 neutralizing activity in CD8+ T cell culture supernatants which had been co-activated with TP. When the TP-induced anti-viral activity was monitored, neutralization of both non-syncytia-inducing (NSI) and syncytia-inducing (SI) patient isolates was enhanced by 38% (NSI, PHA +/- TP), 66% (SI, PHA +/- TP), 28% (NSI, IL-2 +/- TP), and 57% (SI, IL-2 +/- TP) compared with the anti-viral activity present in supernatants from CD8+ T cell cultures stimulated only with PHA or IL-2. Peptide sequence analysis of purified TP showed that the TP mixture predominantly contains peptides with homology to human histone and collagen sequences. Our data demonstrate that CD8+ T cells are additionally activated by a mixture of TP. In this way, the production of HIV-1 neutralizing CD8 factors can be enhanced.

Effect of omeprazole treatment on plasma concentrations of the gastric peptides, xenin, gastrin and somatostatin, and of pepsinogen.

The peptide xenin 25 is a gastric mucosal constituent like gastrin, somatostatin and pepsinogen. Gastrin and pepsinogen plasma concentrations increase when the secretion of gastric acid is reduced by proton pump inhibitors. In the present investigation, treatment with omeprazole led to an increase in fasting and postprandial plasma concentrations of xenin, gastrin and pepsinogens A and C (P < 0.05, in each instance), whereas somatostatin plasma levels remained unchanged. Because subcutaneous injection of pentagastrin did not raise xenin plasma concentrations, a direct effect of gastrin on xenin production seems unlikely. This study indicates that xenin plasma concentrations are regulated by intragastric pH, as are those of gastrin and pepsinogen.

Gastroenteropancreatic effects of xenin in the dog.

Xenin is a 25 amino acid peptide detected in the gastric mucosa of various mammals. It has since been found in low concentrations in other tissues. Xenin plasma concentrations increase after meals. The present study reports some gastroenteropancreatic effects of this peptide in the dog. Intravenous infusion of 64 pmol/kg min synthetic xenin significantly inhibited pentagastrin-stimulated gastric acid secretion and stimulated exocrine pancreatic secretion of volume and protein. Further, intravenous infusion of xenin in a dose of 1.0 pmol/kg min stimulated jejunal motility in the anaesthetized dog. An intravenous infusion of 32 pmol/kg min xenin raised plasma concentrations of pancreatic polypeptide, vasoactive intestinal polypeptide, insulin and glucagon. The present experiments therefore indicate manifold bioactive properties of intravenously infused xenin in the dog, with jejunal motility the most sensitive target. Conclusions as to the physiological role of xenin cannot be drawn from the present experiments. The release of other hormonal peptides indicates a complex action of xenin.

Neurokinetic and myokinetic effects of the peptide xenin on the motility of the small and large intestine of guinea pig.

The effects of xenin 25, a peptide of the neurotensin/xenopsin family, were examined on the motility of the guinea pig jejunum and colon in vitro. In the jejunum, xenin induced a biphasic response: first a small relaxation and then a large contraction. In the colon, xenin induced relaxation. The tenia coli was contracted. Deletion or amidation of the C-terminal leucine inactivated xenin. A peptide sequence of 16 C-terminal amino acids was necessary to elicit a full response in the jejunum, whereas in the colon, the potencies of all fragments of xenin 25, including the 6 C-terminal amino acid sequence (xenin 6), were not significantly different from that of xenin 25. In the jejunum, contraction was abolished or reduced by tetrodotoxin, by atropine, by met-enkephalin, by antagonists to the tachykinin receptors NK1 and NK2 and by the P2-purinoceptor antagonist suramin. L-NNA, phentolamine, methysergide, hexamethonium and apamin had no effect. In the colon, all actions of xenin were tetrodotoxin-resistant; the potency of xenin to relax was reduced by apamin and suramin. The actions of xenin on small and large bowel were attenuated by the neurotensin receptor antagonist SR 48692. The xenin analog neurotensin was significantly less potent than xenin 25 in contracting the jejunum and more potent than xenin 25 in relaxing the colon. We conclude that xenin exerts excitatory effects on a neuronal subtype receptor in the jejunum, with participation of muscarinic, purinergic and tachykinin-related mechanisms. Xenin exerts relaxing effects on the colon by interaction with a myokinetic subtype receptor involving Ca(++)-dependent K+ channels and the P2-purinoceptor.

Identification of proxenin as a precursor of the peptide xenin with sequence homology to yeast and mammalian coat protein alpha.

Proxenin a precursor of the bioactive peptide xenin, was isolated from canine pancreas by HPLC and identified by mass spectrometry and sequence analysis as a pentatriacontapeptide with a molecular weight of 4035: Met Leu-Thr Lys-Phe-Glu-Thr-Lys-Ser-Ala-Arg-Val-Lys-Gly-Leu-Ser- Phe-His-Pro-Lys-Arg-Pro-Trp.Ile-Leu-Thr-Ser-Leu-His-Asn-Gly-Val-Ile-Glo- Leu-OH. Treatment with pepsin cleaved off 10 C-terminal amino acids and released xenin. Data base search showed amino acid sequence homology of xenin and proxenin with the sequence of coal protein alpha of yeast (62%) and humans (100%). Concentration of the coatomer complex from rabbit liver led to an equimolar enrichment of extractable proxenin. We conclude, therefore, that xenin and proxenin are peptide sequences highly conserved during evolution within the alpha-subunit of the coatomer.

The role of CCK and its analogues in the organogenesis of the fetal rat pancreas.

The gastrointestinal peptide cholecystokinin (CCK) has been shown to stimulate pancreatic growth in the adolescent and adult rat. However, little is known about the role of gastrointestinal hormones in the regulation of organ formation during fetal development. We therefore examined the effects of the CCK receptor antagonist devazepide (25 micrograms/h) and an antigastrin/CCK monoclonal immunoglobulin G on the maternal and fetal rat pancreas. These substances were infused subcutaneously with minipumps in female rats during the entire period of gestation. At the end of gestation, the rats were killed and the pancreata of the dams and their litter were examined for DNA and protein. In the dams, the receptor antagonist and the antibody against CCK/gastrin had no effect. In the newborns, the CCK receptor antagonist led to a significant reduction of the protein and DNA concentration [protein in controls, 105.0 +/- 3.75 micrograms/mg pancreatic tissue; in the antagonist group, 91.9 +/- 4.2 micrograms/mg pancreatic tissue (p < 0.05); DNA in controls, 1.28 +/- 0.19 micrograms/mg pancreatic tissue; in the antagonist group, 0.48 +/- 0.06 micrograms/mg pancreatic tissue (p < 0.05) (mean +/- SEM)]. Immune neutralization of CCK/gastrin in the maternal-fetal circulation induced a reduction of the protein concentration in the fetal pancreas (85.3 +/- 3.06 micrograms/mg pancreatic tissue; p < 0.01) but had no effect on fetal pancreatic DNA. Additional experiments indicated effective concentrations of the CCK receptor antagonist in fetal pancreatic tissue and free binding sites of the circulating antibody. In conclusion, the study provides evidence that CCK and its analogues are involved in fetal pancreatic organogenesis.

Distribution, formation, and molecular forms of the peptide xenin in various mammals.

In the present investigation we isolated the recently discovered pentacosapeptide xenin from gastric mucosa of man, dog, pig, guinea pig, rat, and rabbit. HPLC, mass spectrometry, and amino acid sequence analysis showed xenin-25 in concentrations of 54-144 pmol/g tissue in gastric mucosa of each species. Extraction with 2% TFA followed by analytical C18 HPLC revealed 0.02-84 pmol/g xenin-25 also in hypothalamus, lung, liver, heart, kidney, adrenal gland, pancreas, testicle, skin, and duodenal, jejunal, ileal, and colonic mucosa of dog and man, respectively. Digestion of these acid extracts with pepsin liberated xenin-25 in concentrations from 2 up to 166 pmol/g tissue. Gel chromatography revealed a large molecular weight precursor of xenin-25 and evidence for an endogenous acid protease coeluting with pepsinogen capable of releasing xenin-25 from its precursor. Maximal concentrations of xenin-25 were obtained when canine gastric mucosa was incubated with 2% TFA at room temperature for 2 h. Longer incubation times led to a decline of xenin-25 concentration and to formation of xenin-16 and xenin-9, both C-terminal fragments of xenin-25. We conclude that xenin-25 is present not only in human gastric mucosa but also in the stomach of various other mammals. Xenin-25 is further present in low concentrations in many other organs where a pepsin-like protease generates xenin-25 from a large precursor and processes it to smaller fragments.

Identification of xenin, a xenopsin-related peptide, in the human gastric mucosa and its effect on exocrine pancreatic secretion.

One of the peptides previously discovered in amphibians is the octapeptide xenopsin. As immunohistochemistry has also indicated the presence of xenopsin immunoreactivity in man, we extracted in the present investigation xenopsin-immunoreactive material from human gastric mucosa and purified it to homogeneity with several high performance liquid chromatography (HPLC) reverse phase and ion exchange chromatographic steps. The eluates were monitored with a radioimmunoassay for amphibian xenopsin. Determination of the amino acid sequence revealed a 25-amino acid peptide having 6 C-terminal amino acids in common with amphibian xenopsin. The sequence of this peptide, termed xenin 25, is M-L-T-K-F-E-T-K-S-A-R-V-K-G-L-S-F-H-P-K-R-P-W-I-L. The peptide was custom-synthesized. Mass spectrometry of the synthetic and the extracted peptide revealed identical molecular mass. Purification of 250 ml of human postprandial plasma with Sep-Pak C18 cartridges, reverse phase HPLC, and ion exchange chromatography demonstrated circulating xenin immunoreactivity at a retention time identical to xenin 25. The amount of xenin immunoreactivity at the position of xenin 25 on C18-HPLC increased significantly after a meal. A radioimmunoassay utilizing antibodies to xenin 25 and a 125I-labeled analogue of xenin 25 was used to measure immunoreactive xenin in the plasma of 10 volunteers. There was a significant rise of xenin immunoreactivity in the plasma after a meal. Intravenous infusion of the synthetic peptide in dogs stimulated exocrine pancreatic secretion beginning at a dose of 4 pmol/kg/min. The maximal effect was seen with 64 pmol/kg/min. We have detected, therefore, a new peptide, xenin 25, in human gastric mucosa; we have provided evidence for the presence of this peptide in the human circulation, and have shown a rise of plasma xenin concentrations after a meal. This peptide stimulates exocrine pancreatic secretion. Its physiologic role deserves further investigation.