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Background/aim: Helicobacter pylori (Hp) infection affects a substantial proportion of the world population and is a major risk factor of gastric cancer (GC). The caveats of common Hp-tests can be evaded by a serological biomarker test (GastroPanel®, Biohit Oyj, Helsinki), the most comprehensive Hp-test on the market. The clinical validation of Helicobacter pylori IgG ELISA of the new-generation GastroPanel® test is reported. The aim of the study is to validate the clinical performance of the Helicobacter pylori IgG ELISA test in diagnosis of biopsy-confirmed Hp-infection in gastroscopy referral patients., Patients and Methods: A cohort of 101 patients (mean age=50.1 years) referred for gastroscopy at the outpatient Department of Gastroenterology (SM Clinic, St. Petersburg) were examined by two test versions to validate the new-generation GastroPanel®. All patients were examined by gastroscopy and biopsies, which were stained with Giemsa for specific identification of Hp in the antrum (A) and corpus (C)., Results: Biopsy-confirmed Hp-infection was found in 64% of patients, most often confined to antrum. The overall agreement between Hp IgG ELISA and gastric biopsies in Hp-detection was 91% (95%CI=84.1-95.8%). Hp IgG ELISA diagnosed biopsy-confirmed Hp (A&C) with sensitivity (SE) of 92.3%, specificity (SP) of 88.6%, positive predictive value (PPV) of 93.8% and negative predictive value (NPV) of 86.1%, with AUC=0.904 (95%CI=0.842-0.967). In ROC analysis for Hp detection (A&C), Hp IgG ELISA shows AUC=0.978 (95%CI=0.956-1.000)., Conclusion: The Hp IgG ELISA test successfully concludes the clinical validation process of the new-generation GastroPanel® test, which retains the unrivalled diagnostic performance of all its four biomarkers, extensively documented for the first-generation test in different clinical settings., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Numerous studies have demonstrated that in vitro controlled enzymatic hydrolysis of fish and shellfish proteins leads to bioactive peptides. Ultrafiltration (UF) and/or nanofiltration (NF) can be used to refine hydrolysates and also to fractionate them in order to obtain a peptide population enriched in selected sizes. This study was designed to highlight the impact of controlled UF and NF on the stability of biological activities of an industrial fish protein hydrolysate (FPH) and to understand whether fractionation could improve its content in bioactive peptides., Results: The starting fish protein hydrolysate exhibited a balanced amino acid composition, a reproducible molecular weight (MW) profile, and a low sodium chloride content, allowing the study of its biological activity. Successive fractionation on UF and NF membranes allowed concentration of peptides of selected sizes, without, however, carrying out sharp separations, some MW classes being found in several fractions. Peptides containing Pro, Hyp, Asp and Glu were concentrated in the UF and NF retentates compared to the unfractionated hydrolysate and UF permeate, respectively. Gastrin/cholecystokinin-like peptides were present in the starting FPH, UF and NF fractions, but fractionation did not increase their concentration. In contrast, quantification of calcitonin gene-related peptide (CGRP)-like peptides demonstrated an increase in CGRP-like activities in the UF permeate, relative to the starting FPH. The starting hydrolysate also showed a potent antioxidant and radical scavenging activity, and a moderate angiotensin-converting enzyme (ACE)-1 inhibitory activity, which were not increased by UF and NF fractionation., Conclusion: Fractionation of an FPH using membrane separation, with a molecular weight cut-off adapted to the peptide composition, may provide an effective means to concentrate CGRP-like peptides and peptides enriched in selected amino acids. The peptide size distribution observed after UF and NF fractionation demonstrates that it is misleading to characterize the fractions obtained by membrane filtration according to the MW cut-off of the membrane only, as is currently done in the literature., (Copyright (c) 2010 Society of Chemical Industry.)

Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.

Gastric acid production is important in intestinal iron absorption. The peptide hormone gastrin exists in both amidated and non-amidated forms, which stimulate and potentiate gastric acid secretion, respectively. Since non-amidated gastrins require ferric ions for biological activity in vitro, this study investigated the connection between iron status and gastrin by measurement of circulating gastrin concentrations in mice and humans with hemochromatosis. Gastrin concentrations are increased in the plasma and gastric mucosa of Hfe(-/-) mice, and in the sera of humans with HFE-related hemochromatosis. The discovery of a relationship between iron status and circulating gastrin concentrations opens a new perspective on the mechanisms of iron homeostasis.

Amidated and nonamidated progastrin-derived peptides have distinct biological activities that are mediated by a range of receptor subtypes. The objective was to determine the nature of the stored and secreted progastrin-derived peptides and to investigate whether progastrin release is regulated by gastric acidity. Using an antiserum directed to the C terminus of progastrin for identification and to monitor purification, C-terminal flanking peptides (CTFP) of progastrin (prog(76-83), prog(77-83), and prog(78-83) in approximately equivalent amounts) were isolated and identified from extracts of sheep antrum using ion exchange, HPLC, and mass spectrometry. Only trace amounts of full-length progastrin were present. Progastrin CTFP was the predominant progastrin-derived peptide in the antrum [progastrin CTFP/gastrin amide (Gamide) = 3]. Similarly, progastrin CTFP was the major circulating form in the antral (CTFP, 710 +/- 62 pmol/liter; Gamide, 211 +/- 35 pmol/liter) and jugular (CTFP, 308 +/- 16 pmol/liter; gastrin amide, 32 +/- 3 pmol/liter) veins. Alteration of gastric acidity in sheep by iv infusion of a H/K-adenosine triphosphatase inhibitor or somatostatin or by intragastric infusion of HCl demonstrated that the CTFP concentrations changed, although to a lesser extent than the changes in circulating gastrin amide. We conclude that the CTFP of progastrin is the major stored and circulating species of the gastrin gene, and that it is secreted in a regulated fashion rather than constitutively. Because full-length progastrin is bioactive, but is only a minor antral and secreted form, determination of the biological activity of the C-terminal flanking peptides will be important for a complete understanding of gastrin endocrinology.

To identify the teleost gastrin, CCK/gastrin family genes were isolated from puffer and flounder. The cDNA of puffer gastrin, CCK1 and CCK2 were 678, 752 and 533bp, respectively. Puffer gastrin gene consists of three exons, in contrast, CCKs consist of four exons. Flounder gastrin mRNA (526bp) was expressed in the intestine but not in the brain. It was developed synchronously with the stomach differentiation in the larval stage. The phylogenetic analysis shows that puffer and flounder gastrin classified into the vertebrate gastrin cluster and two types of CCK were probably produced by the genome duplication occurred in teleost phylogeny.

Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human progastrin(6-80) and to investigate its structure and biological activities in vitro. Human progastrin(6-80) was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin(6-80) was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin(6-80) at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin(6-80) did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin(6-80) suggested that progastrin(6-80) stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin(6-80) is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone.

The turnover of the epithelium of the gastrointestinal tract is regulated by a balance between cell multiplication and cell loss. We examined the effects of starvation on apoptosis in endocrine and other epithelial cells of rat antropyloric mucosa. Apoptosis was determined by the TUNEL reaction combined with immunocytochemical staining for gastrin and somatostatin. Apoptotic cell morphology was determined by bisbenzimide staining for DNA. Both gastrin and somatostatin cells showed a significantly lower apoptotic index than the general epithelium. This agrees with the longer turnover kinetics of gastric endocrine cells. On starvation, the apoptotic index of the general epithelium and of the gastrin but not of the somatostatin, cells increased significantly. This was prevented by the nitric oxide synthase (NOS) inhibitor L-NAME but not by its inactive stereoisomer D-NAME. Immunoreactive neuronal NOS was present in somatostatin cells, in nonendocrine cells predominating in the surface and pit epithelium, and in rare nerve fibers. Endothelial cell NOS was present in vessels, whereas the inducible isoform was barely detectable. Thus, endogenous NOS isoforms participate in regulating antropyloric epithelial apoptosis during starvation. The close paracrine relation between somatostatin cells and gastrin cells suggests that the former regulates apoptosis of the latter through release of NO.

The distribution of cholecystokinin and gastrin-like immunoreactive cell bodies and fibers in the nervous system of 2 annelid worms, Lumbricus terrestris and Eisenia fetida, has been studied by means of immunohistochemistry. The cerebral ganglion contains 170-250, the subesophageal ganglion contains 120-150, and the ventral ganglia contain 50-75 cholecystokinin immunoreactive cells, that represent 8-12%, 8-10% and 4-5% of the total cell number, respectively. The anti-gastrin serum stained 330-360 nerve cells in the cerebral, 32-46 in the subesophageal and 7-25 in the ventral cord ganglia, representing 15-16%, 2-3% and 0.5-2% of the total cell number. Immunopositivity was found with both antisera in the enteric nervous system, where the stomatogastric ganglia and the enteric plexus contain immunoreactive cells and fibers. Immunopositive cells were found in the epithelial and subepithelial cells, as well as in nerve cells innervating the muscular layer of the gastrointestinal tube. Various epidermal sensory cells also displayed strong immunoreactivity. According to our findings and the results of several functional studies, it is suggested that in annelids cholecystokinin- and gastrin-like peptides may be involved in digestive regulation, sensory processes and central integrating processes.

Gastrin-like immunoreactive peptides were extracted from the gastric antrum of the American alligator (Alligator mississippiensis) and purified by fractionation using C18 Sep-Paks, Sephadex G-50, pH stable C8 reversed-phase HPLC, and C18 reversed-phase HPLC. Three major immunoreactive peaks were purified and found to correspond to 49, 45, and 34 residue peptides by microsequence analysis. The amino acid sequence of the largest peptide was DWLASLSQDQ KHLISKFLPH IYGELAN QEN YWQEDDALHD HDYPGWMDF-amide. The two smaller peptides corresponded to carboxyl-terminal 45 and 34 residue fragments of the 49 residue peptide. The putative proteolysis of the 49 residue peptide to the two shorter peptides occurs at cleavage sites that are unusual in biosynthetic processing. Mass spectral analysis confirmed the molecular weights that were predicted from the amino acid sequences, thus revealing the absence of any post-translational modifications, such as sulfation. Although the three alligator gastrins resemble mammalian cholecystokinin in having a tyrosine residue in the seventh position from the carboxyl terminus, this tyrosine is apparently nonsulfated as in turtle gastrin. When tested by radioreceptor assay, a synthetic replicate of alligator gastrin-49 exhibited a gastrin-like pattern of biological activity on mammalian CCK-A and CCK-B receptors. Comparison of the amino acid sequences of known peptides revealed that alligator gastrin is most similar to turtle gastrin (76% identical), followed by frog gastrin (51% identical), chicken gastrin (49% identical), and human gastrin (12% identical). These similarities closely reflect vertebrate phylogeny and support the hypothesis that functionally distinct gastrins evolved from CCK in early tetrapods. However, gastrin evolved via different mechanisms in the mammalian lineage (mechanism unknown) versus the amphibian and reptilian/avian lineages, in which two different single nucleotide base changes can account for the separate evolution of amphibian gastrin and reptilian/avian gastrin., (Copyright 1997 Academic Press. Copyright 1997 Academic Press)

Cholecystokinins from brain and small intestine of the bullfrog (Rana catesbeiana) and red-eared slider turtle (Pseudomys scripta) were isolated. The purifications were monitored by an antiserum specific for the common C-terminus of mammalian cholecystokinin and gastrin. The peptide structures were identified by sequence analysis of the intact peptides and proteolytic fragments, mass spectrometry, and amino acid analysis. Brain and small intestine of both species contained cholecystokinin-8 and substantial amounts of cholecystokinin-7. Furthermore, the small intestine of both frog and turtle contained a major fraction of the immunoreactive material as large peptides consisting of 69 residues and 70 residues, respectively. The structure for frog cholecystokinin-69 is ASSSAQLKPFQRIDGTSDQKAVIGAMLAKYLQTRKAGSSTGRYAVLPNRPVIDPTHRINDRDYMGWMDF .NH2 and the structure for turtle cholecystokinin-70 is VPSSAGQLKPIQRLDGNVDQKANIGALLAKYLQQARKGPTGRISMMGNRVQNIDPTHRINDRDYMGWMD F.NH2. All the isolated peptides were tyrosine sulfated at the seventh last residue. The peptides are highly similar to each other and to mammalian cholecystokinins (70% mutual identity and more than 50% identity with human cholecystokinin). Thus, they are clearly related to the known mammalian cholecystokinins. Both peptides include the monobasic and dibasic cleavage sites giving rise to cholecystokinins-33, -39, and -58 in mammals. However, only a small amount of turtle cholecystokinin-40 (corresponding to mammalian cholecystokinin-39) was isolated. This confirms that post-translational processing is highly species dependent. Recently, we isolated peptides from frog and turtle antrum. Following their origin they were named gastrins in spite of their C-terminal cholecystokinin-like structure. Thus, two different cholecystokinin/gastrin peptides exist in frog and turtle justifying the choice of two names. This finding of two members of the cholecystokinin/gastrin family in frog shows that the divergence of cholecystokinin and gastrin occurred simultaneously with or earlier than the appearance of amphibia during phylogenesis. Frog cholecystokinin and gastrin show sufficient similarity along the whole sequence to support the notion of a gene duplication of a common ancestor.

Chicken antrum was found to contain 7 nmol/g of carboxyamidated gastrin/CCK-like peptides. The predominant chicken gastrin (so named due to the antral origin) contained 53 amino acid residues: DWPEPPSQEQ QQRFISRFLP HVFAELSDRK GFVQGNGAVE ALHDHFYPDW MDF-NH2. Three smaller (less abundant) forms corresponded to the 30-, 21-, and 7-residue carboxyamidated C-terminal fragments. The major part was sulfated at the tyrosine residue in position seven from the C-terminus. A lower isoelectric point and abrupt termination of the sequencing suggest that some of the peptides had an isoAsp-Gly bond instead of an Asn-Gly bond. The three shorter forms were all derived from the precursor by post-Phe cleavages. This cleavage pattern suggests a processing enzyme specific for bonds between Phe and moderately hydrophobic residues.

Following the curative resection of a pancreatic gastrinoma in a cat, gastrin peptides were purified from the tissue and sequenced. The sequence of cat gastrinoma G17 (18-34) confirms the previously published sequence. The sequence of cat G34 (1-34) is reported for the first time. The NH2-terminal portion of cat G34 differs from that of dog by having a Q (Gln) for L (Leu) at position 10 from the NH2-terminus.

So far the only CNS neurons found to express gastrin have been hypothalamohypophyseal. Using a library of radioimmunoassays specific for different sequences of porcine progastrin in combination with chromatography and enzyme cleavages, 21 or 24 porcine cerebelli was now found to contain progastrin or its products. Sixteen cerebelli processed progastrin via glycine-extended intermediates to bioactive, carboxyamidated gastrin-17 and -34, half of the gastrins being tyrosine O-sulfated. The mean concentration of carboxyamidated gastrins was 0.8 pmol/g tissue (range less than 0.1-2.1 pmol/g), of glycine-extended intermediates 0.1 pmol/g (range less than 0.1-0.3 pmol/g) and of progastrin 0.4 pmol/g (range less than 0.1-1.0 pmol/g). The results show that the gastrin gene is expressed in more than a single region of the brain.

Binding of 125I-[Nle15]gastrin to albumin purified from porcine serum, from porcine gastric mucosal cytosol, and from bovine serum has been demonstrated by covalent cross-linking and ultracentrifugation. Binding was enhanced in the presence of Zn2+, Ni2+, Cu2+, Co2+, and Cd2+, but not Ca2+, Mg2+, or Mn2+. The best fit to the binding data for bovine serum albumin was obtained with a model assuming two nonequivalent binding sites. The affinity of both sites for gastrin was increased in the presence of 100 microM Zn2+ or Ni2+ ions. The highest association constant observed was 2.3 X 10(5) M-1 in the presence of 100 microM Zn2+ ions. The similarity of the Zn(2+)-dependence of binding for bovine and porcine serum albumins, despite the replacement of His3 by Tyr, suggested that the N-terminal metal ion-binding site was not involved. Although all gastrin affinities were reduced by 50% in the presence of 150 mM NaCl, the Zn(2+)-dependence of binding was retained. We therefore propose that the ternary complex of gastrin, Zn2+ ions, and albumin may play a physiological role in the serum transport of Zn2+ ions and in the uptake of Zn2+ ions from the lumen of the gastrointestinal tract.

Four gastrin/cholecystokinin-like peptides (G/CCK) which cross-react with a specific C-terminal gastrin/CCK antiserum have been isolated from the stomach of the marine crustacean Nephrops norvegicus. The molecular weight of the four peptides was estimated between 1000 and 2000 Da by molecular sieving. By radioimmunoassay, the cross-reactivity of these peptides with human gastrin 17-I was found to be around 0.03%. Pure peptidic fractions were recovered after four successive steps of HPLC. Amino-acid analysis suggested a similarity between the four peptides identified which may belong to a new family. A limited homology between the C-terminus of one Nephrops peptide and vertebrate G/CCK was found after sequencing. Two of the peptides exhibited secretagogue effects on crustacean isolated midgut glands. The Nephrops peptides, although structurally distinct from the vertebrate G/CCKs, appear to serve similar biological functions in crustaceans.

Gastrin, the primary hormonal mediator of postprandial gastric acid secretion, is produced from its precursor progastrin by a series of posttranslational processing reactions including dibasic residue cleavage and carboxyl-terminal alpha-amidation. Progastrin contains three dibasic cleavage signals, Arg57Arg58, Lys74Lys75, and Arg94Arg95, that appear to be cleaved differently in different tissues. Differential processing is a potential means by which the production of biologically active peptides may be regulated in a tissue-specific manner. To study these reactions further, we used the pZipNeo SV(X) retroviral vector to express human gastrin cDNA in a heterologous cell line (MTC 6-23) known to be capable of processing other peptide precursors. The psi 2 packaging cell line transfected with the gastrin cDNA-retroviral construct (pSVXgas) produced progastrin, but no substantial amounts of processed amidated gastrin were detected. amounts of processed amidated gastrin were detected. In contrast, MTC 6-23 cells infected with the viral stock obtained from the supernatant of pSVXgas-transfected psi 2 cells produced carboxyl-terminally amidated gastrin in all of its standard molecular forms, including sulfated and nonsulfated forms of tetratriacontagastrin (G-34), heptadecagastrin (G-17), and tetradecagastrin (G-14). These studies indicate that heterologous endocrine cell lines infected with a retroviral-peptide cDNA construct can serve as useful models for peptide hormone posttranslational processing.

The biological activity of natural chicken gastrins isolated from the "antrum"-like region of the chicken gut have been studied on gastric secretion in chickens, turkeys, and rats, pancreatic secretion in turkeys and rats, and gallbladder contraction in chickens and guinea pigs. Natural chicken gastrin was shown to be approximately 85% sulfated on the tyrosine that occurs at position 7 from the COOH-terminus. In both avian and mammalian systems, chicken gastrins were found to be potent stimulants of acid secretion but were virtually inactive as stimulants of pancreatic secretion and gallbladder contraction. Peptides with the COOH-terminal tetrapeptide amide of CCK and a sulfated tyrosine at position 7 from the COOH-terminus are usually potent stimulants of pancreas and gallbladder. However, although chicken gastrin has a CCK-like structure, it has a gastrin-like spectrum of biological actions. A proline immediately adjacent to the sulfated tyrosine may produce a steric effect that lowers the activity of chicken gastrin on pancreas and gallbladder. Evidently, then, the factors that determine specificity of action of CCK and gastrin are different in birds and mammals.

The predominant immunoreactive cholecystokinin (CCK) forms in acid extracts of rat intestine eluted from reverse-phase high-performance liquid chromatography columns in the positions of CCK-8, CCK-33/39, and CCK-58. Control experiments indicated that smaller CCK forms did not arise from artifactual degradation of large CCK forms. Less than 10% of CCK-8 added to and extracted from intestine was recovered in acid; CCK-8 could not be recovered by subsequent alkaline extraction, but subsequent urea extraction yielded approximately 25% of the added peptide. This suggests that CCK binds to proteins during acid extraction and that the preponderance of large CCK forms in acid extracts is not due to inhibition of CCK degradation but results from poor extraction of small CCK forms. No evidence for a CCK-22-like peptide was found in acid or subsequent urea extracts of rat intestine, suggesting CCK posttranslational processing in adult rats is like that in humans and dogs.

The precursor of the acid-stimulating hormone gastrin is processed in pyloric antral gastrin cells by steps involving sulfation, phosphorylation, cleavage, and amidation. We describe here changes in posttranslational processing in dogs with a surgically excluded antrum; in the preparation we used there was an intact pylorus but antral mucosa was excluded from the normal influence of the luminal contents. Three to five months after the operation, basal plasma gastrin increased from 30.1 +/- 4.0 to 66.1 +/- 16.1 pmol/l, and concentrations of gastrin in the excluded mucosa were 9.23 +/- 1.75 compared with 3.2 +/- 0.56 nmol/g in control antral mucosa. Calculations based on the metabolic clearance rate and plasma and tissue gastrin concentrations suggest two-fold lower fractional release rates from the excluded G-cells compared with normal G-cells. Radioimmunoassay of tissue extracts using antisera specific for the extreme COOH-terminus of progastrin, for glycine-extended G-17, and for the COOH-terminus of G-17, combined with gel filtration and ion exchange chromatography, indicated normal endopeptidase cleavage of progastrin. However there was significantly reduced phosphorylation of the COOH-terminal tryptic fragment of progastrin, and there was also decreased conversion of Gly-extended intermediates to the biologically active COOH-terminally amidated forms of gastrin. Thus, in spite of hypergastrinaemia, the excluded antral mucosa showed evidence of decreased secretory rates associated with decreased progastrin phosphorylation and amidating enzyme activity. The results suggest that contact of antral mucosa with the luminal contents is able to modulate the posttranslational processing of progastrin and so determine the production of biologically active hormone.

Urinary and plasma gastrin immunoreactivities in normal subjects were studied by radioimmunoassays using three region-specific antisera. Urinary excretion of NH2-terminal big gastrin immunoreactivity (NT G-34-IR) in the fasting state (0.79 +/- 0.17 pmol/kg/h, mean +/- SE) was several hundred times as much as that of either COOH-terminal gastrin or gastrin/cholecystokinin immunoreactivity. Urinary NT G-34-IR increased significantly after feeding, and correlated closely with integrated plasma NT G-34-IR. Renal clearance of NT G-34-IR was 62.4 +/- 7.9 ml/min and about one hundred times greater than that of any other gastrin immunoreactivities, indicating that there exist different catabolic pathways of gastrin peptides in the kidney. Gel filtration of urine extract revealed that a single giant peak of NT G-34-IR eluted in a later position than NH2-terminal heptadecapeptide of big gastrin. These results suggest that most of plasma NT G-34-IR is excreted in urine, and urinary excretion of NT G-34-IR reflects well-integrated plasma NT G-34-IR. Therefore, urinary NT G-34-IR may serve as a feasible indicator for gastrin secretion.

1. "Little" gastrins from most mammalian species are 17 amino acid peptides and the precursor "big" gastrins are 34 amino acid peptides. 2. "Little" gastrins of the New World hystricomorphs, guinea-pig and chinchilla, are 16 amino acid peptides due to deletion of a glutamic acid in the region 6-9 from their NH2-terminus and the corresponding "big" gastrins are 33 amino acid peptides. 3. Antral gastrins from the opossum, a New World marsupial, have a glutamic acid deletion in the same region as the hystricomorph gastrins. 4. Opossum "big" gastrin is a 33 amino acid peptide with the following sequence: less than ELGPQDLPYLTADLSKKQGPWLEEEEAYGWMDF#.

The present studies were undertaken to confirm reports of high concentrations of the C-terminal tetrapeptide of gastrin in hog antral mucosa. A method was developed whereby synthetic tetrapeptide added to boiling water extracts of hog antral mucosa could be purified to homogeneity by adsorption to Amberlite XAD2 resin, ion exchange chromatography on DEAE cellulose, and reverse phase HPLC. The product had the amino acid composition of gastrin tetrapeptide. When the same method was used on antral mucosa without prior addition of synthetic G4, several small peaks of material with C-terminal immunoreactivity could be found in DEAE column eluates but none could be unequivocally identified as the tetrapeptide. In the same column runs there was a relatively large peak of immunoreactivity eluting later than the tetrapeptide. This material was purified to homogeneity by HPLC and on the basis of its amino acid composition and sequence was identified as the C-terminal hexapeptide of gastrin.

Two peptides which copurified from a human gastrinoma were found to correspond to the amino acid sequence deduced for the amino terminal portion of human and porcine progastrin. The sequence of peptide A is Ser-Trp-Lys-Pro-Arg-Ser-Gln-Gln-Pro-Asp-Ala-Pro-Leu-Gly-Thr-Gly-Ala-Asn- Arg-Asp-Leu-Glu-Leu which is identical to an amino terminal portion of human progastrin. The sequence of peptide. B is identical to that of peptide A except it is missing the first five amino acids. If peptide A corresponds to the amino terminus of progastrin, the signal peptidase cleaves at an Ala-Ser bond.

The heptadecapeptide form the rabbit gastrin was extracted from 16 rabbit antra and purified by a combination of DEAE Sephadex, C-18 SEP PAK cartridges, fast performance liquid chromatography (FPLC) and reverse phase high pressure liquid chromatography (HPLC) steps. After the HPLC purification, a sharp, single peak of gastrin-like immunoreactivity was detected that had the same absorption to immunoreactivity ratio as human gastrin. An amino terminal pyrrolidone carboxylic acid blocking group was removed by incubation with pyrrolidone carboxylic peptidase. The amino acid analysis, microsequence analysis and mass spectrometry all confirmed the structure of rabbit gastrin being pQGPWLQEEEEAYGWMDFamide. This sequence is identical to human gastrin-17 except for glutamine in position 6 which replaces glutamate in human gastrin. Both sulfated and unsulfated rabbit gastrin-17 were characterized by mass spectrometry.

Heptadecapeptide gastrins (G17) have been purified and sequenced from a variety of species. However, progastrin (G34) sequences have been determined only for pig and human from purified peptides and for rat from cDNA. Since G34 in most species accounts for only approximately 5% of total antral gastrin, micropurification techniques must be employed to avoid the need for large quantities of antral tissue. Efficient purification methodology yielded 1.5 and 1.3 nmol of G34 from the antrum of a single goat and of a single dog, respectively. The N-terminal pyroglutamyl residues were enzymatically removed and the peptides were sequenced through to the proximity of their COOH-termini. The COOH-terminal sequences of goat and dog G34 were confirmed by sequencing the corresponding deblocked G17 from each animal. The previously published dog G17 sequence was shown to be incorrect. The sequences for dog and goat G34 are: Dog less than ELGLQGPPQLVADLSKKQGPWMEEEEAAYGWMDF# Goat less than ELGLQDPPHMVADLSKKQGPWVEEEEAAYGWMDF# Dog and goat gastrins differ in 3 sites in the 17 amino acid NH2-terminus and only a single site in G17 (the sites of differences are underlined). The ratio for sulfated to non-sulfated antral G17 is 9:1 for the goat and 1:9 for the dog.

The pathogenesis of peptic ulceration cannot be explained by an abnormal capacity to secrete acid, for ulcers develop in patients who secrete acid normally. Duodenal and gastric ulcers have a common cause. The location of an ulcer in each individual is primarily determined by his capacity to secrete acid at that time. There is a difference between the mechanisms which heal an ulcer and cure a patient of his disease. Procedures that reduce an individual's capacity to secrete acid, heal an ulcer by moving the focus of the ulcerogenic forces to a more proximal site. It is necessary to remove an antral factor if in addition the patient is to be cured of his disease. It is postulated that this antral factor is the gastrin (G17) which is released in abnormal amounts into gastric juice in patients with ulcers and with gastrinomas. The abnormal amount of G17 in gastric juice may be responsible for releasing abnormal amounts of G34 into the circulation from the duodenum and from gastrinomas. The abnormal release of gastrin develops as a result of an impaired response to duodenal acidification manifest in part by an impaired release of secretin. It is postulated that the abnormal stimulation of antral gastrin release may on occasions give rise to antral G-cell hyperplasia, and that the abnormal secretion of gastrin into gastric juice may on occasions give rise to gastrinomas. These abnormalities may cause ulcers by producing an uncontrolled secretion of acid and an abnormal exposure to bile.

The procedures of radioimmunoassays for gastrin are presented in this paper. Techniques are described and evaluated with special reference to preparation of radioiodinated gastrin, purification of monoiodinated gastrin, characterisation of the immunochemical properties of radiolabelled gastrin, raising and characterisation of specific antibodies, incubation conditions, separation of bound and free hormone and treatment of data. Factors contributing to discrepancies of radioimmunoassay results and problems encountered are discussed. Methods employed by the authors in gastrin radioimmunoassay are described. Commercially available gastrin kits are also compared.

A successful synthesis of human gastrin I in 60% overall yield based on the first residue attached to a benzhydrylamine-resin was achieved by the stepwise solidphase method. The synthesis was carried out on a 1% crosslinked polystyrene support, using conventional benzyl-based side chain protecting groups and final deprotection with different acidic protocols. Several improvements in this general approach were applied, including new scavengers, new resin attachment and especially a new technique that allows the strong acid reactions to occur by an SN2 mechanism.

A pair of gastrin tridecapeptides (;minigastrins') have been isolated from Zollinger-Ellison tumour tissue; they correspond to the fragment 5-17 of the heptadecapeptides isolated from the same source. In one (type II) the tyrosine residue present is sulphated, in the other (type I) it is not. The proportion of types I and II is approximately 2:1 similar to that for the ;big' gastrins and the heptadecapeptides isolated from the same source and from human antral mucosa. Both minigastrins are potent stimulants of gastric acid secretion. Immunological evidence exists to indicate that both are present as circulating forms of the hormone gastrin in patients with the Zollinger-Ellison syndrome and in fed normal subjects.

Samples of rat striatum and synthetic sulphated cholecystokinin octapeptide were extracted by different procedures and the solubilised cholecystokinin-like immunoreactivity analysed by gel filtration and ion-exchange chromatography. Ice-cold 90% methanol extraction gave the greatest recovery of tissue immunoreactivity without any major modification of the extracted components. The 33-amino acid form of cholecystokinin was poorly recovered by this extractant . Boiling water or a combined boiling water/acetic acid extraction gave efficient recovery of tissue immunoreactivity but chemically modified a substantial part of the octapeptide-like component. Boiling in acetic acid alone also produced this modification but in addition resulted in poor recovery of the octapeptide-like component. The combined water/acetic acid extraction gave reasonable to good recovery of all added cholecystokinins and gastrins, including the 33-amino acid form of cholecystokinin. Ice-cold 0.1 M HCl was less efficient than 90% methanol at solubilising tissue immunoreactivity and resulted in a substantial modification of the octapeptide component distinct from that produced by boiling extractions, possibly desulphation . The results show that more than one extraction procedure is needed to study all the cholecystokinin components in brain tissue and demonstrates the necessity of using at least two chromatographic systems for such studies.

It was confirmed that histamine, cholinergic drugs and gastrin separately produce acid secretory effects of isolated parietal cells. However, the acid secretory response of isolated parietal cells to gastrin stimulation is known to be weaker than that to histamine or cholinergic drugs, which is different from the acid secretory response found in vivo studies. Tetragastrin and pentagastrin were less potent than histamine and carbachol in stimulating acid secretion in our previous study performed using parietal cells isolated from guinea-pigs. In this study, the effect of gastrin extracted from the pyloric antrum of the guinea-pig on parietal cells isolated from the guinea-pig was investigated based on the assumption that the acid secretory response of isolated parietal cells to gastrin stimulation will be especially good if gastrin isolated from the same species is used.

The history of the discovery of gastrointestinal hormones and the modern methods of their isolation are discussed in the first chapter. The origin of structural relationships of gastrointestinal hormones and the possible ways of evolution are analyzed in the second chapter. The methods used for isolation and identification of secretin and cholecystokinin are dealt with in detail; the isolation of vasoactive intestinal peptide, gastrin, "gastric inhibitory peptide" and enteroglucagon is discussed but briefly in the first chapter. The second chapter is of speculative character. It deals with the structural resemblances between gastrin-type (gastrin, cholecystokinin-pancreozyzimin, caerulein) and secretin-type (secretin, vasoactive intestinal peptide, glucagon, "gastric inhibitory peptide") hormones. The resemblances are explained by different evolutionary mechanisms.

Gastrin antisera were raised by immunization of rabbits or guinea pigs with synthetic human gastrin I conjugated to bovine serum albumin by carbodiimide. Radioiodination of SHG: 2-17 was performed by the chloramine T-method and by an enzymatic procedure. AE-cellulose was used to get a monoiodinated tracer hormone. Antibody reactions with the different forms of gastrin and with CCK-PZ was characterized. Precision (VK = 6-8%) and reproducibility (VK less than 15%) of the gastrin values were comparable to the insulin assay. Gastrin stimulates the parietal cell and has trophic effects on gastric mucosa. Hypergastrinaemia in combination with hypersecretion exhibits clinical significance in patients suffering from Zollinger-Ellison-syndrome or excluded antrum-syndrome which are due to autonomous gastrin release. Some findings suggest a pathogenetic role of gastrin in duodenal ulcer disease. Those disease in which gastrin determinations are of clinical value are discussed.

Biosynthetic incorporation of labeled amino acids into the major gastrins in rat antrum, Component I, gastrin 34 ("big" gastrin), and gastrin 17-like peptides ("little" gastrin) was demonstrated after in vitro incubation of antral mucosal slices. The antral gastrin synthesis was stimulated by fundectomy, which ablates gastric acid secretion and thereby increases the gastrin concentration in plasma 20-fold. Tyrosine O-sulfation of gastrin was also demonstrated by incorporation of [35S]sulfate. Sulfate-labeled peptides precipitated by gastrin antisera coeluted with sulfated gastrin immunoreactivity on ion-exchange chromatography. Aryl-sulfatase treatment removed the [35S]sulfate from labeled gastrin and resulted in a change in elution pattern of the immunoreactivity to that of the unsulfated gastrins. The presence of 35S-labeled tyrosine O-sulfate residues was directly demonstrated by two-dimensional thin-layer electrophoresis after alkaline hydrolysis of [35S]sulfate-labeled gastrin. All the antral gastrins incorporated [35S]sulfate.

Only two 34 amino acid gastrin precursors have previously been purified and sequenced, those of pig and of human. The larger molecular form generally accounts for only about 5% of antral gastrin in most species. This report describes the purification of "big gastrin" from guinea pig (GP) antra. Two hundred grams of antra were defatted with acetone and the acetone cakes were extracted with 0.1M NH4HCO3. The extract was concentrated by adsorption onto and batch elution from QA-52 anion exchange cellulose. Fractionation on a mu Bondapak C18 cartridge resolved 3.6 nmol of the larger peptide from 61 nmol of immunoreactive gastrin in the original extract. Two additional HPLC steps brought the peptide to final purity. GP big gastrin is a 33 amino acid peptide with the following sequence: less than ELGPQVPAHLRTDLSKKQGPWAEEEAAYGWMDF# The GP peptide is different from pig G34 in 6 of the 17 NH2-terminal amino acids as well as in the previously reported deletion of a glutamic acid in the COOH-terminus.

Antibodies to the extreme C-terminal tryptic (nona-) peptide fragment of porcine progastrin have been used in radioimmunoassay to identify progastrin fragments in dog, ferret and pig antral mucosa extracts and to monitor their purification. In addition to previously characterised phosphorylated and unphosphorylated C-terminal tryptic peptides of porcine progastrin a minor form corresponding to the C-terminal octapeptide (i.e. des-Ser C-terminal nonapeptide) was isolated and characterised. The latter form together with phosphorylated and unphosphorylated forms of the nonapeptides were also isolated and chemically characterised from dog antrum, and the unphosphorylated nonapeptide was characterised from ferret antrum. The primary amino acid sequences of the dog, ferret and pig nonapeptides were identical. In ferret the unphosphorylated nonapeptide predominated, and in dog the phosphorylated form predominated; in pig both forms of the nonapeptide were well represented. Intact progastrin was identified in gel filtration eluates of extracts of all 3 species, but occurred only in relatively low concentrations. The nonapeptides did not stimulate acid secretion in the conscious gastric fistula rat and they did not modify the acid response to G17. Phosphorylation of progastrin-derived peptides is evidently well conserved across a range of species even though there appear to be differences in the relative proportions of phosphorylated and unphosphorylated forms.

Gastric acid secretion by the parietal cell is a single digestive process involving a continuous interplay between nervous and hormonal stimuli. Gastric acid hypersecretion and hypergastrinemia may represent pathologic disturbance of the normal "gastric phase" of acid secretion (excluded antrum syndrome) or abnormal gastrin secretion from a nongastric source as in the Zollinger-Ellison syndrome. Diagnosis of these two syndromes preoperatively is dependent on immunoassay for serum gastrin. A fall in serum gastrin level after the injection of secretin will distinguish the excluded antrum syndrome from the Zollinger-Ellison syndrome. Which hormone or hormones cause the acid hyposecretion of the watery diarrhea hypokalemia achlorhydria syndrome is still uncertain. Potential candidates include secretin, glucagon (alone or combined with gastrin), vasoactive intestinal peptide and gastric inhibitory polypeptide. Secretin has undergone trials as therapy in peptic ulcer whereas glucagon is under investigation for the treatment of acute pancreatitis because of its dual actions as (1) an enterogastrone and (2) an inhibitor of pancreatic secretion.

The fluorogenic chymotrypsin substrate N alpha-(4-carboxybutyryl)-L-phenylalanine (4-methyl-7-coumaryl)amide was converted to a thiol-containing compound via its condensation at the carboxyl function with cystamine followed by reduction of the resulting disulfide compound to a cysteamine derivative. By subsequent reaction of the thiol group with N alpha-maleoyl-beta-alanyl-human-little-gastrin-I-[2-17] a fluorogenic substrate-labeled gastrin, fully immunoreactive against antigastrin antisera, was obtained. This tracer was then applied for developing a fluorescence immunoassay based on separation of bound and free tracer followed by chymotryptic digestion of the fluorogenic substrate in the supernatant. The fluorescence intensity of the extracted fluorophore i.e., 7-amino-4-methyl-coumarin, was found to monitor gastrin concentrations in a reproducible manner. With the model peptide hormone human little-gastrin-I the sensitivity of this alternative immunoassay procedure was well documented.

A peptide identical in structure to the carboxyl-terminal flanking nonapeptide of rat progastrin, predicted by cDNA sequence, was synthesized. The synthetic peptide was used for production of a rabbit antiserum. This antiserum was used to develop a radioimmunoassay specific for rat carboxyl terminal flanking peptide. This assay was used to monitor the purification of immunoreactivity from rat antral extracts. Gel permeation, anion exchange and reverse phase chromatography steps resulted in a single absorbance peak associated with the carboxyl terminal flanking peptide immunoreactivity. The purified peptide eluted in the same position as the synthetic peptide during all three types of chromatography. This material was shown to be identical in mass to Ser-Ala-Glu-Glu-Glu-Asp-Gln-Tyr-Asn, the predicted sequence of the carboxyl terminal nonapeptide of rat progastrin.