A Possible Flow Cytometry-Based Viability and Vitality Assessment Protocol for Pathogenic Vibrio cholerae O1 and O139 Postexposure to Simulated Gastric Fluid.

During the intake of contaminated water, for diarrheal disease to occur, Vibrio cholerae must survive through the bactericidal digestive secretion of gastric fluid during passage through the stomach. Determining the viability of these bacteria is challenging, with the standard cultivation methods for viability being time-consuming and unable to culture cells that may still function accordingly. This study assessed the use of enzyme action and membrane integrity as alternatives for determining vitality and viability, respectively, in gastric acid-stressed pathogenic Vibrio cholerae O1 and O139, using fluorescent probes thiazole orange (TO) for viability based on membrane integrity, carboxyfluorescein diacetate (CFDA) with acetoxymethyl ester (AM) for vitality based on metabolic activity, and propidium iodide (PI) for cell death/damage due to loss of membrane integrity, with flow cytometry. Simulated gastric fluid-treated bacterial cells were labelled with blends of TO+PI and CFDA-AM+PI, and these stained cells were separated into heterologous populations based on their fluorescence signal. The gastric acid exposed cells presented with high green fluorescence signals after staining with the metabolic probe CFDA-AM, which indicated intact (live) cells due to being metabolically active, whereas when the same cells were stained with the DNA probe (TO), these appeared to be in a "stressed state" due to loss of membrane integrity. Damaged cells (dead cells) showed high red fluorescence levels after staining with PI probe. The use of flow cytometry with fluorescent probes is a favorable method for evaluating the vitality and viability of bacteria when cells are labelled with a combination of CFDA-AM+PI.

Consumption of medicines used for gastric acid-related disorders in Australia and South Korea: a cross-country comparison.

PURPOSE: The study's aim was to compare the use of proton pump inhibitors (PPIs), histamine 2-receptor antagonists (H2RAs) and mucoprotective medicines (MPs) used for gastric acid-related disorders (GARD) in Australia and South Korea (Korea) from 2004 to 2017. METHODS: Prescription data for PPIs, H2RAs and MPs for Australian outpatients were extracted from the Australian Statistics on Medicines annual reports, with dose-specific and expenditure data obtained from Medicare. Similar data were obtained from Korean National Health Insurance Service claims data. We analysed the volume and expenditure of medicines use annually using the defined daily dose per 1,000 population per day. We calculated which medicines accounted for 90% of use and estimated the proportions of use for low- and high-dose PPIs. RESULTS: While total utilisation for GARD medicines increased over time in both countries, patterns of use differed. Overall, use was somewhat higher in Australia but increased more rapidly in Korea. PPIs were used more extensively in Australia, while more MPs and H2RAs were used in Korea. Expenditure and use of low-dose PPIs is escalating in Korea. CONCLUSION: There were substantial differences in the use of GARD medicines in Australia and Korea over 14 years. Both countries face similar challenges to promote rational medicines use and contain medical care costs. The discrepant prescribing patterns can be attributed to differences in healthcare systems, pharmaceutical policies and demographics. This study provides a baseline to influence more rational use of these medicines. It provides insight into medicines policies for other countries that face similar challenges.

Non-invasive method for the assessment of gastric acid secretion.

Methods for the measure of gastric acid secretion include invasive and non-invasive tests. The gold-standard to measure the acid output is the collection of gastric after in basal condition (Basal Acid Output, B.A.O.) and after an i.m. injection of pentagastrin (Maximal Acid Output, M.A.O.). However, direct measurement of gastric acid production is out of order in clinical practice, but many GI symptoms are claimed to be related with acid disorders and empirically cured. Hypochlorhydria is associated with precancerous conditions such as chronic atrophic gastritis (CAG). Acid measurement with non-invasive methods (pepsinogens) is supported by international guidelines.

Assessment of IgE Reactivity of ß-Casein by Western Blotting After Digestion with Simulated Gastric Fluid.

Cow's milk allergy is defined as an immunologically mediated adverse reaction to cow's milk proteins and it is usually, along with hen's egg allergy, the first food allergy identified in childhood.One of the main aspects to consider when evaluating the allergenic potential of food proteins is the effect of gastric digestion. It is known that allergens are usually able to survive the harsh acidic environment of the stomach, tolerate the presence of surfactants, and resist digestion by pepsin. They might also be digested into high molecular weight peptide fragments, which retain the same, or sometimes increased, IgE-binding. In this respect, western blotting is a highly sensitive and efficient technique that we have used to detect IgE-binding to the digests of milk and egg proteins. Given the importance of the resistance of food proteins to gastric digestion in their capacity to modulate the immune response, we describe in this chapter the assessment of IgE reactivity of a relevant cow's milk allergen, ß-casein, by western blotting after simulated digestion under relevant physiological conditions.

Low cost of gastric acid secretion during digestion in ball pythons.

Due to their large metabolic responses to digestion (specific dynamic action, SDA), snakes represent an interesting animal group to identify the underlying mechanisms for the postprandial rise in metabolism. The SDA response results from the energetic costs of many different processes ranging over prey handling, secretions by the digestive system, synthesis of enzymes, plasticity of most visceral organs, as well as protein synthesis and nitrogen excretion. The contribution of the individual mechanisms, however, remains elusive. Gastric acid secretion has been proposed to account for more than half of the SDA response, while other studies report much lower contributions of the gastric processes. To investigate the energetic cost of gastric acid secretion, ball pythons (Python regius) were fed meals with added amounts of bone meal (up to 25 g bone meal kg(-1) snake) to achieve a five-fold rise in the buffer capacity of the meals. Direct measurements within the stomach lumen showed similar reduction in gastric pH when buffer capacity was increased, but we found no effects on the rise in oxygen consumption over the first three days of digestion. There was, however, a slower return of oxygen consumption to resting baseline. We conclude that gastric acid secretion only contributes modestly to the SDA response and propose that post-absorptive processes, such as increased protein synthesis, are likely to underlie the SDA response.

Prediction of pH dependent absorption using in vitro, in silico, and in vivo rat models: Early liability assessment during lead optimization.

Weakly basic compounds which have pH dependent solubility are liable to exhibit pH dependent absorption. In some cases, a subtle change in gastric pH can significantly modulate the plasma concentration of the drug and can lead to sub-therapeutic exposure of the drug. Evaluating the risk of pH dependent absorption and potential drug-drug interaction with pH modulators are important aspects of drug discovery and development. In order to assess the risk around the extent of decrease in the systemic exposure of drugs co-administered with pH modulators in the clinic, a pH effect study is carried out, typically in higher species, mostly dog. The major limitation of a higher species pH effect study is the resource and material requirement to assess this risk. Hence, these studies are mostly restricted to promising or advanced leads. In our current work, we have used in vitro aqueous solubility, in silico simulations using GastroPlus™ and an in vivo rat pH effect model to provide a qualitative assessment of the pH dependent absorption liability. Here, we evaluate ketoconazole and atazanavir with different pH dependent solubility profiles and based on in vitro, in silico and in vivo results, a different extent of gastric pH effect on absorption is predicted. The prediction is in alignment with higher species and human pH effect study results. This in vitro, in silico and in vivo (IVISIV) correlation is then extended to assess pH absorption mitigation strategy. The IVISIV predicts pH dependent absorption for BMS-582949 whereas its solubility enhancing prodrug, BMS-751324 is predicted to mitigate this liability. Overall, the material requirement for this assessment is substantially low which makes this approach more practical to screen multiple compounds during lead optimization.

Simulated gastrointestinal digestion of Pru ar 3 apricot allergen: assessment of allergen resistance and characterization of the peptides by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry.

RATIONALE: Non-specific lipid transfer proteins (ns-LTPs) are major food allergens of the Rosaceae family. The severity of allergic reactions often relates to resistance of the allergen to digestion. Thus, it is important to evaluate the digestibility of these proteins and characterise the peptides generated in the gastrointestinal tract. METHODS: Simulated gastrointestinal digestion of purified allergen Pru ar 3 was performed using pepsin for the gastric phase in aqueous HCl at pH = 2 and chymotrypsin and trypsin for the intestinal phase in aqueous NH(4)HCO(3) at pH = 7.8. The peptide mixture obtained was analysed by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry (UPLC/ESI-MS). Peptide sequences were identified by comparing their molecular mass to that obtained by in silico digestion, and were confirmed by the ions obtained by in-source fragmentation. Semi-quantification was performed for the intact protein by comparison with internal standards. RESULTS: The resistance to gastrointestinal digestion of Pru ar 3 allergen was evaluated to be 9%. This value is consistent with that found for grape LTP, but much lower than the resistance found for peach LTP (35%). All the peptides generated were identified by ESI-MS on the basis of their molecular mass and from the ions generated from in-source fragmentation. Apart from low molecular mass peptides, five high molecular mass peptides (4500-7000 Da) containing disulphide bridges were identified. ESI-MS of the intact protein indicated a less compact folded structure when compared to that of the homologous peach LTP. CONCLUSIONS: An extensive characterisation of the peptides generated from the gastrointestinal digestion of Pru ar 3 allergen was performed here for the first time via UPLC/ESI-MS analysis. The digestibility of the allergen was evaluated and compared with that of other LTPs, demonstrating that only a small amount of undigested protein remains, and that specific proteolytic action involves immunodominant epitopes. These data might explain the lower allergenicity of apricot LTP compared to peach LTP, despite their high sequence homology.

Flow cytometric assessment of the protectants for enhanced in vitro survival of probiotic lactic acid bacteria through simulated human gastro-intestinal stresses.

The aim of this study was to apply flow cytometric (FCM) analysis to assess the use of sucrose and lecithin vesicles for the protection of probiotic lactic acid bacteria in response to the challenge of gastric acidity and bile salts. FCM analysis in combination with fluorescent probes carboxyfluorescein (cF) and propidium iodide was used to reveal the physiological heterogeneity in the stressed bacteria population. Three subpopulations (intact, stressed, and damaged) were differentiated by FCM in all six examined strains. Significant changes were observed in the presence of the selected protectants. The addition of 20 mM sucrose in the simulated gastric fluid substantially increased the number of intact cells over 20 folds and reduced the damaged subpopulation by half. The presence of 2 % (w/v) lecithin vesicles was shown to protect 50 % more intact cells from the challenge of bile salts. The improved survival as evaluated by FCM analysis was further assessed for the proliferation capacity by sorting a number of cells from each subpopulation on nutrient agar plate. The result confirmed conformity between the proliferation-based cultivability and the probe-indicated viability in the samples of the intact and the damaged subpopulations. However, it also revealed the complexities of the stressed (injured) subpopulation. In conclusion, FCM analysis confirmed that the selected protectants could improve the survival of the probiotic strains in the simulated GI environments. The FCM analysis also proved to be a useful analytical tool for the probiotics research.

Prevention of gastrointestinal bleeding due to stress ulceration: a review of current literature.

Our objective was to audit our current stress ulcer prophylaxis protocol (routine prescription of ranitidine and early enteral feeding) by identifying whether routine prescription of histamine-2 receptor antagonists or proton pump inhibitors as prophylaxis against stress-related mucosal disease and subsequent upper gastrointestinal bleeding is supported in the literature. We also aimed to ascertain what literature evidence supports the role of early enteral feeding as an adjunctive prophylactic therapy, as well as to search for burn-patient specific evidence, since burn patients are at high risk for developing this condition, with the aim of changing our practice. PubMed and Cochrane databases were searched for relevant articles, yielding seven randomised controlled trials comparing histamine-2 receptor antagonists and proton pump inhibitors in the prevention of upper gastrointestinal bleeding associated with stress-related mucosal disease and three separate meta-analyses. Despite level 1 clinical evidence, no significant difference in efficacy between histamine-2 receptor antagonists and proton pump inhibitor treatment groups was demonstrated. No significant difference was demonstrated in the incidence of nosocomial pneumonia between the two drugs given in this indication. However, enteral feeding was found to be safe and effective in preventing clinically significant upper gastrointestinal bleeding. Patients able to tolerate feeds demonstrated no additional benefit with concomitant pharmacological prophylactic therapy. Since all burn patients at the Royal Adelaide Hospital are fed from very early in their admission, the literature suggests that we, like our intensive care unit colleagues, should abolish our reliance on pharmacological prophylaxis, the routine prescription of which is not supported by the evidence.

Functional and histologic assessment of rat gastric mucosa after chronic treatment with sulphurous thermal water.

The effect of a chronic (4 weeks) administration of sulphurous thermal water on gastric acid secretion and mucosal defense was investigated in rats. Animals were randomized to receive daily intake of tap water or of thermal water obtained from a local spa center (Tabiano, Parma, Italy). Rats were followed for one month as for water and food consumption, body weight and general conditions. At the end of the watering period, the following study protocols were carried out: (a) study of basal and stimulated gastric acid secretion under general anesthesia, and (b) study of the gastric mucosal resistance against the damage induced by ethanol and indomethacin in conscious rats. Basal acid secretion and the acid response to pentagastrin or to histamine were similar in rats assuming ordinary drinking water or thermal water. As for resistance to gastric damage, histological, but not macroscopic, evaluation revealed that rats which assumed thermal water were slightly more resistant to the gastrolesive effect of ethanol (either absolute or diluted). Again, when indomethacin was used as a noxious stimulus, no difference was noted between the two groups as for macroscopic damage; only a nonsignificant reduction of damage was observed histologically in stomachs of rats assuming thermal water. In conclusion, these results indicate that chronic treatment of rats with thermal water, rich in sulphur compounds, may have only minimal effects on the rat gastric mucosa and did not significantly affect mucosal defense mechanisms. The observed tendency to gastroprotection would possibly need further investigation with longer periods of administration.

Quantitation of (beta)N-Alkanoyl-5-hydroxytryptamides in coffee by means of LC-MS/MS-SIDA and assessment of their gastric acid secretion potential using the HGT-1 cell assay.

A straightforward stable isotope dilution analysis (SIDA) for the reliable quantitative determination of (beta)N-C(18:0)- to (beta)N-C(24:0)-alkanoyl-5-hydroxytryptamides (C5HTs) in coffee powder and beverages by means of LC-MS/MS was developed. The developed SIDA showing accuracy values of 92.6-107% and precision between 0.5 and 7% relative standard deviation for the individual derivatives allowed the sensitive and selective quantification of the target compounds in coffee beverages. Depending on the type of coffee, quantitation revealed C5HT levels between 65 and 144 microg/L in filtered coffee and up to 3500 microg/L in a French press beverage, thus indicating that about 0.3 or 7.2% of the C5HTs were extracted from the coffee powder into the beverage when using the cellulose filter method or the French press, respectively. To estimate the potential contribution of the C5HTs to the phenomenon of stomach irritation after ingestion of coffee brew, in vitro cell studies were performed with pure individual 5-hydroxytryptamides and a mixture of the predominating derivatives in ratios matching those found in coffee. All substances tested induced a decrease in the intracellular proton index (IPX) coined as an indicator of stomach acid secretion. While the biomimetic C5HT mixture was highest in its inducing effect, the individual stearic acid, oleic acid, and linoleic acid 5-hydroxytryptamide did not differ significantly from each other, but showed a less pronounced effect compared to arachinic acid 5-hydroxytryptamide. In conclusion, not the grade of saturation seems to determine the C5HT's mode of action in driving the stomach acid secretion, rather than the fatty acid chain length.

Costs and risks in the management of patients with gastric acid hypersecretion.

GOALS: To define both risks and costs of optimal care of patients with gastric acid hypersecretion. BACKGROUND: The management of Zollinger-Ellison syndrome and other gastric acid hypersecretory disorders remains challenging. The optimal strategy for follow-up including gastric acid analysis, laboratory studies, and endoscopy is unknown but important given the potential complications from uncontrolled acid secretion. STUDY: Over the last 18 years, patients with gastric acid hypersecretory disorders have been followed prospectively with gastric acid analysis and endoscopy titrating oral lansoprazole and evaluating for complications. Protocol driven charges were calculated using the most recent information available. RESULTS: After 1 year of treatment optimization, 19 of 67 patients had 43 relapses, (once only in 10 patients). Risk markers for relapse included: (1) antrectomy, 67% relapsed versus 21% in unoperated patients; (2) basal acid output >5 mmol/h (risk=5.17); and (3) poor compliance. On treatment, 79% of 58 intact patients (excluding antrectomy) were lesion-free; 11% had only 1 relapse. Thus 90% were well managed with optimized lansoprazole alone. Protocol driven charges exceeded $25,000 the first year and $7000 annually thereafter. CONCLUSIONS: Relapse is infrequent and generally mild with acid secreting status closely monitored. The ideal strategy to balance costs and testing awaits further study.

Rabeprazole: a review of its use in the management of gastric acid-related diseases in adults.

Rabeprazole (Aciphex, Alfence, Pariet) is a proton pump inhibitor (PPI) used for the treatment of adults with conditions requiring a reduction of gastric acid secretion such as erosive or ulcerative gastro-oesophageal reflux disease (GORD), non-erosive reflux disease (NERD), duodenal and gastric ulcers, and pathological hypersecretory conditions including Zollinger-Ellison syndrome (ZES). It is also used as part of combination therapy for the eradication of Helicobacter pylori, a pathogen frequently implicated in the development of gastric and duodenal ulcers. Rabeprazole has a well established efficacy and safety profile in the treatment of gastric acid-related diseases. Rabeprazole is a useful, well tolerated and cost-effective option for the treatment of GORD, NERD, peptic ulcer and other gastric acid-related diseases (including ZES), and provides an appropriate alternative to other currently available PPIs, with the added benefits of having a consistent efficacy profile and low drug interaction potential due to its predominantly nonenzymatic metabolism.

[Comparative assessment of the strength properties of the mucous membrane of the stomach in patients with peptic ulcer and the effect of quamatel on ulcer cicatrization in experiment].

The article reviews the research work of the authors on the strength properties of the mucous membrane of the stomach in patients with peptic ulcer and in experiment with quamatel application. Experiments were performed in laboratory animals and resected stomachs of patients with duodenal or stomach ulcer and complications requiring scheduled surgical treatment. The results of the research into the maximum tension (durability) of the stomach mucous membrane, antrum, and the periulcer area are described. For both localizations of the ulcer, the mucous membrane of the antrum was found to exhibit the least durability, while the highest durability was exhibited by the mucous membrane of the periulcer area. In the case of bulbar ulcer, the durability of the mucous membrane was shown to decrease with an increase in the number of aggravations. An inverse relationship between the strength properties and the intensity of hydrochloric acid production was observed.

[Quantitative-qualitative assessment of duodenogastric reflux at 24-h pH-metry].

AIM: To study characteristics (other than duration) of duodenogastric reflux (DGR), correlations of secretory function and DGR characteristics with gastroduodenal disorders (ulcer, chronic hyperacid gastritis). MATERIAL AND METHODS: A total of 110 patients were examined with 24-h pH-metry: 68 patients with duodenal ulcer (DU), 15 patients with gastric ulcer (GU), 27 patients with chronic hyperacid gastritis (CHG). Mean levels of pH and duration of hyperacidity in the body and an antral part of the stomach, duration of DGR, pH in the body and antral part of the stomach depending on DGR severity were studied. RESULTS: DGR was registered almost in all the patients with DU, GU and CHG. Groups of the patients differed by duration and height" of the DGR. CONCLUSION: Patients with DU are characterized by low refluxes which do not reach gastric body.

Efficiency of potent gastric acid inhibition.

The evidence regarding the efficiency of potent gastric acid inhibition is exposed after a systematic search and a critical evaluation of its quality, using a specific score. The aim was to review alternative options, in economic terms, especially related with gastro-oesophageal reflux disease. The results show that the superiority of the proton pump inhibitors over the histamine H2 receptor antagonist is clear in moderate and severe oesophagitis and in patients with persistent or severe symptoms. This evidence is clearly related with the intensity of the gastric inhibition. An associated benefit is the improvement of the quality of life obtained with this potent gastric acid inhibition profile.

Proton pump inhibitors for acid suppression in the intensive care unit: formulary considerations.

PURPOSE: The rationale for limiting the proton pump inhibitor (PPI) products included in an institutional formulary, factors to consider when making formulary decisions about PPI products, the results and limitations of cost-effectiveness analyses of PPI therapy in critically ill patients, the role of clinical practice guidelines in improving PPI use in the intensive care setting, and how these guidelines can be developed are discussed. SUMMARY: Therapeutic interchange may make it possible to limit the number of PPI products included in the formulary and reduce costs without compromising the efficacy or safety of drug therapy. The results of studies comparing the pharmacokinetics, pharmacodynamics, and efficacy of different PPI dosage forms and routes of administration; practical considerations; safety; and costs are among the factors to consider when making formulary decisions. Some of the newer oral PPI products offer advantages over older ones in improved palatability and ease of preparation, storage, and administration. The cost-effectiveness of intravenous (i.v.) PPIs for preventing the recurrence of peptic ulcer bleeding has been demonstrated, but the cost-effectiveness of oral therapy for this indication and both oral and i.v. therapy for preventing stress-related mucosal bleeding has not been well established. CONCLUSION: Intravenous PPIs are cost-effective for patients at risk for the recurrence of peptic ulcer bleeding. The introduction of new oral PPI products that can be administered as a suspension has expanded the therapeutic options for critically ill patients. The use of clinical practice guidelines can optimize the use of PPIs in the intensive care setting.

Long-term outcomes of endoluminal gastroplication: a U.S. multicenter trial.

BACKGROUND: Endoluminal gastroplication has shown promise for the treatment of GERD in short-term studies. Until now, long-term outcome data have been lacking. METHODS: A prospective, multicenter trial enrolled 85 patients with GERD to be treated with endoluminal gastroplication. Inclusion criteria were 3 or more heartburn or regurgitation episodes per week, >4.2% time in 24 hours with esophageal pH < 4, and dependency on antisecretory medications. Exclusion criteria were the presence of varices, achalasia, aperistalsis, or previous gastric resection. Patients underwent manometry, 24-hour pH monitoring, and symptom severity scoring before and after the procedure. Patient diaries were used to assess medication use and to estimate annual medication cost. RESULTS: At 1- and 2-year follow-up, patients had significant reductions in median heartburn symptom scores (72 at baseline [interquartile range (IQR) 90-48] vs. 4 at 12 months [IQR 43-0] and 16 at 24 months [IQR 53-3.5]; p < 0.0001 vs. baseline) and median regurgitation symptoms (2 at baseline [IQR 3-1] vs. 0 at 12 months (IQR 1-0) and 1 at 24 months [IQR 1-0]; p < 0.0001 vs. baseline). Of all patients, 59% and 52% showed heartburn symptom resolution at 12 and 24 months, respectively ( p < 0.0001 vs. baseline). Also, 83% and 77% had regurgitation symptom resolution at 12 and 24 months, respectively (p < 0.0001 vs. baseline). Proton pump inhibitor use also was significantly reduced at 12 and 24 months after the procedure. At 2-year follow-up, median annualized medication costs were reduced by 88% (1381 US dollars) (p < 0.0001). Endoluminal gastroplication significantly reduced the duration and the number of episodes of esophageal acid exposure (p < 0.0001 vs. baseline). Only 7 patients experienced adverse events. CONCLUSIONS: Endoscopic gastroplication is safe and effective, and is associated with symptom reductions in patients with GERD for at least 24 months.

[Clinical and pharmacoeconomic aspects of the antisecretory therapy of gastroesophageal refluxed disease]. / Klinicheskie i farmakologicheskie aspekty antisekretornoi terapii gastroézofageal'noi refliuksnoi bolezni.

The article discloses the results of an open prospective randomized study of the antisecretory therapy efficiency conducted among 20 patients with gastroesophageal refluxed disease (erosive form, non-complicated course). The selection of the optimal antisecretory drug was substantiated from the point of view of the clinic-diagnostic and pharmacoeconomic assessment of the results of the above-mentioned therapy conducted with the application of rabeprosole (pariet) and its generic omeprosole (omez).