Kinetic Role of Reactive Intermediates in Controlling the Formation of Chlorine Dioxide in the Hypochlorous Acid-Chlorite Ion Reaction.

An advanced experimental protocol is reported for studying the kinetics and mechanism of the complex redox reaction between chlorite ion and hypochlorous acid under acidic condition. The formation of ClO2 is followed directly by the classical two-component stopped-flow method. In sequential stopped-flow experiments, the target reaction is chemically quenched using NaI solution and the concentration of each reactant and product is monitored as a function of time by utilizing the principles of kinetic discrimination. Thus, in contrast to earlier studies, not only the formation of one of the products but the decay of the reactants was also directly followed. This approach provides a firm basis for postulating a detailed mechanism for the interpretation of the experimental results under a variety of conditions. The intimate details of the reaction are explored by simultaneously fitting 78 kinetic traces, i.e., the concentration vs. time profiles of ClO2-, HOCl, and ClO2, to an 11-step kinetic model. The most important reaction steps were identified, and it was shown that two reactive intermediates have a pivotal role in the mechanism. While chlorate ion predominantly forms via the reaction of Cl2O, chlorine dioxide is exclusively produced in reaction steps involving Cl2O2. This study leads to clear conclusions on how to control the stoichiometry of the reaction and achieve optimum conditions to produce chlorine dioxide and to reduce the formation of the toxic chlorate ion in practical applications.

3Rs implementation in veterinary vaccine batch-release testing: Current state-of-the-art and future opportunities. A webinar and workshop report.

Regulatory authorities require veterinary batch-release testing to confirm vaccine potency and safety, but these tests have traditionally relied on large numbers of laboratory animals. Advances in vaccine research and development offer increasing opportunities to replace in vivo testing, and some stakeholders have made significant progress in incorporating 3Rs elements in quality control strategies. A three-part event series entitled "3Rs Implementation in Veterinary Vaccine Batch-Release Testing: Current state-of-the-art and future opportunities" was jointly organized by the Animal-Free Safety Assessment Collaboration, HealthforAnimals, and the International Alliance of Biological Standardization. Two webinars and a workshop aimed to outline the state-of-the-art non-animal approaches for veterinary batch-release testing. The events included information on the state of the deletion of obsolete safety testing and the current initiatives implemented by European, North American, and Asian-Pacific stakeholders on 3Rs implementation and regulatory acceptance. The events contributed to a better understanding of the barriers to 3Rs implementation. Participants highlighted the need for open communication, continued collaboration between stakeholders, and international harmonization of regulatory requirements to help accelerate acceptance. Despite the challenges, the countries represented at this three-part event have shared their commitments to advancing the acceptance of alternative methods.

Genetic diversity and substructuring of the Hungarian merino sheep breed using microsatellite markers.

The Hungarian Merino sheep breed (Ovis aries) is the most significant animal resource of the Hungarian sheep sector which, unfortunately, has gone through a huge reduction in number during the last decades and became endangered in 2014. A modern molecular genetic survey is now becoming more than necessary in order to characterize the within-breed genetic diversity and structure. For that reason, six Hungarian Merino flocks were genotyped in 16 microsatellite markers. In total, 144 different alleles were found and the mean values of observed and expected heterozygosity were 0.714 and 0.705, respectively, suggesting a noticeable genetic variability of the breed. The genetic differentiation of the Hungarian flocks was generally low, as reflected by the estimated total FST value (0.036), the extended pattern of admixture in Structure analysis, as well as, by the noticeable level of genetic clustering in UPGMA and FCA analyses. However, two out of the six studied flocks tended to be genetically more distant. The outcome of our study could be a starting point for a planned breeding strategy of the Hungarian Merino breed, by keeping the within-flock genetic variability in priority, as well as, by preserving the potential genetic uniqueness with close monitoring of the inbreeding.

Synthesis and Characterization of 1,10-Phenanthroline-mono-N-oxides.

N-oxides of N-heteroaromatic compounds find widespread applications in various fields of chemistry. Although the strictly planar aromatic structure of 1,10-phenanthroline (phen) is expected to induce unique features of the corresponding N-oxides, so far the potential of these compounds has not been explored. In fact, appropriate procedure has not been reported for synthesizing these derivatives of phen. Now, we provide a straightforward method for the synthesis of a series of mono-N-oxides of 1,10-phenanthrolines. The parent compounds were oxidized by a green oxidant, peroxomonosulfate ion in acidic aqueous solution. The products were obtained in high quality and at good to excellent yields. A systematic study reveals a clear-cut correlation between the basicity of the compounds and the electronic effects of the substituents on the aromatic ring. The UV spectra of these compounds were predicted by DFT calculations at the TD-DFT/TPSSh/def2-TZVP level of theory.

The Chlorination of N-Methyl Amino Acids with Hypochlorous Acid: Kinetics and Mechanisms.

The formation and decomposition kinetics of N-chloro-N-methyl amino acids were studied to predict the fate and impact of these compounds in water treatment technologies and biological systems. These compounds form in fast second-order reactions between N-methyl amino acids and hypochlorous acid. The comparison of the activation parameters for the reactions of N-methyl substituted and nonsubstituted branched-chain amino acids reveals the transition-state features less organized structure and stronger bonds between the reactants in the reactions with the N-methyl derivatives. This is due to a combined positive inductive effect of the N-methyl group and the alkyl side chain as well as to the steric effects of the substituents. N-Methyl-N-chloro amino acids decompose much faster than the nonsubstituted compounds. The reaction rates do not depend on the pH, and the same final product is formed in the entire pH range. N-Chlorosarcosine is an exception, as it decomposes via competing paths, kdobs = kd + kdOH[OH-], yielding different final products. This feature is most likely due to the lack of an alkyl substituent on the α-carbon atom. Under physiological pH, aldehydes and methylamine form in these reactions, which are not particularly toxic.

Determination of chlorine species by capillary electrophoresis - mass spectrometry.

The applicability of CZE with mass spectrometric detection for the determination of four chlorine species, namely chloride and three stable chlorine oxyanions, was studied. The main aspects of the proper selection of BGE and sheath liquid for the CE-MS determinations of anions with high mobility were demonstrated, pointing out the importance of pH and the mobility of the anion in the BGE. The possibility of using uncoated fused silica capillary and common electrolytes for the separation was shown and the advantage of using extra pressure at the inlet capillary end was also presented. The linear range was found to be 1-100 µg/mL for ClO3- and ClO4- , 5-500 µg/mL for ClO2- , and 25-500 µg/mL for Cl- , but the sensitivity can be greatly improved if larger sample volume is injected and electrostacking effect is utilized. The LOD for ClO3- in drinking water was 6 ng/mL, when very large sample volume was injected (10 000 mbar·s was applied).

Outstanding Activity and Selectivity of Controlled Size Pt Nanoparticles Over WO3 Nanowires in Ethanol Decomposition Reaction.

Pt nanoparticles with controlled size of 1.5 and 6.5 nm were anchored onto the surface of WO3 nanowires (WO3NW) as well as on MCF-17 silica. In the case of WO3NW and MCF-17 supported nanoparticles, 1.5 nm Pt nanoparticles were more active in ethanol decomposition reaction at 533 K. 6.5 nm Pt/WO3NW catalyst showed ~6 times higher activity compared to MCF-17 supported 6.5 nm Pt nanoparticles. While MCF-17 supported catalysts produced hydrogen, methane, carbon-monoxide and acetaldehyde, the tungsten-oxide supported Pt nanoparticles produced a huge amount of acetone as well as ethene with a high acetaldehyde selectivity besides H2, CH4 and CO. The hydrogen formation was significantly higher when the Pt size was 1.5 nm. The metallic nanoparticles, the acid sites and the oxidized centers of support play important role in the formation of decomposition products of ethanol.

Size-Dependent H2 Sensing Over Supported Pt Nanoparticles.

Catalyst size affects the overall kinetics and mechanism of almost all heterogeneous chemical reactions. Since the functional sensing materials in resistive chemical sensors are practically the very same nanomaterials as the catalysts in heterogeneous chemistry, a plausible question arises: Is there any effect of the catalyst size on the sensor properties? Our study attempts to give an insight into the problem by analyzing the response and sensitivity of resistive H2 sensors based on WO3 nanowire supported Pt nanoparticles having size of 1.5±0.4 nm, 6.2±0.8 nm, 3.7±0.5 nm and 8.3±1.3 nm. The results show that Pt nanoparticles of larger size are more active in H2 sensing than their smaller counterparts and indicate that the detection mechanism is more complex than just considering the number of surface atoms of the catalyst.

Formation of 1,10-Phenanthroline-N,N'-dioxide under Mild Conditions: The Kinetics and Mechanism of the Oxidation of 1,10-Phenanthroline by Peroxomonosulfate Ion (Oxone).

This paper confirms the unexpected formation of 1,10-phenanthroline-N,N'-dioxide (phenO2) when 1,10-phenanthroline (phen) is oxidized by peroxomonosulfate ion (PMS) in a neutral aqueous solution. The kinetics of oxidation of phen by PMS features a complex pH dependence. In 1.00 M H2SO4, 1,10-phenanthroline-mono-N-oxide (phenO) is the sole product of the reaction. The rate of the N-oxidation is highly dependent on pH with a maximum at pH ∼6.7. The formation of phenO occurs via two parallel pathways: the rate constant of the oxidation of phen (k = 3.1 ± 0.1 M(-1) s(-1)) is significantly larger than that of Hphen(+) [k = (4.1 ± 0.3) × 10(-3) M(-1) s(-1)] because the two N atoms are open to oxidative attack in the deprotonated substrate while an internal hydrogen bond hinders the oxidation of the protonated form. With an excess of PMS, four consecutive oxidation steps were found in nearly neutral solutions. In the early stage of the reaction, the stepwise oxidation results in the formation of phenO, which is converted into phenO2 in the second step. The formation of phenO2 was confirmed by (1)H NMR and ESI-MS methods. The results presented here offer the possibility of designing an experimental protocol for preparing phenO2.

Decomposition of N-chloroglycine in alkaline aqueous solution: kinetics and mechanism.

The decomposition kinetics and mechanism of N-chloroglycine (MCG) was studied under very alkaline conditions ([OH(-)] = 0.01-0.10 M). The absorbance change is consistent with two consecutive first-order processes in the 220-350 nm wavelength range. The first reaction is linearly dependent on [OH(-)] and interpreted by the formation of a carbanion from MCG in an equilibrium step (KOH) and a subsequent loss of chloride ion from this intermediate: kobs1 = KOH k1 = (6.4 ± 0.1) × 10(-2) M(-1) s(-1), I = 1.0 M (NaClO4), and T = 25.0 °C. The second process is assigned to the first-order decomposition of N-oxalylglycine, which is also formed as an intermediate in this system: kobs2 = (1.2 ± 0.1) × 10(-3) s(-1). Systematic (1)H and (13)C NMR measurements were performed in order to identify and follow the concentration changes of the reactant, intermediate, and product. It is confirmed that the decomposition proceeds via the formation of glyoxylate ion and produces N-formylglycine as a final product. This compound is stable for an extended period of time but eventually hydrolyses into formate and glycinate ions. A detailed mechanism is postulated which resolves the controversies found in earlier literature results.

Fluorescence of a Histidine-Modified Enhanced Green Fluorescent Protein (EGFP) Effectively Quenched by Copper(II) Ions. Part II. Molecular Determinants.

The histidine-modified EGFP was characterized as a sensing element that preferentially binds nanomolar concentrations of Cu(2+) in a reversible manner (Kd = 15 nM). This research aims to determine the causes of nanomolar-affinity of this mutant by investigating significant structural and energetic alterations of the chromophore in the presence of different copper ion concentrations. In order to reveal the unknown parts of the quenching mechanism we have elaborated a specific approach that combines theoretical and experimental techniques. The theoretical experiment included the modeling of potential distortions of the chromophores and the corresponding changes in energy using quantum mechanical calculations. Differences between the modeled energy profiles of planar and distorted conformations represented the energies of activation for the chromophore distortions. We found that some values of the experimental activation energies, which were derived from fluorescence lifetime decay analysis (ex: 470 nm, em: 507 nm), were consistent with the theoretical ones. Thus, it has been revealed similarity between the theoretical activation energy (50 kJmol(-1)) for 40° phenolate-ring distortion and the experimental activation energy (52.17 kJmol(-1)) required for histidine-modified EGFP saturation with copper. This chromophore conformation was further investigated and it has been found that the large decrease in fluorescence emission is attributed to the significant charge transfer over the molecule which triggers proton transfer thereby neutralizing the cromophore.

Rational design of α-helix-stabilized exendin-4 analogues.

Exendin-4 (Ex4) is a potent glucagon-like peptide-1 receptor agonist, a drug regulating the plasma glucose level of patients suffering from type 2 diabetes. The molecule's poor solubility and its readiness to form aggregates increase the likelihood of unwanted side effects. Therefore, we designed Ex4 analogues with improved structural characteristics and better water solubility. Rational design was started from the parent 20-amino acid, well-folded Trp cage (TC) miniprotein and involved the step-by-step N-terminal elongation of the TC head, resulting in the 39-amino acid Ex4 analogue, E19. Helical propensity coupled to tertiary structure compactness was monitored and quantitatively analyzed by electronic circular dichroism and nuclear magnetic resonance (NMR) spectroscopy for the 14 peptides of different lengths. Both (15)N relaxation- and diffusion-ordered NMR measurements were established to investigate the inherent mobility and self-association propensity of Ex4 and E19. Our designed E19 molecule has the same tertiary structure as Ex4 but is more helical than Ex4 under all studied conditions; it is less prone to oligomerization and has preserved biological activity. These conditions make E19 a perfect lead compound for further drug discovery. We believe that this structural study improves our understanding of the relationship between local molecular features and global physicochemical properties such as water solubility and could help in the development of more potent Ex4 analogues with improved pharmacokinetic properties.

Estimation of sampling uncertainty for pesticide residues in root vegetable crops.

The sampling uncertainty for pesticide residues in carrots, parsley leaves and selected medium size crops was estimated with simple random sampling by applying range statistics. The primary samples taken from treated fields consisted of individual carrots or a handful of parsley leaves. The samples were analysed with QUEChERs extraction method and LCMS/MS detection with practical LOQ of 0.001 mg/kg. The results indicate that the average sampling uncertainties estimated with simple random sampling and range statistics were practically the same. The confidence interval for the estimated sampling uncertainty decreased with the number of replicate samples taken from one lot and the number of lots sampled. The estimated relative ranges of sampling uncertainty are independent from the relative standard deviation of the primary samples. Consequently the conclusions drawn from these experiments are generally applicable. There is no optimum for sample size and number of lots to be tested for estimation of sampling uncertainty. Taking a minimum of 6 replicate samples from at least 8-12 lots is recommended to obtain a relative 95% range of sampling uncertainty within 50%. The cost of sampling/analyses, the consequences of wrong decision should also be taken into account when a sampling plan is prepared.

Nature of the field-to-field distribution of pesticide residues.

The supervised trial datasets (1950), consisting of a minimum of five residue values and selected by the experts of FAO/WHO Joint Meeting on Pesticide Residues for recommending maximum residue levels between 1997 and 2011, were evaluated to obtain information on the typical spread of residue values in individual datasets. The typical relative standard deviation, CV, of field-to-field variation of pesticide residues was about 80%. The spread of residues in datasets is independent from the chemical structure of pesticides, residue level, pre-harvest interval and number of values in the datasets. The CV ranges within the Codex commodity groups and between groups overlapped and their difference were not statistically significant. The number of residues below the limit of quantification (LOQ) affects the CV at various extents depending on the ratio of LOQ/R mean. The combined uncertainty of the highest residue in a dataset significantly affects the CV of the dataset. The lowest and intermediate ones have less influence. The residues in different fields receiving the same treatment vary within large range: 55%, 72%, 78%, 86% and 89% of the 25,766 residues values were, respectively, within 3, 4, 5, 6 and 7 times the median value of the corresponding dataset.

Oxidation of branched chain amino acids by HOCl: Kinetics and mechanism.

The kinetics of the chlorination of leucine, isoleucine, and valine (BCAAs) was studied in excess HOCl by stopped-flow and spectrophotometric methods (25 ◦C, I = 1.0 M NaClO4). The intermediates and products were identified and monitored by 1H NMR spectroscopy. It was established that these reactions are fully analogous and proceed according to distinct mechanisms under alkaline and neutral conditions. At high pH, the formation and subsequent rate determining decomposition of N-monochloroamino acid control the process. The decomposition occurs via competing pH-independent and OH--assisted reaction paths and the sequence of chlorination, dichlorination and decarboxylation steps leads to the formation of N-chloroimines and their carbanionic forms, which are in fast acid - base equilibria. The dechlorination of the carbanions yields nitriles as the main products. The hydration of the N-chloro imines produces chloramine and aldehydes which are involved in further oxidation reactions with HOCl. The formation of chloroform and chloroacetaldehyde was confirmed in each system. At pH 7.0, the N-chloro derivatives of BCAAs form immediately and are converted into the corresponding N,N-dichloro species within a few seconds after mixing the reactants. In this reaction, the reactive form of the oxidant is Cl2O. The first-order decomposition of the dichloroamino acids occurs on stopped-flow timescale (k = 0.5 - 0.7 s-1) and yields N-chloroimines which slowly decompose with a characteristic first-order rate constant on the order of a few times 10-5 s-1. The main products are the corresponding nitriles that account for about 80% and 60% of the original amounts of amino acids under neutral and alkaline (cOH- = 5.00 × 10-2 M) conditions, respectively. Aldehydes, carboxylic acids, chloroform and NCl3 were also identified as by-products. The results unequivocally confirm that harmful chlorinated species may form from amino acids long after the chlorination step in water treatment technologies that deteriorates the quality of the finished water. ENVIRONMENTAL IMPLICATION: In source waters, amino acids account for about 75% of the total dissolved nitrogen. Therefore, it is an essential issue how the reactions of these compounds with hypochlorite ion can be controlled to avoid the formation of toxic compounds. The compounds formed from BCAAs are considered to be harmful both under alkaline and neutral conditions (chloroacetaldehyde, chloroform, nitriles). However, some of the intermediates have extended lifetime in these systems and they may also react with other components of raw water during water treatment processes.

Fluorescence of a histidine-modified enhanced green fluorescent protein (EGFP) effectively quenched by copper(II) ions.

Two histidines were introduced by site-directed mutagenesis into the structure of Enhanced Green Fluorescent Protein, replacing the serine at position 202 and the glutamine at position 204 for increasing the sensitivity of the protein towards different metal ions by creating possible metal binding sites near the chromophore group. There is no appreciable difference between the absorbance and fluorescence spectra of the two proteins (wild type and the double-histidine mutant) indicating that the mutation does not change the environment of the fluorophore. Fluorescence quenching was measured at different pH (6.5-8) and temperatures (20-45 °C) varying the concentration of metal ions. Under optimal conditions (pH = 7.5, 20 °C) the mutant's Kd is 16 nM, it binds copper more than 200fold stronger than the wild type EGFP.

The chlorination of glycine and α-alanine at excess HOCl: Kinetics and mechanism.

The chlorination of the two simplest amino acids at HOCl excess was studied by stopped-flow, conventional spectrophotometric and time resolved 1H NMR kinetic methods at 25 °C. These reactions show distinct characteristics under neutral and alkaline conditions. At high pH, the common feature of the two systems is that the N-dichloroamino carboxylate ion does not form and the overall process is controlled by the initial decomposition of the N-monochloro derivative. Under such conditions, carbanions form in equilibrium acid - base processes and open alternative reaction paths, resulting in enhanced complexity of the corresponding mechanisms. In the case of α-alanine, the formation of acetonitrile and N-chloro acetamide as main products; acetate ion, acetaldehyde, chloroacetaldehyde, chloroform as byproducts; acetamide and N-chloro ethanimine as intermediates was confirmed. In the case of glycine, the final products are formamide and OCN-. Under neutral conditions, monochloroamino acid forms immediately upon mixing the reactants, and subsequently it is converted into dichloroamino acid by Cl2O in a fast process. In considerably slower further reaction steps, acetonitrile and acetate ion form as final products in the α-alanine system, while the chlorination of glycine proceeds to full mineralization. The detailed mechanisms suggested for these reactions postulate the formation of various imines and N-chloro imines which are involved in decarboxylation, dechlorination, hydration and hydrolytic reaction steps.

Assessment of Human Mycotoxin Exposure in Hungary by Urinary Biomarker Determination and the Uncertainties of the Exposure Calculation: A Case Study.

Urinary biomarkers of mycotoxin exposure were evaluated in the case of healthy people (n = 41) and coeliac patients (n = 19) by using a multi-biomarker LC-MS/MS immunoaffinity based method capable to analyse biomarkers of nine mycotoxins, i.e., fumonisin B1 (FB1), fumonisin B2 (FB2), deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA), Aflatoxin B1 (AFB1), T-2 toxin, HT-2 toxin and Nivalenol (NIV). Urinary biomarker concentrations were used to calculate the probable daily intake (PDI) of fumonisin B1, deoxynivalenol, zearalenone and ochratoxin A and compared with their tolerable daily intake (TDI). The human urinary excretion rate values reported in the literature and the 24 h excretion rate measured in piglets were used to estimate and compare the PDI values of the four mycotoxins. The highest mean biomarker concentrations were found for DON (2.30 ng/mL for healthy people and 2.68 ng/mL for coeliac patients). Mean OTA concentration was significantly higher (p < 0.001) in healthy people compared to coeliac patients. PDI calculated with piglets excretion data exceeded the TDI values by a much smaller percentage than when they were calculated from human data, especially for FB1. The uncertainties arising from the different calculations can be well perceived on the basis of these data.

The decomposition of N-chloro amino acids of essential branched-chain amino acids: Kinetics and mechanism.

The formation of N-chloro-amino acids is of outmost importance in water treatment technologies and also in vivo processes. These compounds are considered as secondary disinfectants and play important role in the defense mechanism against invading pathogens in biological systems. Adversary effects, such as apoptosis or necrosis are also associated with these compounds and the intermediates and final products formed during their decomposition. In the present study, the decomposition kinetics of the N-chloro derivatives of branched chain amino acids (BCAAs) - leucine, isoleucine, valine - were studied. On the basis of spectrophotometric measurements, it was confirmed that the decomposition proceeds via a spontaneous and an OH- assisted path in each case: kobs = k + kOH[OH-]. 1H, 13C NMR and MS experiments were also performed to identify the products and to monitor the progress of the reactions. It was established that the pH independent and the [OH-] dependent paths lead to the formation of the same aldehyde in each system (isovaleraldehyde, 2-methyl-butyraldehyde, and isobutyraldehyde) as a primary product. Under alkaline conditions, a portion of the aldehydes are converted into the corresponding Schiff-bases by the excess amino acid in a reversible process. A common mechanism was proposed for these reactions which postulates the formation of imines and hemiaminals as reactive intermediates.

Use of Proton Pump Inhibitors in Hungary: Mixed-Method Study to Reveal Scale and Characteristics.

BACKGROUND: Due to their efficacy and tolerability, utilization of proton pump inhibitors (PPI) has significantly increased worldwide. Parallel to the clinical benefits, potential long-term side effects have been observed, which, along with increasing medical expenses and potential drug interactions, justifies the analysis of the trends of utilization. OBJECTIVE: The aim of the present study was to show the level, pattern, and characteristics of PPI use. METHODS: We assessed the nationwide use of proton pump inhibitors in ambulatory care based on aggregated utilization data from the National Health Insurance database. The annual PPI utilization was expressed as the number of packages and as number of DDDs per 1,000 inhabitants and per year. For 2018, we estimated PPI exposure as the number of packages and as the number of DDDs per user per year. The annual reimbursement costs of proton pump inhibitors were also calculated. Moreover, three patient-level surveys were carried out in non-gastroenterological inpatient hospital departments to reveal characteristics of proton pump inhibitor use, namely dose, duration, and indication. RESULTS: The PPI utilisation increased from 5867.8 thousand to 7124.9 thousand packages and from 41.9 to 50.4 DDD per 1,000 inhabitants and per day between 2014 and 2018. Nationwide data showed that 14% of the adult population was exposed to proton pump inhibitors in 2018, while among hospitalized patients, the prevalence of proton pump inhibitor use was between 44.5% and 54.1%. Pantoprazole was the most frequently used active ingredient, both in the nationwide data and in the patient-level surveys. In the patient-level survey in majority of patients (71.5%-80.0%) proton pump inhibitors were prescribed for prophylaxis. Many inpatients (29.4%-36.9%) used 80 mg pantoprazole per day. The average number of PPI packages per user was 6.5 in 2018 in the nationwide data. The duration of PPI therapy was typically between 1 and 5 years in the patient-level surveys and nearly 20% of the inpatients had been taking proton pump inhibitors for more than 5 years. CONCLUSIONS: Our data suggests that Hungarian patients receive proton pump inhibitors in high doses and for a long time. Use of proton pump inhibitors beyond their recommended indications was also found.