Pepsin Concentration Research Articles

Cytotoxicity and Induction of Inflammation by Pepsin in Acid in Bronchial Epithelial Cells

Introduction. Gastroesophageal reflux has been associated with chronic inflammatory diseases and may be a cause of airway remodelling. Aspiration of gastric fluids may cause damage to airway epithelial cells, not only because acidity is toxic to bronchial epithelial cells, but also since it contains digestive enzymes, such as pepsin. Aim. To study whether pepsin enhances cytotoxicity and inflammation in airway epithelial cells, and whether this is pH-dependent. Methods. Human bronchial epithelial cells were exposed to increasing pepsin concentrations in varying acidic milieus, and cell proliferation and cytokine release were assessed. Results. Cell survival was decreased by pepsin exposure depending on its concentration (F = 17.4) and pH level of the medium (F = 6.5) (both P < 0.01). Pepsin-induced interleukin-8 release was greater at lower pH (F = 5.1; P < 0.01). Interleukin-6 induction by pepsin was greater at pH 1.5 compared to pH 2.5 (mean difference 434%; P = 0.03). Conclusion. Pepsin is cytotoxic to bronchial epithelial cells and induces inflammation in addition to acid alone, dependent on the level of acidity. Future studies should assess whether chronic aspiration causes airway remodelling in chronic inflammatory lung diseases.

High concentrations of pepsin in bronchoalveolar lavage fluid from children with cystic fibrosis are associated with high interleukin-8 concentrations

BackgroundGastro-oesophageal reflux is common in children with cystic fibrosis (CF) and is thought to be associated with pulmonary aspiration of gastric contents. The measurement of pepsin in bronchoalveolar lavage (BAL)...

Digestion of peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6: A comparative in vitro study and partial characterization of digestion‐resistant peptides

There are differences in stability to pepsin between the major allergens in peanut; however, data are from different reports using different digestion models. This study provides a comprehensive comparison of the digestibility of the major peanut allergens. Peanut allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6 were incubated with pepsin to mimic the effect of gastric digestion. Samples were analyzed using SDS-PAGE. To further investigate resistance to digestion, Ara h 2 was additionally subjected to digestion with trypsin and residual peptides were characterized. Ara h 1 and Ara h 3 were rapidly hydrolyzed by pepsin. On the contrary, Ara h 2 and Ara h 6 were resistant to pepsin digestion, even at very high concentrations of pepsin. In fact, limited proteolysis could only be demonstrated by SDS-PAGE performed under reducing conditions, indicating an important role for the disulfide bridges in maintaining the quaternary structure of Ara h 2 and Ara h 6. Trypsin digestion of Ara h 2 similarly resulted in large residual peptides and these were identified. Ara h 2 and Ara h 6 are considerably more stable towards digestion than Ara h 1 and Ara h 3.

The refluxate: The impact of its magnitude, composition and distribution

The pathogenesis of gastrointestinal reflux disease is multifactoral. Integral to the disease process is the refluxate itself. The characteristics and composition of the refluxate are dependent on several physiological variables. The refluxate may contain varying concentrations of acid, pepsin, gas, or contents of duodenal reflux (such as bile acid and pancreatic enzymes). Characteristics such as volume and proximal extent of the refluxate, and the chemical content of this refluxate can strongly influence the risk of symptom perception. Strong acid (pH<4) and duodeno-gastro-oesophageal reflux are also implicated in the development of mucosal damage in the form of oesophagitis, Barrett's metaplasia and oesophageal adenocarcinoma. The manifestation of disease, however, is not entirely reliant on the refluxate, which must be considered in the context of the genetic, environmental and psychological susceptibility of the individual.

Correlation of pepsin-measured laryngopharyngeal reflux disease with symptoms and signs

Objective Pepsin detection in throat sputum has been posited as a reliable biological marker of laryngopharyngeal reflux disease (LPRD). This study was designed to further correlate pepsin concentration with symptoms and signs of LPRD. Study Design Cross-sectional study. Setting Nanfang Hospital of Southern Medical University. Subjects and Methods Fifty-six laryngitis patients were divided into a reflux laryngitis group and a chronic laryngitis group based on the reflux symptom index (RSI), reflux finding score (RFS), and proton pump inhibitor treatment for two weeks. Oral and hypopharyngeal secretions from the study patients and from 15 healthy subjects were collected. Thirty-six obstructive sleep apnea (OSA) patients were divided into a mild-moderate group and a severe group by the apnea-hypopnea index (AHI). Bedtime and first-morning oral secretions were collected. Enzyme-linked immunosorbent assay was used to measure the pepsin concentration. Results In laryngitis patients, the total score of RSI and RFS ( P < 0.05), and the symptoms, including clearing throat often, coughing, and sensing a lump in the throat ( P < 0.006), were more severe in the pepsin-positive group. No significant differences were found between the oral and hypopharyngeal secretions. In OSA, pepsin levels in the first-morning oral secretions were correlated with AHI, mean SaO 2, and mini SaO 2 ( P < 0.01). However, RSIs were not significantly correlated with these indicators. Conclusion Higher levels of pepsin in sputum were associated with higher RSI and RFS in cases of laryngitis. There was no relationship between pepsin levels and RSI in cases of OSA. There were no differences of pepsin concentration in sputum collection methods or in collection timing.

Gastric Juice From Patients “On” Acid Suppressive Therapy Can Still Provoke a Significant Inflammatory Reaction by Human Bronchial Epithelial Cells

Patients with reflux-related respiratory symptoms are frequently treated with proton pump inhibitors (PPI). It is unclear whether aspiration of gastric juice (GJ) from patients "on" PPI can provoke a similar bronchial inflammatory reaction than that observed in patients "off" medication. The goal of this study was to evaluate the effect of GJ from patients with and without PPI treatment on production of IL-8 by human primary bronchial epithelial cells (PBEC). PBEC were exposed during 24 hours to GJ (1/1000) from patients "on" (n=10) and "off" (n=13) PPI and to nonacidic gastric components (pepsin and bile acids). IL-8 concentration in supernatant was measured with enzyme-linked immunosorbent assay. Endotoxin level in GJ samples was analyzed with a LAL assay. Exposure of PBEC to GJ from patients "on" PPI provoked a higher production of IL-8 than GJ from patients "off" PPI [279 pg/mL (36 to 498) vs. 11 pg/mL (9 to 27)]. A correlation was found between pH of GJ and IL-8 production (r=0.659, P=0.0006). No correlation was found between IL-8 production and concentration of bile acids or pepsin. Filtration (0.20 [mu]m) of GJ from patients "on" PPI reduced IL-8 production. A positive correlation was found between IL-8 production and endotoxin levels of GJ samples (1/1000) (r=0.654, P=0.0007). Exposure of bronchial epithelial cells to GJ from patients "on" PPI is able to induce high IL-8 production. These results suggest that aspiration of GJ in patients treated with PPI might still be able to provoke a significant bronchial inflammatory reaction.

Effect of methanolic extract of Terminalia arjuna against Helicobacter pylori 26695 lipopolysaccharide-induced gastric ulcer in rats

Helicobacter pylori lipopolysaccharide (HP-LPS) is a potent virulence factor in the causation of gastric ulcer and gastritis. H. pylori-induced gastric pathology is prevalent throughout the world. Herbal medicines are attracting attention because of their traditional values, popularity and belief, as well as for their advantages such as less toxicity, affordability and medicinal value. The present study aimed to evaluate the anti-ulcer effect of a methanolic extract of Terminalia arjuna (TA) against HP-LPS-induced gastric damage in rats. Ulcers were induced with HP-LPS (50 mug per animal) administered orally daily for 3 days. The efficacy of TA on gastric secretory parameters such as volume of gastric juice, pH, free and total acidity, pepsin concentration, and the cytoprotective parameters such as protein-bound carbohydrate complexes in gastric juice and gastric mucosa was assessed. The protective effect of TA was also confirmed by histopathological examination of gastric mucosa. HP-LPS-induced alterations in gastric secretory parameters were altered favourably in rats treated with TA, suggesting that TA has an anti-secretory role. Furthermore, HP-LPS-induced impairments in gastric defence factors were also prevented by treatment with TA. These results suggest that the severe cellular damage and pathological changes caused by HP-LPS are mitigated by TA; these effects are comparable with those of sucralfate. The anti-ulcer effect of TA may reflect its ability to combat factors that damage the gastric mucosa, and to protect the mucosal defensive factors.

Cascade ultrafiltration bioreactor-separator system for continuous production of F(ab′) 2 fragment from immunoglobulin G

We discuss a continuous two-stage cascade ultrafiltration bioreactor-separator system for fragmentation of immunoglobulin G (IgG) by pepsin and purification of F(ab′) 2 fragment thus generated. F(ab′) 2 is frequently preferred to intact IgG in therapeutic applications due to lower immunogenicity and faster onset of action. The 10 kDa MWCO membrane of the first stage bioreactor retained pepsin, IgG and F(ab′) 2 while allowing degraded Fc sub-fragments through and the 70 kDa membrane of the second stage retained both IgG and F(ab′) 2 while allowing pepsin through. Due to this differential retention behavior, the pepsin concentration within the first stage was significantly higher than that in the feed. The first bioreactor therefore primarily carried out IgG digestion while the second stage primarily served as a separator for pepsin and F(ab′) 2. The two-stage system was first assessed using pure equine IgG as feed. Under optimized feed and sweep flow rates, 97% IgG conversion with 93% pure F(ab′) 2 product were obtained. A mathematical model for the bioreactor system was developed and simulated and experimental data were found to be in good agreement. When the bioreactor system was operated with unpurified equine serum as feed, close to 95% IgG conversion was observed. The F(ab′) 2 product obtained from serum showed a single band on SDS-PAGE. The results demonstrated the suitability of the two-step cascade membrane bioreactor for production of F(ab′) 2 from both IgG and unpurified equine serum.

Fluorescence Labeling and Determination of Pepsin with CdSe Quantum Dots

AbstractBased on the solid phase/liquid deposition CdSe quantum dots (QD) were synthesized using selenium and cadmium‐salt as precursor at room temperature. The average diameter of CdSe QD estimated from the high resolution transmission electron microscopy (HRTEM) graph and absorption spectra was ca. 3–3.5 nm. The mercaptoacetic‐acid stabilized CdSe QD exhibited ultraviolet absorption at 350 and 380 nm and strong fluorescence emission at 481.6 nm, respectively. When conjugated with pepsin, the fluorescence peak intensity of CdSe QD decreased considerably and the fluorescence peak shifted to 467.2 nm. Under optimal conditions, a concentration in 5–50 mg· L−1 of pepsin could be determined on the basis of fluorescence decrease ratio of CdSe QD, with a detection limit 3δ of 0.36 mg·L−1 (n=10). The proposed method was applied to detection of the concentration of pepsin in human gastric juice.

Isolation and some properties of collagen from the backbone of Baltic cod ( Gadus morhua)

Ossein from Baltic cod backbone was obtained after extraction of non-collagenous protein with 0.1 M NaOH solution and demineralization with 1.0 M HCl solution. The extractions were performed at 4 °C for 24, 48 and 72 h using a solid/solution ratio from 1:4 to 1:8 (w/v). After 48 h of extraction in 0.5 M acetic acid only about 25% of collagen was dissolved. After 48 h of extraction at optimal concentration of pepsin (4 mg/g ossein) the collagen was dissolved almost completely. Collagen solubilized in acetic acid could be almost completely precipitated with κ-carrageenan, at protein/polysaccharide ratio 1:1 (w:w). The denaturation temperature of Baltic cod backbone collagen was 14.4 °C and the conversion factor of hydroxyproline to collagen was 15.7.

Pepsin measured in induced sputum—a test for pulmonary aspiration in children?

Purpose The purpose of this study was to investigate if pepsin measured in sputum is a useful marker of pulmonary aspiration secondary to gastroesophageal reflux (GER) in children. It is possible that the induced sputum procedure could cause GER and invalidate the results. The hypothesis stated that healthy children (those without history of respiratory or gastroesophageal symptoms) would not have pepsin detected in induced sputum. Methods Children attending surgical outpatients in the Royal Belfast Hospital for Sick Children (Belfast, Northern Ireland) were recruited. After spirometry, sputum was obtained by induction with hypertonic 3% saline. Spirometry was repeated, and complications were noted. An “in-house” enzyme-linked immunosorbent assay was used to measure pepsin concentration in sputum. The lower limit of detection of pepsin was 1.19 ng/mL. Results Children (n = 21) aged 4 to 16 years were recruited. Twenty children completed the study. No adverse effects were reported. Pepsin was detected in 17 (85%) of 20 sputum samples. Conclusions The act of sputum induction appears to induce physiologic GER in a healthy childhood population. The analysis of pepsin in sputum obtained by sputum induction is therefore not useful in the investigation of reflux-related respiratory disease.

Pepsin immunoassay in the sputum for detection of laryngopharyngeal reflux

To study whether the presence of gastric pepsin in the sputum might be used as a reliable criteria of laryngopharyngeal reflux. Fifty-six patients with the symptoms of laryngopharyngitis and fifteen healthy people were recruited. Fifty-six patients were divided into laryngopharyngeal reflux group and chronic laryngitis group by the reflux symptom index (RSI), by the reflux finding score (RFS) and by their treating experiment taking omeprazole 20 mg bid for 2 weeks. Sputum in all three groups was obtained in the morning. Pepsin in the sputum was measured by enzyme-linked immunoadsorbent assay. The positive rate of pepsin in sputum among LPR group, chronic laryngopharyngitis group and normal group were 93.8% (30/32), 75.0% (18/24), 20.0% (3/15) respectively, and the median concentration of pepsin were 5.3 [1.3; 53.4] ng/ml, 0.8 [0.1; 17.2] ng/ml, 0.0[0.0;0.0] ng/ml (H = 23.29, P = 0.000). Compared with co-diagnosis as gold standard, the sensitivity of RSI, RFS treating experiment and the pepsin immunoassay was 59.4%, 84.4%, 81.3% and 93.8%, and the specificity of those was 87.2%, 61.5%, 95.8% and 46.2% respectively. Detection of pepsin in sputum by immunoassay might provide a high sensitive, noninvasive method for laryngopharyngeal reflux.

황다랑어 복부 껍질을 이용한 콜라겐의 제조 및 물리화학적 특성 해석

Processing of collagen from yellowfin tuna (Thunnus albacares) abdominal skins was optimized by response surface methodology and central composite design. The values of independent variables at optimal conditions were NaOH concentration: 0.5 N, NaOH treatment time: 36.2 hr, pepsin concentration: 1:4.9 ratio (0.245%, w/v), and digestion time: 48.1 hr, respectively. The collagen content estimated under optimal conditions was 33.1%, and the actual experimental collagen content was 32.3%. Physicochemical properties of collagen from yellowfin tuna abdominal skin were investigated by amino acids analysis, SDS-PAGE, FT-IR, viscosity and denaturation temperature. Amino acids content of the collagen was 21.0%. SDS-PAGE pattern of the collagen showed two different <TEX>$\alpha$</TEX>-chain (<TEX>${\alpha}_1$</TEX>- and <TEX>${\alpha}_2$</TEX>- chain), <TEX>$\beta$</TEX>-component and <TEX>$\gamma$</TEX>-component. The spectrum of FT-IR of the collagen showed wavenumber at 3,434, 1,650, 1,542 and <TEX>$1,235\;cm^{-1}$</TEX> representing the regions of amide A, I, II and III, respectively. Relative viscosity of the collagen decreased continuously on heating up to <TEX>$32^{\circ}C$</TEX>, and the rate of decrease was retarded in the temperature range of <TEX>$35-50^{\circ}C$</TEX>. Denaturation temperature (Td) of the collagen solution (0.06%, w/v) was <TEX>$31^{\circ}C$</TEX> and was lower than calf skin collagen (<TEX>$35^{\circ}C$</TEX>).

Suitable condition of enzymatic reactivity for determining the digestibility of feedstuffs phosphorus in vitro by dialysis tube method

In order to develop a new method for determining the phosphorus (P) digestibility in vitro in feedstuffs by dialysis tube, a L32(49) orthogonal experiment with eight factors (4 levels for each factor) and a single factor experiment on the enzymatic reactivity were carried out. The sequence of significance of the eight factors on sample-P dialyzability was as follows: trypsin digestion for 6 h, dialyzing solution at 100 mL, pH of pepsin solution at 2.5, pepsin concentration at 2000 U·mL−1 pepsin digestion for 100 min, at temperature of 35°C, trypsin concentration at 1625 U·mL−1, and pH of trypsin solution at 6.5, respectively. And in vitro dialyzabilities of P in soybean meal, barley, sorghum, peanut meal, and rapeseed meal were (36.91 ± 0.58)%, (27.28 ± 0.94)%, (26.95 ± 0.58)%, (30.51 ± 0.83)%, and (20.82 ± 1.09)%, respectively.

Protein quality of larval feed with increased concentration of hydrolysed protein: effects of heat treatment and leaching

Four heat coagulated early weaning diets with increasing concentrations of pepsin hydrolysed protein, were investigated with regard to the change in protein quality during feed production and exposure to leaching. Water-soluble N, trichloroacetic acid-soluble N and amino acid (AA) profiles were determined in finished diets and in diets leached for 6 min. In vitro diet digestibility was measured and related to increasing inclusion of hydrolysed protein and N leakage. Seventeen to 47% of soluble N in the feed ingredients was made insoluble by heat denaturation during feed production, but the concentration of peptides and free amino acids (FAA) were not influenced. All peptides/FAA and 70–80% of water-soluble protein were lost after exposure to leaching. Increased inclusion of hydrolysed protein increased the loss of crude protein (15–30%). All taurine and 30% of histidine was lost during leaching, no other major changes in AA profile were found. There was no difference in digestibility between diets exposed to leaching. However, leached diets showed reduced digestibility as compared to diets that had not been exposed to leaching. In conclusion micro-bound type diets as used in this study have a low efficiency in delivering soluble N to fish larvae and should be carefully considered for this purpose.

Protein engineering of improved prolyl endopeptidases for celiac sprue therapy

Due to their unique ability to cleave immunotoxic gluten peptides endoproteolytically, prolyl endopeptidases (PEPs) are attractive oral therapeutic candidates for protecting celiac sprue patients from the toxic effects of dietary gluten. Enhancing the activity and stability of PEPs under gastric conditions (low pH, high pepsin concentration) is a challenge for protein engineers. Using a combination of sequence- and structure-based approaches together with machine learning algorithms, we have identified improved variants of the Sphingomonas capsulata PEP, a target of clinical relevance. Through two rounds of iterative mutagenesis and analysis, variants with as much as 20% enhanced specific activity at pH 4.5 and 200-fold greater resistance to pepsin were identified. Our results vividly reinforce the concept that conservative changes in proteins, especially in hydrophobic residues within tightly packed regions, can profoundly influence protein structure and function in ways that are difficult to predict entirely from first principles and must therefore be optimized through iterative design and analytical cycles. Incubation with whole wheat bread under simulated gastric conditions also suggests that some variants have pharmacologically significant improvements in gluten detoxification activity.

Acute effects of antacids on gastric juice components in duodenal ulcer patients

In 10 duodenal ulcer patients gastric juice was aspirated every 10 min for 20 min before and 200 min after sham feeding. One antacid tablet or placebo was given 80 min after the sham feeding. Analyses of the aspirates showed that antacids reduced mean hydrogen ion activity and pepsin concentration significantly for 40 and 60 min, respectively, and increased phospholipid concentration for 30 min, compared with placebo. Highest mean pH was 2.52. The percentage of pH readings at or above pH levels of 2, 3, and 4 during the 2 h period following antacid administration was 29, 10, and 2%, respectively. No significant differences between antacid and placebo were found regarding intragastric concentrations of bile acids and prostaglandin E2. When one antacid tablet was administered 80 min after a real meal, the effect on intragastric pH was similarly weak, but lasted slightly longer. Acid neutralization' alone can hardly explain the ulcer-healing effect of low-dose antacids. Antacid-induced increase in intragastric concentration of phospholipids is a new and potentially important observation.

Establishing objective detection limits for the pepsin digestion assay used in the assessment of genetically modified foods

Rationale: Guidelines for assessing the potential allergenicity of genetically modified (GM) organisms recommend testing the digestibility of the introduced protein by pepsin. Previous studies detailed the digestion procedure but have not described a simple objective measurement of the extent of digestion nor evaluated the impact of variation in pepsin activity. Methods: Samples of eight proteins were digested by pepsin at pH 1.2 and 2.0 using standard conditions (10,000 U of pepsin activity per mg test protein) as well as 5000 and 20,000 units per mg of test protein. An independent digestion assay of hemoglobin was used to verify pepsin activity for each assay. Digestion was stopped in timed samples between 0.5 and 60 min. Digestion samples and undigested protein (10% and 100%) were separated by SDS–PAGE. Residual stained protein bands were measured by image analysis. Results: The differences in pH and pepsin concentration only had minor effects on digestion of intermediately stable proteins: concanavalin A, ovalbumin, and lysozyme, but not on rapidly digested or stable proteins. Conclusions: Verification of pepsin activity and measurement of an objective endpoint of digestion (e.g. (90%) should provide more comparable results for the safety assessment of novel food proteins.

Pepsin, a Marker of Gastric Contents, Is Increased in Tracheal Aspirates From Preterm Infants Who Develop Bronchopulmonary Dysplasia

The objective of this study was to study the association between pepsin in tracheal aspirate samples and the development of bronchopulmonary dysplasia in preterm infants. Serial tracheal aspirate samples were collected during the first 28 days from mechanically ventilated preterm neonates. Bronchopulmonary dysplasia was defined as the need for supplemental oxygen at 36 weeks' postmenstrual age. An enzymatic assay with a fluorescent substrate was used to detect pepsin. Total protein was measured by the Bradford assay to correct for the dilution during lavage. Immunohistochemistry using antibody against human pepsinogen was performed in 10 lung tissue samples from preterm infants. A total of 256 tracheal aspirate samples were collected from 59 preterm neonates. Pepsin was detected in 234 (91.4%) of 256 of the tracheal aspirate samples. Twelve infants had no bronchopulmonary dysplasia, 31 infants developed bronchopulmonary dysplasia, and 16 infants died before 36 weeks' postmenstrual age. The mean pepsin concentration was significantly lower in infants with no bronchopulmonary dysplasia compared with those who developed bronchopulmonary dysplasia or developed bronchopulmonary dysplasia/died before 36 weeks' postmenstrual age. Moreover, the mean pepsin level was significantly higher in infants with severe bronchopulmonary dysplasia compared with moderate bronchopulmonary dysplasia. The mean pepsin level in tracheal aspirate samples from the first 7 days was also lower in infants with no bronchopulmonary dysplasia compared with those who developed bronchopulmonary dysplasia or developed bronchopulmonary dysplasia/died before 36 weeks' postmenstrual age. Pepsinogen was not localized in the lung tissues by immunohistochemistry. The concentration of pepsin was increased in the tracheal aspirate of preterm infants who developed bronchopulmonary dysplasia or died before 36 weeks' postmenstrual age. Recovery of pepsin in tracheal aspirate samples is secondary to gastric aspiration, not by hematogenous spread or local synthesis in the lungs. Chronic aspiration of gastric contents may contribute in the pathogenesis of bronchopulmonary dysplasia.

Bronchoalveolar Pepsin, Bile Acids, Oxidation, and Inflammation in Children With Gastroesophageal Reflux Disease

Gastroesophageal reflux has been suggested as an underlying cause of chronic lung disease. The aim of this study was to assess the value of pepsin and bile acids, both components of GI secretions, in the lungs of children with chronic lung diseases as possible markers for gastroesophageal reflux disease and their relation to oxidation and inflammation. BAL was performed in 96 children with different chronic lung diseases. Gastroesophageal reflux was analyzed by two-channel, 24-h esophageal pH measurements. Lung pepsin and bile acids were measured in BAL enzymatically, interleukin (IL)-8 by enzyme-linked immunosorbent assay, and protein carbonyls by slot blot immunoassay. Sixty-five of the 96 children (68%) had an extensive proximal acidic reflux index. Children with reflux had higher pepsin concentrations in their BAL fluid (BALF), compared to children without reflux despite low specificity. No differences were observed for bile acids. Percentages of neutrophils, levels of protein carbonyls, and levels of IL-8 in BALF correlated with the number of proximal reflux events. Pulmonary microaspiration as demonstrated by pepsin detection in BALF is common in children with chronic lung diseases, suggesting that gastroesophageal reflux may contribute significantly to the disease pathogenesis. BALF pepsin concentration correlates positively with the number of proximal reflux events. Protein oxidation in BALF is higher in children with extensive proximal acidic reflux, suggesting that pulmonary microaspirations contribute to lung damage.