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Activity/Stability of Human Pepsin: Implications for Reflux Attributed Laryngeal Disease
Abstract
Exposure of laryngeal epithelia to pepsin during extra-esophageal reflux causes depletion of laryngeal protective proteins, carbonic anhydrase isoenzyme III (CAIII), and squamous epithelial stress protein Sep70. The first objective of this study was to determine whether pepsin has to be enzymatically active to deplete these proteins. The second objective was to investigate the effect of pH on the activity and stability of human pepsin 3b under conditions that might be found in the human esophagus and larynx.
Prospective translational research study.
An established porcine in vitro model was used to examine the effect of active/inactive pepsin on laryngeal CAIII and Sep70 protein levels. The activity and stability of pepsin was determined by kinetic assay, measuring the rate of hydrolysis of a synthetic pepsin-specific substrate after incubation at various pH values for increasing duration.
Active pepsin is required to deplete laryngeal CAIII and Sep70. Pepsin has maximum activity at pH 2.0 and is inactive at pH 6.5 or higher. Although pepsin is inactive at pH 6.5 and above, it remains stable until pH 8.0 and can be reactivated when the pH is reduced. Pepsin is stable for at least 24 hours at pH 7.0, 37 degrees C and retains 79% +/- 11% of its original activity after re-acidification at pH 3.0.
Detectable levels of pepsin remain in laryngeal epithelia after a reflux event. Pepsin bound there would be enzymatically inactive because the mean pH of the laryngopharynx is pH 6.8. Significantly, pepsin could remain in a form that would be reactivated by a subsequent decrease in pH, such as would occur during an acidic reflux event or possibly after uptake into intracellular compartments of lower pH.
... Pepsin prefers the acidic environment in the stomach (pH < 5.5) (Bardhan et al., 2012;Kahrilas & Kia, 2015). In the more alkaline tracheobronchial environment (pH 7.5), pepsin can maintain its proteolytic function for an undefined time before it becomes inactive, yet it has remained stable to pH 8 (Johnston et al., 2007;Piper & Fenton, 1965). Tracheal pepsin was detected up to 4-6 h in rabbits (Metheny et al., 2004) and oral pepsin was detected up to 90 min in humans (Knight et al., 2005). ...
... Tracheal pepsin was detected up to 4-6 h in rabbits (Metheny et al., 2004) and oral pepsin was detected up to 90 min in humans (Knight et al., 2005). In the laryngopharynx, pepsin becomes inactivated at a pH of 6.5 but remains stable up to 24 h and can reactivate if the pH is decreased, such as a subsequent reflux event (Johnston et al., 2007;Piper & Fenton, 1965). It is theorized that pepsin may reactivate inside of the epithelial cells due to lower intracellular acidity, thereby causing cellular level inflammatory changes (Johnston et al., 2007). ...
... In the laryngopharynx, pepsin becomes inactivated at a pH of 6.5 but remains stable up to 24 h and can reactivate if the pH is decreased, such as a subsequent reflux event (Johnston et al., 2007;Piper & Fenton, 1965). It is theorized that pepsin may reactivate inside of the epithelial cells due to lower intracellular acidity, thereby causing cellular level inflammatory changes (Johnston et al., 2007). Tracheal samples in the parent study were obtained every 12 h, suggesting that we missed microaspiration events. ...
Aim
This study explored relationships between enteral feeding and tracheal pepsin A.
Background
Mechanically ventilated (MV) patients receiving enteral feeding are at risk for microaspiration. Tracheal pepsin A, an enzyme specific to gastric cells, was a proxy for microaspiration of gastric secretions.
Methods
Secondary analysis of RCT data from critically ill, MV adults was conducted. Microaspiration prevention included elevated head of bed, endotracheal tube cuff pressure management, and regular oral care. Tracheal secretions for pepsin A were collected every 12 h. Microaspiration was defined as pepsin A ≥ 6.25 ng/mL. Positive pepsin A in >30 % of individual tracheal samples was defined as abundant microaspiration (frequent aspirator). Chi-squared, Fisher's Exact test, and generalized linear model (GLM) were used.
Results
Tracheal pepsin A was present in 111/283 (39 %) mechanically ventilated patients and 48 (17 %) had abundant microaspiration. Enteral feeding was associated with tracheal pepsin A, which occurred within 24 h of enteral feeding. Of the patients who aspirated, the majority received some enteral feeding 96/111 (86 %), compared to only 15/111 (14 %) who received no feeding. A greater number of positive pepsin A events occurred with post-pyloric feeding tube location (55.6 %) vs. gastric (48.6 %), although significant only at the event-level. Frequent aspirators (abundant pepsin A) had higher pepsin A levels compared to infrequent aspirators.
Conclusions
Our findings confirmed the stomach as the microaspiration source. Contrary to other studies, distal feeding tube location did not mitigate microaspiration. Timing for first positive pepsin A should be studied for possible association with enteral feeding intolerance.
... There is increasing evidence that pepsin, which is present in all refluxate, 55 is partly, if not wholly, responsible for damage and inflammation caused by LPR. 20,39,[56][57][58][59][60] Pepsin is a proteolytic enzyme which is synthesized and secreted as the zymogen pepsinogen by chief cells in the gastric fundus and subsequently cleaved upon introduction to the acidic stomach lumen to produce pepsin. Pepsin is maximally active at pH 2 and retains activity up to pH 6.5. ...
... Although stable at pH 8, pepsin is irreversibly inactivated at higher pH. 58,61 The stomach and esophagus have intrinsic defenses against pepsin (mucus, peristalsis, and bicarbonate secretion), however, laryngeal tissues do not. 62 Pepsin is thought to play a key role in mucosal damage and inflammation during nonacidic reflux. ...
... 62 Pepsin is thought to play a key role in mucosal damage and inflammation during nonacidic reflux. 8,9,58,59,[62][63][64][65][66][67][68][69][70][71][72][73][74] At neutral pH, pepsin is taken up by laryngeal and hypopharyngeal cells by receptor-mediated endocytosis and retained in intracellular vesicles of low pH where it is presumed to be reactivated. 58,67,68 The consequence is chronic inflammation, which in turn, gives rise to symptoms. ...
Objective:
More than 20% of the US population suffers from laryngopharyngeal reflux. Although dietary/lifestyle modifications and alginates provide benefit to some, there is no gold standard medical therapy. Increasing evidence suggests that pepsin is partly, if not wholly, responsible for damage and inflammation caused by laryngopharyngeal reflux. A treatment specifically targeting pepsin would be amenable to local, inhaled delivery, and could prove effective for endoscopic signs and symptoms associated with nonacid reflux. The aim herein was to identify small molecule inhibitors of pepsin and test their efficacy to prevent pepsin-mediated laryngeal damage in vivo.
Methods:
Drug and pepsin binding and inhibition were screened by high-throughput assays and crystallography. A mouse model of laryngopharyngeal reflux (mechanical laryngeal injury once weekly for 2 weeks and pH 7 solvent/pepsin instillation 3 days/week for 4 weeks) was provided inhibitor by gavage or aerosol (fosamprenavir or darunavir; 5 days/week for 4 weeks; n = 3). Larynges were collected for histopathologic analysis.
Results:
HIV protease inhibitors amprenavir, ritonavir, saquinavir, and darunavir bound and inhibited pepsin with IC50 in the low micromolar range. Gavage and aerosol fosamprenavir prevented pepsin-mediated laryngeal damage (i.e., reactive epithelia, increased intraepithelial inflammatory cells, and cell apoptosis). Darunavir gavage elicited mild reactivity and no discernable protection; aerosol protected against apoptosis.
Conclusions:
Fosamprenavir and darunavir, FDA-approved therapies for HIV/AIDS, bind and inhibit pepsin, abrogating pepsin-mediated laryngeal damage in a laryngopharyngeal reflux mouse model. These drugs target a foreign virus, making them ideal to repurpose. Reformulation for local inhaled delivery could further improve outcomes and limit side effects.
Level of evidence:
NA. Laryngoscope, 2022.
... Acid-only treatments may be less successful in confirming reflux as the etiology of a patient's complaint by missing the non-acid and weakly acidic components of the refluxate [5]. Understanding the inflammatory role of the non-acidic components of gastric refluxate including trypsin, bile and, especially, pepsin is an ongoing target of research and has the potential for better symptom control through more targeted treatment regimens in patients with proven LPR [6,7]. ...
... Pepsin, a proteolytic enzyme made active in the acidic environment of the stomach is a primary mediator of the effects that reflux has on the laryngopharyngeal mucosa [7]. While the stomach and esophagus have native protection against pepsin, the upper aerodigestive tract does not [8]. ...
Introduction:
Laryngopharyngeal reflux (LPR) manifests with a constellation of common throat symptoms and inconclusive signs on laryngoscopic exam. It is a diagnosis, often made clinically, that can lead to prescriptions of proton pump inhibitors that are unnecessary and potentially harmful. Glottic insufficiency (GI) and the accompanying hyperfunctional laryngeal behaviors can also present with similar, common throat complaints that may or may not include a qualitative change to the voice.
Methods:
This is a reflection article. It is written to summarize, explain, and support with evidence the opinion of the author on the topic of how symptoms of voice disorders can easily be mistaken for symptoms of LPR. The offered reflection is based on his experience, research and the available literature.
Reflection:
This article intends to explore the similarities between GI and LPR, how to ultimately differentiate them and how to approach treatment with a broader differential diagnosis.
Conclusion:
LPR and GI can present with identical, vague throat, and voice symptoms. Empiric medication trials, behavioral interventions and objective laryngovideostroboscopy, impedance-based reflux, and esophageal motility testing may all be needed, sometimes in a trial and error fashion, to correctly diagnose and treat a patient’s symptoms.
... The discrepancy in the prevalence between studies could be explained by the sample size, as larger samples had lower prevalence rates. 24,43 The meta-analysis showed a significant correlation between the presence of GERD/LPR and CRS, indicating a significant association between the two conditions. In the majority of the studies reviewed, impedance-pH monitoring was used to assess reflux diseases, which is 44 We posit that the diversity in these outcomes could be attributed to a constellation of intricate individual factors. ...
... The activity of pepsin-A in the laryngeal epithelium has been extensively researched. 42,43 In this review, most included studies found high levels of pepsin-A in CRS patients compared to controls, with some discrepancies in pepsin levels in different types of collected samples. These discrepancies could be attributed to the sensitivity of anti-pepsin-A antibodies, the single-detection method used in some studies, or the timing of sample collection. ...
Background
Over the last few decades, reflux diseases, such as laryngopharyngeal reflux (LPR) and gastroesophageal reflux disease (GERD), have been identified as significant contributors to inflammatory upper aerodigestive tract diseases. Establishing a direct relationship between reflux disease and chronic rhinosinusitis (CRS) is challenging due to the high prevalence of both diseases and their potential for independent coexistence.
Objective
The purpose of this study is to review the existing literature and evaluate the evidence of an association between reflux diseases and CRS.
Methods
A comprehensive electronic search was conducted across multiple databases to identify all studies that investigated the relationship between LPR, GERD, and CRS from January 1, 1950, to June 16, 2022. Only studies with English manuscripts involving adult populations were included, while case series, case reports, and in vitro studies were excluded. The risk of bias was evaluated using The Newcastle-Ottawa Scale for case-control studies and the NIH quality assessment tool for observational cohort and cross-sectional studies.
Results
The search strategy yielded a total of 427 articles, out of which 25 studies examined the correlation between reflux diseases and CRS. The meta-analysis indicated a significant association between the presence of GERD and CRS compared to control groups ( P < .001; CI 3.56 [2.25, 5.65]), as well as significantly higher pH values and pepsin detection in CRS patients when compared to healthy individuals ( P = .003). Furthermore, all studies that evaluated proton pump inhibitor (PPI) therapy in CRS patients reported positive outcomes, with 93% of CRS patients showing improvement on PPIs.
Conclusion
The existing literature provides suggestive evidence of an association between reflux diseases and CRS, with regards to both prevalence and treatment. Nonetheless, further studies are required to confirm this relationship.
... Instead, studies indicate that PPIs may increase the concentration and toxicity of nonacid constituents of refluxate [30,32]. In the context of weakly to nonacidic refluxate such as that of patients taking PPIs or in extraesophageal reflux, the gastric enzyme pepsin is active up to pH6.5 and stable up to pH8 and can be endocytosed, leading to inflammatory and carcinogenic changes in the aerodigestive tract mucosa, including in the esophagus [8,[33][34][35][36]. ...
... While much of this work focused on anatomical locations proximal to the esophagus [34,[51][52][53][54][55][56][57][58][59] evidence suggests that pepsin, independent of acid, elicits damage to the esophagus via similar molecular mechanisms. Pepsin harbors its greatest enzymatic activity at pH2, retains 70% of its activity up to pH6.5, and is stable up to pH8 [36]. In a nonacidic environment, pepsin is endocytosed by aerodigestive tract mucosa, including that of the larynx and esophagus. ...
Gastroesophageal reflux disease (GERD) significantly impacts patient quality of life and is a major risk factor for the development of Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC). Proton pump inhibitors (PPIs) are the standard-of-care for GERD and are among the most prescribed drugs in the world, but do not protect against nonacid components of reflux such as pepsin, or prevent reflux-associated carcinogenesis. We recently identified an HIV protease inhibitor amprenavir that inhibits pepsin and demonstrated the antireflux therapeutic potential of its prodrug fosamprenavir in a mouse model of laryngopharyngeal reflux. In this study, we assessed the capacity of amprenavir to protect against esophageal epithelial barrier disruption in vitro and related molecular events, E-cadherin cleavage, and matrix metalloproteinase induction, which are associated with GERD severity and esophageal cancer. Herein, weakly acidified pepsin (though not acid alone) caused cell dissociation accompanied by regulated intramembrane proteolysis of E-cadherin. Soluble E-cadherin responsive matrix metalloproteinases (MMPs) were transcriptionally upregulated 24 h post-treatment. Amprenavir, at serum concentrations achievable given the manufacturer-recommended dose of fosamprenavir, protected against pepsin-induced cell dissociation, E-cadherin cleavage, and MMP induction. These results support a potential therapeutic role for amprenavir in GERD recalcitrant to PPI therapy and for preventing GERD-associated neoplastic changes.
... Dysphonia is one of the most frequent symptoms, accounting for up to 55% of LPR patients [1,2]. Dysphonia may be attributed to pepsin-related injuries, macroscopic and microscopic histological changes on the vocal folds, which may lead to aerodynamic and acoustic measurement impairments [3][4][5]. Because evolutions of non-specific symptoms and findings are still subjective, the identification of objective indicators of the treatment effectiveness remains challenging [1]. ...
... Our data supported that acoustic measurements may be used more specifically in LPR patients with dysphonia, while they are useless in patients without self-reported dysphonia. This thought appears consistent with the basic science and clinical studies that reported macroscopic and microscopic histological changes on the vocal folds of reflux subjects that are clinically highlighted by acoustic measurements [3][4][5][6]. The physiological mechanisms involve the pepsin-related impairments of defense mechanisms of the vocal folds, including mucin production, type III anhydrase carbonic activity, growth factor secretion, which may favor the occurrence of epithelial cell dehiscence, microtraumas, inflammatory infiltrate and macroscopic lesions [3]. ...
Objectives
The objective is to study the usefulness of acoustic measurements as therapeutic outcomes for patients with dysphonia related to laryngopharyngeal reflux (LPR).
Methods
From September 2019 to April 2021, 120 patients with LPR at the hypopharyngeal-esophageal multichannel intraluminal impedance pH-monitoring (HEMII-pH) were prospectively recruited from three University Hospitals. They were divided in two groups regarding the presence of dysphonia. The treatment consisted of a combination of diet, proton-pump inhibitors, magaldrate and alginate for 3–6 months. The following clinical and acoustic evaluations were studied regarding groups at baseline, 3- and 6-month posttreatment: reflux symptom score (RSS), reflux sign assessment (RSA), percent jitter, percent shimmer and noise-to-harmonic ratio (NHR).
Results
A total of 109 patients completed the evaluations, accounting for 49 dysphonic and 60 non-dysphonic individuals. HEMII-pH, gastrointestinal endoscopy, baseline clinical and acoustic features were comparable between groups. RSS and RSA significantly improved from pre- to 3-month posttreatment in both groups. Jitter, Shimmer and NHR significantly improved from pre- to 3-month posttreatment in dysphonic patients, without additional 3- to 6-month posttreatment changes. Acoustic parameters did not change throughout treatment in patients without dysphonia.
Conclusion
Acoustic measurements may be an interesting indicator of treatment in LPR patients who reported dysphonia. In this group of individuals, the evolution of acoustic parameters was consistent with the evolution of symptoms and findings.
... Pepsin is a digestive enzyme with maximal activity at pH 1.5-2.5; however, proteolytic activity has been demonstrated at pH levels as high at 6.5, and the enzyme remains stable up to a pH of 8 [9][10][11]. Tissue-bound pepsin within the larynx may be activated by acidic refluxate, leading to tissue damage and inflammation, while another study has demonstrated evidence of receptor-mediated endocytosis [8][9][10]. ...
... however, proteolytic activity has been demonstrated at pH levels as high at 6.5, and the enzyme remains stable up to a pH of 8 [9][10][11]. Tissue-bound pepsin within the larynx may be activated by acidic refluxate, leading to tissue damage and inflammation, while another study has demonstrated evidence of receptor-mediated endocytosis [8][9][10]. Interestingly and counterintuitively, proton pump inhibitors (PPIs) have been shown to exacerbate the pathogenic properties of pepsin, bile salts, and other gastroduodenal enzymes by creating a more alkaline milieu. ...
The purpose of this article is to review the cornerstone and most recent literature regarding laryngopharynoesophageal reflux (LPR) including epidemiological characteristics, pathophysiology, symptoms, diagnosis, and management. The role of pepsin in the pathophysiology of LPR is highlighted in addition to new diagnostic modalities and pharmacologic therapies that target pepsin.
... 1. Alteration in carbonic anhydrase secretion: Carbonic anhydrase is a defensive enzyme that contributes to endogenous secretion of bicarbonate within epithelial cells [26]. As a result to exposure of the laryngeal mucosa to pepsin and acid, there is a decrease in intracellular carbonic anhydrase secretion with a subsequent decrease in intraepithelial resistance [25,104,105]. Johnston et al. [106] investigated the correlation between pepsin and CA-III depletion in laryngeal biopsies taken from 9 patients with reflux and 12 normal controls. Using antibodies specific for human pepsins, the authors demonstrated the presence of pepsin in eight out of nine biopsies and a negative correlation between pepsin and CAI-III. ...
This article provides an overview of gastro‐esophageal (GERD) and laryngopharynegal (LPRD) reflux diseases in the context of obesity as a confounding entity. A detailed review of the clinical presentation, pathogenesis, diagnosis, and treatment of laryngopharyngeal reflux disease may be found elsewhere. The association between obesity and gastroesophageal reflux disease has been well established, and a similar Association exists between obesity and laryngopharyngeal reflux disease (LPRD). Reflux should be sought in obese patients with voice complaints, and a weight reduction should be recommended in patients in whom LPRD is diagnosed.
... Pepsin exhibits its highest activity at a pH of 2.0 and becomes inactive at a pH of 6.5, though it remains stable at a pH of 8.0. As a result, it can be reactivated when the pH drops again (12). Beyond its proteolytic function, pepsin plays a critical role as a major component of acidic refluxate (13). ...
Gastroesophageal reflux disease (GERD) is a common gastrointestinal disorder that significantly affects populations in both developing and developed countries. Due to both intrinsic pathology and extrinsic risk factors, the incidence of GERD has risen substantially in recent decades. This disorder results from an imbalance between the esophagus’s defensive mechanisms and the harmful effects of the refluxate. The pepsin, an enzyme secreted exclusively by the stomach, plays a critical role in the pathogenesis of GERD due to its invasiveness effects in acidic environments. By thoroughly understanding the pathogenesis of pepsin-induced GERD, we could better address its diagnostic and therapeutic potential in clinical practice. Although current diagnostic tools are widely used, they have several limitations. As a result, researchers have increasingly focused on the salivary pepsin test, a novel diagnostic method that utilizes the specific pathological mechanisms of pepsin. To overcome the drawbacks of the currently used salivary pepsin test, fluorescence response detection has been integrated with other technologies. Beyond its diagnostic significance, pepsin in saliva may also serve as a target for GERD management in innovative clinical trials. In this review, we summarize the latest advancements in the diagnosis and management of GERD to improve patient outcomes.
... As pepsin is activated in an acidic environment of pH 2.0 and is inactivated again at pH > 6.0, it may remain inactive when refluxed to the laryngopharynx at pH 6.8. However, it can be reactivated by subsequent acid reflux events, increasing proteolytic activity [3]. Pepsin can enter the oral cavity with reflux fluid and mix with the saliva when reflux occurs [4]. ...
In this study, we introduce a novel cleavage reaction lateral flow assay (LFA) based on pepsin activity against a pepsin-susceptible peptide (PSP) substrate to detect salivary pepsin. Two types of cleavage reaction LFAs, the within-tube and on-strip cleavage reactions, were prepared based on the PSP and pepsin reaction location. In the within-tube cleavage reaction LFA, samples were treated in the microtube within a heating block for 30 min separately and subsequently developed with running buffer in the LFA. For the on-strip cleavage reaction, samples were treated on the reaction zone of the strip within the heating zone of the multifunctional strip cassette for 10 min. After developing the running buffer in the LFA, the assay image was obtained using a universal mobile reader with a multifunctional strip cassette. The within-tube cleavage reaction LFA showed high sensitivity (limit of detection [LOD] 1.9 ng/mL), good specificity, and high reproducibility. This assay exhibited better linearity in the log concentration range of pepsin (4–500 ng/mL) than a commercially available dipstick assay. The on-strip cleavage reaction LFA showed a similar sensitivity (LOD 1.4 ng/mL) to that of the within-tube reaction assay. Therefore, we expect these cleavage reaction LFAs using PSP to be utilized as simple and effective tools to detect salivary pepsin.
... It is well known that although pepsin's enzymatic activity decreases as pH levels rise, the enzyme remains stable up to a pH of 8 [34]. While pepsin is enzymatically inactive at neutral pH, it is taken up by airway and esophageal epithelial cells through receptor-mediated endocytosis and is stored in intracellular vesicles of pH 4-5 where it would be reactivated [35,36]. We have previously demonstrated molecular mechanisms of injury by nonacid pepsin in esophageal cells that are consistent with inflammatory and carcinogenic processes attributed to GERD [31,37,38]. ...
Breakthrough symptoms are thought to occur in roughly half of all gastroesophageal reflux disease (GERD) patients despite maximal acid suppression (proton pump inhibitor, PPI) therapy. Topical alginates have recently been shown to enhance mucosal defense against acid-pepsin insult during GERD. We aimed to examine potential alginate protection of transcriptomic changes in a cell culture model of PPI-recalcitrant GERD. Immortalized normal-derived human esophageal epithelial cells underwent pretreatment with commercial alginate-based anti-reflux medications (Gaviscon Advance or Gaviscon Double Action), a matched-viscosity placebo control, or pH 7.4 buffer (sham) alone for 1 min, followed by exposure to pH 6.0 + pepsin or buffer alone for 3 min. RNA sequencing was conducted, and Ingenuity Pathway Analysis was performed with a false discovery rate of ≤0.01 and absolute fold-change of ≥1.3. Pepsin-acid exposure disrupted gene expressions associated with epithelial barrier function, chromatin structure, carcinogenesis, and inflammation. Alginate formulations demonstrated protection by mitigating these changes and promoting extracellular matrix repair, downregulating proto-oncogenes, and enhancing tumor suppressor expression. These data suggest molecular mechanisms by which alginates provide topical protection against injury during weakly acidic reflux and support a potential role for alginates in the prevention of GERD-related carcinogenesis.
... This can occur after it enters a cell or during a reflux episode [6]. This may explain the fact that even weakly acidic refluxate can damage the mucosa [7]. The role of pepsin in damaging the esophageal epithelium was experimentally demonstrated by Goldberg et al. as early as 1969 [8]. ...
The significance of extraesophageal reflux as a risk factor in lung adenocarcinoma has been understudied. In this study, we investigated whether extraesophageal reflux leads to higher pepsin concentrations in bronchoalveolar lavage (BAL) in patients with lung adenocarcinoma compared to controls. Subjects were recruited from non-smoker patients (lifelong non-smokers and ex-smokers with more than 5 years of non-smoking history) who had undergone bronchoscopy due to pulmonary abnormalities on a CT scan and met the inclusion criteria. Based on histological verification of the lung process, the patients were divided into three groups: (1) lung adenocarcinoma, (2) pulmonary metastases, and (3) lung sarcoidosis. Lung adenocarcinoma cases were further categorized as central or peripheral. BAL samples collected during bronchoscopy were quantitatively analyzed by enzyme-linked immunosorbent assay (ELISA) to measure pepsin levels. No statistically significant difference in pepsin concentration was observed between the lung adenocarcinoma group and control groups (p = 0.135). After excluding hemorrhagic BAL samples, the pepsin concentration was significantly the lowest in patients with lung adenocarcinoma (p = 0.023) compared to the control groups. The results of the study do not support the hypothesis of a higher occurrence of extraesophageal reflux (evaluated as the amount of pepsin in BAL) in non-smoker patients with lung adenocarcinoma.
... Although pepsin is inactive at pH values above 6.5, it remains stable until pH 8.0 and can be reactivated when the pH is reduced. 15,16 The current research indicates two pathways through which pepsin harms the pharyngeal mucosal epithelium. One is that pepsin retained in the mucosa damages the intercellular structure and intracellular protective proteins through its hydrolytic activity, leading to the destruction of the mucosal barrier of the pharyngeal epithelium. ...
Objective
To investigate the diagnostic value of the fasting hypopharyngeal salivary pepsin concentration test for laryngopharyngeal reflux disease (LPRD).
Methods
Volunteers were grouped by reflux symptom index scale, reflux finding score scale, and 24‐h hypopharyngeal‐esophageal multichannel intraluminal impedance with pH monitoring results. The study comprised 56, 27, and 20 individuals in the LPRD, asymptomatic laryngopharyngeal reflux (LPR), and asymptomatic non‐LPR groups, respectively. All underwent a fasting hypopharyngeal saliva pepsin testing via enzyme‐linked immunosorbent assay. Statistical analysis determined the optimal diagnostic cutoff value, sensitivity, and specificity of hypopharyngeal salivary pepsin for LPRD. Correlation analysis was performed with reflux scale scores and LPR parameters.
Results
Fasting hypopharyngeal salivary pepsin concentration in the LPRD group was significantly higher than in the asymptomatic control group (Z = −4.724, p < 0.05). The area under the receiver operating characteristic curve (AUC) analysis identified an optimal cutoff value of 29.62 ng/mL for salivary pepsin concentration (AUC, 0.767; sensitivity, 51.8%; and specificity, 93.6%). There was no statistically significant difference in salivary pepsin concentration between the asymptomatic LPR and non‐LPR groups. The concentration was higher in the LPRD group than in the asymptomatic LPR and non‐LPR groups (p < 0.05). Fasting hypopharyngeal salivary pepsin concentration exhibited a weakly positive correlation with vocal cord edema, intralaryngeal mucus adherence, excess throat mucus or postnasal drip, coughing during meals/lying down, troublesome or annoying cough, and liquid and weakly acidic reflux episodes.
Conclusions
The fasting hypopharyngeal salivary pepsin concentration test is a highly specific, simple, and noninvasive method with significant clinical diagnostic value for LPRD.
... Pepsin may remain inactive when refluxed to the laryngopharynx, as its pH is 6.8. However, it can remain stable in the epithelium for at least 24 h, even at a pH of 7.0, and can be activated when acid reflux occurs later, resulting in its resumed proteolytic activity 16 . Furthermore, pepsin can contribute to the inflammatory response of laryngopharyngeal mucosa, even in a non-acidic environment. ...
To analyze the predictive value of salivary pepsin for treatment outcomes in laryngopharyngeal reflux (LPR) using multivariate analysis that includes various associated factors. This prospective cohort study was conducted between August 2020 and August 2022. Patients with LPR who had symptoms lasting more than 1 month and a reflux symptom index (RSI) of 14 or higher were enrolled. The participants received a 2-month regimen of proton pump inhibitors (PPIs) treatment and lifestyle modification. Salivary pepsin was checked using fasting saliva before treatment. Salivary pepsin was detected more frequently in the good treatment response group (61.1%), compared to 14.3% in the poor response group. Similarly, patients with higher compliance to lifestyle modifications (> 90%) had a higher chance of a good response (91.7%) compared to those with lower compliance, who had a 53.8% chance of a good response. Other clinical factors have no significant association with treatment response. In multivariate analysis, both pretreatment salivary pepsin and higher compliance with lifestyle modification were found to be independent factors for treatment response (OR 14.457, CI 1.075 ~ 194.37 for both). This study found that positive salivary pepsin and strict lifestyle modification are independent predictors of treatment outcomes in LPR.
... While stomach acid may directly damage the airway epithelium (Liu et al., 2020), pepsin has been demonstrated to have a significant role in reflux-related hypopharyngeal trauma. Pepsin is most strongly activated at pH 1.5-2.5, deactivates (but remains stable for up to 24 hours) at 6.5-7.0, and is not irreversibly inactivated until pH 8.0 (Johnston et al., 2007). When in the hypopharynx, pepsin binds to cells, continues to digest proteins, damages cell cohesion, and causes mitochondrial damage (Johnston et al., 2010). ...
Laryngopharyngeal reflux (LPR) is a condition that affects up to 10% of the general population. It contributes to breathing, hearing, voicing, and swallowing problems. By incorporating knowledge of this phenomenon into their practice, clinicians may better address these concerns. This Spotlight on Nature, Assessment, and Management of Laryngopharyngeal Reflux equips clinicians with a current understanding of how and why LPR occurs, and what to do about it. Practical recommendations are provided to support immediate implementation of knowledge, and to improve patient care.
... The performance of the pepsin enzyme is hydrolyze aromatic amino acids, specifically at the N-terminal of an amino acid (Johnston et al., 2007). The pepsin enzyme will work optimally at pH 2, so it is necessary to set these conditions during virtual cutting. ...
Beta-casein in milk is known to be a bioactive peptide producer because of its amino acid sequence. Bioactive peptides have prospected molecules that can adhere with SARS-CoV-2 spike protein, so they can inhibit the virus from hooking up with human cell receptor protein. The research is aimed to find any peptides from goat’s milk beta-casein that are prospective candidates as SARS-CoV-2 spike protein inhibitors. Goat’s milk beta-casein was simulated as being digested by the digestive tract. Pepsin, trypsin, and chymotrypsin enzymes cut the beta-casein amino acids sequence into small peptides. Then, their bioavailability was predicted by Lipinski’s Rules of 5 (Ro5), any most fitted peptides to the rules will be simulated to dock to SARS-CoV2 spike protein besides Curcumin as the control ligand. Peptides with the best bind activity with the spike protein will be selected as inhibitor candidates. Peptide QPK is selected as a SARS-CoV-2 inhibitor candidate because it has better affinity energy than Curcumin or other selected peptides.
... The pepsin activity is related to the pH value of the environment, with the highest enzyme activity at pH 2.0, inactive at pH 6.5, and partially active at pH 2.0-pH 6.5. When the environment's pH drops from a higher level to below 2.0, 68-90% of activity can be recovered [18]. Pepsin enters the cells through endocytosis and is stored in the vesicles after it reaches the throat. ...
The aim of this study was to explore the changes in pH and pepsin concentrations in oral lavage fluid of rabbit reflux model. A total of 18 New Zealand rabbits were randomly divided into two groups. The lower esophageal sphincters (LESs) of the rabbits in the experimental group (EG) were dilated by balloon after the LESs were localized by manometry. The pH levels of the throat and the lower esophagus were monitored 1 week before and 2 weeks after inflation. Oral lavage fluid was collected 1 week before, and 2 and 8 weeks after inflation. The pH monitoring showed that the percentage of reflux time, the number of reflux events, and the longest time of reflux after the dilation (AE) in the EG were significantly higher than before the dilation (P < 0.01). The pepsin concentrations at 2 and 8 weeks AE in the EG were significantly higher than that before and that in the control group (P < 0.05). Based on receiver operating characteristic curve analysis, the best diagnostic threshold value was 30.3 ng/ml. The reflux model constructed by balloon inflation of the LES in rabbits is characterized by a decrease in throat pH and an increase in salivary pepsin concentration.
... Human gastric juices were provided by Department of Biochemistry and Molecular Biology, Qingdao University Medical College. Activity of pepsin in human gastric juice was evaluated by the method of (Johnston et al., 2007) with a slight modification using hemoglobin (HB) as the substrate. A volume of 50 μL human gastric juice was mixed with 250 μL (2.5 mg) of 37°C equilibrated HB substrate (pH 3.0), and incubated at 37°C for 10 min. ...
Nucleic acids (NAs) were recently shown to be digested by pepsin in vitro; however, NAs digestion in human gastric juice in vivo is more complicated because of the complex gastric environment and ingestion of other food components. The purpose of this study was to investigate the digestibility of NAs in real human gastric juices after ingestion of other food components. As a result, DNA digestion was not affected when carbohydrates, proteins, and metal elements were ingested within the recommended dietary intake levels. Separately, protein exerted an inhibitory effect on DNA digestion when the mass ratio of protein:DNA was greater than 40:1. DNA exists in the nucleoprotein, which is closer to the state of DNA in real food, and was digested efficiently in human gastric juice. Meanwhile, DNA digestion was rarely affected even when the concentrations of monovalent ion (Na ⁺ ) and divalent ions (Mg ²⁺ ) were as high as 500 and 100 mM, respectively, and high concentration of Mg ²⁺ ranged from 20 to 100 mM accelerated the digestion. In particular, short‐stranded DNA (<100 nt) and miRNAs (19 ~ 25 nt) were not obviously degraded in human gastric juice. In conclusion, dietary NAs were digested efficiently and were not affected by other food components in human gastric juice, which may facilitate further digestion and utilization of DNA in the intestinal tract.
... 5,[32][33][34][35][36][37] In experimental models, pepsin leads to inflammatory and carcinogenic changes irrespective of pH in vitro and in vivo including altered transcriptomic profiles; promotion of apoptotic resistance, cell migration, anchorage-independent growth and glycolysis; and development of tumors in a hamster cheek model. 27,33,[35][36][37][38][39][40][41] Although at neutral pH, pepsin is transiently inactivated and taken up by respiratory epithelia by receptor-mediated endocytosis where it appears to exert its proinflammatory and carcinogenic effects by initiation of molecular signaling events or intracellular reactivation, 27 pepsin in strong to weak acid (up to pH 6.5) retains enzymatic activity 42 and may degrade cell adhesion molecules resulting in impaired epithelial barrier integrity, 43-45 a hallmark of LPR and GERD thought to contribute to symptom origination. 46,47 The adhesion molecule E-cadherin is inversely correlated with pepsin in LPR 48 and has been demonstrated to undergo regulated intramembrane proteolysis (RIP) in GERD and LPR specimens, releasing biologically active fragments known to promote proinflammatory and cancer-associated signaling, including regulation of tissue-remodeling matrix metalloproteinases (MMPs). ...
Background
Laryngopharyngeal reflux (LPR) causes chronic cough, throat clearing, hoarseness, and dysphagia and can promote laryngeal carcinogenesis. More than 20% of the US population suffers from LPR and there is no effective medical therapy. Pepsin is a predominant source of damage during LPR which disrupts laryngeal barrier function potentially via E‐cadherin cleavage proteolysis and downstream matrix metalloproteinase (MMP) dysregulation. Fosamprenavir (FDA‐approved HIV therapeutic and prodrug of amprenavir) is a pepsin‐inhibiting LPR therapeutic candidate shown to rescue damage in an LPR mouse model. This study aimed to examine amprenavir protection against laryngeal monolayer disruption and related E‐cadherin proteolysis and MMP dysregulation in vitro.
Methods
Laryngeal (TVC HPV) cells were exposed to buffered saline, pH 7.4 or pH 4 ± 1 mg/mL pepsin ± amprenavir (10–60 min). Analysis was performed by microscopy, Western blot, and real time polymerase chain reaction (qPCR).
Results
Amprenavir (1 μM) rescued pepsin acid‐mediated cell dissociation (p < .05). Pepsin acid caused E‐cadherin cleavage indicative of regulated intramembrane proteolysis (RIP) and increased MMP‐1,3,7,9,14 24‐h postexposure (p < .05). Acid alone did not cause cell dissociation or E‐cadherin cleavage. Amprenavir (10 μM) protected against E‐cadherin cleavage and MMP‐1,9,14 induction (p < .05).
Conclusions
Amprenavir, at serum concentrations achievable provided the manufacturer's recommended dose of fosamprenavir for HIV, protects against pepsin‐mediated cell dissociation, E‐cadherin cleavage, and MMP dysregulation thought to contribute to barrier dysfunction and related symptoms during LPR. Fosamprenavir to amprenavir conversion by laryngeal epithelia, serum and saliva, and relative drug efficacies in an LPR mouse model are under investigation to inform development of inhaled formulations for LPR.
... 1 These components lead directly (acid and bile salts) or indirectly (most likely pepsin) to tissue damage causing inflammation and injury of the aerodigestive mucosa. 2,3 On this way, LPR can lead to extraesophageal symptoms with (i) established associations as reflux cough syndrome, reflux-laryngitis syndrome, reflux asthma syndrome, or the reflux dental erosion syndrome; and/or (ii) proposed associations as pharyngitis, sinusitis, idiopathic pulmonary fibrosis, or recurrent otitis media. 4 Today, the prevalence of LPR is estimated from 5 to 30% across different gastroesophageal reflux populations, defining atypical gastroesophageal reflux disease (GERD) manifestations in 10% the principal reason why patients visit their primary care doctors. ...
Up to 30% of patients with gastroesophageal reflux disease (GERD) suffer from laryngopharyngeal reflux (LPR) with symptoms, as chronic cough, laryngitis, or asthma. Besides life-style modifications and medical acid suppression, laparoscopic fundoplication is an established treatment option. Treatment-related side effects after laparoscopic fundoplication have to be weighted against LPR symptom control in 30-85% of patients after surgery. Magnetic sphincter augmentation (MSA) is described as an effective alternative to fundoplication for surgical treatment of GERD. However, evidence on the efficacy of MSA in patients with LPR is very limited. Preliminary data on the results of MSA treating LPR symptoms in patients with acid and weakly acid reflux are promising; showing comparable results to laparoscopic fundoplication by providing the potential of decrease side effects.
... In more alkaline environments, it is inactivated but is still stable and can be reactivated if the pH drops again, i.e., with the next acidic laryngopharyngeal reflux event. Recent studies show that pepsin can also be reactivated within the acidic intracellular environment after receptor-mediated uptake of pepsin by laryngeal epithelial cells, even if the pH in the throat is up to 7.4 [81]. Moreover, the laryngopharyngeal mucosa is still susceptible to pepsin even in a nonacidic environment because inactive pepsin can still stimulate the expression of many proinflammatory cytokines and receptors such as CXCL14, CCL20, IL1A, IL5, IL8, CCR6, CCL26, BCL6, and IL1F10 [82]. ...
Background:
Laryngopharyngeal reflux may affect people of any age; still, most of the accumulated knowledge concerns adults, and evidence regarding pediatric populations remains relatively restricted. This study aims to review the most recent and emerging aspects of pediatric laryngopharyngeal reflux from the last ten years. It also attempts to identify gaps in knowledge and highlight discrepancies that future research should urgently address.
Methods:
An electronic search of the MEDLINE database was conducted, limited to January 2012 through December 2021. Non-English language articles, case reports, and studies that concerned a purely or predominantly adult population were excluded. The information from the articles with the most relevant contribution was initially categorized by theme and subsequently synthesized into a narrative form.
Results:
86 articles were included, of which 27 were review articles, eight were surveys, and 51 were original articles. Our review systematically maps the research done in the last decade and provides an updated overview and the current state-of-the-art in this subject.
Conclusions:
Despite discrepancies and heterogeneity in accumulating research, evidence gathered so far endorses a need for refining an escalating multiparameter diagnostic approach. A step-wise therapeutic plan appears to be the most reasonable management approach, starting with behavioral changes for mild to moderate, uncomplicated cases and escalating to personalized pharmacotherapy options for severe or nonresponsive cases. Surgical options could be considered in the most severe cases when potentially life-threatening symptoms persist despite maximal medical therapy. Over the past decade, the amount of available evidence has been gradually increasing; however, its strength remains low. Several aspects remain markedly under-addressed, and further adequately powered, multicenter, controlled studies with uniformity in diagnostic procedures and criteria are urgently needed.
... 40 Although pepsin is inactivated when pH > 6.5, it remains stable and regains digestive activity when the pH decreases again. 41 Therefore, pepsin taken up by laryngeal epithelial cells is reactivated leading to cell destruction with HREs occurring and more severe epithelial cell damage when the acid HREs occur. ...
Objectives
To investigate the profiles of laryngopharyngeal reflux (LPR) and the relationship between hypopharyngeal-proximal reflux episodes (HREs) and the positive rate of salivary pepsin testing in patients with vocal fold lesions (VFLs).
Methods
The enrolled patients were divided into vocal fold cancer group (VFCG), vocal fold leukoplakia group (VFLG), benign vocal fold lesion group (BVFLG) and control group (CG). Patients benefited from multichannel intraluminal impedance-pH monitoring (MII-pH) and multi-time point salivary pepsin testing (MTPSPT). The LPR profiles of patients with VFLs were studied by analyzing the MII-pH findings. The relationship between HREs and positive rate of MTPSPT was investigated through the nonparametric test.
Results
177 patients were included. According to 24 h MII-pH, the occurrence of HREs tended to increase after meals. 55.75%, 63.98%, 66.82% and 55.77% of the HREs in the VFCG, VFLG, BVFLG and CG occurred within 3 h after meals, respectively. The overall positive rate of MTPSPT was higher in the VFCG than the remaining groups. In the VFCG, there was a significant correlation between overall positive results of MTPSPT and HREs occurring within 3 h after meals.
Conclusion
In the Chinese population, the occurrence of HREs tend to more frequently after meals in patients with VFLs, and most HREs occur within 3 h after meals. By analyzing the 24h MII-pH findings, we can develop a more individualized anti-reflux therapeutic strategy for LPR patients.
... pubs.acs.org/journal/abseba Article produce gels from decellularized matrices, 63 translation to inpatient use for IVD degeneration treatment may be hindered by the reversible inactivation of the pepsin, which regains its activity in acidic environments, 64 such as the degenerated IVD. Pepsin may be irreversibly inactivated at pH ≥8; 65 however, this will affect not only pepsin but the decellularized ECM proteins and potential encapsulated cells as well. ...
Porcine notochordal cell-derived matrix (NCM) has anti-inflammatory and regenerative effects on degenerated intervertebral discs. For its clinical use, safety must be assured. The porcine DNA is concerning because of (1) the transmission of endogenous retroviruses and (2) the inflammatory potential of cell-free DNA. Here, we present a simple, detergent-free protocol: tissue lyophilization lyses cells, and matrix integrity is preserved by limiting swelling during decellularization. DNA is digested quickly by a high nuclease concentration, followed by a short washout. Ninety-four percent of DNA was removed, and there was no loss of glycosaminoglycans or collagen. Forty-three percent of the total proteins remained in the decellularized NCM (dNCM). dNCM stimulated as much GAG production as NCM in nucleus pulposus cells but lost some anti-inflammatory effects. Reconstituted pulverized dNCM yielded a soft, shear-thinning biomaterial with a swelling ratio of 350% that also acted as an injectable cell carrier (cell viability >70%). dNCM can therefore be used as the basis for future biomaterials aimed at disc regeneration on a biological level and may restore joint mechanics by creating swelling pressure within the intervertebral disc.
... Bulk resistance measures the overall effect of gastric content resisting probe movement. During gastric digestion, the acidity in the stomach enhanced food softening by promoting food degradation through acid and enzymatic hydrolysis (Johnston et al., 2007). The textural softening and the disintegration of food occurred causing a reduction of the bulk resistance. ...
The objective of this study was to investigate the influence of particle size, texture, and gastric viscosity on food gastric disintegration and emptying rates. In vitro dynamic gastric digestion was conducted for carrots using a dynamic gastric simulation model. The changes in the texture of carrots and other foods as indicated by bulk resistance, the percent solids emptied, and the particle size distribution were used as parameters to study disintegration kinetics and emptying patterns. The influence of viscosity on food emptying was studied on both indigestible particles (amberlite beads) and digestible solids (carrots). The results indicated medium‐size carrot particles (1.40–2.00 mm) had a greater disintegration and a higher amount of emptying rate when compared to larger (2.00–3.34 mm) and smaller (1.14–1.40 mm) carrot particles. A high correlation exists between the final bulk resistance after 120 min digestion and the amount of solids emptied. Increasing viscosity up to values 8.20 Pa·s improved the particle dispersion for amberlite and carrots and increased rates of solids emptying, while further increase in viscosity hindered emptying of amberlite and carrot solids. The variable emptying rates of both indigestible and digestible solids with viscosity were described with a mathematical model based on particle‐emptying coefficient. A dynamic in vitro gastric digestion model was used to investigate how food particle size and texture, and gastric viscosity affect food disintegration and emptying. A mathematic model was proposed to predict the change in the emptying rate of solids with viscosity.
... It was found that the use of a head-over-heels rotation increased the bioaccessibility of sterols, compared with the commonly used orbital agitation at higher speed, due to a greater homogenization of the digestate. Furthermore, as the pepsin added in our trial was lower and its optimal activity is at pH 2 (much closer to the pH of the adult than that of the elderly), 35 at the end of digestion there will still be more protein to digest. This greater amount of protein could increase the bioaccessibility of PSs, as occurred with other lipophilic compounds. ...
Elderly people suffer from a higher cardiovascular risk. Thus, the fortification of foods with plant sterols (PSs), which have a cholesterol-lowering function, could be of great interest for this target group. To date, no studies have analyzed how the gastrointestinal conditions of the elderly affect PS bioaccessibility. Therefore, this study evaluated the impact of the adaptation of the gastric phase alone and in combination with the intestinal phase on sterol bioaccessibility. For this purpose, the standardized INFOGEST 2.0 method previously adapted for sterol bioaccessibility evaluation in healthy adults was applied to PS-enriched milk-based fruit beverages, examining changes in enzyme activity, incubation time, agitation and pH, based on elderly physiology. The results suggest that the specific gastrointestinal conditions of the elderly could increase absorption of PSs, since their bioaccessibility (%) in a PS-enriched milk-based fruit beverage was significantly increased compared with that in adults (14.95 ± 0.33 vs. 7.96 ± 0.26), also indicating that these conditions increase the bioaccessibility of the beverage's own cholesterol (61.25 ± 2.91 vs. 20.86 ± 2.79). These data support the recommendation of foods of this type for the elderly who can benefit from the increase in bioaccessibility of PSs to have an improved potential cholesterol lowering effect, thus decreasing their risk of cardiovascular disease. However, the performance of subsequent in vivo tests to confirm these results is necessary.
... We collected the supernatant by 30 min centrifugation at 4000× g. After filtering the supernatant through 0.2 μm bottle filters, we lowered the pH to 2 (optimum pH for pepsin function 25 ), added pepsin to the supernatant at a final concentration of 0.01 mg/mL, and incubated it at 4°C overnight for pepsin to digest the pepsin-sensitive impurities. Next, we concentrated the voluminous crude protein solution by cross-flow filtration. ...
... Five millilitre of bacterial culture grown at OD 600 = 1 was suspended in 0.5 ml synthetic gastric juice (150 mM of HCl, 15 mM of KCl and 0.5 mg ml À1 of pepsin) (Atkins, 1998), adjusted to pH 2.0 and mixed in a shaker under a rotation speed of 150 rpm min À1 at 37°C for 10 h. Since the enzymatic activity of pepsin is inactive at pH 6.5 or higher (Johnston, et al., 2007), the pH value was readjusted back to 7 and centrifuged to remove the precipitate. The supernatant was saved and served as the extract for testing bactericidal activity. ...
The lactoferricin expressed in Bacillus subtilis is relatively low in yield, making it hard to apply in industrial settings. We constructed a six tandem repeat of lactoferricin cDNA driven by promoter PtrnQ. After transformation, two transformants P245 and P263 possessing a stable inheritance of plasmid and high expression of lactoferricin were selected. The bactericidal activities, 1 μl of aliquot of a total 5.5 ml of solution extracted from 5 ml of cultured P245 and P263, were equivalent to the efficacy of 238.25 and 322.7 ng of Ampicillin against Escherichia coli, respectively, and 366.4 and 452.52 ng of Ampicillin against Staphylococcus epidermidis respectively. These extracts were able to kill an Ampicillin‐resistant E. coli strain. The bactericidal activities of P245 and P263 equivalent to the efficacy of Tetracycline against Vibrio parahaemolyticus and V. alginolyticus were also determined. Moreover, the bactericidal activities of P245 and P263 were 168.04 and 249.94 ng of Ampicillin against Edwardsiella tarda, respectively, and 219.7 and 252.43 ng of Tetracycline against Streptococcus iniae respectively. Interestingly, the survival rate of E. tarda‐infected tilapia fry fed the P263 extract displayed a significantly greater than that of the fry‐fed control strain. Collectively, these B. subtilis transgenic strains are highly promising for use in animal husbandry during a disease outbreak.
... It enters cells through endocytosis and is stored in vesicles or transported to other complex organelles (such as the Golgi apparatus), causing mitochondrial damage and promoting the expression of many tumor-related genes in a cell environment with a low pH [28]. (3) Pepsin reduces the expression of CA III and attenuates the neutralization of CA III on acid [29]. (4) It is involved in the stress response mediated by squamous epithelium stress proteins (Sep), leading to impaired laryngopharyngeal mucosal cell function [30]. ...
Purpose
Laryngopharyngeal reflux disease (LPRD) is a general term for the reflux of gastroduodenal contents into the laryngopharynx, oropharynx and even the nasopharynx, causing a series of symptoms and signs. Currently, little is known regarding the physiopathology of LPRD, and proton pump inhibitors (PPIs) are the drugs of choice for treatment. Although acid reflux plays a critical role in LPRD, PPIs fail to relieve symptoms in up to 40% of patients with LPRD. The influence of other reflux substances on LPRD, including pepsin, bile acid, and trypsin, has received increasing attention. Clarification of the substances involved in LPRD is the basis for LPRD treatment.
Methods
A review of the effects of acids, pepsin, bile acids, and trypsin on laryngopharyngeal reflux diseases was conducted in PubMed.
Results
Different reflux substances have different effects on LPRD, which will cause various symptoms, inflammatory diseases and neoplastic diseases of the laryngopharynx. For LPRD caused by different reflux substances, 24-h multichannel intraluminal impedance combined with pH-metry (MII-pH), salivary pepsin, bile acid and other tests should be established so that different drugs and treatment courses can be used to provide patients with more personalized treatment plans.
Conclusion
This article summarizes the research progress of different reflux substances on the pathogenesis, detection index and treatment of LPRD and lays a theoretical foundation to develop target drugs and clinical diagnosis and treatment.
Nowadays, gastroesophageal reflux disease (GERD) has emerged as one of the major hazards to the health of the upper gastrointestinal tract, and there is an urgent need for a low‐cost, user‐friendly, and non‐invasive detection method. Herein, a paper‐based sensor (CP sensor) for the non‐invasive screening of GERD is proposed. The sensor is structured as a specially shaped cellulose paper strip embedded with fluorescent colloids, which are self‐assembled from a cleavable synthetic fluorescent polymer (P4). Benefiting from the introduction of amide bonds and the unique assembled structure of the nanocolloids, the pepsin in the sample solution will hydrolyze the water‐soluble branches in the micellar shell during detection, resulting in a corresponding output of the fluorescent signal. This responsiveness, which can be observed by the naked eye, is so sensitive with a minimum detectable concentration for pepsin as low as 0.3 ng·mL⁻¹. Clinical trials have further demonstrates that the designed paper sensor is capable of providing improved accuracy in the early diagnosis of GERD.
Objective
The pathogenic mechanism underlying the effects of acidic pepsin in laryngeal cancer remains unclear. This study investigated whether acidic pepsin influences Glut‐1 expression and glycolytic activity in laryngeal carcinoma cells and whether it plays a role in the growth and migration of these cells through glycolysis.
Study Design
In vitro study.
Setting
A university‐affiliated hospital.
Methods
Laryngeal carcinoma TU 212 and TU 686 cells were treated with acidic pepsin and 2‐deoxy‐ d ‐glucose (2‐DG), then transfected with Glut‐1 small interfering RNA (siRNA). Glucose uptake was detected by a radioimmunoassay counter, lactate secretion was detected by a lactic acid kit, and Glut‐1 expression was detected by western blotting. Cell viability, migration and invasion, and clonal formation were assessed using the Cell Counting Kit‐8, Transwell chamber, and clonal formation assays, respectively.
Results
Acidic pepsin significantly increased Glut‐1 expression in laryngeal carcinoma cells compared with the control group ( P < .01). It also significantly enhanced ¹⁸ F‐fluorodeoxyglucose ( C in / C out ) uptake, lactate secretion, cell viability, migration, invasion, and clonal formation in laryngeal carcinoma cells compared with the control group ( P < .01). The glycolytic inhibitor 2‐DG and Glut‐1 siRNA significantly reversed the effects of acidic pepsin on laryngeal carcinoma cells ( P < .01).
Conclusion
Acidic pepsin enhances the growth and migration of laryngeal carcinoma cells by upregulating Glut‐1, thus promoting glycolysis.
A propeptide is removed from a precursor protein to generate its active or mature form. Propeptides play essential roles in protein folding, transportation, and activation and are present in about 2.3% of reviewed proteins in the UniProt database. They are often found in secreted or membrane‐bound proteins including proteolytic enzymes, hormones, and toxins. We identified a variety of globular and nonglobular Pfam domains in protein sequences designated as propeptides, some of which form intramolecular interactions with other domains in the mature proteins. Propeptide‐containing enzymes mostly function as proteases, as they are depleted in other enzyme classes such as hydrolases acting on DNA and RNA, isomerases, and lyases. We applied AlphaFold to generate structural models for over 7000 proteins with propeptides having no less than 20 residues. Analysis of residue contacts in these models revealed conformational changes for over 300 proteins before and after the cleavage of the propeptide. Examples of conformation change occur in several classes of proteolytic enzymes in the families of subtilisins, trypsins, aspartyl proteases, and thermolysin‐like metalloproteases. In most of the observed cases, cleavage of the propeptide releases the constraints imposed by the covalent bond between the propeptide and the mature protein, and cleavage enables stronger interactions between the propeptide and the mature protein. These findings suggest that post‐cleavage propeptides could play critical roles in regulating the activity of mature proteins.
Objective
This study aimed to evaluate the role of pepsin inhibitors in the inflammatory response and their effects on laryngeal mucosal integrity during gastroesophageal reflux (GERD) under in vivo conditions.
Methods
A surgical model of GERD was used, in which mice were treated with pepstatin (0.3 mg/kg) or darunavir (8.6 mg/kg) for 3 days. On the third day after the experimental protocol, the laryngeal samples were collected to assess the severity of inflammation (wet weight and myeloperoxidase activity) and mucosal integrity (transepithelial electrical resistance and paracellular epithelial permeability to fluorescein).
Results
The surgical GERD model was reproduced. It showed features of inflammation and loss of barrier function in the laryngeal mucosa. Pepstatin and darunavir administration suppressed laryngeal inflammation and preserved laryngeal mucosal integrity.
Conclusion
Pepsin inhibition by the administration of pepstatin and darunavir improved inflammation and protected the laryngeal mucosa in a mouse experimental model of GERD.
Level of Evidence
NA Laryngoscope , 2024
Objective: To review the current findings of the literature on the existence of several profiles of laryngopharyngeal reflux (LPR) patients and to propose personalized diagnostic and therapeutic approaches. Methods: A state-of-the art review of the literature was conducted using the PubMED, Scopus, and Cochrane Library databases. The information related to epidemiology, demographics, clinical presentations, diagnostic approaches, and therapeutic responses were extracted to identify outcomes that may influence the clinical and therapeutic courses of LPR. Results: The clinical presentation and therapeutic courses of LPR may be influenced by gender, age, weight, comorbidities, dietary habits and culture, anxiety, stress, and saliva enzyme profile. The clinical expression of reflux, including laryngopharyngeal, respiratory, nasal, and eye symptoms, and the hypopharyngeal–esophageal multichannel intraluminal impedance-pH monitoring profile of patients are important issues to improve in patient management. The use of more personalized therapeutic strategies appears to be associated with better symptom relief and cures over the long-term. The role of pepsin in LPR physiology is well-established but the lack of information about the role of other gastrointestinal enzymes in the development of LPR-related mucosa inflammation limits the development of future enzyme-based personalized diagnostic and therapeutic approaches. Conclusion: Laryngopharyngeal reflux is a challenging ear, nose, and throat condition associated with poor therapeutic responses and a long-term burden in Western countries. Artificial intelligence should be used for developing personalized therapeutic strategies based on patient features.
Laryngopharyngeal reflux (LPR) is an inflammatory condition of the upper respiratory tract tissues resulting from the direct or indirect effects of gastroduodenal content reflux. Although LPR shares similarities with gastroesophageal reflux disease (GERD) in pathophysiology, its symptoms and signs differ considerably. There remains a lack of consensus regarding diagnostic criteria, and no gold standard diagnostic method has been established, leading to ongoing research on standardizing diagnosis and treatment. Epidemiological data on LPR prevalence are scarce due to the absence of a definitive diagnostic technique, and the non-specific nature of its symptoms. Pathophysiologically, LPR can cause damage and irritation to the upper respiratory tract mucosa through direct and indirect mechanisms involving pepsin, bile acids, and vagal reflexes. Diagnosis remains challenging and relies on clinical evaluation, laryngopharyngoscopy, and pH-monitoring. Various clinical tools, such as the Reflux Symptom Index (RSI), Reflux Finding Score (RFS), and Reflux Sign Assessment (RSA), have been developed to aid diagnosis. Treatment approaches for LPR include lifestyle modifications, medical therapy, and surgical interventions. This book chapter provides an extensive and analytical overview of LPR, focusing on its epidemiology, pathophysiology, symptoms, signs, differential diagnosis, diagnostic methods, and treatment options.
Background:
Non-acid reflux is the most common form of laryngopharyngeal reflux (LPR). However, the damage caused by non-acid reflux to the laryngeal mucosa is weaker than that caused by acid reflux.
Aims:
To evaluate whether pepsin immunohistochemical (IHC) staining of laryngeal lesions can accurately diagnose acidic and non-acidic LPR.
Materials and methods:
Hypopharyngeal-esophageal multichannel intraluminal impedance-pH monitoring was performed, and the patients were divided into acid reflux and non-acid reflux groups. Pathological sections of laryngeal lesions were examined using pepsin IHC staining, which was positive when pepsin was detected in the cytoplasm.
Results:
The study included 136 patients, with 58 in the acid reflux group, 43 in the non-acid reflux group, and 35 in the without reflux group. There were no significant differences in the positive rate of pepsin IHC staining between the non-acid and acid reflux groups (p = .421). The sensitivity of pepsin IHC staining for the diagnosis of acid and non-acid reflux was 94.8% and 90.7%, respectively.
Conclusions:
The sensitivity of pepsin IHC staining for laryngeal lesions in the diagnosis of non-acidic LPR is satisfactory.
Significance:
Pepsin IHC staining is suitable for LPR screening of patients with laryngeal lesions as it is economical, non-invasive, and highly sensitive.
This review article seeks to define and describe aerodigestive disease in dogs, and review current and emerging methods of diagnostic evaluation. Aspiration of gastric contents into the respiratory tract is associated with the development and progression of numerous respiratory diseases in humans. In veterinary medicine the term “aspiration” is considered synonymous with “aspiration pneumonia” which, while frequently encountered, does not accurately reflect the breadth of aspiration associated respiratory syndromes (AARS). In the clinical veterinary literature, the effect of alimentary dysfunction on respiratory disease and vice versa (aerodigestive disease) is rarely investigated despite evidence in the human literature, animal models, and some studies and case reports linking alimentary and respiratory disease in small animals. Current methods of investigating aerodigestive diseases in veterinary patients are limited by inadeqate sensitivity or specificity, potential for bias, cost, and availability. This necessitates investigations into advanced diagnostics to identify potentially underrecognized animals with AARS. Additionally, similarities in anatomy, physiology, and several disorders between dogs and humans, make experimental and naturally occurring canine models of AARS integral to translational research. Thus, evaluating dogs with aerodigestive disease might represent an area of substantial clinical relevance in human as well as veterinary medicine.
Some of the main traits of urbanization are transition in macronutrient consumption and change in eating habits. Driven by cash economy, marketing, and other socio-cultural factors, urban dwellers have shifted toward the consumption of fast and processed food rich in fat and sugar. This transition in diet has affected health adversely aggravating many chronic illnesses such as diabetes, hypertension, gastro-esophageal reflux disease, and others. Gastro-esophageal reflux disease (laryngopharyngeal reflux disease) is a known and common culprit in patients with voice problems. High fat diet and the intake of refluxogenic products cause dysfunction of the lower and upper esophageal sphincters. As a result, there is back flow of gastro-duodenal contents into the larynx and pharynx leading to a constellation of symptoms including hoarseness. The transition in diet also has been linked to body weight. Obesity is known to impact voice by impairing respiration and increasing intra-abdominal pressure. It also causes marked alteration in vocal tract configuration and anatomy. This chapter reviews the impact of urbanization-induced transition in diet on voice. The pathophysiology of dysphonia in the context of macronutrient consumption and eating habits is emphasized.
This chapter provides a description of the gastric environment. Gastric fluids consist of numerous components either secreted by the stomach (gastric acid, enzymes, electrolytes, mucus), swallowed/ingested (saliva, food, liquids), or refluxed from the duodenum in the stomach. Gastric content volume can be described as the resultant volume in the stomach of ingested material, swallowed saliva, gastric secretions, and the emptying of gastric content in the small intestine, whereas total gastric volume also takes into account parts of the stomach void of liquid or solid material. Gastric acidity is crucial for several of the stomach's functions. In fed state, buffer capacity of gastric content is expected to initially be similar to that of the ingested meal, given the typically high meal‐to‐gastric fluid ratio. The chapter also presents the composition of the small intestinal contents under fasting and fed conditions and the luminal environment in the proximal colon.
We studied rheology and nanostructures (by small angle neutron scattering) of heat-set canola seed protein gels, containing cruciferin and napin, prepared at pH 8 and pH 11. We focused on gastric and intestinal digestion of ten mm pieces, mimicking human gastro-intestinal tract. Stronger gels, prepared at pH 11 (above the IEP, isoelectric points, of both proteins), retained local folded and compact conformations, close to native. Preparation at pH 8 (below napin IEP but above cruciferin one), could destabilize conformations due to charge differences of the proteins. For preparation pH 8, proteins were almost unfolded, and the gel softer. In gastric digestion, modulus decreased for pH 8 gels, but surprisingly, increased for pH 11. We propose a competition between unfolding, increasing local interactions (hence the modulus), and enzymatic scission. Scission could be less efficient for pH 11, with less unfolded proteins and higher crosslink density, hindering enzymatic diffusion. Additional interactions could result from crossing one or two IEPs, towards gastric pH 2. For intestinal digestion, the two gels behave similar (proteins re-compaction and modulus decrease). Beyond loss of submicronic connectivity, external erosion of the gel for largest times is observed, but less on SANS, which involves the centre of the piece.
Lactose intolerance in infants can be overcome by adding lactase enzyme formulations to milk, hydrolysing the lactose present. Commonly, such formulations require a significant incubation period after addition to milk to allow time for hydrolysis to take place at desired temperature. Such incubation is often problematic for household conditions, and therefore, formulations with short or, ideally, no incubation period are needed. Development of such formulations requires understanding of the complex process of lactose hydrolysis within the variable temperature and pH conditions in the milk bottle and in the gastrointestinal tract. The current paper describes the application of high-resolution ultrasonic spectroscopy for detailed characterisation of the effect of pH, temperature, and proteases present in the digestive tract on hydrolysis of lactose. The results were employed in the development of a predictive model of lactose hydrolysis within conditions of the infant digestive system. The model was applied for analysis of the whole infant feeding process: from hydrolysis in the bottle, to the stomach and small intestine, and for assessment of the impact of enzyme dosage, the incubation period, and bottle temperature profile on the levels of lactose and galacto-oligosaccharides (produced during hydrolysis), released from the stomach and reaching the large intestine where symptoms of intolerance arise. The results showed that the activity of Ha-Lactase in the stomach and small intestine assists significantly in the reduction of lactose load to 10 % or less of the amount of lactose in the feed, depending on conditions.
To develop and validate an analytical procedure for the quantitation of pepsins and gastricsin in human gastric juice and to assess its potential in a controlled gastric secretory study.
High performance ion-exchange chromatography was used to separate human pepsin 1, 3a, 3b, 3c and gastricsin from gastric juice. Computed chromatographic areas for each enzyme were quantified by relation to a known amount of a secondary standard porcine pepsin. The assay procedure was validated by recovery and analytical precision studies. Gastric secretions after pentagastrin and insulin stimulation from 10 patients with portal hypertension were used to assess the potential of the analytical procedure.
The assay precision varied from 1.5 to 9.0% within batch and 7.5 to 18.1% between batch, with about 100% recoveries of porcine pepsin A from human gastric juice over the assay range 0.025-0.5 mg/ml. A fourfold increase in combined pepsin and gastricsin concentration was observed following pentagastrin and insulin stimulation. The mean percentage content of pepsins 3a, 3b, 3c, and 1 in non-stimulated gastric juice were 4%, 72%, 12% and 1.4%, respectively, and did not change significantly after gastric stimulation. An approximate doubling of the percentage of gastricsin (10% to 20%) relative to the pepsins was observed, however, after both insulin and pentagastrin stimulation.
This procedure for quantifying individual human pepsins and gastricsin in gastric juice is simple and reliable. It may be of considerable importance in determining the mechanisms involved in the control and secretion of these digestive enzymes in man, including the effect of anti-ulcer drugs and our understanding of the pathophysiology of peptic ulcer disease.
Esophageal epithelium has intrinsic antireflux defenses, including carbonic anhydrases (CAs I to IV) that appear to be protective against gastric reflux. This study aimed to investigate the expression and distribution of CA isoenzymes in laryngeal epithelium. Laryngeal biopsy specimens collected from the vocal fold and interarytenoid regions were analyzed by Western blotting and immunofluorescence. Carbonic anhydrases I and II were expressed by the majority of samples analyzed. In contrast, CA III was differentially expressed in the interarytenoid samples and was not detected in any vocal fold samples. The expression of CA III was increased in esophagitis as compared to normal esophageal tissue. Carbonic anhydrase I and III isoenzymes were distributed cytoplasmically in the basal and lower prickle cell layers. The laryngeal epithelium expresses some CA isoenzymes and has the potential to protect itself against laryngopharyngeal reflux. Laryngeal tissue may be more sensitive to injury due to reflux damage than the esophageal mucosa because of different responses of CA isoenzymes.
This is the second annual report of an international collaborative research group that is examining the cellular impact of laryngopharyngeal reflux (LPR) on laryngeal epithelium. The results of clinical and experimental studies are presented. Carbonic anhydrase (CA), E-cadherin, and MUC gene expression were analyzed in patients with LPR, in controls, and in an in vitro model. In patients with LPR, we found decreased levels of CAIII in vocal fold epithelium and increased levels in posterior commissure epithelium. The experimental studies confirm that laryngeal CAIII is depleted in response to reflux. Also, cell damage does occur well above pH 4.0. In addition, E-cadherin (transmembrane cell surface molecules, which have a key function in epithelial cell adhesion) was not present in 37% of the LPR laryngeal specimens. In conclusion, the laryngeal epithelium lacks defenses comparable to those in esophageal epithelium, and these differences may contribute to the increased susceptibility of laryngeal epithelium to reflux-related injury.
Objectives
The objectives of this study were to define the conditions that give rise to a stress protein response in laryngeal epithelium and to investigate whether and how stress protein dysfunction contributes to reflux-related laryngeal disease.
Methods
Western analysis was used to measure stress protein (squamous epithelial proteins Sep70 and Sep53 and heat shock protein Hsp70) and pepsin levels in esophageal and laryngeal tissue specimens taken from both normal control subjects and patients with pH-documented laryngopharyngeal reflux (LPR) who had documented lesions, some of whom had laryngeal cancer. A porcine organ culture model was used to examine the effects of low pH and pepsin (0.1% porcine pepsin A) on stress protein levels. A laryngeal squamous carcinoma (FaDu) cell line was used to examine uptake of human pepsin 3b-tetramethyl-5 and -6 isothiocyanate.
Results
Sep70, Sep53, and Hsp70 were found to be expressed at high levels, and pepsin was not detected, in esophageal and laryngeal specimens taken from normal control subjects and in esophageal specimens taken from LPR patients. The patients with LPR were found to have significantly less laryngeal Sep70 (p = .027) and marginally less laryngeal Sep53 (p = .056) than the normal control subjects. Laryngeal Hsp70 was expressed at high levels in the LPR patients. The patients with laryngeal cancer had significantly lower levels of Sep70, Sep53 (p < .01), and Hsp70 (p < .05) than the normal control subjects. A significant association was found between the presence of pepsin in laryngeal epithelium from LPR patients and depletion of laryngeal Sep70 (p < .001). Using the organ culture model, we demonstrated that laryngeal Sep70 and Sep53 proteins are induced after exposure to low pH. However, in the presence of pepsin, Sep70 and Sep53 levels are depleted. Confocal microscopy analysis of cultured cells exposed to labeled pepsin revealed that uptake is by receptor-mediated endocytosis.
Conclusions
These findings suggest that receptor-mediated uptake of pepsin by laryngeal epithelial cells, as may occur in LPR, causes a change in the normal acid-mediated stress protein response. This altered stress protein response may lead to cellular injury and thus play a role in the development of disease.
The human oesophageal epithelium is subject to damage from thermal stresses and low extracellular pH that can play a role in the cancer progression sequence, thus identifying a physiological model system that can be used to determine how stress responses control carcinogenesis. The classic heat shock protein HSP70 is not induced but rather is down-regulated after thermal injury to squamous epithelium ex vivo; this prompted a longer-term study to address the nature of the heat shock response in this cell type. An ex vivo epithelial culture system was subsequently used to identify three major proteins of 78, 70, and 58 kDa, whose steady-state levels are elevated after heat shock. Two of the three heat shock proteins were identified by mass spectrometric sequencing to be the calcium-calmodulin homologue transglutaminase-3 (78 kDa) and a recently cloned oesophageal-specific gene called C1orf10, which encodes a 53-kDa putative calcium binding protein we have named squamous epithelial heat shock protein 53 (SEP53). The 70-kDa heat shock protein (we have named SEP70) was not identifiable by mass spectrometry, but it was purified and studied immunochemically to demonstrate that it is distinct from HSP70 protein. Monoclonal antibodies to SEP70 protein were developed to indicate that: (a) SEP70 is induced by exposure of cultured cells to low pH or glucose starvation, under conditions where HSP70 protein was strikingly down-regulated; and (b) SEP70 protein exhibits variable expression in preneoplastic Barrett's epithelium under conditions where HSP70 protein is not expressed. These results indicate that human oesophageal squamous epithelium exhibits an atypical heat shock protein response, presumably due to the evolutionary adaptation of cells within this organ to survive in an unusual microenvironment exposed to chemical, thermal and acid reflux stresses.
Occult (silent) gastroesophageal reflux disease (GER, GERD) is believed to be an important etiologic factor in the development of many inflammatory and neoplastic disorders of the upper aerodigestive tract. In order to test this hypothesis, a human study and an animal study were performed. The human study consisted primarily of applying a new diagnostic technique (double‐probe pH monitoring) to a population of otolaryngology patients with GERD to determine the incidence of overt and occult GERD. The animal study consisted of experiments to evaluate the potential damaging effects of intermittent GER on the larynx.
Two hundred twenty‐five consecutive patients with otolaryngologic disorders having suspected GERD evaluated from 1985 through 1988 are reported. Ambulatory 24‐hour intraesophageal pH monitoring was performed in 197; of those, 81% underwent double‐probe pH monitoring, with the second pH probe being placed in the hypopharynx at the laryngeal inlet. Seventy percent of the patients also underwent barium esophagography with videofluoroscopy.
The patient population was divided into seven diagnostic subgroups: carcinoma of the larynx (n = 31), laryngeal and tracheal stenosis (n = 33), reflux laryngitis (n = 61), globus pharyngeus (n = 27), dysphagia (n = 25), chronic cough (n = 30), and a group with miscellaneous disorders (n = 18).
The most common symptoms were hoarseness (71%), cough (51%), globus (47%), and throat clearing (42%). Only 43% of the patients had gastrointestinal symptoms (heartburn or acid regurgitation). Thus, by traditional symptomatology, GER was occult or silent in the majority of the study population.
Twenty‐eight patients (12%) refused or could not tolerate pH monitoring. Of the patients undergoing diagnostic pH monitoring, 62% had abnormal esophageal pH studies, and 30% demonstrated reflux into the pharynx. The results of diagnostic pH monitoring for each of the subgroups were as follows (percentag with abnormal studies): carcinoma (71%), stenosis (78%), reflux laryngitis (60%), globus (58%), dysphagia (45%), chronic cough (52%), and miscellaneous (13%). The highest yield of abnormal pharngeal reflux was in the carcinoma group and the stenosis group (58% and 56%, respectively).
By comparison, the diagnostic barium esophagogram with videofluoroscopy was frequently negative. The results were as follows: esophagitis (18%), reflux (9%), esophageal dysmotility (12%), and stricture (3%).
All of the study patients were treated with antireflux therapy. Follow‐up was available on 68% of the patients and the mean follow‐up period was 11.6 ± 12.7 months. After 6 months of treatment, symptoms had resolved in 85% and medical therapy had failed in 15%. Subsequently, an additional 20% experienced medical treatment failure. Fifteen percent of patients underwent Nissen fundoplication, and all subsequently had resolution of symptoms.
To further investigate the role of gastroesophageal reflux in the development of laryngeal damage, experiments mimicking the effects of intermittent reflux (of acid and pepsin) on the canine larynx were performed. The results of these experiments revealed: 1 . Intermittent reflux (three episodes per week) can result in severe laryngeal damage if there is prior mucosal injury; 2 . pepsin, and not hydrochloric acid, is the principal injurious agent of the refluxate; and, 3 . severe laryngeal damage can occur even when the pH of the refluxate is 4.0.
The manuscript describes the limitations and advantages of standard diagnostic procedures and of 24‐hour pH monitoring. The differences between gastroenterology and otolaryngology patients with GERD are emphasized and specific new diagnostic and therapeutic recommendations are made.
The hydrolysis of the chromogenic peptide Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond by nine aspartic proteinases of animal origin and seven enzymes from micro-organisms is described [Phe(4-NO2) is p-nitro-L-phenylalanine]. A further series of six peptides was synthesized in which the residue in the P3 position was systematically varied from hydrophobic to hydrophilic. The Phe-Phe(4-NO2) bond was established as the only peptide bond cleaved, and kinetic constants were obtained for the hydrolysis of these peptide substrates by a representative selection of aspartic proteinases of animal and microbial origin. The value of these water-soluble substrates for structure-function investigations is discussed.
Esomeprazole, the S isomer of omeprazole, has been shown to have higher healing rates of erosive esophagitis than omeprazole. This study compared esomeprazole with lansoprazole for the healing of erosive esophagitis and resolution of heartburn.
This United States multicenter, randomized, double blind, parallel group trial was performed in 5241 adult patients (intent-to-treat population) with endoscopically documented erosive esophagitis, which was graded by severity at baseline (Los Angeles classification). Patients received 40 mg of esomeprazole (n = 2624) or 30 mg of lansoprazole (n = 2617) once daily before breakfast for up to 8 wk. The primary efficacy endpoint was healing of erosive esophagitis at week 8. Secondary assessments included proportion of patients healed at week 4, resolution of investigator-recorded heartburn, time to first and time to sustained resolution of patient diary-recorded heartburn, and proportion of heartburn-free days and nights.
Esomeprazole (40 mg) demonstrated significantly higher healing rates (92.6%, 95% CI = 91.5-93.6%) than lansoprazole (30 mg) (88.8%, 95% CI = 87.5-90.0%) at week 8 (p = 0.0001, life-table estimates, intent-to-treat analysis). A significant difference in healing rates favoring esomeprazole was also observed at week 4. The difference in healing rates between esomeprazole and lansoprazole increased as baseline severity of erosive esophagitis increased. Sustained resolution of heartburn occurred faster and in more patients treated with esomeprazole. Sustained resolution of nocturnal heartburn also occurred faster with esomeprazole. Both treatments were well tolerated.
Esomeprazole (40 mg) is more effective than lansoprazole (30 mg) in healing erosive esophagitis and resolving heartburn. Healing rates are consistently high with esomeprazole, irrespective of baseline disease severity.
Frequency occurrence of nonacidic and nonliquid reflux events in the pharynx has not been systematically studied. The aim of the present study was to characterize the physical (liquid, gas, and mixed gas/liquid) and pH properties of the gastroesophagopharyngeal refluxate.
We performed a total of 31 24-h simultaneous ambulatory pharyngoesophageal impedance and pH recordings in 11 GERD patients, 10 patients with reflux-attributed laryngitis, and 10 healthy controls.
On average, the total number of reflux events (all kinds) in the pharynx was less than half of that in the proximal esophagus (18 +/- 4 vs 50 +/- 4, p < 0.01). Most of the pharyngeal reflux events were gas events and were observed in all three studied groups. Prevalence of these gas reflux events ranged between 0 and 74. The number of gas reflux events accompanied by a minor pH drop in laryngitis patients (1 (0-36)) was significantly higher than those in GERD and controls (0 (0-2) and 0 (0-1), respectively, p < 0.05). There was no significant difference in the number of nonacidic gas reflux events among the three groups (GERD: 10 (2-57), laryngitis: 11.5 (0-51), controls: 10.5 (0-27)). Impedance recording identified a total number of 566 events in the pharynx. Of these, a total of 563 events were compatible with gas reflux events, 101 events were accompanied by minor drops in intrapharyngeal pH, whereas 460 events were not accompanied by any pharyngeal pH change.
Concurrent impedance and pH recordings detect significantly more events qualifying as reflux in the pharynx than pH recordings alone. A substantial majority of these events are gaseous refluxes both with and without minor pH drops. Gas reflux events with weak acidity appear to be more common among patients with reflux-attributed laryngeal lesions compared to GERD patients and controls.
Proton pump inhibitors are the mainstay of medical management in gastroesophageal reflux disease. Although they provide relief from most symptoms, reflux may persist. We hypothesize that omeprazole does not reduce the total amount of gastroesophageal reflux but simply alters its pH characteristics. Six asymptomatic volunteers had combined 24-hour impedance pH monitoring before and after 7 days of omeprazole (20 mg BID). Multichannel intraluminal impedance was used to identify reflux episodes, which were classified as acid (pH < 4), weak acid (pH > 4 but decrease > 1 pH unit) and nonacid (pH > 4 and decrease < 1 pH unit) by pH measurements 5 cm above the lower esophageal sphincter (LES). A gastric pH sensor located 10 cm below the LES was used to verify the action of omeprazole. Impedance detected a total of 116 reflux episodes before and 96 episodes after omeprazole treatment. The median number of reflux episodes (18 versus 16, P = 0.4), median duration of reflux episodes (4.7 versus 3.6 minutes, P = 0.5), and total duration of reflux episodes (27.2 versus 42.4 minutes, P = 0.5) per subject were similar before and after omeprazole. Acid reflux episodes were reduced from 63% before to 2.1% after omeprazole (P < 0.0001), whereas nonacid reflux episodes increased (15% to 76%, P < 0.0001). Weak acid reflux episodes did not change (22.4% to 21.8%, P = 1.0). The proportion of reflux episodes greater than pH 4 increased from 37% to 98% (P < 0.0001). In normal subjects, omeprazole treatment does not affect the number of reflux episodes or their duration; rather it converts acid reflux to less acid reflux, thus exposing esophagus to altered gastric juice. These observations may explain the persistence of symptoms and emergence of mucosal injury white on proton pump inhibitor therapy.
The objective was to investigate the potential use of pepsin and carbonic anhydrase isoenzyme III (CA-III) as diagnostic markers for laryngopharyngeal reflux disease.
Prospective cell biological investigation was conducted of laryngeal biopsy specimens taken from 9 patients with laryngopharyngeal reflux disease and 12 normal control subjects using antibodies specific for human pepsin (produced in the authors' laboratory within the Department of Otolaryngology at Wake Forest University Health Sciences, Winston-Salem, NC) and CA-III.
Laryngeal biopsy specimens were frozen in liquid nitrogen for Western blot analysis and fixed in formalin for pepsin immunohistochemical study. Specimens between two groups (patients with laryngopharyngeal reflux disease and control subjects) were compared for the presence of pepsin. Further analyses investigated the correlation between pepsin, CA-III depletion, and pH testing data.
Analysis revealed that the level of pepsin was significantly different between the two groups (P < .001). Secondary analyses demonstrated that presence of pepsin correlated with CA-III depletion in the laryngeal vocal fold and ventricle (P < .001) and with pH testing data in individuals with laryngopharyngeal reflux disease.
Pepsin was detected in 8 of 9 patients with laryngopharyngeal reflux disease, but not in normal control subjects (0 of 12). The presence of pepsin was associated with CA-III depletion in the laryngeal vocal fold and ventricle. Given the correlation between laryngopharyngeal reflux disease and CA-III depletion, it is highly plausible that CA-III depletion, as a result of pepsin exposure during laryngopharyngeal reflux, predisposes laryngeal mucosa to reflux-related inflammatory damage.
The significance of gastroesophageal reflux disease (GERD) in laryngeal cancer is controversial due to disparate studies. To evaluate the overall strength of the association of GERD with laryngeal cancer, we performed meta-analysis of the original studies in literature.
Meta-analysis.
All studies cited on Ovid Medline (1966-June 2004), EMBASE (1980-June 2004), and Cochrane database describing GERD and laryngeal cancer were eligible for inclusion. The inclusion criteria for the study included original controlled study design and a clear documentation of the reflux prevalence in cases and controls. Statistical analysis was performed by NCSS software.
Fifteen original studies were identified. Eight studies did not have control groups, two studies did not clearly document GERD prevalence in controls, and two studies were published using the same data, one of which was included in this meta-analysis. Thus, four studies qualified for inclusion for the meta-analysis. The methodologic quality in the studies was heterogeneous, not only in the evaluation of confounding risk factors such as smoking and alcohol but also in the mode of GERD diagnosis. There was also significant heterogeneity of effect of reflux among the studies (P = .001). The pooled odds ratio on the basis of fixed-effects model was 2.86 (95% CI, 2.73-2.99), and on the basis of random-effects model was 2.37 (95% CI 1.38-4.08).
Our meta-analysis suggests that GERD may be a significant risk factor for laryngeal cancer. However, given the heterogeneity of the published data, future prospective controlled studies are needed.
Airway symptoms are often caused by aspiration of refluxed materials into the larynx. In this study we sought to define the frequency, character, and proximal extent of refluxed contents - including nonacid reflux-in normal subjects using intraluminal impedance to improve our understanding of the relationship between reflux and aspiration. Ten subjects, who had no symptoms of gastroesophageal reflux disease or airway disease, underwent impedance/pH monitoring with a catheter that allowed simultaneous esophageal and pharyngeal monitoring. Impedance detected 496 gastroesophageal reflux episodes in the 10 subjects during 240 hours of study. The majority, 399 (81% of the total) were acid reflux episodes (pH < 4). Ninety-seven were nonacid (pH > 4). Most reflux episodes (348 of 496) reached the mid esophagus (9 cm above lower esophageal sphincter). There were 51 reflux episodes that reached the pharynx (PR). Only 13 (25%) of PR were acidic (pH < 4), while 38 were nonacid. Twenty-six PR episodes were liquid and 25 were mixed (liquid and gas). The median number of PR episodes measured with impedance was 5 (0-10). In asymptomatic subjects, most episodes of gastroesophageal reflux are acidic and reach the midesophagus. Reflux into the PR appears to be more common than previously believed, and most of these episodes are not acidic. Thus, traditional 24-hour pH monitoring may underestimate the presence of pharyngeal reflux. The combination of impedance with pH monitoring markedly enhances our ability to accurately detect potential microaspiration.
Empiric proton pump inhibitor (PPI) trials have become increasingly popular leading to gastroenterologists frequently evaluating gastro-oesophageal reflux disease (GORD) patients only after they have "failed" PPI therapy. Combined multichannel intraluminal impedance and pH (MII-pH) monitoring has the ability to detect gastro-oesophageal reflux (GOR) episodes independent of their pH and evaluate the relationship between symptoms and all types of GOR. Using this technique, we aimed to characterise the frequency of acid and non-acid reflux (NAR) and their relationship to typical and atypical GOR symptoms in patients on PPI therapy.
Patients with persistent GORD symptoms referred to three centres underwent 24 hour combined MII-pH monitoring while taking PPIs at least twice daily. Reflux episodes were detected by impedance channels located 3, 5, 7, 9, 15, and 17 cm above the lower oesophageal sphincter (LOS) and classified into acid or non-acid based on pH data from 5 cm above the LOS. A positive symptom index (SI) was declared if at least half of each specific symptom events were preceded by reflux episodes within five minutes.
A total of 168 patients (103 (61%) females and 65 (39%) males; mean age 53 (range 18-85) years) underwent combined MII-pH monitoring while taking PPIs at least twice daily. One hundred and forty four (86%) patients recorded symptoms during the study day and 24 (15%) patients had no symptoms during testing. Sixty nine (48%) symptomatic patients had a positive SI for at least one symptom (16 (11%) with acid reflux and 53 (37%) with NAR) and 75 (52%) had a negative SI. A total of 171 (57%) typical GORD symptoms were recorded, 19 (11%) had a positive SI for acid reflux, 52 (31%) for NAR, and 100 (58%) had a negative SI. One hundred and thirty one (43%) atypical symptoms were recorded, four (3%) had a positive SI for acid reflux, 25 (19%) had a positive SI for NAR, and 102 (78%) had a negative SI.
Combined MII-pH identifies the relation of reflux of all types to persistent symptoms and the importance of NAR in patients taking PPIs.