Association of pepsin and DNA damage in laryngopharyngeal reflux-related vocal fold polyps.

PURPOSE: This study aimed to evaluate if laryngopharyngeal reflux (LPR) plays a role as a risk factor for vocal fold polyps (VFPs), and if pepsin is associated with higher oxidative DNA damage of VFPs in the presence of LPR. METHODS: Thirty patients with VFPs were recruited between 2017 and 2018. Prior to surgery, a laryngoscopy was performed on all subjects to evaluate VFPs. Polyp tissue and saliva samples were obtained scrupulously. Hematoxylin-eosin staining was performed for pathologic analysis. Immunohistochemistry and ELISA were used to detect pepsin in tissue and saliva of VFP patients. 8-OHdG and p-H2AX expression was detected to measure oxidative DNA damage in tissue. DNA damage was investigated in human immortalized laryngeal epithelial cells exposed to pepsin. RESULTS: The pepsin concentration in saliva was significantly higher (t = 2.38, P = .024) in the pepsin positive group. There was no significant difference in pepsin expression at different sites and pathological subtypes of VFPs. The levels of 8-OHdG and p-H2AX were significantly higher in the pepsin positive group and positively correlated with the tissue expression of pepsin. The concentration of pepsin in saliva also showed a significant correlation with 8-OHdG levels. Expression of 8-OHdG and p-H2AX, and tail moment of the comet assay were elevated in human immortalized laryngeal epithelial cells following treatment with pepsin. CONCLUSION: Patients with VFPs have higher levels of oxidative DNA damage in the presence of pepsin reflux. Pepsin may induce DNA damage in laryngeal epithelial cells and participate in the pathogenesis of VFPs.

A non-interventional, observational study of a fixed combination of pepsin and amino acid hydrochloride in patients with functional dyspepsia.

BACKGROUND: Functional dyspepsia (FD) is a gastrointestinal disorder characterized by recurrent and diverse symptoms and pathophysiology that remains unexplained following routine clinical investigation. Enzynorm®f is a pharmaceutical preparation comprising fixed amounts of pepsin of biological origin and organically bound acid in the form of amino acid hydrochloride. It is traditionally used as a mild agent to support gastric function and to stimulate the stomach's proteolytic activities in FD. METHODS: In a non-interventional, observational, post-marketing surveillance study, patients with an established diagnosis of FD were treated with a fixed combination of pepsin and amino acid hydrochloride taken as tablets three times daily for 6 weeks. The primary objective of this study was to assess the change in symptoms using the validated Gastrointestinal Symptom Score (GIS©). Secondary objectives included patients' assessment of their gastrointestinal symptoms as well as treatment safety and tolerability. RESULTS: A total of 97 patients (mean age 58.4 ± 13.9 years; 63.2% females) were included in the study, with 72 data having GIS© score data at baseline and at 6 weeks, and 34 also at 3 weeks. The overall GIS© sum score decreased by 4.1 (p < 0.0001) from 11.6 (±4.8) at baseline to 7.4 (± 4.6) reflecting an improvement of clinical symptomatology after 6 weeks of treatment. In a subgroup of 70 patients who had FD meeting the Rome III criteria a GIS© score reduction of ≥50% was observed after 3 weeks treatment in 24% and in 30.8% after 6 weeks. Adverse events were mostly gastrointestinal in nature and consistent with the underlying disease; no unexpected adverse reactions were reported. Twenty-seven patients discontinued the study, mostly because of gastrointestinal symptoms. CONCLUSION: The results of this study support the efficacy of a fixed combination of pepsin and amino acid hydrochloride for the treatment of patients with FD and also suggest good to moderate treatment tolerability. These findings should be further explored in a randomised, placebo-controlled clinical trial. CLINICAL TRIAL REGISTRATION: This study has been retrospectively registered in the ClinicalTrials.gov registry, trial identifier NCT03076411 .

RNA-Seq Reveals Protective Mechanisms of Mongolian Medicine Molor-Dabos-4 on Acute Indomethacin-Induced Gastric Ulcers in Rats.

This study aimed to apply transcriptomics to determine how Molor-Dabos-4 (MD-4) protects healthy rats against indomethacin (IND)-induced gastric ulcers and to identify the mechanism behind this protective effect. Rats were pretreated with MD-4 (0.3, 1.5, or 3 g/kg per day) for 21 days before inducing gastric ulcers by oral administration with indomethacin (30 mg/kg). Unulcerated and untreated healthy rats were used as controls. Effects of the treatment were assessed based on the ulcer index, histological and pathological examinations, and indicators of inflammation, which were determined by enzyme-linked immunosorbent assay. Transcriptomic analysis was performed for identifying potential pharmacological mechanisms. Eventually, after identifying potential target genes, the latter were validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). After pretreatment with MD-4, gastric ulcers, along with other histopathological features, were reduced. MD-4 significantly (p < 0.05) increased the superoxide dismutase (SOD) levels in ulcers and reduced pepsin, TNF-α, and IL-6 levels. RNA-seq analysis identified a number of target genes on which MD-4 could potentially act. Many of these genes were involved in pathways that were linked to anti-inflammatory and antioxidant responses, and other protective mechanisms for the gastric mucosa. qRT-PCR showed that altered expression of the selected genes, such as Srm, Ryr-1, Eno3, Prkag3, and Eef1a2, was consistent with the transcriptome results. MD-4 exerts protective effects against IND-induced gastric ulcers by reducing inflammatory cytokines and pepsin and increasing the expression of SOD levels. Downregulation of Srm, Ryr-1, Eno3, Prkag3, and Eef1a2 genes involved in regulating arginine and proline metabolism, calcium signaling pathway, HIF-1 signaling pathway, oxytocin signaling pathway, and legionellosis are possibly involved in MD-4-mediated protection against gastric ulcers.

Role of ATP in the ROS-mediated laryngeal airway hyperreactivity induced by laryngeal acid-pepsin insult in anesthetized rats.

The pathogenetic mechanisms of laryngeal airway hyperreactivity (LAH) in patients with extraesophageal reflux are unclear. We recently reported that a laryngeal acid-pepsin insult produces LAH that is mediated through sensitization of the capsaicin-sensitive laryngeal afferent fibers by reactive oxygen species (ROS) in rats. Since ROS may promote the release of ATP from cells, we hypothesized that activation of P2X purinoceptors by ATP subsequent to an increase in ROS induces LAH in an inflamed larynx that has been insulted by acid-pepsin or H(2)O(2) (a major type of ROS). The larynxes of 208 anesthetized rats were functionally isolated while the animals breathed spontaneously. Ammonia vapor was delivered into the larynx to measure laryngeal reflex reactivity. Laryngeal insult with acid-pepsin or H(2)O(2) produced LAH with similar characteristics. The H(2)O(2)-induced LAH was prevented by laryngeal pretreatment with dimethylthiourea (a hydroxyl radical scavenger), suggesting a critical role for ROS. The LAH induced by both insults were completely prevented by ATP scavengers (a combination of apyrase and adenosine deaminase) or a P2X receptor antagonist (iso-pyridoxalphosphate-6-azophenyl-2',5'-disulfonate). Laryngeal application of a P2X receptor agonist (alpha,beta-methylene-ATP) also produced LAH. An insult with either acid-pepsin or H(2)O(2) similarly promoted an increase in the levels of ATP, lipid peroxidation, and inflammation in the larynx. Our findings suggest that laryngeal insult with acid-pepsin or H(2)O(2) induces inflammation and produces excess ROS in the rat's larynx. The latter may in turn promote the release of ATP to activate P2X receptors, resulting in sensitization of capsaicin-sensitive laryngeal afferent fibers and LAH.

Lactobacillus rhamnosus GG Protects the Epithelial Barrier of Wistar Rats from the Pepsin-Trypsin-Digested Gliadin (PTG)-Induced Enteropathy.

Celiac disease (CD) is a chronic immune-mediated disorder, characterized by enhanced paracellular permeability across the intestinal epithelium. The complex system of intercellular junctions, including tight junctions (TJs) and adherens junctions (AJs), seals together the epithelial cells to form a continuous layer. The improvements in barrier integrity have been related to modifications in intercellular junction protein expression. Polyamines (spermidine, spermine, and putrescine) actively participate in the modulation of the AJ expression. Both in vitro and in vivo studies have demonstrated that also probiotics can promote the integrity and the function of the intestinal barrier. On these bases, the present work investigated the protective effects exerted by Lactobacillus rhamnosus GG (L.GG) against the pepsin-trypsin-digested gliadin (PTG)-induced enteropathy in jejunal tissue samples of Wistar rats. In particular, the probiotic effects have been evaluated on the intestinal mucosal architecture, polyamine metabolism and intercellular junction protein expression (ZO-1, Occludin, Claudin-1, ß-catenin and E-cadherin). The results from this study indicate that L.GG protects the intestinal mucosa of rats from PTG-induced damage, by preventing the reduction of the expression of the intercellular junction proteins. Consequently, a role for L.GG in the therapeutic management of the gluten-related disorders in humans could be hypothesized.

Neural and hydroxyl radical mechanisms underlying laryngeal airway hyperreactivity induced by laryngeal acid-pepsin insult in anesthetized rats.

Laryngopharyngeal or gastroesophageal reflux is associated with laryngeal airway hyperreactivity (LAH), but neither the cause-effect relationship nor the underlying mechanism has been elucidated. Here we established a rat model with enhanced laryngeal reflex reactivity induced by laryngeal acid-pepsin insult and investigated the neural and hydroxyl radical (*OH) mechanisms involved. The laryngeal segments of 103 anesthetized rats were functionally isolated while animals breathed spontaneously. Ammonia vapor was delivered into the laryngeal segment to measure laryngeal reflex reactivity. We found that the laryngeal pH 5-pepsin treatment doubled the reflex apneic response to ammonia, whereas laryngeal pH 7.4-pepsin, pH 2-pepsin, and pH 5-denatured pepsin treatment had no effect. Histological examination revealed limited laryngeal inflammation and epithelial damage after pH 5-pepsin treatment and more severe damage after pH 2-pepsin treatment. In rats that had received the laryngeal pH 5-pepsin treatment, the apneic response to ammonia was abolished by either denervation or perineural capsaicin treatment (PCT; a procedure that selectively blocks capsaicin-sensitive afferent fibers) of the superior laryngeal nerves, but was unaffected by perineural sham treatment. LAH was prevented by laryngeal application of either dimethylthiourea (DMTU; a *OH scavenger) or deferoxamine (DEF; an antioxidant for *OH), but was unaltered by the DMTU vehicle or iron-saturated DEF (ineffective DEF). LAH reappeared after recovery from PCT, DMTU, or DEF treatment. We conclude that 1) laryngeal insult by pepsin at a weakly acidic pH, but not at acidic pH, can produce LAH; and 2) LAH is probably mediated through sensitization of the capsaicin-sensitive laryngeal afferent fibers by a *OH mechanism.

Occupational rhinitis due to pepsin.

A woman presented work-related rhinoconjunctivitis due to inhalation of pepsin used in a slaughterhouse. Prick tests and conjunctival challenge were positive to pepsin. Serum specific IgE to pepsin was 5.58 kU/L and an IgE-binding band of 43 kDa was detected in SDS-PAGE Immunoblotting. Rhinoconjunctivitis improved clearly when the patient was assigned to another place without contact with pepsin. Pepsin has been previously reported to cause occupational allergic asthma on three occasions. As far as we know, this is the first reported case in which an IgE-immunoblot has been performed.

Bioadhesive oesophageal bandages: protection against acid and pepsin injury.

The rate of acid and pepsin diffusion through solutions of sodium alginate was measured using in vitro techniques. Previous work has demonstrated that solutions of alginate may adhere to the oesophagus for up to 60 min; this work measured their ability to protect the oesophageal epithelial surface from damage caused by refluxed acid and pepsin. Franz diffusion cells were used to measure the rate of acid and pepsin diffusion through an alginate layer. The effect of the type of alginate, alginate concentration and depth of alginate applied were investigated. The rate of both acid and pepsin diffusion was significantly reduced (ANOVA analysis; P<0.05) in the presence of an alginate solution compared to the control. A 2% (w/v) alginate solution with a high guluronic acid component, in a layer of 0.44 mm depth, demonstrated the greatest reduction in acid diffusion with a permeation coefficient 14% than that of a control value. All three alginates demonstrated significant reductions in acid diffusion with both increasing depth and increasing concentration, as expected. Pepsin diffusion was also significantly reduced as the depth and concentration of applied alginate increased. This study demonstrates that an adhesive layer of alginate present within the oesophagus will limit the contact of refluxed acid and pepsin with the epithelial surface.

c-Fos immunoreactivity in the brain after esophageal acid stimulation.

The goal of this study was to use c-Fos immunohistochemistry to establish a rat model for studying the central projection of the esophageal afferent neurons during acid exposure. A cannula was placed in the esophagus of anesthetized Wistar rats with the tip approximately 2 cm above the lower esophageal sphincter (LES). Hydrochloric acid (0.1 N HCl, 50 mmol/L) with pepsin (3,200-4,500 U/mL), at pH 1.6, was then perfused into the esophagi of the experimental rats (n = 8) at 10 mL/hr continuously for 50 minutes. Normal saline solution (0.9% NaCl) was used in control rats (n = 6), and home cage control animals (n = 6) were given no stimulation. Thirty minutes after the perfusion, the rat was killed and the brain was removed and processed for c-Fos immunohistochemistry. A transverse section of the esophagus, 2 cm above the LES, was stained with hematoxylin and eosin stain for light microscopy. c-Fos immunoreactivity was significantly increased in a number of brain regions in the rats receiving the acid plus pepsin perfusion. These areas included the central amygdala, the Kölliker-Fuse nucleus, the nucleus of the solitary tract (NTS), the medial part of the NTS, the interstitial part of the NTS, the commissural part of the NTS, the paratrigeminal nucleus, the ambiguus nucleus, and the rostroventrolateral recticular nucleus. Perfusion with acid-pepsin solution also resulted in morphologic changes in the esophagus on light microscopy. This study suggests that acid plus pepsin perfusion of esophagus results in both neural activation in areas of the central nervous system and damage to the esophagus in an animal model.

In-vitro cytotoxic activity of cross-linked protein microcapsules.

Microcapsules (5-100 microns) were prepared through interfacial cross-linking of various proteins (human serum albumin, lysozyme, haemoglobin, casein, pepsin) with glutaraldehyde or terephthaloylchloride. Surprisingly they all showed an inhibitory effect on cultured cells in a concentration range of 100 micrograms ml-1 to 10 mg ml-1. This effect seemed non-specific, reversible and to depend on contact with the cell plasma membrane. The electric charges of microcapsules could be involved in the inhibition phenomenon.

Function of the eustachian tube after weekly exposure to pepsin/hydrochloric acid.

OBJECTIVE: To determine the effects of repeated pepsin/hydrochloric acid (HCl) exposure on the eustachian tube (ET). STUDY DESIGN AND SETTING: ET function was studied in 22 rats. Group I (control) rats received transtympanic phosphate buffered saline solution; groups II (0.5 mg/ml) and III (2.0 mg/ml) received transtympanic pepsin/HCl. Test solutions were applied on day 0 with ET function evaluated on days 1, 2, 3, and 7 after exposure. Each 7-day period represents 1 cycle; all groups underwent 4 cycles. ET function was evaluated using passive opening and closing pressure, and active clearance of positive and negative pressure tests. RESULTS: Rats exposed to pepsin/HCl had elevated passive opening pressures and a decreased ability to clear positive and negative pressure. A temporal relationship exists. CONCLUSION: The results suggest middle ear exposure to pepsin/HCl leads to ET dysfunction in rats, and that this dysfunction is enhanced with repeated exposures. SIGNIFICANCE: Gastroesophageal reflux may induce ET dysfunction.

Pepsin hydrolysis of the adherent mucus barrier and subsequent gastric mucosal damage in the rat: effect of diosmectite and 16,16 dimethyl prostaglandin E2.

BACKGROUND AND OBJECTIVE: The gastroduodenal mucus layer is progressively eroded at its luminal surface as a consequence of pepsin mucolysis. Diosmectite binds to gastric mucus and modifies its rheological properties. Prostaglandins are well-known mucus secretagogues. The aim of this study is to describe interactions of diosmectite and 16,16 dimethyl prostaglandin E2 on adherent gastroduodenal mucus and pepsin mucolysis in the rat. METHODS: Instillation of pepsin (1 or 2 mg.mL-1 at pH1 or pH2) into the pylorus ligated stomach of anaesthetised rats resulted in progressive disruption of the adherent mucus layer and a large, significant, increase in soluble degraded mucin compared to that following instillation of HCl pH1 or pH2. Pepsin (2 mg.mL-1), instillation over 2 hours, but not HCl alone, consistently resulted in small focal, haemorrhagic mucosal lesions, significant bleeding into the lumen and histologically, localised punctate ulcers in an otherwise intact epithelium. Diosmectite (500 mg.kg-1) and 16,16 dimethyl prostaglandin E2 were given by oro-gastric intubation. RESULTS: Diosmectite, given 30 minutes beforehand, inhibited breakdown of the adherent gastric mucus barrier by pepsin in vivo. When administered up to 16 hours prior to the experiment, diosmectite prevented pepsin induced gastric mucosal haemorrhage and histological epithelial damage. Substantial amounts of diosmectite (39.6 micrograms/mm2, equivalent in volume to layer 93 microns thick) were bound to the gastric mucosa 30 minutes after administration. Diosmectite (100:1 by weight to enzyme) completely inhibited pepsin hydrolysis of protein in vitro. Topical 16,16 dimethyl prostaglandin E2, 5 micrograms.kg-1 increased the thickness of the adherent mucus layer by two-fold. Both doses of the prostaglandin prevented pepsin induced gastric mucosal haemorrhage and histological epithelial damage. CONCLUSIONS: These results show that both diosmectite and prostaglandin increase the effectiveness of the mucus barrier against mucosal damage by pepsin in vivo.

Animal model of acid-reflux esophagitis: pathogenic roles of acid/pepsin, prostaglandins, and amino acids.

Esophagitis was induced in rats within 3 h by ligating both the pylorus and transitional region between the forestomach and glandular portion under ether anesthesia. This esophageal injury was prevented by the administration of acid suppressants and antipepsin drug and aggravated by exogenous pepsin. Damage was also aggravated by pretreatment with indomethacin and the selective COX-1 but not COX-2 inhibitor, whereas PGE2 showed a biphasic effect depending on the dose; a protection at low doses, and an aggravation at high doses, with both being mediated by EP1 receptors. Various amino acids also affected this esophagitis in different ways; L-alanine and L-glutamine had a deleterious effect, while L-arginine and glycine were highly protective, both due to yet unidentified mechanisms. It is assumed that acid/pepsin plays a major pathogenic role in this model of esophagitis; PGs derived from COX-1 are involved in mucosal defense of the esophagus; and some amino acids are protective against esophagitis. These findings also suggest a novel therapeutic approach in the treatment of esophagitis, in addition to acid suppressant therapy. The model introduced may be useful to test the protective effects of drugs on esophagitis and investigate the mucosal defense mechanism in the esophagus.

Chronic pepsin exposure promotes anchorage-independent growth and migration of a hypopharyngeal squamous cell line.

OUTCOME OBJECTIVES: (1) Investigate the role of reflux, specifically pepsin, in laryngopharyngeal carcinogenesis. (2) Evaluate effects of chronic pepsin exposure on cell migration, apoptosis, and colony-forming ability in hypopharyngeal cells. STUDY DESIGN: Translation research. SETTING: Academic research laboratory. METHODS: Human hypopharyngeal squamous carcinoma FaDu cells were chronically exposed to nonacidic pepsin (exposed for 24 hours, 4 times over 2 weeks at the following concentrations: 0.01 mg/mL, 0.1 mg/mL, or 1 mg/mL). Precise wounds were created in confluent cell plates, and rates of cell migration into wounds were quantified. Separately, cell viability of chronic pepsin-exposed FaDu cells acutely treated with paclitaxel was measured. Finally, a clonogenic assay was performed on these cells to measure effects of chronic pepsin exposure on colony-forming ability. RESULTS: An increased rate of relative wound density was observed in chronic pepsin-treated (0.01 mg/mL, 0.1 mg/mL) cells compared with control (P < .001), suggesting greater rates of cell migration. Pepsin-treated (0.1 mg/mL) cells demonstrated on average greater cell viability compared with control after exposure to paclitaxel, suggesting possible apoptotic resistance; however, this was not statistically significant. Chronic pepsin exposure (0.1 mg/mL, 1 mg/mL) was associated with a dose-dependent increase in colony-forming ability relative to control (P < .001). CONCLUSION: Hypopharyngeal squamous cell line chronically exposed to pepsin demonstrated increased cell migration and colony-forming ability relative to control cells. These experiments indicate that chronic pepsin exposure acts as a promoter of tumorigenesis and metastasis of airway epithelium, suggesting a role for pepsin in laryngopharyngeal carcinogenesis attributed to gastric reflux.

Prevention of dentine erosion by brushing with anti-erosive toothpastes.

OBJECTIVE: This in vitro study aimed to investigate the preventive effect of brushing with anti-erosive toothpastes compared to a conventional fluoride toothpaste on dentine erosion. MATERIALS AND METHODS: Bovine dentine specimens (n=12 per subgroup) were eroded in an artificial mouth (6 days, 6×30 s/day) using either citric acid (pH:2.5) or a hydrochloric acid/pepsin solution (pH:1.6), simulating extrinsic or intrinsic erosive conditions, respectively. In between, the specimens were rinsed with artificial saliva. Twice daily, the specimens were brushed for 15 s in an automatic brushing machine at 2.5 N with a conventional fluoride toothpaste slurry (elmex, AmF) or toothpaste slurries with anti-erosive formulations: Apacare (NaF/1% nHAP), Biorepair (ZnCO3-HAP), Chitodent (Chitosan), elmex Erosionsschutz (NaF/AmF/SnCl2/Chitosan), mirasensitive hap (NaF/30% HAP), Sensodyne Proschmelz (NaF/KNO3). Unbrushed specimens served as control. Dentine loss was measured profilometrically and statistically analysed using two-way and one-way ANOVA followed by Scheffe's post hoc tests. RDA-values of all toothpastes were determined, and linear mixed models were applied to analyse the influence of toothpaste abrasivity on dentine wear (p<0.05). RESULTS: Dentine erosion of unbrushed specimens amounted to 5.1±1.0 µm (extrinsic conditions) and 12.9±1.4 µm (intrinsic conditions). All toothpastes significantly reduced dentine erosion by 24-67% (extrinsic conditions) and 21-40% (intrinsic conditions). Biorepair was least effective, while all other toothpastes were not significantly different from each other. Linear mixed models did not show a significant effect of the RDA-value of the respective toothpaste on dentine loss. CONCLUSION: Toothpastes with anti-erosive formulations reduced dentine erosion, especially under simulated extrinsic erosive conditions, but were not superior to a conventional fluoride toothpaste.

Review article: reflux and its consequences--the laryngeal, pulmonary and oesophageal manifestations. Conference held in conjunction with the 9th International Symposium on Human Pepsin (ISHP) Kingston-upon-Hull, UK, 21-23 April 2010.

BACKGROUND: Gastro-oesophageal reflux disease (GERD) is one of the commonest diseases of Western populations, affecting 20 to 30% of adults. GERD is multifaceted and the classical oesophageal symptoms such as heartburn and regurgitation often overlap with atypical symptoms that impact upon the respiratory system and airways. This is referred to as extra-oesophageal reflux disease (EERD), or laryngopharyngeal reflux (LPR), which manifests as chronic cough, laryngitis, hoarseness, voice disorders and asthma. AIM: The 'Reflux and its consequences' conference was held in Hull in 2010 and brought together a multidisciplinary group of experts all with a common interest in the many manifestations of reflux disease to present recent research and clinical progress in GERD and EERD. In particular new techniques for diagnosing reflux were showcased at the conference. METHODS: Both clinical and non-clinical key opinion leaders were invited to write a review on key areas presented at the `Reflux and its consequences' conference for inclusion in this supplement. RESULTS AND CONCLUSION: Eleven chapters contained in this supplement reflected the sessions of the conference and included discussion of the nature of the refluxate (acid, pepsin, bile acids and non-acid reflux); mechanisms of tissue damage and protection in the oesophagus, laryngopharynx and airways. Clinical conditions with a reflux aetiology including asthma, chronic cough, airway disease, LPR, and paediatric EERD were reviewed. In addition methods for diagnosis of reflux disease and treatment strategies, especially with reference to non-acid reflux, were considered.

Laryngeal mucosa: its susceptibility to damage by acid and pepsin.

OBJECTIVES/HYPOTHESIS: Exposure of pig laryngeal mucosa to pepsin and acid will have a differential damaging effect depending on the anatomical site, mirroring the effects seen in the human larynx in laryngopharyngeal reflux (LPR). This study aims to quantitate damage caused to laryngeal tissue by acid alone, and acid and pepsin, and also to determine if the extent of this damage depends on the tissue site. STUDY DESIGN: Prospective translational research study. METHODS: An excised porcine laryngeal damage model in a small Ussing chamber was used to measure the effect of pepsin and acid on five sites (ventricles, vocal folds, posterior commissure, supraglottic, and subglottic mucosa). The tissue samples were incubated on the lumenal side for 1 hour with pH 2 and 4 HCl, pH 2 plus 1 mg/mL pepsin, and pH 4 plus 1 mg/mL pepsin. Damage was assessed by changes in absorbance of the bathing solution at optical density (OD) 260 nm and OD 280 nm and by measurement of released DNA compared to tissues bathed in pH 7.4 buffer. Damage was also assessed histologically. RESULTS: Based on histology, all the tissues were resistant to pH 4.0 except the subglottic mucosa. Only the posterior commissure was not damaged by pH 2.0 plus pepsin. Similar patterns were observed with absorbance changes and DNA release. CONCLUSIONS: The subglottic mucosa was the most susceptible to damage and the posterior commissure the least. Laryngeal tissues are essentially resistant to damage at pH 4.0, but are damaged when pepsin is present. This suggests that in LPR, pH 4.0 or above refluxate would only be damaging if it contains pepsin.