Objectives: Studies using combined multichannel intraluminal impedance with pH monitoring reveal a role for nonacidic reflux in laryngopharyngeal symptoms and injury. We have discovered that pepsin is taken up by laryngeal epithelial cells by receptor-mediated endocytosis. This finding reveals a novel mechanism by which pepsin could cause cell damage, potentially even in nonacidic refluxate. The objective of this study was to determine whether pepsin, at pH 7.4 and thus in nonacidic refluxate, causes cell damage. Methods: Cultured hypopharyngeal epithelial (FaDu) cells were exposed to human pepsin (0.1 mg/mL) at pH 7.4 for either 1 hour or 12 hours at 37°C and analyzed by electron microscopy, cytotoxicity assay, and SuperArray. Results: We report mitochondrial and Golgi complex damage in cells exposed to pepsin at neutral pH, observed by electron microscopy. We also report cell toxicity of pepsin at pH 7.4, measured by a cytotoxicity assay. Furthermore, using SuperArray, we found that pepsin at pH 7.4 significantly alters the expression levels of multiple genes implicated in stress and toxicity. Conclusions: These findings are perhaps the first to explain why many patients have symptoms and injury associated with nonacidic reflux, and could have important implications for the development of new therapies for reflux, such as pepsin receptor antagonists and/or irreversible inhibitors of peptic activity.

Diagnosis of extraesophageal reflux (EER) currently relies on tools designed for diagnosis of gastroesophageal reflux. Such tools lack the sensitivity and reproducibility to detect the less frequent and mildly acidic reflux associated with upper airway disease. Pepsin has been posited to be a reliable biological marker of EER. Our aim was to present a comprehensive literature review of the use of pepsin as a diagnostic marker of EER. Two methods are typically used for detection of pepsin in the airways: enzymatic and immunologic. The limitations, advantages, and examples of use of each are discussed. Pepsin assay has been used to identify refluxate in trachea, lung, sinus, middle ear, combined sputum and saliva, and breath condensate. An immunologic pepsin assay of combined sputum and saliva was determined to be 100% sensitive and 89% specific for detection of EER (based on pH-metry), and an enzymatic test of nasal lavage fluid (100% sensitivity and 92.5% specificity) demonstrated an increased incidence of EER in patients with chronic rhinosinusitis. Pepsin assay identified tracheal pepsin to be an indicator of bronchopulmonary dysplasia and related mortality risk in ventilated preterm infants. Pepsin assay is a useful tool for correlation of reflux with airway disease and is a reliable diagnostic marker of EER.

Objectives: We undertook to 1) obtain unequivocal evidence to confirm or rebut our initial observations that pepsin is taken up by hypopharyngeal epithelial cells by receptor-mediated endocytosis, 2) investigate whether uptake of pepsin at pH 7, in nonacidic refluxate, is of pathological significance, and 3) test our hypothesis that inactive but stable pepsin (<pH 8) taken up by hypopharyngeal epithelial cells causes damage by becoming reactivated inside the cell. Methods: Human posterior cricoid biopsy specimens and cultured hypopharyngeal FaDu epithelial cells were used to perform competitive binding studies and to investigate colocalization of pepsin with clathrin, Rab-9, and TRG-46. FaDu cells were exposed to pepsin (both irreversibly and reversibly inactivated) in the presence and absence of wortmannin and dimethyl amiloride and analyzed by electron microscopy, MTT cytotoxicity assay, and Stress and Toxicity SuperArray. Results: Pepsin is unequivocally taken up by hypopharyngeal epithelial cells by receptor-mediated endocytosis. Uptake of pepsin at pH 7, in nonacidic refluxate, causes mitochondrial damage and changes the expression of several genes implicated in stress and toxicity. Irreversible, but not reversible, inhibition of peptic activity prevents these changes. Conclusions: Pepsin, at pH 7, in nonacidic refluxate, causes damage by becoming reactivated inside the cell. Irreversible inhibitors of peptic activity hold promise as a new therapy for reflux.

Objectives: We sought to document the mucin gene profile in normal human laryngeal epithelium and compare it with that in patients with reflux-attributed laryngeal injury or disease. We also investigated the effect of low pH with or without pepsin on mucin messenger RNA levels in vitro. Methods: Laryngeal biopsy specimens were obtained from 3 patients with clinically diagnosed laryngopharyngeal reflux and from 2 control subjects who had no signs or symptoms of reflux. Signs and symptoms were assessed by the Reflux Finding Score and the Reflux Symptom Index, respectively. Reverse transcription–polymerase chain reaction (RT-PCR) was performed to establish the mucin gene profile. Human hypopharyngeal epithelial cells were exposed to pH 7, 5, 4, and 2 with and without pepsin (0.1 mg/mL) for 20 minutes at 37°C, and expression of selected mucins was analyzed via real-time RT-PCR. Results: Mucin 1–5, 7, 9, 13, 15, 16, and 18–20 transcripts were detected in normal laryngeal epithelium, whereas mucin 6, 8, and 17 transcripts were not. Mucins 2, 3, and 5 were expressed at reduced levels in patients with reflux-attributed laryngeal injury or disease. These mucin genes were up-regulated after exposure to low pH in vitro (p < 0.005). Pepsin inhibited this up-regulation (p < 0.001). Conclusions: Reflux laryngitis is associated with down-regulation of mucin gene expression.