Activity/Stability of Human Pepsin: Implications for Reflux Attributed Laryngeal Disease

Laryngopharyngeal reflux (LPR), defined as the backflow of gastric contents into the laryngopharynx, contributes to several otolaryngologic inflammatory disorders and neoplastic diseases. It is has been reported in up to 10% of patients referred to otolaryngologists for treatment and in 50% of patients with laryngeal and voice disorders. Damage to the laryngeal epithelium is believed central in the development of reflux-attributed laryngeal disease, thought to occur after a breakdown in defense. Despite recent advances in diagnosing LPR, very little is known regarding its effect on the biology and biochemistry of the laryngeal epithelium.

For several years, a presumed relationship between LPR and laryngeal lesions and disease contributed to a focus on reflux pH as the principal diagnostic and therapeutic target for LPR. Double-probe pH monitoring is commonly used to diagnose acid reflux into the laryngopharynx; however, this does not address the role of non/weak acid reflux, which is now known to occur, causing symptoms and mucosal injury. Proton pump inhibitors (PPIs), the most potent form of acid suppression therapy available, are widely prescribed to treat LPR, yet even high doses can often be inadequate to treat many patients with LPR-attributed symptoms and disease. This is because PPI therapy decreases the hydrogen ion concentration of the refluxed fluid but not the number or duration of reflux events.  With new evidence that symptoms and injury can be caused by non/weak acid reflux (using combined multichannel intraluminal impedance pH), it is clear that PPI therapy alone has limited ability to protect patients from LPR-induced damage.

It is likely that patients with incomplete response to acid suppression may have significant involvement from other components of the gastric refluxate, such as pepsin and bile acids, both of which can injure the laryngeal/esophageal epithelium at non/weakly acidic pH. Pepsin is the major proteolytic enzyme produced in the stomach by gastric chief cells. We have demonstrated that pepsin is present in the laryngeal epithelium of patients with reflux-attributed laryngeal disease but is absent in normal control subjects. Furthermore, we have determined a significant association between the presence of pepsin in laryngeal epithelia of LPR patients and the depletion of two laryngeal protective proteins, carbonic anhydrase isoenzyme III (CAIII) and squamous epithelial stress protein Sep70.  Using an established porcine in vitro model, we have demonstrated that exposure of laryngeal mucosa to pepsin, although not to low pH alone, causes depletion of CAIII and Sep70 protein levels. These findings suggest that the pepsin present in laryngeal epithelia of LPR patients is likely to be the causal factor for the observed depletion of CAIII and Sep70 proteins.

The objectives of this study were to 1) determine whether enzymatically active pepsin is required to deplete laryngeal CAIII and Sep70 protective proteins and 2) establish whether pepsin, present in the laryngeal epithelium of LPR patients (which is presumably inactive because the mean pH of the laryngopharynx is pH 6.8, and pepsin is maximally active at pH 2.0 and inactive at pH 6.5), is stable and thus could be reactivated after a decrease in pH, as would occur by a subsequent acidic reflux event. To accomplish these objectives, the affect of exposure of laryngeal mucosa to active/inactive pepsin (porcine pepsin A, 1 mg/mL,  + pepstatin, 23 μg/mL) on CAIII and Sep70 protein levels was investigated using the established porcine in vitro model.9 The effect of pH on activity and stability of human pepsin 3b was measured by kinetic assay by measuring the rate of hydrolysis of a specific synthetic substrate for human pepsin 3b. Hydrolysis of the peptide substrate was measured at pH 1.5 to pH 8.0 to determine peptic activity at each pH. The stability of pepsin was measured by incubating the enzyme at pH 2.0 to pH 8.0 for 24 hours at 37°C, after which the rate of hydrolysis of the substrate was measured at pH 3.0.

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Nikki Johnston, PhD; Peter W. Dettmar, PhD; Bimjhana Bishwokarma, MS; Mark O. Lively, PhD; Jamie A. Koufman, MD

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