title: Activity/stability of human pepsin: implications for reflux attributed laryngeal disease.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  Prospective translational research study.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Otolaryngology and Communication Sciences, Medical College of Wisconsin, Milwaukee 53226, USA. njohnston@mcw.edu
authors.forename: Nikki
authors.initials: N
authors.lastname: Johnston
authors.forename: Peter W
authors.initials: PW
authors.lastname: Dettmar
authors.forename: Bimjhana
authors.initials: B
authors.lastname: Bishwokarma
authors.forename: Mark O
authors.initials: MO
authors.lastname: Lively
authors.forename: Jamie A
authors.initials: JA
authors.lastname: Koufman
mesh_terms: D015153:Blotting, Western; D004945:Esophagoscopy; D005744:Gastric Acid / Q000502:physiology; D005764:Gastroesophageal Reflux / Q000150:complications* / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007818:Laryngeal Diseases / Q000209:etiology* / Q000473:pathology; D007820:Laryngeal Mucosa / Q000378:metabolism / Q000473:pathology; D010434:Pepsin A / Q000737:chemistry* / Q000378:metabolism*; D011446:Prospective Studies
chemical_list: D018840:HSP70 Heat-Shock Proteins; C533662:SEP70 protein, human; D010434:Pepsin A