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Pepsin Detection in Digestive and Respiratory Fluids
Peter W. DettmarRD BioMed Ltd, Hull, UK
OESO Post-graduate CourseBergamo, Italy, February 24-25, 2012
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Disclosure
Peptest™ is a trade name for a pepsin diagnostic medical device owned by RD BioMed Ltd
Peter Dettmar is a Director of RD BioMed Ltd
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What’s in Gastric Refluxate?
Acid – HCl from the stomach Pepsin – digestive enzyme from the stomach Bile acids – detergents from the small intestine Proteases – pancreatic enzymes from the small
intestine Mucus – protective gel from the stomach Bicarbonate – HCO3
- ions that neutralise acid Food and drink
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What Causes Damage?
The most damaging component of the gastric refluxate is not acid but PEPSIN
It is well documented that acid alone is unable to produce damage to the esophagus and larynx
Data presented by many different research groups over nearly 40 years to support this.
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An Introduction to Pepsin
Major component of gastric refluxate Family of isoenzymes Pepsin 3 complex = 80% Main activity pH 2 to pH 4 Active up to pH 6.5 Denatured pH 7.8 Basal secretion in normals
126 mg/hr 0.9 mg/ml
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Separation of individual pepsins in human gastric juice by High Performance
Ion Exchange Chromatography
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Reflux – What is Normal?
Regurgitation of gastric contents into esophagus is a daily occurrence
Up to 50 acidification episodes (pH<4) per day is normal
Esophagus well equipped to handle physiological reflux
When normal physiological reflux reaches the poorly protected laryngopharynx, clinical manifestations occur.
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Evidence of Pepsin Reflux
Pepsin, a marker for reflux, has been detected in:Larynx Knight et al. (2005) Laryngoscope 115:1473
Potlurri et al. (2003) Dig Dis Sci 48:1813Gill et al. (2005) Ann Otol Rhinol Laryngol
114:913Trachea Krishnan et al. (2002) J Pediatr Gastro Nutr 35:303
Meert et al. (2002) Pediatr Crit Care Med 3:19Metheny et al. (2002) Am J Crit Care
11:150Farhath et al. (2006) J Pediatr Gastroenterol
Nutr 43:336 Metheny et al. (2006) Crit Care Med 34:1007
Lung Ward et al. (2005) Thorax 60:872Stovold et al. (2007) Am J Respir Care Med
175:1298 Farrell et al. (2006) J Pediatr Surg 41:289Middle Ear Tasker et al. (2002) Laryngoscope 112:1930
Lieu et al. (2005) Otolaryngol HNS 133:357 Saliva/Sputum Strugala et al (2007) Gastroenterology 132(4
S2):A99Strugala et al (2007) Gut 56(SIII):A212Strugala et al (2008) J Clin Gastroenterol
42(S1):S8
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Protection Mechanisms
Stomach Esophagus
Pepsin does not digest the gastric epithelium as it is protected by a mucus layer and pH gradient
The esophageal epithelium is unprotected and pepsin in the refluxate causes damage
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Reflux of Pepsin
Reflux of pepsin into the esophagus and larynx causes mucosal damage No mucus protection Squamous cells pH not high enough to denature enzyme
Pepsin disrupts protective defence mechanisms Carbonic anhydrase Heat shock proteins
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Diagnosis of Extra-esophageal reflux (EER)
The current ‘gold standard’ for the diagnosis of reflux disease is 24 hour double-probe ambulatory pH monitoring. Detects reflux events with a drop in pH to below 4 Invasive procedure Prevents normal activity and reflux frequency Long waiting list for assessment & high cost Abnormal if pH < 4 for 4% of 24hr period Any reflux event above the UES is abnormal Acid alone is not damaging
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Larynx versus Esophagus
The larynx is more sensitive to pepsin damage than the esophagus
50 reflux events normal in the esophagus 3 reflux events per week abnormal in the larynx It can be argued that any reflux event above the
upper esophageal sphincter is abnormal Pepsin has been shown to cause significant
damge to the laryngeal epithelium Up to pH 6
Koufman (1991) Laryngoscope 101(Suppl 53):1-78 Ludemann et al. (1998) J. Otolaryngol. 27:127-131
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Pepsin in Laryngeal Epithelial
Detected in laryngeal epithelia from 26/27 patients with Laryngopharyngeal Reflux (LPR)
(p<0.0001)
Not detected in 18/19 esophageal epithelia from LPR patients
(p<0.001)
Detected in 0/19 esophageal and laryngeal specimens from normal control subjects
(p<0.001)
Johnston et al. Laryngoscope 2004; 114 (12): 2129-2134Johnston et al. Ann Otol Rhinol & Laryngol 2006; 115 (1):47-58
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Why are PPIs not effective in LPR? PPIs do not reduce the total number of reflux events
simply alters its pH characteristics converting acid reflux to weak acid reflux
Pepsin still detected Nocturnal acid breakthrough
approx 70% of patients. PPI half-life Even 1 reflux event is abnormal into the larynx
Rebound acid hypersecretion Not licensed for treatment of LPR These observations may explain the persistence of
symptoms and emergence of mucosal injury while patients are on PPI therapy
Nzeako & Murray (2002) APT 16:1309Tamhankar et al (2004) J GI Surg 8:888Hemmink et al (2008) Am J Gastro 103:2446
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Pepsin identified in wide spectrum of conditions
GERDEERLPR
Chronic coughAsthma
SinusitusCystic fibrosis
Lung allograph rejectionOtitis media with effusion
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Saliva Sputum Tracheal aspirate Esophageal aspirate Exhaled breath condensate Bronchoalveolar lavage fluid Middle ear effusions Nasal lavage fluid Laryngeal biopsy
Pepsin is detected in a wide range of clinical samples:-
Saliva collection
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Methods used for Pepsin Detection
Enzymatic Activity Assays Fibrinogen digestion Anson’s TCA precipitation assay TNBS N-terminal Assay FITC-casein + TCA ppt (fluorimetry)
Issues to be aware of: pH dependent Substrate specific Low sensitivity Lab based, skilled process
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Methods used for Pepsin Detection
Immunological Methods ELISA
Direct Sandwich Chromogenic detection Fluorimetric detection
Western blotting Issues to be aware of:
Highly skilled techniques Very time consuming Sensitive & quantitative
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Antibodies
Polyclonal Antibodies Antiserum (commercial or custom pepsin abs) Mixed population of antibodies specific for multiple
antigens Antisera will run out
exact match can not be remade Monoclonal Antibodies
Highly specific binding Monospecific, Homogenous, Identical Unlimited supply Custom pepsin Mabs
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Future of Pepsin Detection
Detection of pepsin as a diagnostic tool for reflux is now established
Need for simple rapid diagnostic test Can be utilised by healthcare professional Without specific lab skills needed Without need for sophisticated lab equipment Can give rapid results Does not require invasive procedures
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Lateral Flow Device (LFD)
In Vitro Diagnostic Medical Device
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Pepsin standard curve
0200400600800
100012001400160018002000220024002600280030003200
0 50 100 150 200 250 300 350 400 450 500
pepsin (ng/ml)
LFD
Inte
nsity
@ 1
5 m
ins
Pepsin standard
0 ng/ml
25 ng/ml
100 ng/ml
250 ng/ml
500 ng/ml
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QuantificationVisualisation
Sample Pepsin Detection
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Extra-esophageal reflux vs Controls
Pepsin was detected in 71% of patients suspected of having extra-esophageal reflux
Pepsin was not detected in control subjects p=0.0003
Pepsin more likely to be detected if sample is provided following symptoms (82% vs 35%)
p=0.0238
Strugala et al. Gastroenterology 2007; 132(4S2):A99-A100
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Cystic Fibrosis patients 25 patients with Cystic Fibrosis Provided 4 saliva samples each
Bed, meal, physiotherapy, symptoms Positive for pepsin in over half of all samples
bed 32%, meal 60%, physio 58%, symptoms 63% Most common in post meal, post
physiotherapy samples or when symptomatic (coughing)
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Pepsin detected in the saliva of 82% symptomatic LPR patients, not detected in control subjects
Pepsin detected in 93% of saliva samples from symptomatic chronic cough patients
Pepsin detected in cystic fibrosis patients
Pepsin detected in regurgitation samples from children experiencing reflux
Pepsin detected in patients experiencing persistent symptoms on PPI therapy Pepsin detected in 89% EBC
samples from patients presenting with reflux associated chronic cough
Clinical Summary
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Detection of Pepsin in specific patient groups
Infants (0 to 6 months) ICU patients Lung disease / transplant patients Pre / post fundoplication COPD (4% of popn / 60% related to reflux)
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COPD Exacerbations
Reflux is strongly linked to COPD patients with repeated exacerbations Frequent flyers
COPD patients hospitalised with acute exacerbations Reflux in 54% Frequent refluxers more likely to have exacerbations O.R. 6.55 for reflux symptoms causing
exacerbationsTerada K, et al (2008) Thorax 63(11):951-955Rogha M, et al (2010) J Gastrointestin Liver Dis 19(3):253-256
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Exhaled Breath Condensate
EBC Common non-invasive sampling technique
in respiratory medicine Detects markers of lung disease There is potential for using EBC to detect
pepsin refluxed into respiratory tissue
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The new exhaled breath condenser device
Rapid, disposable device for EBC collection Optimised to allow pepsin measurement in EBC
condensatecollection pot
venting tube
mouthpiecefiller cap
condensing spiral
space filled with water/ice
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Exhaled Breath Condensate (EBC) in Chronic Cough Patients
34 EBC samples from 10 patients EBC collected when symptomatic with cough
Portable EBC device used Measurement of pepsin using Peptest™
30 / 34 positive for pepsin (88%)
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Conclusions Pepsin detected in a range of clinical samples
and pathological conditions Rapid, simple, non-invasive pepsin test has
been developed - Peptest™ Peptest™ has wide application as a diagnostic
in a variety of clinical conditions Simple, disposable EBC collection device
developed Pepsin detected in EBC Pepsin EBC detection has potential for use in
respiratory patients
