What is Peptest™ Peptest is a new, accurate, painless and inexpensive saliva test that tells you conclusively if you have reflux. Peptest can be ordered by both doctors and patients direct. Your body produces Pepsin in the stomach to break down protein. If Pepsin is found outside of the stomach you are suffering from Reflux. We take samples of your saliva and test them in our laboratory for Pepsin. If Pepsin is present the concentration in your sample is measured to show the severity of Reflux and give a benchmark to assess the success or failure of any treatment. Retesting can be used at any future treatment point.

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Pepsin Detection in Digestive
and Respiratory Fluids
Peter W. Dettmar
RD BioMed Ltd, Hull, UK
OESO Post-graduate Course
Bergamo, 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 48:1813
Gill et al. (2005) Ann Otol Rhinol Laryngol 114:913
Trachea Krishnan et al. (2002) J Pediatr Gastro Nutr 35:303
Meert et al. (2002) Pediatr Crit Care Med 3:19
Metheny et al. (2002) Am J Crit Care 11:150
Farhath et al. (2006) J Pediatr Gastroenterol Nutr 43:336
Metheny et al. (2006) Crit Care Med 34:1007
Lung Ward et al. (2005) Thorax 60:872
Stovold et al. (2007) Am J Respir Care Med 175:1298
Farrell et al. (2006) J Pediatr Surg 41:289
Middle 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):A99
Strugala et al (2007) Gut 56(SIII):A212
Strugala 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): 2129-2134
Johnston 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 16:1309
Tamhankar et al (2004) J GI Surg 8:888
Hemmink et al (2008) Am J Gastro 103:2446

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Pepsin identified in wide spectrum of
conditions
GERD
EER
LPR
Chronic cough
Asthma
Sinusitus
Cystic fibrosis
Lung allograph rejection
Otitis 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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How it works

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MHRA registered / CE Marked

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Clinical sample application

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Pepsin standard curve
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
0 50 100 150 200 250 300 350 400 450 500
pepsin (ng/ml)
LFDIntensity@15mins
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
exacerbations
Terada K, et al (2008) Thorax 63(11):951-955
Rogha 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
condensate
collection pot
venting tube
mouthpiece
filler 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