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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Peptest - Pepsin detection in digestive and respiratory fluids
1. www.rdbiomed.com
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
3. www.rdbiomed.com
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
4. www.rdbiomed.com
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.
5. www.rdbiomed.com
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
7. www.rdbiomed.com
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.
8. www.rdbiomed.com
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
10. www.rdbiomed.com
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
11. www.rdbiomed.com
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
12. www.rdbiomed.com
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
13. www.rdbiomed.com
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
14. www.rdbiomed.com
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
15. www.rdbiomed.com
Pepsin identified in wide spectrum of
conditions
GERD
EER
LPR
Chronic cough
Asthma
Sinusitus
Cystic fibrosis
Lung allograph rejection
Otitis media with effusion
16. www.rdbiomed.com
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
17. www.rdbiomed.com
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
18. www.rdbiomed.com
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
19. www.rdbiomed.com
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
20. www.rdbiomed.com
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
27. www.rdbiomed.com
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
28. www.rdbiomed.com
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)
29. www.rdbiomed.com
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
30. www.rdbiomed.com
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)
31. www.rdbiomed.com
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
32. www.rdbiomed.com
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
33. www.rdbiomed.com
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
34. www.rdbiomed.com
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%)
35. www.rdbiomed.com
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