lecture 2 Flashcards
what has gastric juice in peptic ulcer disease caused?
- enhanced ability to degrade the mucosal barrier
what pH is pepsinogen stable up to?
- 10 to 11
what pH is pepsin stable to?
- around pH 7
how is pepsin activation decided?
- pepsin taken and incubated overnight at different pHs
- return to pH 2 and see if any activity is found
what causes the dip at pH 2?
- will digest itself as a protein so some activity is lost
what pH is pepsin completely denatured?
- pH 7
(after pH7 its irreversibly denatured)
what can extend the denaturation profile of pepsin?
- gastric juice
- extends to around pH 7.5 and 8
what diseases are associated with reflux?
- GORD (gastro-oesophageal reflux disease)
- OME (otitis media with effusion, glue ear)
- LPR (laryngo pharyngeal reflux)
- Rhinitis and sinusitis
- VFL (vocal fold epithelial cell hyperplasia)
- larangomalacia (incomplete development of larynx in children, collapses)
what occurs in glue ear?
- where middle part of ear canal fills up with fluid
- hole made so can suck out contents
- fit a grommet in ear
how can we tell the diseases are linked with reflux?
- pepsin is measured on the tissue but pepsin shouldn’t be there
what is the standard for diagnosis of reflux?
- 24 hour double probe pH monitoring
where are the probes?
- oesophageal probe (5cm above lower oesophageal sphincter)
- pharyngeal probe (pH probe, 1cm above upper oesophageal sphincter)
what is regarded as a positive pharyngeal reflux event?
- drop below pH 4.0 preceded by precipitous drop at distal probe
- tells us low pH gastric juice has come out stomach through oesophagus
why is it damaging?
- a pH of 3.5 added on a tissue causes cell death
what happened in pepsin detection with laryngo pharyngeal reflux?
- pepsin levels in sputum were measured
what is a slot blot?
- nitrocellulose membrane and vacuum system
- can suck sample onto membrane
- to measure the pepsin levels
- can slot and measure area under curve for pepsin amount
what is another method used to measure pepsin?
- activity assay
what occurs in this method?
- 200ul of specimen + 1ml of acidified haemoglobin
- incubated at 37 degrees for 10 mins
- add 3ml of 6.2% TCA (trichloroacetic acid)
- incubate for 10 min at 37 degrees
- filter
- read at 280nm
what does TCA do?
- precipitates the protein
- any undigested haemoglobin is precipitated
- leaves only digested protein (peptides and amino acids) in solution
what were the results for LPR patients?
- no reflux event = still pepsin present
- 1 reflux event = pepsin present
- more than 1 reflux event = pepsin present
what does this tell us?
- pH4 isn’t best to measure as shouldn’t be reflux event at that pH but pepsin is present meaning reflux event has taken place
- contradictory results
what happens if stayed at pH 4?
- gives just over 1 reflux event
what happens when shifting to pH 4.1?
- get 4 reflux events
what happens at ph 4.5?
- 10 reflux events
what happens at pH 5?
- almost 30 reflux events
what is needed for a lower pH buffer?
- glycine HCl buffer system
what are the conclusions from this experiment?
- pepsin is present in sputum of patients with normal pharyngeal probe testing
- pepsin from human gastric juice is capable of enzymatic cavity at pH 5
what are the laryngeal damage models?
- watertight seal made between glass tube and larynx
- larynx suspended in krebs ringer solution
how is tissue damage measured?
- DNA release is measured at different pHs
what type of tissue is the sub glottic?
- columnar epithelium
what type of tissue is the supra glottic?
- squamous epithelium
what are the potential damage solutions?
- untreated
- ph7.4 krebs ringers
- ph4 krebs ringers
- ph2 krebs ringers
- ph4 krebs ringers + 1mg.ml porcine pepsin
- ph2 krebs ringers + 1mg/ml porcine pepsin
what is shown in sub glottic mucosa?
- damage with every reagent apart from untreated and ph7.4 krebs ringers
what is shown in vocal fold mucosa (squamous)?
- no real evidence of damage until ph2 krebs ringers + 1mg/ml porcine pepsin
what is shown in supra glottic mucosa?
- damage from ph2 krebs ringers onwards
what is shown at posterior commissure mucosa?
- no damage
- resistant to pepsin digestion
what is sown at ventricle mucosa?
- damage shown only with addition of 1mg/ml porcine pepsin
which tissue is damaged most based?
- sub glottic
what are the mechanisms of mucosal protection?
- stomach has mucus layer and bicarbonate secretion
- oesophagus has squamous. epithelium
- larynx has some squamous and some columnar, carbonic anhydrase
what is the laryngopharyngeal reflex?
- larynx more sensitive so less protected than oesophagus
- hoarseness, chronic cough, globus pharyngeus (lump in throat), heartburn
