lecture 2

Question 1

what has gastric juice in peptic ulcer disease caused?

Answer 1

• enhanced ability to degrade the mucosal barrier

Question 2

what pH is pepsinogen stable up to?

Answer 2

• 10 to 11

Question 3

what pH is pepsin stable to?

Answer 3

• around pH 7

Question 4

how is pepsin activation decided?

Answer 4

• pepsin taken and incubated overnight at different pHs
• return to pH 2 and see if any activity is found

Question 5

what causes the dip at pH 2?

Answer 5

• will digest itself as a protein so some activity is lost

Question 6

what pH is pepsin completely denatured?

Answer 6

• pH 7
  (after pH7 its irreversibly denatured)

Question 7

what can extend the denaturation profile of pepsin?

Answer 7

• gastric juice
• extends to around pH 7.5 and 8

Question 8

what diseases are associated with reflux?

Answer 8

• 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)

Question 9

what occurs in glue ear?

Answer 9

• where middle part of ear canal fills up with fluid
• hole made so can suck out contents
• fit a grommet in ear

Question 10

how can we tell the diseases are linked with reflux?

Answer 10

• pepsin is measured on the tissue but pepsin shouldn’t be there

Question 11

what is the standard for diagnosis of reflux?

Answer 11

• 24 hour double probe pH monitoring

Question 12

where are the probes?

Answer 12

• oesophageal probe (5cm above lower oesophageal sphincter)
• pharyngeal probe (pH probe, 1cm above upper oesophageal sphincter)

Question 13

what is regarded as a positive pharyngeal reflux event?

Answer 13

• drop below pH 4.0 preceded by precipitous drop at distal probe
• tells us low pH gastric juice has come out stomach through oesophagus

Question 14

why is it damaging?

Answer 14

• a pH of 3.5 added on a tissue causes cell death

Question 15

what happened in pepsin detection with laryngo pharyngeal reflux?

Answer 15

• pepsin levels in sputum were measured

Question 16

what is a slot blot?

Answer 16

• 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

Question 17

what is another method used to measure pepsin?

Answer 17

• activity assay

Question 18

what occurs in this method?

Answer 18

• 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

Question 19

what does TCA do?

Answer 19

• precipitates the protein
• any undigested haemoglobin is precipitated
• leaves only digested protein (peptides and amino acids) in solution

Question 20

what were the results for LPR patients?

Answer 20

• no reflux event = still pepsin present
• 1 reflux event = pepsin present
• more than 1 reflux event = pepsin present

Question 21

what does this tell us?

Answer 21

• 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

Question 22

what happens if stayed at pH 4?

Answer 22

• gives just over 1 reflux event

Question 23

what happens when shifting to pH 4.1?

Answer 23

• get 4 reflux events

Question 24

what happens at ph 4.5?

Answer 24

• 10 reflux events

Question 25

what happens at pH 5?

Answer 25

• almost 30 reflux events

Question 26

what is needed for a lower pH buffer?

Answer 26

• glycine HCl buffer system

Question 27

what are the conclusions from this experiment?

Answer 27

• 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

Question 28

what are the laryngeal damage models?

Answer 28

• watertight seal made between glass tube and larynx
• larynx suspended in krebs ringer solution

Question 29

how is tissue damage measured?

Answer 29

• DNA release is measured at different pHs

Question 30

what type of tissue is the sub glottic?

Answer 30

• columnar epithelium

Question 31

what type of tissue is the supra glottic?

Answer 31

• squamous epithelium

Question 32

what are the potential damage solutions?

Answer 32

• 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

Question 33

what is shown in sub glottic mucosa?

Answer 33

• damage with every reagent apart from untreated and ph7.4 krebs ringers

Question 34

what is shown in vocal fold mucosa (squamous)?

Answer 34

• no real evidence of damage until ph2 krebs ringers + 1mg/ml porcine pepsin

Question 35

what is shown in supra glottic mucosa?

Answer 35

• damage from ph2 krebs ringers onwards

Question 36

what is shown at posterior commissure mucosa?

Answer 36

• no damage
• resistant to pepsin digestion

Question 37

what is sown at ventricle mucosa?

Answer 37

• damage shown only with addition of 1mg/ml porcine pepsin

Question 38

which tissue is damaged most based?

Answer 38

• sub glottic

Question 39

what are the mechanisms of mucosal protection?

Answer 39

• stomach has mucus layer and bicarbonate secretion
• oesophagus has squamous. epithelium
• larynx has some squamous and some columnar, carbonic anhydrase

Question 40

what is the laryngopharyngeal reflex?

Answer 40

• larynx more sensitive so less protected than oesophagus
• hoarseness, chronic cough, globus pharyngeus (lump in throat), heartburn