Physiology of the Pancreas
3.1 Introductive notions
The Pancreas is an annex gland of the digestive tract, located retroperitoneal, at the
level of L1-L2 vertebrae. In mammals, the pancreas has a double secretion: exocrine
and endocrine. The pancreatic juice is the secretion product of the exocrine
pancreas. Endocrine secretion is produced by the islets of Langerhans and is
represented by hormones such as: insulin, glucagon, somatostatin, pancreatic
polypeptide, gastrin, etc. The histological structure of the pancreas is similar to that of
the salivary glands, being represented by acinar cells and a canalicular system,
which end in a main Wirsung canal and, sometimes, in a Santorini accessory canal.
Wirsung s canal spills into the duodenum at the level of the ampulla of Vater, near
the common bile duct. The exocrine secretion of the pancreas has two components:
Hydrelatic secretion (hydrolatic), rich in water and sodium bicarbonate, with a
reduced quantity of enzymes. It is produced mainly in canalicular epithelia and
is secretin-dependent.
The ecbolic secretion is a low quantity secretion, rich in enzymes and is
produced mainly by the cells of glandular acini. It is dependent on
cholecystokinin, serotonin and vagal stimulation.
The pancreatic juice carries out important functions in the digestion of food. Through
its rich content of bicarbonate, it contributes in neutralizing the acidity of the
semiliquid gastric mass and creates optimal conditions for the activation and action
of the digestive enzymes. It also contains enzymes that have an intense proteolytic,
lipolytic and glycolytic activity, being able to partially or completely hydrolyze most of
the ingested food.
Deficiencies found in the pathology of exocrine pancreatic secretion have important
effects over digestion, consequences being maldigestion, malabsorption and
progressive malnutrition.
3.3.1. Physical and chemical characteristics of the pancreatic juice
Appearance: clear or slightly opalescent liquid, inodorous, with low viscosity
Quantity: in adults, averaging 700 1500 ml/24 hours; maximum quantity 4000
ml\24 hours. Secretion is discontinuous, secretionary flow being more abundant
postprandially, with a duration of approximately 3 hours.
Density: 1,008 1,014 g/cm3.
1
Cryoscopic point: -0,55 down to 0,63 oC,
hypertonic compared to plasma.
the pancretic juice being slightly
pH: 7,5 8. An increase in the secretion rate of pancreatic juice leads to an
elevation in pH levels up to 9. It is the most alkaline liquid in the organism. Alkalinity
is produced by the large content of sodium bicarbonate.
Pancreatic juice composition: is variable, depending on the type of food or the type
of stimulant responsible for triggering the secretion:
water 98,5%
dry residue 1,5%, out of which 50-60% are inorganic substances, and 4050% organic substances.
Inorganic substances
sodium bicarbonate: 25 150 mEq/l
chlorides : 4 - 129 mEQ/l
phosphates, potasium, sodium, calcium, mangesium, sulphur, copper, zinc
Concentration of cations is constant, regardless of the secretion rate. Concentration
of anions, in the secretion period is however, variable. The bicarbonate content grows
according to the secretionary flow with a hyperbolic curve, and the chloride content
drops with a hyperbolic curve, the opposite way, symmetrically to the bicarbonate
curve. As a result, the sum of the concetratrions of the two components remains
almost constant, regardless of the secretionary flow ( 154 mEq/l).
Organic substances: The main organic compounds are proteins, in variable
quantities, from 1 g/l up to 30 g/l , according tot the stimulant and type of secretion.
The main proteins are enzymes, synthesized in the acinar cells and
intracellularly, in zymogen granules.
deposited
Proteolytic enzymes:
Endopeptidases: trypsin, chymotrypsin, elastase, collagenase. They hydrolyze
the peptide bonds from inside the peptide chains.
Exopeptidases: carboxypeptidases. They act at the end of polypeptide chains.
Nucleases: ribonucleases and deoxyribonucleases. They break the internal
links of nucleoproteins, releasing oligonucleotides.
Proteolytic enzymes are sythesized as proenzymes, being activated in the
duodenal lumen. Trypsinogen is activated by enterokinase, an enzyme
secreted by the duodenal mucus, resulting in active trypsin, which acts
autocatalytically, as well as upon the other proteolytic enzymes, activating
them.
Lipolytic enzymes:
Lipase : Acts on the emulsioned lipides, in the presence of bilious salts
and Ca2+
Phospholipases
Cholesterol Esterase.
They are secreted actively in the pancreatic juice
Glycolityc enzymes:
Pancreatic amylase : is secreted actively in the pancreatic juice and
acts upon amidone, producing hydrolyisis into dextrin and maltose.
3.1.2. Sampling the pancreatic juice
Experimental sampling:
Permanent pancreatic fistulas are made for sampling the pancreatic juice
directly from Wirsungs canal.
Sampling of pancreatic juice in humans:
Normally, the pancreatic juice is collected through duodenal tubing with an
Einhorn probe, introduced 60- 70 cm up to the duodenum. Through this
method duodenal juice is sampled, which is a mixture of gastric,
pancreatic, intestinal and bilious juice. To obtain a pancreatic juice with a
purer composition, double or triple lumen probes are used ( in which the
components have unequal lengths), permitting the separate suction of
gastric and duodenal juice. Modern techniques achieve the sampling of
pancreatic juice by
endoscopic catheterization of Wirsungs canal.
Pancreatic secretion can be stimulated using food, secretin,
cholecystokinin etc.
3.2 Practical assignments
3.2.1. Dosage of the enzymatic activity of trypsin through the Gross- Michaelis
method.
Method principle:
Casein ( proteinic substratum) is hydrolyzed up to amino acids, under the influence of
trypsin, at a temperature of 37 oC. Unhydrolyzed casein will become unclear, by the
addition of an acid-alcohol solution.
Required materials:
test tube support with test tubes
pipettes
NaCl solution 9%
biological product: pancreatic juice
casein solution 1%
acid - alcohol solution ( glacial acetic acid and alcohol)
3
buffer solution
thermostat adjusted to 37oC
laboratory clock
Technique:
Using 10 tubes there are made increasing dilutions of the investigated liquid
(pancreatic juice). Proceed as follows: in the first tube put 2 cm 3 of pancreatic juice
and in the others about 1 cm 3 NaCl 9% solution. 1 cm 3 is taken from the first test tube
and put in the second tube it is mingled, afterwards 1 cm3 is taken from this tube and
put in the third tube and so on. Thus are obtained dilutions of pancreatic juice in ratio
of 1/1; 1/2; 1/4; 1/8; 1/16; 1/32; 1/64; 1/128; 1/256; 1/512. In each tube add 2 cm3 of
1 casein solution (2 mg of casein) and 1 cm3 of buffer solution (pH = 7.8 - 8).
Specimens are held one hour at 37C, in order to create optimal working conditions
for the enzyme. Once you remove the tubes from the thermostat, add 2-3 drops of
alcohol - acid. The aim is the content of the tubes starting from the first one. Note the
last tube, whose content is still clear (the amount of trypsin was sufficient to
hydrolyse the whole casein from the tube, 2 mg of casein to be exact.)
Tubes
10
Pancreatic
juice
2
cm3
NaCl
solution
9
1
cm3
1
cm3
1
cm3
1
cm3
1
cm3
1
cm3
1
cm3
1
cm3
1
cm3
From first tube take 1 cm3 D From the first tube take 1 cm3 pancreatic juice and put in the
second tube, mix and from from this pmixture take 1 cm3 and pute in the 3rd tube and so on.
Casein
solution
1
2
cm3
2
cm3
2
cm3
2
cm3
2
cm3
2
cm3
2
cm3
2
cm3
2
cm3
2
cm3
Buffer
solution
(pH = 7,8 8)
1
cm3
1
cm3
1
cm3
1
cm3
1
cm3
1
cm3
1
cm3
1
cm3
1
cm3
1
cm3
Agitate all tubes and leave them at the thermostat at 37oC,for an hour.
alcool
acid
23
23
23
23
23
23
23
23
23
23
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Tabelul nr. 3.1. Substances used in reactions
Results:
The result is expressed in triptych units. Triptych unit is the amount of enzyme
able to hydrolyze 1 mg of casein in one hour at 37 C. For Example: tube no. 4, the
original quantity of 1 cm3 pancreatic or duodenal juice was diluted to 1/8, hydrolysing
2 mg of casein. Trypsin contained in a cm3 of undiluted pancreatic juice will be 8 x 2 =
16 units tryptichs
3.2.4. Amylolytic activity dosage using the Wohlgemuth method
Principle:
Pancreatic amylase activity is highlighted through its ability to hydrolyse
starch, in standard conditions, into intermediary products (dextrine) and final products
(maltose), by using a Lugol solution.
Materials:
Blood serum or urine
NaCl 9 solution
Starch solution 1
Lugol solution (iodine iodide 1%)
10 tubes stand
Pipettes
Thermostat set at 37oC
Technique:
Take a 10 stand tubes, in which make increasing dilutions of blood serum or
urine. In the first tube put 2 cm 3 of urine (or blood serum), and in the other 9 tubes put
1 cm3 of NaCl 9 (saline solution). From the first tube take 1 cm 3 of urine put in the
second tube, mix, take 1 cm 3 and put in the third tube and so on. Are obtained the
following dilutions: 1/2; 1/4; 1/8; 1/16; 1/32; 1/64; 1/128; 1/256; 1/512. In each tube
add 2 cm3 of starch solution 1 (which contain 2 mg of starch). Wag the tubes and
leave them at 37oC for 30 minutes in the thermostat, while the urines amylase (or
blood serum) acts on starch. After that, the tubes are cooled down using a cold water
jet, and in each tube are added 1-2 drops of Lugol solution. Tubes containing
unhydrolysed starch will be turning blue, tubes containing partial hydrolysed starch till
amylo or eritrodextrine will turn purple or red, and tubes with fully hydrolysed starch
will be colorless. Write down the tubes number, before reaching the blue one. In this
tube, the quantity of starch was adequate to hidrolyse 2 mg of starch.
5
Eprubeta
Urine or
blood
2 cm3
NaCl
Solution 9
1 cm3
1 cm3
1 cm3
1 cm3
1 cm3
1 cm3
1 cm3
10
1 cm3
cm3
From the first tube put 1 cm3 of urine in the second tube, mix it up, and from this mixture take 1 cm3 and put in the third tube and
so one.
Amidon
solutuion
2 cm3
2 cm3
2 cm3
2 cm3
2 cm3
2 cm3
2 cm3
2 cm3
2 cm3
2 cm3
12
12
Shake all tubes and leave them at the thermostat for 30 minutes
After thermostat holding, the tubes must be cooled
Lugol
solution
12
12
12
12
12
12
12
12
(drops)
Tabel nr. 3.2. Substances from the reaction tubes
Results:
Results are presented in Wohlgemuth units. A wohlgemuth unit is the quantity
of amilase that may hydrolise 1 mg of starch, to dextrine, in 30 minutes at 37 oC. In
each tube there are 2 mg of starch, there for in the numbered tube there are 2
Wohlgemuth units.
Exemple: Number 6 tube was noted, in which the urine dilution (or blood serum) is
1/32. If in 1 cm3 of this solution there are 2 Wohlgemuth units, in 1 cm 3 of undiluted
urine (or blood serum) there are
2 u.W. x 32 = 64 u.W.
Amylase determination is made from total urine, collected in 24 hours and
cold conserved (because the amilazurie is different in various moments in 24 hours,
especially due to nutrition)
Normal Values:
Amylase 16 32 u.W./cm3 blood serum
Amilazurie 16 64 u.W./cm3 urine
Amiloclastic Somogy method: is a more precise method than the Wohlgemuth method,
because it uses starch solutions with more precise concentrations.
Convert specific methods for the determination of amylolytic activity is as
follows:
1 u Wohlgemuth = 0,0775 u.Somogy = 0,01425 International Units
3.3 Pancreatic Secretions Disorders
Pancreatic ducts obstruction (due to internal or external causes), determine the
quantity growth of pancreatic enzymes in bloodstream and urine, paralel with their
decrease in the pancreatic juice.
Acute pancreatitis ia an inflammatory process of the pancreas, in which the activation
of proteolytic enzymes takes place in pancreatic interstitium, and the pancreatic
enzymes pass into the bloodstream. Pancreatic enzymes dosage in the
bloodstream, urine, duodenal juice, ascites fluid, pleural fluid indicates high values,
according to the dramatic clinical picture of this disease.
Chronic pancreatitis is a slow growing disease, such as secretory exocrine
pancreas insufficiency. Enzymes dosage in duodenal juice, bloodstream and urine,
indicates low values, and the stimulation tests of pancreatic secretion shows the
decrease of the pancreatic juice, late and short answer, with low values of the
pancreatic enzymes. Decrease pancreatic enzymes results in poor digestion of
proteins, fats and carbohydrates. Thus, the remaining undigested proteins are
eliminated in increased amount, in the faeces (creatorrhea), as well as the remaining
undigested fat resulting in steatorrhea.
Modern exploration methods used in exocrine pancreas function:
Direct stimulation of pancreatic secretion by secretin administration. Secretin is
administered intravenously at a dose of 1 clinical unit/kg and duodenal fluid is
collected through duodenal tubing. Pancreatic secretory response is proportional to
the mass of functional pancreatic tissue. Reduced secretory response corresponds to
chronic pancreatitis. Normal Values:
Duodenal juice volume > 2ml/kg body/h
Bicarbonates dosage > 80 mmol/l
Bicarbonates flow > 10 mmol/h
Direct stimulation of pancreatic secretion by secretin and cholecystokinin allows
quantitative measurement of pancreatic juice secretion and pancreatic enzyme
dosage. Low values indicate an advanced destruction of acini cells.
Indirect stimulation of pancreatic secretion by administration of food as lunch Lundh.
Lundh test lunch implies fat, carbohydrates and protein in quantities necessary for
the release of cholecystokinin. Determination of enzymes in duodenal juice shows
low values in pancreatic secretory insufficiency.
Benzoil-tirozil-p-aminobenzoic test (Bz-Ti-PABA, bentiromide). This synthetic peptide
is split by chymotrypsin, releasing PABA , which is absorbed inside the blood, and a
PABA metabolite is excreted in the urine , where it is dosed.
Pancreolauril test. Fluorescein Dilaurate is hydrolyzed by pancreatic chymotrypsin
and fluorescein is determined in urine. The test gives information on pancreatic
enzyme synthesis.
Determination of intraluminal digestion products through microscopic examination of
faeces. Observed in the microscope existence undigested muscle fibers, fat and
unhydrolyzed starch. Their presence indicates a lack of secretion of pancreatic
enzymes.
Determination of nitrogen in faeces. Reduced secretion of proteolytic enzymes
affects protein digestion and increases fecal nitrogen.
Determination of pancreatic enzymes chymotrypsin and elastase in faeces. These
enzymes are eliminated unchanged by faeces and is an indicator of pancreatic
secretion of proteolytic enzymes.
Schilling test double marked. Labeled cobalamin is released from contact with
substances substrate by proteolytic enzymes and is excreted in the urine. Is an
indicator of exocrine pancreas secretion failure.
Clearance report amylase / creatinine.
Determination izoamylaze P, synthesized in the pancreas, is a sensitive test for the
diagnosis of acute pancreatitis.
Determination of serum lipase shows elevated values in acute pancreatitis.
Serum trypsin-like immunoreactivity is increased in acute pancreatitis and low in
chronic pancreatitis with steatorrhoea.
Pancreatic polypeptide is reduced in chronic pancreatitis.
These tests have variable sensitivity and significance in the clinical context
and interpreted only in conjunction with other tests because exocrine pancreas is
characterized by significant functional reserve. Tests to stimulate exocrine pancreatic
function give reliable results in the destruction of over 60% of exocrine pancreas and
for the appearance of fat and protein maldigestion, pancreatic tissue must be
inoperative within 80-90%.
Consulting a medical dictionary or teaching materials, please define the
following terms: pancreatitis, obstruction, enterokinase, fistula or other terms found in
this chapterand you have no knowledge of them.
.4 Questions
1. What are the pancreas structure components that produce ecbolic secretion?
2. What are the pancreas structure components that produce hydrolactic
secretion?
3. What is the composition of pancreatic juice?
4. What is the normal level of pancreatic juice pH?
5. Please list proteolytic enzymes present in pancreatic juice.
6. Please list lipolytic enzymes present in pancreatic juice.
7. Please list glicolytic enzymes present in pancreatic juice.
8. What is trypsin?
9. What is lipase?
10. What is amylase?
11. What is enterokinase?
12. What is the trypsins activation mechanism?
13. What is secretin?
14. What is cholecytokinin?
15. How does pancreatic stimulation reacts at vagal stimulation?
16. What is pancreatic polypeptid?
17. What is glucagon?
18. What are the hormones involved in the regulation of exocrine pancreas
hormonal secretion ?
19. List foods that are substrates of trypsin action.
20. List foods that are substrates of lipase action.
21. List foods that are substrates of amylase action.
22. List the exploration methods of the exocrine pancreas function.
23. What is creatorrhea?
24. What is steatorrhea?
25. What does eliminate muscle fibers, starch and undigested fat in faeces
mean?
26. Nervous mechanism regulating pancreatic secretion.
27. Hormonal mechanism regulating pancreatic secretion.
