MSc Medical Biochemistry,
The pancreas secretes 1500–3000 mL of isosmotic
alkaline (pH >8) fluid per day containing about 20
The pancreatic secretions provide the enzymes needed
to effect the major digestive activity of the
gastrointestinal tract and provide an optimal pH for
the function of these enzymes.
Regulation of Pancreatic Secretion
The exocrine pancreas is influenced by intimately interacting
hormonal and neural systems.
Gastric acid is the stimulus for the release of secretin from
the duodenum, which stimulates the secretion of water and
electrolytes from pancreatic ductal cells.
Release of cholecystokinin (CCK) from the duodenum and
proximal jejunum is largely triggered by long-chain fatty
acids, certain essential amino acids
(tryptophan, phenylalanine, valine, methionine), and gastric
CCK evokes an enzyme-rich secretion from acinar cells in the
Water and Electrolyte Secretion
Bicarbonate is the ion of primary physiologic importance within
The ductal cells secrete bicarbonate predominantly derived from
plasma (93%) more than from intracellular metabolism (7%).
Bicarbonate enters through the sodium bicarbonate cotransporter
with depolarization caused by chloride efflux through the cystic
fibrosis transmembrane conductance regulator (CFTR).
Secretin and VIP, both of which increase intracellular cyclic
AMP, act on the ductal cells opening the CFTR in promoting
CCK, acting as a neuromodulator, markedly potentiates the
stimulatory effects of secretin.
Acetylcholine also plays an important role in ductal cell secretion.
Bicarbonate helps neutralize gastric acid and creates the
appropriate pH for the activity of pancreatic enzymes and bile
The acinar cell is highly compartmentalized and is concerned
with the secretion of pancreatic enzymes.
Proteins synthesized by the rough endoplasmic reticulum
are processed in the Golgi and then targeted to the
Amylolytic enzymes such as amylase, hydrolyze starch to
oligosaccharides and to the disaccharide maltose.
The lipolytic enzymes include lipase, phospholipase A2, and
cholesterol esterase. Bile salts inhibit lipase in isolation, but
colipase, another constituent of pancreatic secretion, binds
to lipase and prevents this inhibition. Bile salts activate
phospholipase A and cholesterol esterase.
Proteolytic enzymes include endopeptidases
(trypsin, chymotrypsin), which act on internal peptide bonds
of proteins and polypeptides; exopeptidases
(carboxypeptidases, aminopeptidases), which act on the free
carboxyl- and amino-terminal ends of peptides, respectively;
The proteolytic enzymes are secreted as inactive precursors
and packaged as zymogens.
Ribonucleases (deoxyribonucleases, ribonuclease) are also
Enterokinase, an enzyme found in the duodenal
mucosa, cleaves the lysine-isoleucine bond of trypsinogen to
Trypsin then activates the other proteolytic zymogens and
phospholipase A2 in a cascade phenomenon.
All pancreatic enzymes have pH optima in the alkaline range.
The nervous system initiates pancreatic enzyme secretion.
The stimulatory neurotransmitters are acetylcholine and
These neurotransmitters activate calcium-dependent second
messenger systems, resulting in the release of zymogen
Autoprotection of the Pancreas
Autodigestion of the pancreas is prevented by the packaging
of pancreatic proteases in precursor form and by the
synthesis of protease inhibitor [i.e., pancreatic secretory
trypsin inhibitor (PSTI) (SPINK1) ], which can bind and
inactivate about 20% of trypsin activity.
Mesotrypsin, chymotrypsin c, and enzyme y can also lyse
and inactivate trypsin.
These protease inhibitors are found in the acinar cell, the
pancreatic secretions, In addition, low calcium concentration
within the cytosol of acinar cells in the normal pancreas
promotes the destruction of spontaneously activated trypsin.
Loss of any of these protective mechanisms leads to zymogen
activation, autodigestion, and acute pancreatitis.
Insulin appears to be needed locally for secretin and CCK to
promote exocrine secretion; thus, it acts in a permissive role for
these two hormones.
Enteropancreatic Axis and Feedback Inhibition
The available evidence supports the concept that the duodenum
contains a peptide called CCK-releasing factor that is involved in
stimulating CCK release.
It appears that serine proteases inhibit pancreatic secretion by
inactivating a CCK-releasing peptide in the lumen of the small
Acidification of the duodenum releases secretin, which stimulates
vagal and other neural pathways to activate pancreatic duct
cells, which secrete bicarbonate.
Pancreatic inflammatory disease may be classified as (1)
acute pancreatitis or (2) chronic pancreatitis.
The pathologic spectrum of acute pancreatitis varies from
interstitial pancreatitis, which is usually a mild and self-
limited disorder, to necrotizing pancreatitis, in which the
extent of pancreatic necrosis may correlate with the
severity of the attack and its systemic manifestations.
Hypertriglyceridemia is the cause of acute pancreatitis in 1.3–
3.8% of cases.
Any factor (e.g., drugs or alcohol) that causes an abrupt
increase in serum triglycerides to levels >11 mmol/L (1000
mg/dL) can precipitate a bout of acute pancreatitis.
Finally, patients with a deficiency of apolipoprotein CII have
an increased incidence of pancreatitis; apolipoprotein CII
activates lipoprotein lipase, which is important in clearing
chylomicrons from the bloodstream.
Approximately 2–5% of cases of acute pancreatitis are drug
Several recent studies have suggested that pancreatitis is a disease
that evolves in three phases.
The initial phase is characterized by intrapancreatic digestive
enzyme activation and acinar cell injury.
Trypsin activation appears to be mediated by lysosomal
hydrolases such as cathepsin B that become colocalized with
digestive enzymes in intracellular organelles; it is currently
believed that acinar cell injury is the consequence of trypsin
The second phase of pancreatitis involves the activation,
chemoattraction, and sequestration of leukocytes and
macrophages in the pancreas, resulting in an enhanced
intrapancreatic inflammatory reaction.
The third phase of pancreatitis is due to the effects of activated
proteolytic enzymes and cytokines, released by the inflamed
pancreas, on distant organs.
Activated proteolytic enzymes, especially trypsin, not only digest
pancreatic and peripancreatic tissues but also activate other enzymes
such as elastase and phospholipase A2.
The active enzymes and cytokines then digest cellular membranes and
cause proteolysis, edema, interstitial hemorrhage, vascular
damage, coagulation necrosis, fat necrosis, and parenchymal cell
Cellular injury and death result in the liberation of bradykinin
peptides, vasoactive substances, and histamine that can produce
vasodilation, increased vascular permeability, and edema with profound
effects on many organs, most notably the lung.
The systemic inflammatory response syndrome (SIRS) and acute
respiratory distress syndrome (ARDS) as well as multiorgan failure may
occur as a result of this cascade of local as well as distant effects.
There appear to be a number of genetic factors that can
increase the susceptibility and/or modify the severity of
pancreatic injury in acute pancreatitis.
Four susceptibility genes have been identified:
(1) cationic trypsinogen mutations ,
(2) pancreatic secretory trypsin inhibitor ,
(3) CFTR, and
(4) monocyte chemotactic protein (MCP-1).
Experimental and clinical data indicate that MCP-1 may be
an important inflammatory mediator in the early pathologic
process of acute pancreatitis, a determinant of the severity of
the inflammatory response, and a promoter of organ failure.
Pancreatic Secretory Trypsin Inhibitor (Psti) Gene Mutations
PSTI, or SPINK1, is a 56-amino-acid peptide that specifically
inhibits trypsin by physically blocking its active site. SPINK1
acts as the first line of defense against prematurely activated
trypsinogen in the acinar cell.
Recently, it has been shown that the frequency of SPINK1
mutations in patients with idiopathicchronic pancreatitis is
markedly increased, suggesting that these mutations may be
associated with pancreatitis.
The diagnosis of acute pancreatitis is usually established by
the detection of an increased level of serum amylase and
Values threefold or more above normal virtually clinch the
diagnosis if gut perforation, ischemia, and infarction are
Serum lipase activity increases in parallel with amylase
A threefold elevated serum lipase value is usually diagnostic
of acute pancreatitis; these tests are especially helpful in
patients with nonpancreatic causes of hyperamylasemia.
Hypocalcemia occurs in 25% of patients. Leukocytosis occurs
Hyperbilirubinemia [serum bilirubin >68 mol/L (>4.0
mg/dL)] occurs in 10% of patients.
Hypertriglyceridemia occurs in 5–10% of patients,
Finally, the electrocardiogram is occasionally abnormal in
acute pancreatitis with ST-segment and T-wave
abnormalities simulating myocardial ischemia.
Chronic Pancreatitis and Pancreatic Exocrine Insufficiency
Chronic pancreatitis is a disease process characterized by
irreversible damage to the pancreas as distinct from the
reversible changes noted in acute pancreatitis.
The condition is best defined by the presence of histologic
abnormalities, including chronic inflammation, fibrosis, and
progressive destruction of both exocrine and eventually
alcoholism is the most common cause of clinically apparent
chronic pancreatitis, while cystic fibrosis is the most frequent
cause in children.
Recent investigations have indicated that up to 15% of
patients with idiopathic pancreatitis may have pancreatitis
due to genetic defects .
mutations of CFTR.
This gene functions as a cyclic AMP–regulated chloride
In patients with cystic fibrosis, the high concentration of
macromolecules can block the pancreatic ducts.
Patients with chronic pancreatitis seek medical attention
predominantly because of two symptoms: abdominal pain or
maldigestion and weight loss.
In contrast to acute pancreatitis, the serum amylase and
lipase levels are usually not strikingly elevated in chronic
The diagnostic test with the best sensitivity and specificity is
the hormone stimulation test utilizing secretin. It becomes
abnormal when 60% of the pancreatic exocrine function has
been lost. This usually correlates well with the onset of
chronic abdominal pain.
Assessment of pancreatic
1. Measurement of pancreatic enzymes :
Amylase or alpha 1,4 –glucosidase is the major enzyme
which digests starch.
The serum amylase contains the P ( pancreatic ) &
S ( salivary) isoenzymes.
These two can be distinguised by the inhibition test.
Normal amylase level in serum is 50 – 120 Units.
The level rises within 5 hr of the onset of acute pancreatitis.
The level reaches a peak(4-6 fold) within 12 hours.
Within 2-4 days of the attack, the level returns to normal.
As the serum amylase level starts falling, urinary amylase
Amylase level in blood is mildly increased in cases of
cholecystitis, peptic ulcer, diseases of mesentry &
obstruction of intestine.
Chronic pancreatitis : no change or only mild elevation is
CLEARANCE RATIO :
If the sample is collected too early, the serum amylase
levels may not show the expected rise.
If the sample is collected too late, again serum amylase
may be low due to necrosis of the pancreatic tissue.
Calculation of clearance ratio will avoid these defects.
CR = Urine amylase level x Scr x 100
Serum amylase level Ucr
( Scr & Ucr is serum & urinary creatinine level )
In patients with acute pancreatitis, the ratio varies from
The normal ratio is 1-4.4%
serum lipase :
Major lipolytic enzyme which hydrolyzes glycerol esters
of long chain fatty acids.
Normal level in serum : 30-235 U/L
The level in blood is highly elevated(2-50 fold) in acute
pancreatitis.This persists for 7-14 days.
Thus lipase remains elevated longer than amylase.
Lipase is not elevated in salivary diseases.
Lipase estimation has advantage over amylase.
In macroamylasemia, amylase circulates in the blood in a polymer form
too large to be easily excreted by the kidney. Patients with this condition
demonstrate an elevated serum amylase value, a low urinary amylase
value, and a Cam/Ccr ratio of <1%. The presence of macroamylase can be
documented by chromatography of the serum. The prevalence of
macroamylasemia is 1.5% of the nonalcoholic general adult hospital
population. Usually macroamylasemia is an incidental finding and is not
related to disease of the pancreas or other organs.
Macrolipasemia has now been documented in a few patients with
cirrhosis or non-Hodgkin's lymphoma. In these patients, the pancreas
appeared normal on ultrasound and CT examination. Lipase was shown
to be complexed with immunoglobulin A. Thus, the possibility of both
macroamylasemia and macrolipasemia should be considered in patients
with elevated blood levels of these enzymes.
The secretin test, used to detect diffuse pancreatic disease, is
based on the physiologic principle that the pancreatic secretory
response is directly related to the functional mass of pancreatic
In the standard assay, secretin is given IV in a dose of 0.2 g/kg of
synthetic human secretin as a bolus. Normal values for the
standard secretin test are (1) volume output >2 mL/kg per
hour, (2) bicarbonate (HCO3
–) concentration >80 mmol/L, and (3)
– output >10 mmol/L in 1 hour.
The most reproducible measurement, giving the highest level of
discrimination between normal subjects and patients with
chronic pancreatic exocrine insufficiency, appears to be the
maximal bicarbonate concentration.
There may be a dissociation between the results of the
secretin test and other tests of absorptive function. For
example, patients with chronic pancreatitis often have
abnormally low outputs of HCO3
– after secretin but have
normal fecal fat excretion. Thus the secretin test measures
the secretory capacity of ductular epithelium, while fecal fat
excretion indirectly reflects intraluminal lipolytic activity.
It must be noted that, an abnormal secretin test result
suggests only that chronic pancreatic damage is present.
The amount of human elastase (normal level=175-1500µg/g of
stool reflects the pancreatic output of this
proteolyticenzyme. Decreased elastase activity in stool(<100
µg/g of stool) is an excellent test to detect severe pancreatic
exocrine insufficiency in patients with chronic pancreatitis
and cystic fibrosis provided that the stool specimen is solid.
The fecal elastase-1 and small bowel biopsy are useful in the
evaluation of patients with suspected pancreatic steatorrhea.
The fecal elastase level will be abnormal and small bowel
histology will be normal in such patients.
Fat balance studies:
The astimation of fat in stool is done
When feces contains split fatty acids, it points to a normal
pancreatic function,but defective absorption.
If fat excreated is neutral fat, is due to defective
digestion, & is more in favour of pancreatic disease
Steatorrhea does not occur until intraluminal levels of
lipase are markedly reduced, underscoring the fact that
only small amounts of enzymes are necessary for
intraluminal digestive activities.
Normal fat level in stool is
Children upto 6yrs of age -<2g/d
Thereafter – 2-6g/d
Estimation of sweat electrolytes
In pancreatic fibrocystic disease,sodium & chloride are
increased in sweat.
The disease is characterized by thick viscous secretions
of exocrine glands.
Sweat chloride levels of >60mmol/L,on two separate
occations, is diagnostic of cystic fibrosis.