Trypsin-Based Laboratory Methods

Marja-Leena Kylänpää-Bäck1, Esko Kemppainen1, Pauli Puolakkainen1,2
1Department of Surgery, Helsinki University Central Hospital. Helsinki, Finland. 2The Hope Heart
Institute and the University of Washington. Seattle, WA, USA
Acute pancreatitis is a common disease
varying widely in severity. At present, there is
no “gold standard” for the diagnosis of acute
pancreatitis. Currently, the diagnosis of acute
pancreatitis is based on measurements of
serum amylase and/or lipase activity, which
are considered unsatisfactory due to their low
level of accuracy. Early identification of acute
pancreatitis and especially detection of
patients with a severe form of the disease is of
utmost importance.
Premature intrapancreatic activation of
trypsinogen is a crucial early event in the
pathophysiology of acute pancreatitis. The
conversion of trypsinogen to active trypsin is
mediated by the release of its activation
peptide (TAP). The active trypsin is then able
to activate other pancreatic zymogens (i.e.
procarboxypeptidase) leading to tissue
damage and eventually to autodigestion of the
pancreas. To improve the laboratory
diagnostics of AP, new methods have been
developed to measure this primary pancreatic
proteolytic insult.
Here we review the current knowledge and
clinical implications of trypsin based
laboratory methods and carboxypeptidase
activation peptide (CAPAP) in the diagnosis
and severity assessment of acute pancreatitis.
Acute pancreatitis (AP) is a common
emergency presentation. The incidence rate of
AP varies considerably, however, in different
countries. A low incidence has been reported
in England (10/100,000) [1, 2] and in
Germany (15/100,000) [3]. In contrast, in the
USA (40-80/100,000) and in Finland
(70/100,000) the incidence is high [4, 5]. AP
has many distinct etiologies, though
approximately 80% of all cases can be
attributed to either gallstones or alcohol [6].
Also, the frequency of AP of different
etiologies varies markedly in different
countries [5, 7, 8, 9].
The severity of AP forms a continuum. Most
of the cases are mild and conservative
treatment results in rapid recovery. However,
severe AP constitutes 15–20% of all cases [9,
10]. In severe AP the inflammatory process of
the pancreas is often violent with frequent
involvement of regional tissues and remote
organ systems [11, 12, 13]. In recent decades,
the mortality rate from severe AP has
decreased from 30-80% to 15-20% [14].
Severe AP is now recognized to be a two-
phase systemic disease. In the first phase,
extensive pancreatic inflammation and/or
necrosis are followed by a systemic
inflammatory response syndrome (SIRS) that
may lead to multiple organ dysfunction
syndrome (MODS) within the first week.
About 50% of deaths occur during the first
week of the attack, mostly from MODS [15,
16, 17, 18, 19, 20, 21]. Unless the first phase
is arrested and reversed by natural defenses or
therapeutic intervention, the second phase
usually ensues after the second week of onset
and includes the development of infected

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pancreatic necrosis or fluid collection with
possible progression to overt sepsis, MODS
and death [22, 23, 24, 25].
Organ failure is present in half of the patients
with pancreatic necrosis, but the extent of
pancreatic necrosis does not influence the
development of remote organ complications
[26, 27]. With an increasing number of failing
organ systems involved in AP, the associated
mortality rises [28]. The mortality associated
with MODS varies between 30 and 100% [20,
26, 29, 30, 31].
The major function of pancreatic acinar cells
is the synthesis, storing and secretion of
powerful digestive enzymes and their inactive
procarboxypeptidases A and B and
prophospholipase A2) [32, 33]. These
zymogens are synthesized in the endoplasmic
reticulum and then packaged into secretory
granules. Following acinar cell stimulation,
the content of these granules is discharged by
exocytosis into the acinar lumen and passes
via the pancreatic ductal system into the
duodenum [34, 35]. One of the precursors,
serine protease precursor trypsinogen, is the
main protease in human pancreatic fluid. The
conversion of trypsinogen to active trypsin, a
24-kDa protease, is normally catalyzd in the
duodenum by intestinal enterokinase [34, 35,
36]. Trypsinogen is activated by proteolytic
cleavage of a peptide called trypsinogen
activation peptide (TAP) [37, 38]. Trypsin is
the key enzyme for the rapid activation of all
the proenzymes, including its own
proenzyme, trypsinogen [39]. There are two
major isoenzymes of trypsinogen: cationic
trypsinogen-1 and anionic trypsinogen-2 [40,
41]. In healthy subjects, the ratio of
trypsinogen-1 to trypsinogen-2 in pancreatic
fluid is nearly fourfold and trypsinogen-1 and
trypsin-1-alpha-1-antitrypsin are the major
forms in serum [42].
Owing to their potent proteolytic and lipolytic
functions, the secretory enzymes represent a
considerable degradative (autodigestive)
transport and synthesis of secretory enzymes
as inactive zymogens represent protective
mechanisms against this degradation [43, 44].
The pancreatic acinar cells also synthesize the
protease pancreatic secretory trypsin inhibitor
(PSTI) which is considered to be the first line
of defense. PSTI, a 56-amino acid
polypeptide, can immediately neutralize
potentially harmful trypsin intracellularly,
thereby, maintaining a stable state [43]. It has
been shown that even if no active trypsin was
found in unstimulated pancreatic acini, there
can be comparable amounts of active
intracellular proteases [45]. This may be due
to spontaneous intracellular protease
activation. It may, however, also indicate that
intracellular trypsin is immediately
neutralized by local protease inhibitors.
In the normal state, only a minor proportion
of the total trypsinogen production leaks into
the circulation [46]. When active trypsin
reaches the circulation, the major trypsin
inhibitors (alpha-1-antitrypsin and alpha-2-
macroglobulin) inactivate it [43]. Trypsin-
alpha-1-antitrypsin complexes transfer the
enzyme to alpha-2-macroglobulin before its
elimination [47]. In human subjects, free
alpha-2-macroglobulin has a half-life of over
100 hours [48]. Instead, the trypsin-alpha-2-
macroglobulin complex is cleared from the
circulation by the reticular endothelial system
within 10 minutes or less [49, 50].
The pathogenesis of AP is only partially
known. The initial phase involves triggering
events, which are, for the most part,
extrapancreatic in origin. Clinically, the most
important of these are either passage of a
biliary tract stone or ingestion of ethanol.
Although the clinical association of AP with
biliary disease and with ethanol has been
firmly established, mechanistic explanations
for these associations have proven elusive
[51]. In experimental AP, microscopic
examination of pancreatic tissue obtained
after common bile-pancreatic duct ligation
indicates that the earliest signs of cell injury
involve acinar cells [52]. The severity of
experimental AP has been directly related to
the duration of duct obstruction [53].

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More than 100 years ago, the premature
intrapancreatic activation of trypsinogen and
the subsequent activation of zymogens
leading to autodigestion of the pancreas was
suggested to be an essential event in the
pathogenesis of AP [54]. Trypsin can be
found in the normal pancreas. However, the
pathological intrapancreatic activation of
trypsinogen to trypsin overwhelming the
inhibitory potential of PSTI leads to a more
general activation of digestive enzymes in the
pancreas [46, 55]. The activation of
trypsinogen, in and around the pancreas,
followed by the activation of other pancreatic
zymogens, occurs early in the course of AP,
in proportion to the extent of pancreatic injury
[56, 57]. Intracellular activation of
trypsinogen may be triggered by the abnormal
colocalization of digestive enzyme precursors
and lysosomal hydrolases [58, 59, 60].
Trypsinogen activation mediated by
lysosomal hydrolase cathepsin B has been
shown to be an early, as well as a critical
event, leading to cell injury [61]. However,
the extent of colocalization does not seem to
correlate with the severity of AP and
colocalisation of enzyme precursors and
hydrolases has also been shown in normal
acinar cells [62]. Another alternative for
trypsinogen activation during secretory
blockade is autoactivation, which can be
considered unique for human trypsinogen [36,
63]. The disruption of the acinar cell follows
premature activation of the proteases as a
result of interaction between the digestive and
lysosomal enzymes, and activated proteases
then escape into the interstitium of the
pancreas [51]. In an animal model of AP, it
has been shown that there are significant
quantities of uncleaved trypsinogen in the
interstitial compartment suggesting that
possible activation of this extracellular
trypsinogen leads to autodigestion of the
gland [64, 65]. Once released into the
pancreatic interstitium, retroperitoneum,
peritoneal cavity, and circulation, the active
enzymes cause necrotising tissue damage
through a variety of events, including local
autodigestion by lipase and proteases
eventually resulting in AP [66, 67, 68].
Currently, there is evidence that some cases
of AP can be hereditary. Patients with
hereditary AP have a mutation in the
trypsinogen-1 gene, which makes trypsin-1
resistant to proteolytic inactivation by other
intrapancreatic proteases resulting in the self-
destruction of the content of the zymogen
granules [69].
Pancreatic digestive enzymes explain only
part of the pathogenesis of complicated AP.
The release of various inflammatory
mediators is another important mechanism
[24, 70]. In fact, the pathophysiology of
severe AP resembles other conditions with
SIRS such as sepsis, multitrauma, ischaemia-
reperfusion injury and burns, which do not
involve the release of digestive enzymes from
the pancreas [18].
AP is a disease having a wide clinical
variation. Patients may suffer from a
multitude of symptoms, including upper
abdominal pain, meteorism, abdominal
resistance, fever, nausea and vomiting, ileus
and jaundice [9]. None of these frequent
symptoms are specific for AP or are they
related to the severity of the disease. Rare
clinical findings, such as ecchymosis of the
flank (Grey Turner’s sign) or periumbilical
area (Cullen’s sign), which occur in 1-3% of
patients, also fail to effectively predict the
severity of AP [71]. Within the first days of
admission, patients with severe AP may
develop SIRS characterized by a combination
of fever, tachycardia, and tachypnea [72]. In
summary, clinical findings, though helpful,
are not sufficiently accurate to diagnose AP.
Contrast enhanced computed tomography
(CE-CT) has become the standard imaging
method for diagnosing and staging AP and its
complications [73, 74]. The diagnostic
accuracy of CE-CT findings has proven high,
with specificity approaching 100% [75]. The
use of CT for a primary diagnosis is not
always possible due to its limited availability
and high costs [76, 77]. Furthermore, CT may

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be normal in 8-28 % of patients with AP,
especially in mild forms of the disease [75,
78, 79, 80].
Laboratory Methods for Diagnosing AP
Amylase and Lipase
Traditionally, the biochemical diagnosis of
AP is based on the determination of serum
and/or urinary amylase activity [81], the
activity of which increases in serum within 2-
12 hours of the onset and returns to normal
within 3-5 days [82]. However, up to 19% of
AP patients have a normal amylase value
[83]. Furthermore, it is well-known that
extrapancreatic diseases resulting in a low
specificity for AP [33]. Pancreatic lipase is
synthesized, similarly to amylase, in the
exocrine acinar cells and catalyzes the
hydrolysis of triglycerides into diglycerides
and fatty acids [33]. Wide variation in
sensitivity and specificity has been reported
for serum lipase determination in the
diagnosis of AP, which may partially be due
to different assay methods [84, 85, 86, 87,
88]. Because serum lipase remains elevated
longer than serum amylase, it has been
suggested that it may be useful when there is
a delay between the onset of symptoms and
admission [33, 82, 89, 90]. In all, however,
measurement of amylase and/or lipase activity
is generally considered not to be accurate
enough in detecting AP.
Other Methods
There is a pressing clinical requirement for an
early, simple and accurate test to improve the
biochemical diagnosis of AP. Serum elastase
stays elevated for up to one week after the
onset of AP and may be useful in cases with
delayed admission [33, 90], but the test is not
routinely used. Other serum markers such as
ribonuclease, chymotrypsin, phospholipase A2
and pancreatic isoamylase have been
evaluated, but their use is infrequent because
of limited utility [83, 90, 91, 92, 93, 94, 95].
Severity Assessment
At present, the classification of AP is based
on the internationally recognized Atlanta
criteria [96]. According to the Atlanta
classification, mild AP is associated with
minimal organ dysfunction and an uneventful
recovery, while AP is classified as severe if
systemic and/or local complications are
present. In an emergency setting, the
identification of severe AP remains
problematic and several patients with severe
disease are diagnosed only at autopsy [97]. It
has been shown that patients with severe AP
and delayed transfer to intensive care unit
have higher mortality than those admitted
directly [98]. There is evidence that early
enteral feeding, prophylactic antiobiotics and
emergency endoscopic sphincterotomy in
patients with biliary AP are beneficial in
severe AP [8, 10, 990, 100, 101, 102, 103].
Early diagnosis of patients with severe AP,
especially those with subsequent organ failure
would enable their immediate referral to a
centre having facilities for maximal intensive
care and specialists in the endoscopic,
radiological and surgical management of AP
[104]. Increasing knowledge of the
inflammatory process in AP has led to new
therapeutic strategies aiming at modifying
SIRS [24]. Moreover, since new
immunomodulatory therapies may have
undesirable side effects, it is of utmost
importance to accurately identify patients who
will benefit from immunomodulation [105,
106]. On the other hand, it is also important to
recognize patients with mild course of the
disease to allow them to be treated in lower-
cost hospital beds.
One of the main problems with AP has been
the lack of accurate predictors of disease
severity and the development of organ failure
in the early stages of the disease. On
admission, clinical assessment of severity has
been shown to be unreliable [71, 107, 108].
CE-CT has improved the assessment of the
disease severity by accurately identifying

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areas of necrosis [73, 74, 109, 110, 111]. It
has been reported that necrosis of only the
head of the pancreas is as dangerous as when
the entire pancreas is involved [112].
Magnetic resonance imaging is being
increasingly used for assessing the severity of
AP with promising results [113, 114, 115].
However, organ failure in AP patients is even
more problematic to predict as it occurs in
only half of the patients with pancreatic
necrosis [26].
There are several clinicobiochemical scoring
systems for the assessment of the severity of
AP [116]. The Ranson scoring system
comprises 11 biochemical criteria, which
require up to 48 hours for complete data
collection [117]. According to a recent meta-
analysis, Ranson`s signs show poor predictive
power [118]. The APACHE II illness grading
system is more accurate and can be used
throughout the patient’s hospitalization [119,
120]. However, 12 separate measurements are
needed for the APACHE II score and
additional values for age and chronic health.
clinicobiochemical systems are seldom used
routinely in clinical practice [121].
Laboratory Methods
Much effort has been directed to developing a
single, simple, rapid, affordable and reliable
laboratory test for the severity assessment of
an attack of AP [63]. The severity of AP does
not correlate with the level of serum amylase
and lipase [83, 91, 122]. C-reactive protein
(CRP) is the most commonly used laboratory
test in the assessment of the severity of AP
but it is useful only 48-72 hours after the
onset of the disease while it is insensitive
earlier [33, 38, 123]. However, in the follow-
up during the course of the disease, CRP has
proven to be useful [124]. The more recent
tests, especially those for cytokines are
expensive and/or laborious and time-
consuming to perform.
Trypsin Based Methods
Immunoreactive Trypsin
There are laboratory methods based on the
determination of pancreatic enzymes in serum
and urine, which measure the intrinsic
biological severity of organ damage by
Table 1. Accuracy of prognostic markers for acute pancreatitis on admission to hospital.
Marker and Reference
Urine trypsinogen-2
Hedström et al. 1996 [131]
Serum trypsin-2-AAT
Hedström et al. 1996 [130]
Urine TAP
Gudgeon et al. 1990 [37]
Tenner et al. 1997 [151]
Neoptolemos et al. 2000 [38]
Appelros et al. 2001 [128]
Trypsin-2-AAT: trypsin2-alpha-1-antitrypsin
TAP: trypsinogen activation peptide
CAPAP: carboxypeptidase activation peptide
PPV: positive predictive value
NPV: negative predictive value
PLR: positive likelihood ratio: sensitivity/(100-specificity)
NLR: negative likelihood ratio: (100-sensitivity)/specificity
Pre-test probability: the index of suspicion for severe acute pancreatitis
Post-test probability: the probability of severe disease according to the result of the test [157].

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estimating the degree of the primary
pancreatic proteolytic insult. Levels of
immunoreactive trypsin reflect the leakage of
unactivated proenzymes from injured acinar
cells. The original assays for determination of
measured cationic trypsinogen, i.e.
trypsinogen-1 and abnormal concentrations
were strongly considered to suggest a
pancreatic course of the illness [125, 126,
127]. Recently, Appelros et al. studied
immunoreactive anionic trypsinogen with an
enzyme-linked immunosorbent assay, which
complex with alpha-1-antitrypsin [128]. The
area under the curve (AUC) of the receiver
operating characteristics (ROC) curve
representing the discriminatory power for
severe disease was only 0.796 when
immunoreactive trypsinogen was measured in
urine whereas it was 0.475 in serum [128].
The sensitivity, specificity, positive predictive
value (PPV) and negative predictive value
(NPV) were 58%, 74%, 37%, 88% when
measured in urine and 38%, 58%, 14%, 84%
in serum. Thus, neither the measurement of
immunoreactive trypsinogen in urine or in
serum seems accurate enough for severity
assessment of AP.
Using specific antibodies for trypsinogen-1
and trypsinogen-2 it was found that in AP, the
serum concentrations of trypsinogen-2 were
increased 50-fold and those of trypsinogen-1
only 15-fold [127]. The corresponding
increase in immunoreactive trypsin was also
only 15-fold [127]. Moreover, patients with
AP excrete large amounts of trypsinogen-2
into urine and the concentration rises within
hours of the onset of the disease. The
measurement for trypsinogen-2 both in urine
and serum is a highly accurate marker for AP
[127, 129, 130, 131]. In addition, the
concentration of trypsinogen-2 shows a
marked correlation with the severity of the
disease (Table 1).
Our research group previously introduced a
rapid urinary trypsinogen-2 test strip which is
based on the use of immunochromatography
with monoclonal antibodies [132]. The
trypsinogen-2 strip test can be performed
rapidly in health care centres with limited
laboratory facilities. A modified 5-min
trypsinogen–2 (T-2) dipstick (Medix
Biochemica, Kauniainen, Finland) has
recently been developed with new antibodies
Table 2. Accuracy of urinary trypsinogen-2 (T-2) dipstick in detecting acute pancreatitis on admission to hospital.
Marker and Reference
Urinary trypsinogen-2 (T-2) dipstick
Hedström et al. 1996 [132]
Kemppainen et al. 1997 [134]
Kylänpää-Bäck et al. 2000 [133]
Kylänpää-Bäck et al. in press [158]
Pezzilli et al. 2001[136]
- /0.47
Trypsin-2-AAT: trypsin2-alpha-1-antitrypsin
TAP: trypsinogen activation peptide
CAPAP: carboxypeptidase activation peptide
PPV: positive predictive value
NPV: negative predictive value
PLR: positive likelihood ratio: sensitivity/(100-specificity)
NLR: negative likelihood ratio: (100-sensitivity)/specificity
Pre-test probability: the index of suspicion for severe acute pancreatitis
Post-test probability: the probability of severe disease according to the result of the test [157].

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to detect elevated levels of trypsinogen-2 in
urine. The accuracy of the T-2 dipstick test
[133] proved to be very similar to that of the
preliminary dipstick (sensitivity 96%,
specificity 92%), which has been reported in a
retrospective [132] and a prospective [134]
study (Table 2). Due to the very high
sensitivity (96%) and negative predictive
value (NPV, 99%) of the T-2 dipstick test
strip, AP can be excluded with high
probability with a negative dipstick result. It,
therefore, appears to be suitable as a
screening test for AP in patients with acute
abdominal pain. However, the positive
predictive value (PPV) is relatively low
(54%) indicating that the dipstick result alone
cannot establish the diagnosis of AP, but
additional examinations (laboratory or
radiology) are needed. Earlier, trypsinogen-2
concentrations were also reported to be
elevated in conditions such as hepatobiliary
and pancreatic cancer, and in chronic
pancreatitis [135]. The quantitative
measurements of urinary trypsinogen-2
showed a good agreement with the test strip
result (kappa value equal to 0.86), supporting
the use of the simple and rapid dipstick [133].
In a recent report by Pezzilli et al. the T-2
dipstick test showed high specificity but low
sensitivity in diagnosing AP (Table 2.) [136].
The difference between those results and ours
may be due to different diagnostic criteria
based solely on imaging procedures and also
due to possible difficulties in reading the
dipstick result. In addition, urine excretion of
trypsinogen could be delayed and therefore, if
the time between the onset of pancreatic pain
and the execution of the test is quite short
(less than 6 hours), it is possible that the
results of the dipstick test are negative.
However, in all the studies performed so far,
the dipstick has detected the severe cases of
AP very accurately, which is important in
clinical practice.
Trypsinogen Complexed with Antiproteases
Biochemical indicators of AP and its severity
include the antiproteases and their complexes
with trypsin. High serum concentrations of
immunoreactive trypsin-alpha-1-antitrypsin
complexes have been demonstrated in AP,
and the levels on admission correlate with the
severity of AP [137]. Our study group has
measured trypsin-2 complexed with alpha-1-
antitrypsin using a specific monoclonal
antibody to trypsin-2 and a polyclonal
antibody to alpha-1-antitrypsin [130]. In this
study of 110 patients with AP and 66 patients
with acute abdominal pain, trypsin-2-alpha-1-
antitrypsin complex in serum had the largest
AUC both in differentiating AP from control
patients (0.995) and in detecting mild AP
from the severe disease (0.82) as compared to
CRP, amylase and trypsinogen-2 12 hours
after admission (Table 1) [130]. Recently, the
ratio of trypsin-2-alpha-1-antitrypsin to
trypsinogen-1 in serum was reported to be a
promising new indicator for discriminating
between biliary and alcohol-induced AP
concentrations are found to be significantly
lower in complicated attacks of AP
suggesting its excessive consumption [139,
140, 141, 142, 143]. Additionally, the
concentrations have been shown to increase in
severe AP [144]. However, measurement of
neither form of alpha-2-macroglobulin is in
clinical use, partially due to complicated and
time-consuming assay methods.
Another potential marker for AP is TAP.
Immunoreactive TAP reflects the amount of
pathological intrapancreatic trypsinogen
activation irrespective of whether the
resulting trypsin is active or blocked by
inhibitors [56, 145]. Thus, it is a marker
specifically related to the onset of AP [37].
Free TAP is liberated into the peritoneal
cavity and the circulation, after which,
because of its small size, the peptide is rapidly
cleared by the kidneys and excreted into the
urine [145]. However, the measurement of
TAP in urine is not a perfect test for
diagnosing AP, since Gudgeon et al. reported
in 1990 that 30% of patients with AP had
normal TAP values on admission [37].

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Further, in an endoscopic retrograde
cholangiopancreatography (ERCP) study,
urinary TAP was not useful in predicting mild
post-ERCP AP [146]. It has also been shown
that the concentrations of urinary TAP do not
vary according to the cause of the disease
In an animal model of AP in ascites, urine,
plasma and pancreatic tissue, the TAP
concentration has been shown to correlate
well with the extent of pancreatic necrosis
[37, 147, 148, 149, 150]. The urinary
TAP/creatinine ratio correlates with the
severity of the disease in humans [57].
Concentrations of TAP in urine have been
shown to predict severe AP with a sensitivity
of 100% and a specificity of 85% on
admission to hospital within 48 hours of the
onset of symptoms in an American
multicenter study [151]. In a recent European
multicenter study, urinary TAP showed a
somewhat lower accuracy for the assessment
of the severity of AP as soon as 24 hours after
the onset of symptoms with a sensitivity of
58% and a specificity of 73% [38] (Table 1).
However, when likelihood ratios were
calculated, for example a positive urinary
TAP assay 48 hours after the onset of
symptoms only increased the positive
likelihood ratio of severe AP from 20 to 35%.
When urinary TAP measurement was
combined with CRP, the probability of severe
AP increased from 20 to 55% [152].
Clinically the most favourable feature of
urinary TAP is the capability of
differentiating between severe and mild
disease during the very early phase of the
disease, when other methods are not yet
useful [63]. However, the general accuracy of
urinary TAP alone does not qualify for
clinical decision-making. In plasma, TAP
showed maximal accuracy for distinction
between mild and severe disease within 6
hours after admission with a sensitivity of
70% and specificity of 78%. Thereafter the
prognostic accuracy declined rapidly and TAP
values showed a very variable pattern
possibly due to burst-like secretion [153].
Carboxypeptidase Activation Peptide (CAPAP)
In the normal state, local trypsin inhibitors
inactivate trypsin in and around the pancreas.
However, if trypsin activation exceeds the
capacity of the trypsin inhibitors, the
activation of other pancreatic zymogens
occurs [56]. Therefore, released activation
peptides of zymogens reflect free trypsin
activity. Procarboxypeptidase B has an
activation peptide (CAPAP), which is larger
than other peptides released during
proenzyme activation [154]. The large size
makes it more stable and, thus, suitable for
measurement in serum and urine, and now, a
radioimmunoassay has been developed for
this peptide [155]. It has been reported that
the levels of CAPAP in urine and serum
correlate well with the severity of AP [128,
156] (Table 1). The AUC, representing the
discriminatory power for severe disease, was
0.9422 when CAPAP concentration was
measured in urine and when measured in
serum [126]. However, the study included
only 60 non-consecutive AP patients and only
12 of them had severe disease. Eight of the 60
AP patients (all with mild disease) had
undetectable levels of CAPAP in urine. This
suggests that mild AP can occur without
detectable trypsinogen activation and
activation of other zymogens. It also means
that measurement of CAPAP cannot be used
as a diagnostic test for AP, since many mild
cases would be missed. In a recent study by
Pezzilli et al., though, CAPAP reached a
sensitivity and specificity of 95% for
diagnosing AP [156]. The study population
was limited to 20 patients with AP and 20
controls. As for the accuracy of the CAPAP
assay for the severity assessment of AP,
further prospective clinical studies with
sufficient number of patients are needed.
At present, no single biochemical marker is
ideal for diagnosing and/or the early

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prediction of the severity of AP. Assays based
on trypsin pathophysiology have brought
interesting new alternatives for diagnostics
and severity grading of AP. The available
study results suggest the use of Actim
Pancreatitis® for screening for AP in patients
with acute abdominal pain. Comparison of the
results in different studies is very difficult
partly because there is no “gold standard” for
the diagnosis of AP and also due to
differences in the pre-test probability and
severity grading of the disease. It has been
pointed out that likelihood ratios should be
used to assess the results. Urinary TAP can be
measured relatively easily and has a
prognostic value especially when combined
with CRP and this could help physicians in
clinical practice. Urinary CAPAP seems very
promising as a prognostic marker but should
be studied with a large consecutive series of
AP patients before wider clinical use. It seems
obvious, however, that no single test is ideal
and accurate enough, and a combination of
tests may be needed to predict severe AP and
its systemic complications when new
therapies are planned. In the future, additional
studies with a sufficient number of patients
will be needed to find out the most accurate
set of markers.
Received October 12th, 2001 – Accepted
January 10th, 2002
Carboxypeptidases; Pancreatitis; Peptides;
Abbreviations AP: acute pancreatitis; AUC:
area under the curve; CAPAP:
carboxypeptidase activation peptide; CE-CT:
contrast enhanced computed tomography;
CRP: C-reactive protein; ERCP: endoscopic
MODS: multiple organ dysfunction
syndrome; NPV: negative predictive value;
PPV: positive predictive value; PSTI,
pancreatic secretory trypsin inhibitor; ROC:
receiver operating characteristics; SIRS:
systemic inflammatory response syndrome;
TAP: trypsinogen activation peptide; T-2
dipstick: trypsinogen-2 dipstick
Pauli Puolakkainen
The Hope Heart Institute
1124 Columbia Street
Seattle, WA 98104
Phone: +1-206-903.2035
Fax: +1-206-903.2044
1. Corfield A, Cooper M, Williamson R. Acute
pancreatitis: a lethal disease of increasing incidence.
Gut 1985; 26:724-9. [AN 85259138]
2. Giggs J, Bourke J, Katschinski B. The
epidemiology of primary acute pancreatitis in Greater
Nottingham: 1969-1983. Soc Sci Med 1988; 26:79-89.
[AN 88178172]
3. Assmus C, Petersen M, Gottesleben F, Drüke M,
Lankisch P. Epidemiology of acute pancreatitis in a
defined German population. Digestion 1996; 57:A217.
4. Lankisch P. Epidemiology of acute pancreatitis.
In: Büchler M, Uhl W, Friess H, Malfertheimer P, eds.
Acute Pancreatitis, Novel Concepts in Biology and
Treatment. Berlin-Vienna: Blackwell Wissenschafts-
Verlag, 1999:145-53.
5. Jaakkola M, Nordback I. Pancreatitis in Finland
between 1970 and 1989. Gut 1993; 34:1255-60. [AN
6. Karne S, Gorelick F. Etiopathogenesis of acute
pancreatitis. Surg Clin North Am 1999; 79:699-710.
[AN 99399335]
7. Leese T, Shaw D, Holliday M. Prognostic markers
in acute pancreatitis: can pancreatic necrosis be
predicted? Ann R Coll Surg Engl 1988; 70:227-32.
[AN 88325256]
8. Fan S, Lai E, Mok F, Lo C, Zheng S, Wong J.
Early treatment of acute biliary pancreatitis by
endoscopic papillotomy. N Engl J Med 1993; 328:228-
32. [AN 93116811]
9. Steinberg W, Tenner S. Acute pancreatitis. N Engl
J Med 1994; 330:1198-210. [AN 94187814]
10. Barie PS. A critical review of antibiotic
prophylaxis in severe acute pancreatitis. Am J Surg
1996;172(Suppl 6A):38-43. [AN 97157404]
11. Mann D, Hershman M, Hittinger R, Glazer G.
Multicentre audit of death from acute pancreatitis. Br J
Surg 1994; 81:890-3. [AN 94319876]

Page 10
JOP. J. Pancreas (Online) 2002; 3(2):34-48.
JOP. Journal of the Pancreas – – Vol. 3, No. 2 – March 2002
12. Banerjee A, Kaul A, Bache E, Parberry A, Doran
J, Nicholson M. An audit of fatal acute pancreatitis.
Postgrad Med J 1995; 71:472-5. [AN 96014865]
13. Grönroos J, Nylamo E. Mortality in acute
pancreatitis in Turku University Central Hospital 1971-
1995. Hepatogastroenterology 1999; 46:2572-4. [AN
14. Bank S. Clinical course of acute pancreatitis: what
has changed in recent years? In: Büchler M, Uhl W,
Friess H, Malfertheimer P, eds. Acute Pancreatitis,
Novel Concepts in Biology and Treatment. Berlin-
Vienna: Blackwell Wissenschafts-Verlag, 1999:163-9.
15. Deitch E. Multiple organ failure. Ann Surg 1992;
216:117-34. [AN 92368273]
16. Heath D, Alexander D, Wilson C, Larvin M, Imrie
C, McMahon M. Which complications of acute
pancreatitis are most lethal? A prospective multicentre
clinical study of 719 episodes. Gut 1995; 36:A478.
17. Gullo A, Berlot G. Ingredients of organ
dysfunction or failure. World J Surg 1996; 20:430-6.
[AN 96270254]
18. Nathens A, Marshall J. Sepsis, SIRS, and MODS:
What`s in a name? World J Surg 1996; 20:386-91. [AN
19. Balakrishnan V, Philip M. Cytokines as predictors
of severity in acute pancreatitis. J Gastroenterol
Hepatol 1998; 13:1177-9. [AN 99114980]
20. Neoptolemos JP, Raraty M, Finch M, Sutton R.
Acute pancreatitis: the substantial human and financial
cases. Gut 1998; 42:886-91. [AN 98356961]
21. McKay C, Evans S, Sinclair M, Carter C, Imrie C.
High early mortality rate from acute pancreatitis in
Scotland, 1984-1995. Br J Surg 1999; 86:1302-6. [AN
22. American College of Chest Physicians/Society of
Critical Care Medicine Consensus Conference.
Definitions for sepsis and organ failure and guidelines
for the use of innovative therapies in sepsis. Crit Care
Med 1992; 20:864-74. [AN 92282912]
23. Deitch EA, Goodman ER. Prevention of multiple
organ failure. Surg Clin North Am 1999; 79:1471-88.
[AN 20091434]
24. Osman M, Jensen S. Acute pancreatitis: The
pathophysiological role of cytokines and integrins. Dig
Surg 1999; 16:347-62. [AN 20036980]
25. Schmid S, Uhl W, Friess H, Malfertheiner P,
Büchler M. The role of infection in acute pancreatitis.
Gut 1999; 45:311-6. [AN 99332275]
26. Tenner S, Sica G, Hughes M, Noordhoek E, Feng
S, Zinner M, et al. Relationship of necrosis to organ
failure in severe acute pancreatitis. Gastroenterology
1997; 113:899-903. [AN 97434096]
27. Lankisch P, Pflichthofer D, Lehnick D. No strict
correlation between necrosis and organ failure in acute
pancreatitis. Pancreas 2000; 20:319-22. [AN
28. Knaus W, Draper E, Wagner D, Zimmerman J.
Prognosis in acute organ-system failure. Ann Surg
1985; 202:685-93. [AN 86076129]
29. McFadden D. Organ failure and multiple organ
system failure in pancreatitis. Pancreas 1991; 6:37-43.
[AN 92158980]
30. Marshall J, Cook D, Christnou N, Bernard G,
Sprung C, Sibbald W. Multiple organ dysfunction
score: A reliable descriptor of a complex clinical
outcome. Crit Care Med 1995; 23:1638-52. [AN
31. Erwin P, Lewis H, Dolan S, Tobias P, Schumann
R, Lamping N, et al. Lipopolysaccharide binding
protein in acute pancreatitis. Crit Care Med 2000;
28:104-9. [AN 20128969]
32. Dubick M, Mar G, Mayer A, Majumdar A,
McMahon M, Geokas M. Digestive enzymes and
protease inhibitors in plasma from patients with acute
pancreatitis. Pancreas 1987; 2:187-94. [AN 87317534]
33. Clavien P-A, Burgan S, Moossa AR. Serum
enzymes and other laboratory tests in acute
pancreatitis. Br J Surg 1989; 76:1234-43. [AN
34. Steer M, Meldolesi J, Figarella C. Pancreatitis.
The role of lysosomes. Dig Dis Sci 1984; 29:934-8.
[AN 85003093]
35. Steer M. Primary intracellular events in
pancreatitis. In: Büchler M, Uhl W, Friess H,
Malfertheimer P, eds. Acute Pancreatitis, Novel
Concepts in Biology and Treatment. Berlin-Vienna:
Blackwell Wissenschafts-Verlag, 1999:4-12.
36. Kassel B, Kay J. Zymogens of proteolytic
enzymes. Science 1973; 180:1022-7. [AN 73182838]
37. Gudgeon A, Heath D, Hurley P, Jehanli A, Patel
G, Wilson C, et al. Trypsinogen activation peptides
assay in the early prediction of severity of acute
pancreatitis. Lancet 1990; 335:4-8. [AN 90113538]
38. Neoptolemos J, Kemppainen E, Mayer J,
Fitzpatrick J, Raraty M, Slavin J, et al. Early prediction
of severity in acute pancreatitis by urinary trypsinogen
activation peptide: a multicentre study. Lancet 2000;
355:1955-60. [AN 20315573]
39. Marshall J. Acute pancreatitis. A review with an
emphasis on new developments. Arch Intern Med
1993; 153:1185-98. [AN 93263775]
40. Rinderknecht H, Geokas M. Anionic and cationic
trypsinogens (trypsins) in mammalian pancreas.
Enzyme 1973; 14:116-30.

Page 11
JOP. J. Pancreas (Online) 2002; 3(2):34-48.
JOP. Journal of the Pancreas – – Vol. 3, No. 2 – March 2002
41. Figarella C. Amouric M. Carrere J. Miszczuk-
Jamska B. Guy-Crotte O. Pancreatic proteins in normal
state and pancreatic diseases in pancreatic juice and
blood. Ric Clin Lab 1984; 14:349-59. [AN 85114948]
42. Durie P, Gaskin K, Geokas M, O`Rourke M,
Largman C. Plasma immunoreactive anionic pancreatic
trypsin in cystic fibrosis. J Pediatr Gastroenterol Nutr
1982; 1:337-43. [AN 83293885]
43. Ohlsson K. Acute pancreatitis. Biochemical,
pathophysiological and therapeutic aspects. Acta
Gastroenterol Belg 1988; 51:3-12. [AN 89046402]
44. Glasbrenner B, Adler G. Pathophysiology of acute
pancreatitis. Hepatogastroenterology 1993; 40:517-21.
[AN 94164589]
45. Saluja M, Saluja A, Learh M, Steer M. A plasma
protease which is expressed during supramaximal
stimulation causes in vitro subcellular redistribution of
lysosomal enzymes in rat exocrine pancreas. J Clin
Invest 1991; 87:1280-5. [AN 91185609]
46. Borgström A, Ohlsson K. Studies on the turnover
of endogenous cathodal trypsinogen in man. Eur J Clin
Invest 1978; 8:379-82. [AN 79127668]
47. Balldin G. Laurell CB. Ohlsson K. Increased
catabolism of alpha-Macroglobulins after intravenous
infusion of trypsin-alpha1-antitrypsin complexes in
dogs. Hoppe-Seylers Z Physiol Chem 1978; 359:699-
708. [AN 78216709]
48. Blatrix C. Amouch P. Drouet J. Steinbuch M.
Study on the plasmatic elimination of the alpha2-
macroglobulin-proteinase complexes. Pathol Biol
1973; 21(Suppl):11-4. [AN 74125284]
49. Ohlsson K. Elimination of 125-I-trypsin alpha-
macroglobulin complexes
from blood by
reticuloendothelial cells in dogs. Acta Physiol Scand
1971; 81:269-72. [AN 71161671]
50. Ohlsson K. Laurell CB. The disappearance of
enzyme-inhibitor complexes from the circulation of
man. Clin Sci Mol Med 1976; 51:87-92. [AN
51. Steer M. The early intraacinar cell events which
occur during acute pancreatitis. Pancreas 1998; 17:31-
7. [AN 98330091]
52. Lerch M, Saluja A, Dawra R, Ramarao P, Saluja
M, Steer M. Acute necrotizing pancreatitis in the
opossum: earliest morphological changes involve
acinar cells. Gastroenterology 1992; 103:205-13. [AN
53. Runzi M, Saluja A, Lerch M, Dawra R, Nishino H,
Steer M. Early ductal decompression prevents the
progression of biliary panreatitis: an experimental
study in the opossum. Gastroenterology 1993; 105:157-
64. [AN 93292807]
54. Chiari H. Über selbstverdauung des menschlichen
Pancreas. Z Heilk 1896; 17:69-96.
55. Steer M, Meldolesi J. The cell biology of
experimental pancreatitis. N Engl J Med 1987;
316:144-50. [AN 87090173]
56. Ohlsson K, Balldin G, Bohe M, Borgström A,
Genell S, Lasson C. Pancreatic proteases and
antiproteases in pancreatic disease; biochemical,
pathophysiological and clinical aspects. Int J
Pancreatol 1988; 3(Suppl 1):67-78. [AN 89094006]
57. Heath D, Cruickshank A, Gudgeon A, Jehanli A,
Shenkin A, Imrie C. The relationship between
pancreatic enzyme release and activation and the acute
phase protein response in patients with acute
pancreatitis. Pancreas 1995; 10:347-53. [AN
58. Greenbaum L, Hirschkowitz A. Endogenous
cathepsin activation of trypsinogen in extracts of dog
pancreas. Proc Soc Exp Biol Med 1961;107:74-6.
59. Bialek R, Willemer S, Arnold R, Adler G.
Evidence of intracellular activation of serine proteases
in acute cerulein-induced pancreatitis in rat. Scand J
Gastroenterol 1991; 26:190-6. [AN 91188208]
60. Luthen R, Owen R, Sarbia M, Grendell J,
Niederau C. Premature trypsinogen activation during
cerulein pancreatitis in rats occurs inside pancreatic
acinar cells. Pancreas 1998; 17:38-43. [AN 98330092]
61. Saluja A, Donovan E, Yamanaka K, Yamaguchi
Y, Hofbauer B, Steer M. Cerulein-induced in vitro
activation of trypsinogen in rat pancreatic acini is
mediated by cathepsin B. Gastroenterology 1997;
113:304-10. [AN 97350915]
62. Luthen R, Niederau C, Niederau M, Ferrel L,
Grendell J. Influence of ductal pressure and infusates
on activity and subcellular distribution of lysosomal
enzymes in the rat pancreas. Gastroenterology 1995;
109:965-7. [AN 95340115]
63. Frossard J. Trypsin activation peptide (TAP) in
acute pancreatitis: From pathophysiology to clinical
usefulness. JOP. J Pancreas (Online) 2001; 2(2):69-77.
64. Mithofer K, Fernandez-del Castillo C, Rattner D,
Warshaw A. Subcellular kinetics of early trypsinogen
activation in acute rodent pancreatitis. Am J Physiol
1998; 274:71-9. [AN 98120396]
65. Fernandez del Castillo C, Schmidt J, Warshaw A,
Rattner D. Interstitial protease activation is the central
event in progression to necrotizing pancreatitis.
Surgery 1994; 116:497-504. [AN 73182838]
66. Warshaw A. Damage prevention versus damage
control in acute pancreatitis. Gastroenterology 1993;
104:1216-9. [AN 93216040]

Page 12
JOP. J. Pancreas (Online) 2002; 3(2):34-48.
JOP. Journal of the Pancreas – – Vol. 3, No. 2 – March 2002
67. Gorelick F, Matovcik L. Lysosomal enzymes and
pancreatitis. Gastroenterology 1995; 109:620-5. [AN
68. Warshaw A. Ischemia- and reperfusion-related
injury in pancreatitis. Dig Dis Sci 1996; 41:821-2. [AN
69. Whitcomb D, Gorry M, Preston R, Furey W,
Sossenheimer M, Ulrich C, et al. Hereditary
pancreatitis is caused by a mutation in the cationic
trypsinogen gene. Nat Genet 1996; 14:141-5. [AN
70. Giroir BP. Pancreatitis, cytokines, and SIRS: Dé ja
vu all over again? Crit Care Med 1999; 27:680-1. [AN
71. Büchler M. Objectification of the severity of acute
pancreatitis. Hepatogastroenterology 1991; 38:101-8.
[AN 91310020]
72. Bone R. Toward a theory regarding the
pathogenesis of the systemic inflammatory response
syndrome: What we do and do not know about
cytokine regulation. Crit Care Med 1996; 24:163-72.
[AN 96160719]
73. Kivisaari L, Somer K, Strandertskjöld-Nordenstam
C, Schröder T, Kivilaakso E, Lempinen M. Early
detection of acute fulminant pancreatitis by contrast-
enhanced computed tomography. Scand J
Gastroenterol 1983; 18:39-41. [AN 84196146]
74. Balthazar E, Robinson D, Megibow A, Ranson J.
Acute pancreatitis: value of CT in establishing
prognosis. Radiology 1990; 174:331-6. [AN 90116369]
75. Clavien P-A, Hauser H, Meyer P, Rohner A. Value
of contrast-enhanced computerized tomography in the
early diagnosis and prognosis of acute pancreatitis. Am
J Surg 1988;155:457-66. [AN 88148232]
76. London N, Neoptolemos JP, Lavelle J, Bailey I,
James D. Contrast-enhanced abdominal computed
tomography scanning and prediction of severity of
acute pancreatitis: a prospective study. Br J Surg 1989;
76:268-72. [AN 89248357]
77. Lucarotti M, Virjee J, Alderson D. Patient
selection and timing of dynamic computed tomography
in acute pancreatitis. Br J Surg 1993; 80:1393-5. [AN
78. Balthazar E. CT diagnosis and staging of acute
pancreatitis. Radiol Clin North Am 1989; 27:19-37.
[AN 89058100]
79. Thoeni R, Blankenberg F. Pancreatic imaging.
Radiol Clin North Am 1993; 31:1085-113. [AN
80. Balthazar E, Freeny P, van Sonnenberg E. Imaging
and intervention in acute pancreatitis. Radiology 1994;
193:297-306. [AN 95062956]
81. Elman R, Arneson N, Graham E. Value of blood
amylase estimations in the diagnosis of pancreatic
disease: a clinical study. Arch Surg 1929; 19:943-67.
82. Tietz NW, Shuey DF. Lipase in serum - the
elusive enzyme: an overwiev. Clin Chem 1993;
39:746-56. [AN 93251639]
83. Clavien P-A, Robert J, Meyer P, Borst F, Hauser
H. Acute pancreatitis and normoamylasemia. Not an
uncommon combination. Ann Surg 1989; 210:614-20.
[AN 90055421]
84. Patt H, Kramer S, Woel G, Zeitung D, Seligman
A. Serum lipase determinat

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