Clin Chem Lab Med 2015; 53(1): 65–71
Els N. Dumoulin, Stephanie Van Biervliet, Michel R. Langlois and Joris R. Delanghe*
Proteolysis is a confounding factor in the
interpretation of faecal calprotectin
Abstract
Background: Calprotectin is a 36  kDa calcium and zinc
binding protein. An increased level of calprotectin points
towards inflammatory bowel disease. However, the bio-
marker calprotectin shows 14 potential cleavages sites for
trypsin. Next to trypsin, also the presence of its inhibitor
α1-antitrypsin after a gastrointestinal bleeding may affect
calprotectin testing. In this study, effects of trypsin and α1-
antitrypsin as potential confounders for faecal calprotec-
tin testing are investigated.
Methods: An in vitro model was created. As calprotectin
source, leukocytes were isolated and subsequently lysed
(1% Triton X-100) and diluted in faecal matrix. Trypsin
digestion was carried out by adding trypsin. Incubation
occurred for 24  h or 48  h (37 °C). To study the influence
of α1-antitrypsin on trypsin, the same experiment was
repeated after adding serum containing α1-antitrypsin.
Results: In vitro experiments enabled monitoring of the
faecal calprotectin digestion, leading to loss of immuno-
reactivity. Trypsin activity was a potential confounder in
the interpretation of calprotectin, in particular for proxi-
mal lesions, where exposure of calprotectin to trypsin is
prolonged. Relative calprotectin loss was proportional to
the amount of trypsin. Decrease of calprotectin was more
pronounced after 48 h of incubation in comparison to 24 h
of incubation. Analogue experiments also showed stable
calprotectin values after adding α1-antitrypsin.
Conclusions: Transit time, trypsin activity and addition
of blood as a source of α1-antitrypsin may be regarded as
potential confounders in the interpretation of calprotectin
results. Age-related cut-off values depending on the ana-
tomical localisation of the lesions could improve the diag-
nostic efficiency of calprotectin testing.
*Corresponding author: Joris R. Delanghe, Department of Clinical
Chemistry, Microbiology and Immunology, Ghent University
Hospital, De Pintelaan 185, Ghent, Belgium, Phone: +32 9 3322956,
Fax: +32 9 3324985, E-mail: joris.delanghe@ugent.be
Els N. Dumoulin: Department of Clinical Chemistry, Microbiology
and Immunology, Ghent University Hospital, Ghent, Belgium
Stephanie Van Biervliet: Department of Pediatric Gastroenterology
and Nutrition, Ghent University Hospital, Ghent, Belgium
Michel R. Langlois: Department of Laboratory Medicine, AZ Sint-Jan,
Bruges, Belgium
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Keywords: α1-antitrypsin; calprotectin; faeces; inflamma-
tory bowel disease; proximal gut lesions; trypsin.
DOI 10.1515/cclm-2014-0568
Received May 28, 2014; accepted June 13, 2014; previously published
online July 4, 2014
Introduction
Calprotectin is a 36 kDa calcium and zinc binding protein,
accounting for about 60% of soluble proteins in neutro-
philic cytosol. It is secreted extracellularly by neutrophils
upon stimulation or by cell disruption [1–3]. It can be
measured in faeces and is believed to be resistant to enzy-
matic degradation. Calprotectin is reported to be stable
up to 1 week at room temperature and for several months
when stored at –20 °C [4]. Faecal calprotectin is regarded
as a marker of inflammation in the gastrointestinal tract.
It is used to discern patients with an organic bowel disease
from the ones with functional complaints. In adults and
children above the age of 4, an increased value leads to
the identification of patients with suspicion of inflamma-
tory bowel disease (IBD) and in need of an endoscopy [1,
5, 6]. Calprotectin is also studied as surrogate biomarker
of mucosal improvement in patients with Crohn’s disease
(CD). Endoscopic responders achieved a significant
decrease of faecal calprotectin, whereas in the majority of
the endoscopic non- and partial responders these markers
remained abnormal [7]. Endoscopic score and histological
findings correlated significantly with faecal calprotectin in
ileocolonic and colonic disease. However, ileal endoscopic
scores and histological findings failed to correlate with
faecal markers, such as calprotectin and lactoferrin. In the
study of Siponnen et al. 21 out of 87 patients were classified
as ileal disease. As the majority of these patients had a stric-
turing disease type, larger studies of faecal markers’ behav-
iour in ileal and small bowel disease are necessary [8].
Trypsin (EC 3.4.21.4) is an endopeptidase found in the
intestinal lumen where it hydrolyses proteins. Trypsin is pro-
duced in large amounts by the human pancreas as the inac-
tive proenzyme trypsinogen. The reported enzyme activities
in duodenal juice vary from 20–50 U/mL between meals to
80–180 U/mL in the early postprandial and 60–150  U/mL
66      Dumoulin et al.: Confounders of calprotectin interpretation
in the late postprandial period [9]. Trypsin cleaves peptide
chains mainly at the carboxyl side of the amino acids lysine or
arginine, except when either is followed by proline. Trypsin
activity has been reported to be stable 7 h after faecal sample
collection between 0 and 4 °C [10, 11]. Following release of
trypsinogen, the enzyme reaches its highest activity in the
duodenum. During transit, activity of proteases decreases,
resulting in a residual activity of only 20%–30% in the ter-
minal ileum. For trypsin, its enzymatic activity is preserved
better than its immunoreactivity. Structural integrity may not
be essential for the proteolytic activity [12, 13]. Since calpro-
tectin contains 14 residues which may act as potential cleav-
age sites, (partial) digestion of calprotectin by trypsin can be
postulated (Figure 1).
α1-Antitrypsin (AAT) is a 52  kDa plasma glycoprotein
that inhibits a variety of serine proteinases, such as trypsin.
Normal plasma levels range from 1 g/L to 2 g/L. A common
method used for establishing a protein-losing enteropathy
is the clearance of AAT from plasma. It requires a blood
sample for AAT analysis and a 24  h stool collection to
determine stool volume and stool AAT level. Normal AAT
clearance is   ≤  27 mL/24 h. Blood or protein loss in stool will
thus be accompanied by a variable release of AAT. As AAT
does not undergo proteolysis or degradation in the gas-
trointestinal lumen, it can be regarded as a confounder of
residual trypsin activity in stool samples [14, 15].
Clinical efficiency of calprotectin testing is lower for
proximal lesions [8]. The reasons for these findings are
poorly understood. Therefore, we postulate a potential
role for trypsin and AAT as confounders in calprotectin
test interpretation. In the present study, we have studied
in detail the effects of trypsin and its inhibitor protein AAT
on faecal calprotectin analysis using an in vitro model.
Materials and methods
In vitro model
To create an in vitro model and study the influence of possible
inhibitors in faeces, faecal extracts were prepared according to the
Figure 1 The molecular structure of calprotectin.
Trypsin cleaves peptide chains at the carboxyl side of the amino acids lysine (K) or arginine (R). Fourteen potential cleavage sites are
present, as shown by the arrows.
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manufacturer’s instruction with the Faecal Sample Preparation Kit
(Roche diagnostics, Mannheim, Germany). After adding a standard-
ised amount of faeces to a tube, 5  mL of EliA Calprotectin Extrac-
tion Buffer (Thermo Scientific, Uppsala, Sweden) was added and the
sample was vortexed. After centrifugation the supernatant was used
as a faecal matrix in our experiments. Faecal samples from outpa-
tients consulting the Department of Gastro-Enterology were used.
Calprotectin was prepared from white blood cells (WBC) obtained
from fresh ethylenediaminetetraacetic acid (EDTA) blood samples.
After isolation of WBC with erythrocyte lysis buffer (Qiagen, Hilden,
Germany) from a sample with a high neutrophil count (45.5×103 per
μL), a 1% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) lysis buffer
(TRIS 50 mM, Sigma Aldrich; NaCl 150 mM, Merck Chemicals, Darm-
stadt, Germany) adjusted to pH 7.5 was used to release the cytosolic
calprotectin. Aliquots of 1 mL were frozen.
An optimal dilution in faecal matrix of 1:100 was chosen by
serial dilutions (calprotectin levels of 102–103 mg/kg). Trypsin diges-
tion of calprotectin was carried out by adding trypsin (Sigma Aldrich)
solution (1 mg/mL, 1 mM HCl). Incubation occurred at 37 °C. Samples
were analysed for calprotectin after 24  h or 48  h of incubation. To
evaluate the influence of AAT, routine serum samples were selected
in the normal range. First, incubation of trypsin with a serum sam-
ple was performed at room temperature for 30 min. Second, 100 μL
serum or saline was added to the mixture of trypsin and calprotec-
tin in faecal matrix and incubated under the same conditions as
described above.
Methods and analysis
Calprotectin was assayed on the Phadia ImmunoCAP 250 analyser
(Thermo Scientific) according to the manufacturer’s instructions.
Results were expressed in mg/kg [16]. Trypsin activity was measured
using a colourimetric method with Nα-Benzoyl-L-arginine 4-nitroan-
ilide hydrochloride (BAPNA, Sigma Aldrich) as a substrate for trypsin
(Sigma Aldrich). The amount of 4-nitroaniline released per minute
was measured by increase in absorbance 405 nm at 25 °C. A trietha-
nolamine solution (TEA 0.2 mol/L, Sigma Aldrich; CaCl2 0.02 mol/L,
Sarstedt, Nümbrecht, Germany) adjusted to pH 7.8 was used as buffer.
Results were expressed in BAPNA U/L [10]. AAT concentration was
measured on the Behring Nephelometer Analyzer II (Siemens, Mar-
burg, Germany) according to the manufacturer’s instructions [17].
Statistics
All calculations were made in Excel 2010 and MedCalc (version
11.6.1.0, Mariakerke, Belgium). Results were given as median and

inter-quartile ranges between brackets. Comparisons between condi-
tions were made by using a Wilcoxon test. A value of p < 0.05 was con-
sidered statistically significant. Regression analysis was performed
and box-and-whisker plots were made.
Results
At first effects of trypsin digestion on calprotectin were
evaluated by incubating a 1:100 dilution of calprotectin
in faecal matrix with different added trypsin concentra-
tions dissolved in 1 mM HCl (a serial dilution with trypsin
levels between 12 and 61 U/L). Differences were evaluated
after 24 h and 48 h of incubation at 37 °C. Figure 2 depicts
the effects of these different trypsin concentrations on
calprotectin immunoreactivity in faecal matrix after 24 h
and 48  h of incubation. The more trypsin was added to
the mixture the higher the percentage of calprotectin pro-
teolysis after 24 h. After 48 h of incubation the effect was
still present but the trend towards more proteolysis when
more trypsin was added to the mixture was less clear.
For further evaluation an ideal ratio of 500 μL trypsin
solution (1 mg/mL) and 500 μL 1:100 diluted calprotectin
in faecal matrix was chosen. Table 1 represents the indi-
vidual results. A median baseline trypsin activity in the
used faecal matrix of 6 [0–19] U/L was found. The median
trypsin activity after adding trypsin was 68.5 [62.0–
73.5]  U/L. Following trypsin treatment, relative decrease
of c­alprotectin immunoreactivity showed a broad
­variation. A median absolute decrease of –38.5 [(–68.5)–
(–5.5)] mg/kg is seen after 24 h of incubation, correspond-
ing to a relative decrease of –14.0 [(–28.5)–( –14.0)]%. After
48  h of incubation a median absolute decrease of –57.0
[(–80.0)–(–2.5)] mg/kg was found, equivalent to a relative
0
-10
-20
∆rel calprotectin, %
-30
-40
-50
-60
-70
-80
0 10 20 30 40 50
Trypsin concentration, U/L
Figure 2 Effects of in vitro trypsin digestion (U/L) on calprotectin levels (mg/kg) in faecal matrix.
Relative differences are expressed as compared to the baseline calprotectin levels (%). The upper graph shows the effect after 48 h and the
lower graph after 24 h of incubation at 37 °C. h, hours; Δrel, relative difference.
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Dumoulin et al.: Confounders of calprotectin interpretation      67
decrease of –28.0 [(–42.0)–(0.5)]%. It is, however, clear
that decreases after 48 h of incubation were higher, as also
seen in Figure 3. Differences between decreases after 24 h
and 48 h of incubation were evaluated using a Wilcoxon
test. Both differences, absolute and relative decrease, were
statistically significant (respectively, p < 0.01 and p = 0.02).
We also studied the effect of AAT addition on trypsin
activity. First, 1 mg/mL trypsin solution was added to a
routine serum sample with an AAT concentration of 2 g/L.
50 μL trypsin solution was added to, respectively, 25 μL, 50
μL and 100 μL samples. Incubation at room temperature
was performed for 30 min. Baseline trypsin activity was 90
U/L, after adding AAT activity decreased to, respectively,
37 U/L, 49 U/L, and 48 U/L. Figure 4 shows the decrease
in trypsin activity. Second, analogue incubation experi-
ments were performed to evaluate the influence of AAT in
our in vitro model. To the mixture of 500 μL trypsin solu-
tion and 500 μL 1:100 diluted calprotectin in faecal matrix,
100 μL saline (blank) or 100 μL serum (AAT concentration
of 1.47 g/L) was added. After 24 h and 48 h of incubation
at 37 °C calprotectin values remained stable in the mixture
with AAT, whereas in the blank mixture values decreased
63% after 24 h and even more after 48 h (value below the
limit of detection,  < 15 mg/kg).
Discussion
Calprotectin present in a faecal extract matrix is subject
to trypsin digestion in vitro, leading to loss of immu-
noreactivity. Human calprotectin shows 14 potential
cleavages sites for trypsin. In the present study, trypsin
activities in the range of values typically observed in the
24 h
48 h
60 70
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Table 1 Evaluation of in vitro trypsin digestion [median activity 68.5 (62.0–73.5) U/L] on calprotectin levels (expressed as mg/kg) in faecal
matrix. Relative and absolute differences are expressed as compared to the baseline calprotectin levels (n = 20).

Trypsin activity,  Faecal trypsin  ΔAbs calprotectin,  ΔAbs calprotectin,  ΔRel calprotectin,  ΔRel calprotectin,
U/L activity baseline, U/L mg/kg; 24 h mg/kg; 48 h %; 24 h %; 48 h

74  23  –72  –70  –24  –24

69  30  50  28  12  7
87  30  –12  –38  –8  –26
84  17  40  8  18  4
77  23  –65  –57  –43  –38
73  21  –12  10  –8  7
50  UD  –51  –83  –26  –43
59  UD  –139  –563  –10  –41
68  11  –8  –32  –4  –14
65  5  –33  –61  –18  –34
65  4  –49  –57  –30  –35
65  UD  14  8  13  7
75  6  –19  –9  –6  –3
72  UD  0  4  0  4
71  3  –63  –77  –25  –30
52  7  –128  –156  –36  –56
57  UD  –158  –150  –27  –74
69  UD  –103  –113  –72  –21
66  6  –44  –58  –41  –46
58  UD  –3  –19  –5  –71

Δabs, absolute difference; h, hours; Δrel, relative difference; UD, undetectable.

20 median trypsin activity of 68.5 (62.0–73.5) U/L and incuba-

0
-20
-40
-60
-80
∆rel calprotectin, %; 24 h ∆rel calprotectin, %; 48 h
Figure 3 Distribution of relative decreases of calprotectin after
adding a median trypsin concentration of 68.5 (62–73.5) U/L.
All results are shown separately in Table 1, n = 20. h, hours;
Δrel, relative difference.
gastrointestinal lumen were used. In the proximal part of
the intestinal tract, trypsin activities are maximal as the
pancreas enzymes enter the gastrointestinal tract in the
duodenum. In the more distal segments, trypsin activities
drop rapidly. The experimental conditions of our study [a
tion times of 24 h/48 h] were in the same order as the ones
observed in vivo [18]. Our experiments confirmed an effect
of proteolytic enzymes, such as trypsin on the faecal bio-
marker calprotectin. The results proof trypsin activity to
be a potential confounder in the interpretation of calpro-
tectin results, in particular for proximal lesions, where the
exposure of calprotectin to trypsin is prolonged.
We also showed a statistically significant difference
between absolute and relative proteolysis of calprotectin
after 24  h and 48  h of incubation, confirming that transit
time is also an important confounder in calprotectin results.
It is described that children younger than 4 years have higher
values of faecal calprotectin, the reasons still remain unclear
[19]. Most studies do not include subjects below 4 years of
age or make no distinction of different age groups [20]. Oord
et  al. were the first to establish age-specific cut-off values
using healthy controls (n = 75): 538  mg/kg (1–6 months),
214 mg/kg (6 months–3 years) and 75 mg/kg (3–4 years).
In contrast, a cut-off value of 50 mg/kg is used for children
older than 4 years and adults [21]. Possible explanations for
these differences are the migration of neutrophils through
the mucosal membrane during regulation of the microbial
flora [22]. However, transit time could also be a possible
explanation for this phenomenon.

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0.7
Baseline activitty 25 µL sample
0.6
Absorbance, 405 nm
0.5
0.4
0.3
0.2
0.1
0
0 1 2 3
Figure 4 Influence of α1-antitrypsin (AAT) on trypsin activity after 30 min of incubation at room temperature and adding 25 μL, 50 μL or
100 μL of serum sample (with an AAT concentration of 2 g/L).
Absorbance is monitored in function of time to calculate trypsin activity (a decrease is seen from 90 U/L to respectively, 37 U/L, 49 U/L, and
48 U/L).
Kaiser et al. also reported higher levels of calprotec-
tin in distal inflammation compared with more proximal
inflammation but these were not significant [23]. However,
Siponnen et  al. reported that ileal endoscopic score
and histological findings failed to correlate with faecal
markers, such as calprotectin and lactoferrin [8]. They
also showed that although the good specificity, sensitiv-
ity remained too low to exclude small bowel CD. Despite
small bowel active CD faecal calprotectin values remained
low [24]. These findings may be explained by a major effect
of pancreatic trypsin on the investigated biomarkers. In a
serial dilution experiment we were also able to prove that
the more trypsin is added to calprotectin, the higher the
percentage of calprotectin proteolysis is seen. Proteolysis
is also more pronounced when incubation times are pro-
longed, as demonstrated with our experiments.
Patients with cystic fibrosis (CF) have a marked defi-
ciency of trypsin due to pancreatic exocrine insufficiency.
This insufficiency leads to maldigestion and therefore
malabsorption and gastrointestinal complaints. Bruzz-
ese et  al. reported that faecal calprotectin levels are sig-
nificantly higher in children with CF compared to healthy
controls. Despite the increasing evidence of intestinal
inflammation explaining these findings, the exact under-
lying pathologic mechanism is still unknown. Probiotic
administration reduces faecal calprotectin values and gas-
trointestinal complaints, supporting the presence of intes-
tinal inflammation. As trypsin activities of CF patients are
lower in the gastrointestinal lumen due to pancreas insuf-
ficiency, calprotectin proteolysis is impaired and levels
in stool are higher. In this way, our data are in agreement
with clinical observations [25–27].
Next to the biological variation of trypsin concen-
trations in the faecal matrix, proteolytic effects are
Dumoulin et al.: Confounders of calprotectin interpretation      69
50 µL sample 100 µL sample
4 5 6 7 8 9 10
Time, min
counteracted by a variety of protease inhibitors in the intes-
tinal lumen. Our experiments demonstrate that effects of
trypsin digestion are not always straightforward, which
can be explained by a variable stability of trypsin or the
presence of inhibitors. The occurrence of the endogenous
protease inhibitor protein, AAT, may play a role. Human
plasma contains a variable amount of AAT. This variation
is partly due to the existence of genetic polymorphisms.
Currently, several variants are known, leading to deficient
or non-detectable plasma levels with an increased risk of
lung emphysema. The highest prevalence of the Z variant
peaks is found in southern Scandinavia, Denmark, the
Netherlands, the UK and northern France. The distribu-
tion of the S variant differs and the highest frequency is
found in southern Europe [28]. AAT phenotypes are char-
acterised by their own reference range, PiS shows about
60% of the normal level, PiW 80%, PiZ only 15%, and Pi
null 0% [29]. The proposed interim reference range for
Caucasians is 0.9–2 g/L [30]. The modulation of AAT syn-
thesis by the acute phase response is another important
factor. AAT could be present in the gastrointestinal lumen
after a minor or major bleeding at a proximal lesion or in
case of a protein-loosing enteropathy [15]. In these cases,
AAT is normally present in the excreted stool, mostly
complexed to pancreatic trypsin and elastase. This could
lead to inactivation of the present trypsin, at least when
patients are not deficient for AAT. Our first experiments
demonstrated the inactivation of trypsin by AAT in serum.
However, also when testing the effect of AAT in our in
vitro model, we proved that calprotectin values remained
stable after inactivating trypsin by addition of AAT.
As trypsin shows a limited stability, our findings do
not imply major pre-analytical precautions following sam-
pling [10, 11]. In most cases, residual trypsin activity in
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70      Dumoulin et al.: Confounders of calprotectin interpretation
faeces is very low as demonstrated in our results, which is
almost negligible versus the huge concentrations observed
in the duodenum [9]. Addition of protease inhibitors to the
sample will therefore only have a marginal effect on test
results. Post-sampling stability of calprotectin has also
been confirmed in other pre-analytical studies dealing
with calprotectin [1]. Taking these different facts into
account, numerous caveats should be taken into account
when interpreting calprotectin test results. Transit time,
trypsin activity and addition of blood as a source of AAT
may be regarded as potential confounders. Age-related
cut-off values depending on the anatomical localisation
of the lesions could therefore improve the diagnostic effi-
ciency of calprotectin testing.
Acknowledgments: The authors would like to thank
Thermo Scientific (Uppsala, Sweden) for providing the
necessary calprotectin reagents. We also wish to thank
Elke Lecocq, Jonas Himpe and Mieke Hutsebaut for their
technical support.
Author contributions: All the authors have accepted
responsibility for the entire content of this submitted
manuscript and approved submission.
Financial support: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Role of sponsor: The funding organisation(s) played no
role in the study design; in the collection, analysis, and
interpretation of data; in the writing of the report; or in the
decision to submit the report for publication.
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