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Int J Colorectal Dis (2014) 29:485491

DOI 10.1007/s00384-013-1817-3

ORIGINAL ARTICLE

Fecal calprotectin and lactoferrin as predictors of relapse


in patients with quiescent ulcerative colitis
during maintenance therapy
Takayuki Yamamoto & Manabu Shiraki &
Takuya Bamba & Satoru Umegae & Koichi Matsumoto

Accepted: 4 December 2013 / Published online: 17 December 2013


# Springer-Verlag Berlin Heidelberg 2013

Abstract
Purpose This prospective study was to evaluate the significance of fecal calprotectin and lactoferrin for the prediction of
ulcerative colitis (UC) relapse.
Methods Eighty UC patients in remission for 3 months on
mesalamine as maintenance therapy were included. At entry,
stool samples were collected for the measurement of
calprotectin and lactoferrin. All patients were followed up
for the following 12 months. To identify predictive factors
for relapse, time-dependent analyses using the Kaplan-Meier
graphs and Cox's proportional hazard model were applied.
Results During the 12 months, 21 patients relapsed. Mean
calprotectin and lactoferrin levels were significantly higher
in patients with relapse than those in remission
(calprotectin173.7 vs 135.5 g/g, P =0.02; lactoferrin
165.1 vs 130.7 g/g, P =0.03). A cutoff value of 170 g/g
for calprotectin had a sensitivity of 76 % and a specificity of
76 % to predict relapse, while a cutoff value of 140 g/g for
lactoferrin had a sensitivity of 67 % and a specificity of 68 %.
In a multivariate analysis, calprotectin (170 g/g) was a
predictor of relapse (hazard ratio, 7.23; P =0.002). None of
the following parameters were significantly associated with
relapse: age, gender, duration of UC, number of UC episode,
severity of the previous episode, extent of UC, extraintestinal
manifestation, and lactoferrin level.
Conclusions Fecal calprotectin showed a higher sensitivity
and specificity than fecal lactoferrin for predicting UC relapse.
Fecal calprotectin level appeared to be a significant predictor
of relapse in patients with quiescent UC on mesalamine as
maintenance therapy.
T. Yamamoto (*) : M. Shiraki : T. Bamba : S. Umegae :
K. Matsumoto
Inflammatory Bowel Disease Center, Yokkaichi Social Insurance
Hospital, 10-8 Hazuyamacho, Yokkaichi, Mie 510-0016, Japan
e-mail: nao-taka@sannet.ne.jp

Keywords Calprotectin . Fecal marker . Lactoferrin .


Mesalamine . Ulcerative colitis

Introduction
Ulcerative colitis (UC) is a chronic disease characterized by
mucosal inflammation in the colon and the rectum [1]. The
course of UC is characterized by spontaneous remission,
which may be followed by a relapse, and is typically unpredictable. The main goal of medical therapy in patients with
UC is effective and sustained suppression of intestinal inflammation in order to induce and maintain clinical remission.
Identifying patients at a significant risk of relapse during
quiescent UC should help to determine a strategy for maintenance therapy during remission. Previous studies have reported that younger age, multiple previous episodes of relapse,
extraintestinal manifestations, a low-fiber diet, seasonal factors, and a long-term perceived stress were risk factors for
future relapse in patients with quiescent UC [25]. These
factors are of limited value in clinical setting, and therefore
more objective parameters that may predict future relapse
would be desirable in our clinical practice.
The presence of active intestinal inflammation in patients
with UC is associated with an acute phase reaction and migration of leucocytes to the gut, and this leads to the release of
several neutrophil proteins, which may be measured in stool
samples [6]. Fecal markers fulfill all the criteria of being
noninvasive, simple, inexpensive, sensitive, and specific
markers to detect gastrointestinal inflammation. Calprotectin
is a calcium-binding protein, representing up to 60 % of the
cytosolic proteins in neutrophils [7]. Since calprotectin is
primarily derived from neutrophils, its concentration is directly proportional to the concentration of neutrophils in the
colonic/rectal mucosa [8]. It is resistant to bacterial

486

degradation in the gut and is stable in the stool for up to 1 week


at room temperature [9, 10]. Lactoferrin is an iron-binding
glycoprotein and a major component of the secondary
granules of polymorphonuclear neutrophils [11, 12].
During intestinal inflammation, leucocytes invade the
mucosa, which may lead to an increase in the excretion
of lactoferrin into the stool. Lactoferrin can be measured
by using simple and inexpensive techniques since it is
stable in the stool for up to 4 days [13, 14].
Several studies [1518] have compared fecal markers with
disease activity indices and endoscopic/histological evaluations to monitor intestinal inflammation in patients with inflammatory bowel disease (IBD). Levels of fecal calprotectin
and lactoferrin have a proportional correlation to the degree of
inflammation of the intestinal mucosa. Fecal calprotectin and
lactoferrin may have a role in monitoring disease activity in
patients with IBD [1924]. With this background in mind, the
present study was designed to rigorously evaluate whether or
not these neutrophil proteins are clinically relevant for
predicting relapse in patients with quiescent UC. Further, we
included a homogeneous group of patients who were receiving mesalamine as maintenance therapy.

Patients and methods

Int J Colorectal Dis (2014) 29:485491


Table 1 Baseline characteristics of 80 eligible patients
Age at entry (mean SE)
Male/female (n)
Duration of UC before entry (mean SE)
Number of UC episode (n)
14
5
Severity of the previous episode (n)
Mild (DAI score 4)
Moderate (5DAI score 8)
Severe (DAI score 9)
Extent of UC (n)
Left-sided colitis
Extensive colitisa
Extraintestinal manifestations (n)
Arthritis
Pyoderma gangrenosum
Induction therapy for the previous episode (n)
Mesalamine (Pentasa 4 g/day)
Corticosteroids (prednisolone 3060 mg/day)
Leukocytapheresis
Immunosuppressants (Tacrolimus)
Biologics (Infliximab)

54
26
28
41
11
60
20
11
3
63
47
48
10
2

UC ulcerative colitis, DAI Disease Activity Index [25]


a

Study design

35.10.8 years
49:31
50.02.7 months

Involvement extends proximal to the splenic flexure

This was a prospective, single-center study undertaken at the


Yokkaichi Social Insurance Hospital, a referral center treating
a large number of patients with IBD in the Mie Prefecture of
Japan. The study was conducted in accordance with the principle of good clinical practice and the Helsinki Declaration.
Our study protocol was reviewed and approved by our Institutional Review Board.

clinical remission with medical treatment. As remission induction therapy, high-dose mesalamine (Pentasa, 4 g/day) was
used in 63 patients, prednisolone (3060 mg/day) in 47 patients, therapeutic leucocytapheresis (Adacolumn, JIMRO,
Takasaki, Japan) in 48 patients, tacrolimus (Prograf) in 10
patients and infliximab (Remicade, 5 mg/kg at weeks 0, 2,
and 6, and then at 8-week intervals) in 2 patients.

Patients

Remission maintenance therapy

Patient inclusion criteria were: (1) age between 20 and 75, (2)
having endoscopic and histologic diagnosis of UC, (3) UC
confined to the colon and the rectum, and (4) patient in clinical
remission (normal stool frequency and no rectal bleeding) on
oral mesalamine for 3 months. Exclusion criteria were: (1)
patients who had received corticosteroids, immunosuppressants, or biologic agents at entry; (2) had received mesalamine
enema; and (3) had received nonsteroidal anti-inflammatory
drugs, antidiarrheal (loperamide or codeine), or antispasmodic
medications at entry.
A total of 80 patients who met the inclusion criteria were
included in this study. The baseline characteristics of the 80
eligible patients are presented in Table 1. The severity of
previous UC episodes was mild in 28 patients, moderate in
41 patients, and severe in 11 patients. All patients achieved

During the study period, all patients were given oral


mesalamine (Pentasa, 1.53.0 g/day). No patient received
mesalamine enema (oral, intravenous, intramuscular, or rectal)
corticosteroids, immunosuppressants (tacrolimus, azathioprine, and 6-mercaptopurine), or biologic agents except patients who developed clinical relapse.
Follow-up
All patients were reviewed in our clinic every 2 up to
12 months after entry. Patients were advised to record their
symptoms in a diary every day. At the clinic visits, patient's
compliance with medication, adverse effects, general wellbeing, stool frequency, stool consistency and presence or
absence of abdominal pain, tenderness, tenesmus, rectal

Int J Colorectal Dis (2014) 29:485491

487

bleeding, and mucus in stool were recorded. Stool frequency


and rectal bleeding were scored according to the Disease
Activity Index (DAI) system listed in Table 2 [25]. Clinical
remission was defined as normal stool frequency (= score 0)
and no rectal bleeding (= score 0). When patients developed
symptoms suggestive of a flare-up, endoscopic examination
was immediately undertaken. Endoscopic score was according to the mucosal appearance section of the DAI. A relapse
was defined as a worsening of stool frequency and/or rectal
bleeding with an endoscopic score of 2 or 3 [25].
In our clinic, peripheral blood samples were collected for
measurement of white blood cell count (WBC), hemoglobin
(Hb), platelet count, C-reactive protein (CRP), total protein,
albumin, creatinine, urea, sodium, potassium, chloride, alanine aminotransferase, aspartate aminotransferase, alkaline
phosphatase, lactic dehydrogenase, total bilirubin, and
cholesterol.
Measurement of fecal calprotectin and lactoferrin
At entry, patients provided a stool sample for the measurement
of calprotectin and lactoferrin. Fecal calprotectin was measured by a quantitative enzyme immunoassay (Human
Calprotectin ELISA Kit, Cell Sciences Inc., Massachusetts,
USA). Lactoferrin was measured by a colloidal gold agglutination reagent (Auto Lf-Plus, Alfresa Pharma Corp., Osaka,
Table 2 Disease activity index (DAI) scoring system
Score
Stool frequency
Normal number of stools
12 stools/day greater than normal
34 stools/day greater than normal
5 stools/day greater than normal
Rectal bleeding
No blood seen in stool
Streaks of blood with stools less than half the time
Obvious blood with stools most of the time
Blood alone passed
Mucosal appearance
Normal mucosa or inactive disease
Mild inflammatory changes (erythema, decreased vascular
patterns, mild friability)
Moderate inflammatory changes (marked erythema, absent
vascular patterns, friability, erosions)
Severe inflammatory changes (spontaneous bleeding and
ulcerations)
Physician's overall assessment of disease severity
Normal
Mild disease
Moderate disease
Severe disease

0
1
2
3
0
1
2
3
0
1
2
3

0
1
2
3

Japan) by using a high-throughput discrete clinical chemistry


analyzer (Hemo Techt NS-Plus C, Alfresa Pharma Corp.). As
a normal control group, fecal samples were obtained from 80
age- and gender-matched healthy volunteers with no history
of gastrointestinal disorder. Laboratory personnel were
blinded to the clinical data.
Statistical analysis
Continuous data are presented as the meanSE values. Comparisons of frequencies were done by using the chi-square test
with Yates' correction. The average values between two
groups are compared by unpaired t test. For comparisons
involving more than two groups, the one-way analysis of
variance was applied. Correlations were calculated by using
the Spearman's r test. The cumulative relapse rate was
calculated by the Kaplan-Meier method, and was compared
between the groups by using the log-rank test. Predictors of
relapse were determined by multivariate analysis using Cox's
proportional hazard model. P <0.05 was considered statistically significant.

Results
Fecal calprotectin and lactoferrin levels in UC and controls
Both fecal calprotectin and lactoferrin levels were markedly
higher in the UC vs the control groups. The mean fecal
calprotectin level was 145.57.0 g/g in the UC group vs
4.51.0 g/g in the control group (P <0.001), representing
more than 30-fold increase in patients with UC while in
remission relative to healthy controls. Similarly, the mean
fecal lactoferrin level was 139.76.9 g/g in the UC group
vs 9.20.7 g/g in the control group (P <0.001).
Fecal markers vs clinical and laboratory measurements
In the patient group, there was a significant correlation between fecal calprotectin and lactoferrin levels (r =0.779,
P <0.001). Fecal calprotectin and lactoferrin levels showed
no significant correlation with the following parameters: age
at entry, gender, duration of UC before entry, number of UC
episodes, severity of previous episodes, extent of UC, and
extraintestinal manifestations (Table 3). Similarly, laboratory
measurements including WBC, Hb, platelet count, albumin,
and CRP at entry did not correlate with fecal markers
(calprotectin vs WBC, r =0.060, P =0.60; Hb, r =0.174,
P =0.12; platelet count, r =0.120, P =0.29; albumin, r =0.125,
P =0.27; CRP, r =0.128, P =0.26; lactoferrin vs WBC,
r = 0.034, P = 0.77; Hb, r = 0.120, P = 0.29; platelet
count, r =0.127, P =0.26; albumin, r =0.125, P =0.27;
CRP, r =0.165, P =0.14).

488

Int J Colorectal Dis (2014) 29:485491

Table 3 Relationship between


fecal calprotectin and lactoferrin,
and clinical parameters
Age at entry
<35 years (n =40)
35 years (n =40)
Gender
Male (n =49)

The mean SE values are


presented

Female (n =31)
Duration of UC before entry
<50 months (n =41)
50 months (n =39)
Number of UC episodes
14 (n =54)
5 (n =26)
Severity of the previous episodes
Mild (n =28)
Moderate (n =41)
Severe (n =11)
Extent of UC
Left-sided colitis (n =60)
Extensive colitis (n =20)
Extraintestinal manifestations
Yes (n =14)
No (n =66)

Relapse rates during maintenance therapy


During this study, the dose of orally administered mesalamine
(Pentasa) was 3.0 g/day in 50 patients, 2.25 g/day in 24
patients, and 1.5 g/day in 6 patients. During the
12 months follow-up, UC relapse was observed in 12
(24 %) of the 50 patients on 3.0 g/day mesalamine, 7
(29 %) of the 24 patients on 2.25 g/day mesalamine,
and 2 (33 %) of the 6 patients on 1.5 g/day mesalamine
(P =0.82). The cumulative relapse rate was not significantly different among the three groups (P =0.73).
Predictors of UC relapse
As stated above, 21 of 80 patients developed relapse during
the 12-month follow-up. As seen in Fig. 1, both fecal
calprotectin and lactoferrin levels were significantly higher
in patients who relapsed as compared with patients who
maintained remission (P =0.02 for calprotectin and P =0.03
for lactoferrin). A cutoff value of 170 g/g fecal calprotectin
had a sensitivity of 76 % and a specificity of 76 % to predict
disease relapse, while a cutoff value of 140 g/g lactoferrin
had a sensitivity of 67 % and a specificity of 68 % to predict
UC relapse. As shown in Fig. 2a, the cumulative relapse rate
was significantly higher in patients with elevated fecal
calprotectin (170 g/g) as compared with those with low

Calprotectin (g/g)

Lactoferrin (g/g)

139.69.7
151.410.1

0.40

136.89.2
142.710.5

0.68

149.311.0

0.67

148.69.6

0.31

144.311.1
146.88.4

0.86

144.110.6
135.28.9

0.52

148.59.0
139.410.9

0.55

140.68.6
137.911.9

0.86

133.111.0
152.210.5
152.316.3

0.43

134.814.0
141.68.6
145.417.1

0.86

142.27.8
155.615.4

0.41

136.88.0
148.714.0

0.46

147.320.9
145.27.3

0.91

141.515.6
139.47.8

0.91

143.19.1

134.19.5

fecal calprotectin level (<170 g/g, P <0.001). Similarly,


Fig. 2b shows that the cumulative relapse rate was significantly higher in patients with elevated fecal lactoferrin level
(140 g/g) vs those with low fecal lactoferrin level
(<140 g/g, P =0.004).
The following clinical parameters did not significantly affect the cumulative relapse rate: age (<35/35 years,
P =0.80), gender (P =0.96), duration of UC before entry
(<50/50 months; P =0.82), number of UC episodes (14/5;
P =0.90), severity of the previous episodes (mild/moderate/
severe; P =0.41), and extraintestinal manifestation (P =0.80).
However, the cumulative relapse rate was significantly higher
in patients with extensive colitis than those with left-sided
colitis (P =0.03).
In a multivariate analysis, calprotectin (170 g/g) was a
significant predictor of disease relapse (P =0.002). None of
the following parameters were significantly associated with
relapse: age, gender, duration of UC, number of UC episodes,
the severity of previous episode, extent of UC, extraintestinal
manifestation, and fecal lactoferrin level (Table 4).

Discussion
Given that both fecal calprotectin and lactoferrin are known to
be elevated during intestinal inflammation in patients with

Int J Colorectal Dis (2014) 29:485491

489

Fig. 1 The mean SE values of


fecal calprotectin and lactoferrin
levels were significantly higher in
patients with relapse than those in
remission (calprotectin173.7
15.9 vs 135.57.2 g/g, P =0.02;
lactoferrin165.117.0 vs
130.76.9 g/g, P =0.03)

Calprotectin

g/g
200

Lactoferrin

g/g
200

180

180

160

160

140

140

120

120

100

100

80

80

60

60

40

40

20

20

P=0.03

P=0.02
0

Yes

No

Yes

Relapse

The proportion of patients


in remission

Relapse

involving the Kaplan-Meier plots. Likewise, the prediction of


disease relapse was by applying widely accepted statistical
models.
Determination and validations of biomarkers, which can
identify patients at a high risk of IBD relapse should have
significant appeal in clinical settings for designing strategies
to prevent or delay disease relapse during remission by medical interventions. For example, in this study, patients relapsed
while on maintenance mesalamine, but it would be interesting
to see if a small dose of corticosteroid can sustain the remission in such patients. Previous studies reported that several
factors like young age at onset, multiple previous episodes of
relapse, and extraintestinal manifestations were risk factors for
disease relapse in patients with quiescent UC [25]. In contrast, in the present study, age, gender, duration of UC, the
number and severity of past UC episodes, the extent of UC,
and extraintestinal manifestation were not associated with UC

IBD [1518], our major interest in this study was to see if the
elevated fecal levels of these biomarkers relate to clinical
relapse in patients with UC. A major strength of this study is
that we included a well-defined and homogeneous group of
patients with quiescent UC who were on mesalamine as
maintenance therapy, and without any other medication for
UC. This allowed us to evaluate the significance of these
biomarkers in the absence of likely interference from multiple
medications patients with UC may receive. In line with this
assertion, corticosteroids are known to prolong neutrophil
survival [26]. Being the source of calprotectin and lactoferrin,
any drug effect on neutrophils potentially could impact the
fecal levels of these biomarkers. Other strengths of our study
might include a relatively large number of patients we could
include for this study, diligent monitoring of disease activity,
and measurement of two biomarkers in the same test samples.
The relapse rate was estimated by time-dependent analyses

Fig. 2 The cumulative relapse


rate was significantly higher in
patients with elevated calprotectin
(170 g/g) than in those with
low calprotectin level (<170 g/
g). Similarly, the cumulative
relapse rate was significantly
higher in patients with elevated
lactoferrin level (140 g/g) than
in those with low lactoferrin level
(<140 g/g)

No

Calprotectin
< 170 g/g

1.0

Lactoferrin
1.0

0.8

0.8

0.6

0.6

< 140 g/g

140 g/g

0.4

170 g/g

0.2

0.4
0.2

P<0.001

P=0.004

0
0

10

12

Months

10

12

Months

Patients at risk
170 g/g 50

50

50

49

49

47

45

Patients at risk
140 g/g 47

47

47

46

44

42

40

170 g/g 30

30

29

24

20

16

14

140 g/g 33

33

32

27

25

21

19

490
Table 4 Predictors for disease
relapse determined by multivariate analysis

CI confidence interval

Int J Colorectal Dis (2014) 29:485491

Hazard ratio

95 % CI

Age at entry: 35 years


Gender: male
Duration of UC before entry: 50 months
Number of UC episodes: 5
Severity of the previous episodes: moderate (vs mild)

0.84
0.72
0.72
2.28
1.37

0.292.40
0.252.12
0.242.16
0.677.71
0.424.42

0.75
0.56
0.56
0.19
0.60

Severity of the previous episodes: severe (vs mild)


Extent of UC: extensive colitis
Extraintestinal manifestations
Calprotectin level: 170 g/g
Lactoferrin level: 140 g/g

2.27
2.86
0.80
7.23
1.03

0.4312.05
0.899.21
0.222.89
2.1124.79
0.323.35

0.34
0.08
0.73
0.002
0.96

relapse. Likewise, laboratory measurements including WBC,


Hb, platelet count, albumin, and CRP did not correlate with
the fecal markers. However, as indicated above, both
calprotectin and lactoferrin are derived from polymorphonuclear neutrophils, primarily from those, which infiltrate the
colonic mucosa [614]. It has been reported that when the
concentration of neutrophils in the mucosa reaches a threshold
level, the patient may experience a clinical relapse [27]. Accordingly, an elevated amount of calprotectin or lactoferrin in
the stool is secondary to mucosal inflammation, so much that
mucosal inflammation may be another predictor of relapse.
Further, occult disease in patients in morphologic remission
may identify patients at a high risk of an impending relapse.
Based on the results of this study, fecal calprotectin should be
a simpler and more reliable predictor of relapse than any
pathologic manifestation of IBD.
In this study, inclusion of a large number of healthy individuals as a control group enabled us to see that even during
remission phase of UC, fecal level of calprotectin was up to
30-fold higher in UC patients than in healthy controls. The
elevated level of calprotectin provides a unique opportunity
for diagnostic application of calprotectin as a biomarker of
underlying UC disease activity. Additionally, these fecal
markers are stable, convenient to measure at a very low cost,
and may be used as a noninvasive approach to monitor mucosal inflammation and predict subsequent relapse in patients
with IBD. This should spare the patients from complicated
endoscopic procedures. We found that both fecal calprotectin
and lactoferrin levels were significantly higher in patients who
relapsed as compared with those who remained in remission.
For the prediction of UC relapse, a cutoff value of 170 g/g
for fecal calprotectin had a sensitivity of 76 % and a specificity
of 76 %, while a cutoff value of 140 g/g for lactoferrin had a
sensitivity of 67 % and a specificity of 68 %. Therefore, based
on the findings of this study, fecal calprotectin measurement
appeared to provide higher sensitivity and specificity than
fecal lactoferrin in our mathematical models to predict relapse.
The cumulative relapse rate was significantly higher in patients with elevated calprotectin (170 g/g) than in those

with low calprotectin level (<170 g/g). Also, the cumulative


relapse rate was significantly higher in patients with elevated
lactoferrin level (140 g/g) than in those with low lactoferrin
level (<140 g/g).
In conclusion, this study showed that both fecal
calprotectin and lactoferrin levels were very significantly increased in patients with UC even during remission as compared with healthy controls. Further, among UC patients, fecal
calprotectin level 170 g/g was associated with a clinical
relapse. To our knowledge, this is the first study which has
measured calprotectin and lactoferrin head on in the same
samples from the same patients. Fecal calprotectin showed a
higher sensitivity and specificity than fecal lactoferrin for
predicting UC relapse. Assay of fecal calprotectin should
serve as low cost and noninvasive biomarker to predict UC
relapse during maintenance therapy. We believe that a future
study should look at medical intervention to sustain remission
based on the measurement of fecal calprotectin.

Competing interests None.


External funding None.

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