Faecal calprotectin concentrations in gastrointestinal diseases

Abstract

Objective

To quantify faecal calprotectin concentrations in a variety of gastrointestinal disorders in order to determine its diagnostic value.

Methods

Patients with gastrointestinal symptoms undergoing upper or lower endoscopy and healthy control subjects provided stool samples. Calprotectin was quantified by enzyme-linked immunosorbent assay.

Results

The study recruited 210 patients with definitively diagnosed gastrointestinal diseases and 50 control subjects. Calprotectin concentrations were significantly higher in patients with ulcerative colitis or Crohn’s disease compared with controls, or patients with colorectal polyps or irritable bowel syndrome. The faecal calprotectin concentration significantly differentiated between inflammatory bowel diseases (IBD) and non-IBD (area under ROC curve 0.949). Calprotectin concentrations were significantly higher in patients with oesophageal polyps or gastric neoplasms than in those with chronic gastritis, stomach ulcers, duodenal ulcers or acute pancreatitis.

Conclusion

Calprotectin may be a useful noninvasive marker for the diagnosis of IBD.

Introduction

Common diagnostic tests for diseases of the gastrointestinal tract include endoscopy, barium X-radiography, contrast radiography, computed tomography and biopsy, but these are limited by high cost and invasiveness. The calcium and zinc binding protein calprotectin is a member of the S-100 protein family with apoptotic and antimicrobial properties. ¹ Calprotectin is resistant to bacterial degradation and is stable for up to 1 week in faecal samples. ² The value of calprotectin as a laboratory marker has been shown in chronic inflammatory bowel disease^3,4 and ulcerative colitis, ⁵ but the significance of calprotectin in other gastrointestinal diseases remains unclear. The aim of this study, therefore, was to investigate faecal concentrations of calprotectin in a wide range of gastrointestinal diseases, in order to evaluate its clinical significance.

Patients and methods

Study population

The study recruited patients with gastrointestinal symptoms requiring upper or lower endoscopy, who were admitted to the Department of Gastroenterology, Jingling Hospital, Nanjing, China, between June 2011 and August 2011. Patients were required to have a definitive diagnosis, made on the basis of standard clinical, endoscopic, radiological and histological criteria. Exclusion criteria were serious cardiopulmonary, hepatic, renal, neurological or psychiatric diseases.

Healthy volunteers attending Jingling Hospital, Nanjing, China for routine screening were recruited as control subjects.

The study was conducted with the approval of the Jingling Healthcare local ethics committee, and all participants provided written informed consent.

Calprotectin ELISA

Patients provided a faecal sample on the day before scheduled colonoscopy; control subjects provided a sample ≤48h after enrolment. Faeces was stored at 4℃ until use. Faeces (50–100 mg) was diluted 1 : 50 (w/v) with extraction buffer (Bühlmann Laboratories AG, Basel, Switzerland), homogenized for 30 min, then stored at 4℃ overnight prior to analysis. Calprotectin was quantified via enzyme-linked immunosorbent assay (ELISA) (Bühlmann Laboratories AG).

Statistical analyses

Data were presented as mean ± SD, and compared using one-way analysis of variance. Data from patients with ulcerative colitis or Crohn's disease were combined to form the IBD group; data from patients with colonic polyps or irritable bowel syndrome were combined to form the non-IBD group. Logistic regression analysis was used to construct receiver operating characteristic (ROC) curves for calprotectin sensitivity and specificity in these groups, and cut-off values were calculated. Statistical analyses were performed with SPSS® software, version 17.0 (SPSS Inc., Chicago, IL, USA), and P-values < 0.05 were considered statistically significant.

Results

The study included 210 patients with gastrointestinal symptoms (72 males/138 females; mean age 46.3 ± 22.5 years; age range 18–65 years) and 50 healthy control subjects (25 males/25 females; mean age 40.9 ± 27.3 years; age range 18–70 years). The patient group included 28 patients with ulcerative colitis, 44 with Crohn’s disease, 24 with colorectal polyps, 32 with irritable bowel syndrome (IBS), 20 with oesophagitis, 10 with oesophageal polyps, 12 with chronic gastritis, 11 with gastric ulcers, 10 with duodenal ulcers, seven with acute pancreatitis and 12 with gastric neoplasms.

Data regarding faecal calprotectin concentrations are shown in Table 1. Calprotectin was significantly elevated in patients with ulcerative colitis or Crohn’s disease, compared with controls or patients with colorectal polyps or irritable bowel syndrome (P < 0.01 for all comparisons). In addition, calprotectin was significantly elevated in patients with oesophageal polyps/gastric neoplasm compared with controls or patients with chronic gastritis/stomach ulcer/duodenal ulcer/acute pancreatitis (P < 0.01). There were no other statistically significant between-group differences.

Table 1.

Faecal calprotectin concentrations in healthy control subjects and patients with gastrointestinal disease, stratified according to diagnosis.

Group	n	Calprotectin µg/g faeces
Healthy controls	50	33.94 ± 13.64
Patients
Ulcerative colitis	28	1914.79 ± 2638.80abc
Crohn’s disease	44	3276.03 ± 2259.42abc
Colorectal polyps	24	106.54 ± 70.50
Irritable bowel syndrome	32	125.37 ± 155.97
Oesophagitis	20	743.78 ± 1811.11
Oesphageal polyps/gastric neoplasm	22	1918.71 ± 2567.46 a d
Chronic gastritis/stomach ulcer/duodenal  ulcer/acute pancreatitis	40	85.87 ± 80.88

Data presented as mean ± SD.

a

P < 0.01 vs control, ^bP < 0.01 vs colonic polyps, ^cP < 0.01 vs irritable bowel syndrome; one-way analysis of variance.

d

P < 0.01 vs chronic gastritis/stomach ulcer/duodenal ulcer/acute pancreatitis.

The results of ROC analyses in the IBD and non-IBD groups are shown in Figure 1. The faecal calprotectin concentration significantly differentiated between IBD and non-IBD (P < 0.001; area under curve 0.949, 95% confidence interval 0.911, 0.988). A cut-off value of 45.40 µg/g resulted in a sensitivity of 0.944 and specificity of 0.643; a cut-off of 110.65 µg/g resulted in a sensitivity of 0.944 and specificity of 0.429.

Figure 1.

Receiver operating characteristic analysis of the faecal calprotectin concentration in the differentiation between patients with inflammatory and noninflammatory bowel diseases.

Discussion

Calprotectin is a calcium-binding protein that makes up around 60% of the total cytosolic protein content of neutrophils and mononuclear cells. ⁶ It is a heterocomplex protein comprising two heavy (L1H) chains and one light (L1L) chain which are noncovalently linked,^7–9 and is an important regulatory protein in inflammatory reactions.^1,10,11 Studies in paediatric and adult cohorts have demonstrated a correlation between faecal calprotectin concentrations and the severity of mucosal inflammation.^2,12 Faecal calprotectin has also been shown to correlate with changes in C-reactive protein and erythrocyte sedimentation rate in active inflammation.^13–18 The stability of calprotectin at room temperature adds to its value as a practical and convenient marker in intestinal inflammation, and for the differential diagnosis of IBD and IBS.^6,10,19

Studies have found significantly higher faecal calprotectin concentrations in some gastrointestinal disorders (including oesophageal/gastric carcinoma, Crohn’s disease, ulcerative colitis and colorectal carcinoma) than in others (Barrett’s oesophagus, gastric ulcer, gastritis/duodenitis, colorectal polyps and adenoma). ²⁰ In accordance with the findings of others, ⁶ faecal calprotectin concentrations were significantly higher in patients with IBD than in those with IBS or colorectal polyps in the present study. In addition, ROC curve analysis confirmed that the faecal calprotectin concentration effectively differentiated between IBD and IBS, with increased calprotectin being associated with a heightened risk of IBD. The elevated levels of calprotectin seen in patients with oesophageal polyps and gastric neoplasms in the present study (in contrast to those with chronic gastritis, stomach ulcer, duodenal ulcer or acute pancreatitis) may be explained by the synthesis of calprotectin by squamous epithelial cells, granulocytes and macrophages, ²¹ or by the large wound surface present on these tissues.

In conclusion, findings of the present study indicate that faecal calprotectin may be a useful noninvasive marker for differentiating between IBD and IBS, and we also should note that faecal calprotectin can have high expression in other patients, excluding IBD patients.