Clinica Chimica Acta 381 (2007) 63–68www.elsevier.com/locate/clinchim

Invited critical review

Enzymes in feces: Useful markers of chronic inflammatory bowel disease

Imerio Angriman a,⁎, Marco Scarpa a, Renata D'Incà b, Daniela Basso c, Cesare Ruffolo a,Lino Polese a, Giacomo C. Sturniolo b, Davide F. D'Amico a, Mario Plebani c

a Clinica Chirurgica I, Dipartimento di Scienze Chirurgiche e Gastroenterologiche, University of Padova, Italyb Gastroenterologia, Dipartimento di Scienze Chirurgiche e Gastroenterologiche, University of Padova, Italy

c Medicina di Laboratorio, Dipartimento di Scienze Diagnostiche e Terapie Speciali, University of Padova, Italy

Received 22 January 2007; accepted 13 February 2007Available online 21 February 2007

Abstract

Background: Ulcerative colitis and Crohn's disease are characterized by a chronic intestinal inflammation. Since the precise etiology is stillunknown, current therapies are aimed at reducing or eliminating inflammation.Methods: Endoscopy and histology on biopsy specimens remain the gold standard methods for detecting and quantifying bowel inflammation.These technique are expensive, invasive and not well tolerated by patients since the need of repeated examinations affects their quality of life.Although disease activity scores and laboratory inflammatory markers are widely used they showed unreliable relations with endoscopy andhistology. Fecal markers have been investigated in inflammatory bowel disease (IBD) by many authors for diagnostic purposes, to assess diseaseactivity and of risk of complications, to predict relapse or recurrence, and to monitor the effect of therapy. Many inflammatory mediators havebeen detected in the feces such as leukocytes, cytokines and proteins from neutrophil activation. Some of these, particularly lactoferrin andcalprotectin, have been demonstrated to be useful in detecting active inflammatory bowel disease, in predicting recurrence of disease after surgeryor monitoring the effects of medical therapy. Calprotectin and lactoferrin are remarkably stable and easily detect in stool using ELISA so theyappear to be equally recommendable as inflammation markers in the lower gastrointestinal tract especially in IBD patients.Conclusion: Fecal markers are non-invasive, simple, cheap, sensitive and specific parameters and are useful to detect strointestinal inflammation.© 2007 Elsevier B.V. All rights reserved.

Keywords: Ulcerative colitis; Crohn's disease; Lactoferrin; Calprotectin; C reactive protein

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632. Fecal markers in chronic inflammatory bowel disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643. Neutrophil-derived proteins in the feces in IBD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 654. Fecal enzymes in the clinical management of inflammatory bowel disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

⁎ Corresponding author. Clinica Chirurgica I, Dipartimento di ScienzeChirurgiche e Gastroenterologiche, Policlinico Universitario, Università diPadova, via Giustiniani 2, 35128 Padova, Italy. Tel.: +39 049 821 2235; fax: +39049 65 61 45.

E-mail address: imerio.angriman@unipd.it (I. Angriman).

0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.cca.2007.02.025

1. Introduction

Crohn's disease (CD) and ulcerative colitis (UC), collec-tively known as inflammatory bowel disease (IBD), are chronicillnesses that affect the gastrointestinal tract. The incidence rate

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of UC varies between 0.5 and 24.5/105 inhabitants/y, while thatof CD varies between 0.1 and 16/105 inhabitants/year, withprevalence rates of IBD reaching up to 396/105 inhabitants.Recent data from South Europe [1–3], East Europe [4] and Asia[5] in the mid-1990s report a rise of the incidence of IBD and insome areas it is already comparable to rates reported in NorthernEurope or North America [6]. The anatomic location and degreeof inflammation determine the predominant symptoms thatinclude rectal bleeding, diarrhea and abdominal pain. In theabsence of rectal bleeding it can be difficult to differentiatebetween functional disorders of the lower GI tract and therefore,the diagnosis needs invasive and expensive testing such asendoscopy with biopsy for histological examination, smallbowel series or barium enema. One of the major problems ofclinical management of UC and CD is the lack of univocaldiagnostic tool.

Several laboratory markers are used in the diagnosis of IBDand to monitor of IBD disease activity. These include ery-throcyte sedimentation rate (ESR), C-reactive protein (CRP),acute phase protein (albumin), and platelets; however, none ofthese markers is specific for inflammation of the gastrointestinaltract [7].

The pathogenesis of inflammatory bowel diseases impliesthe loss of the barrier function and the loss of the toleranceagainst luminal and self antigens and both these phenomenoncause the recruitment of leukocytes in the intestinal wall [8,9].Activated leucocytes infiltrate the mucosa and can be detectedin feces due to shedding in the intestinal lumen [10,11]. Themost important leukocyte population in the intestinal wall inIBD are the polymorphonuclear cells. In fact, high fecalneutrophil levels were detected in inflammatory bowel diseasepatients and may be used as surrogate markers of active disease[12,13]. Nevertheless neutrophil determination in the stools israther inefficient because of their brief lifetime that implies thatthe sample should be examined within a few hours of itscollection. On the contrary calprotectin, lactoferrin and otherproteins are produced in significant amounts by inflammatorycells and both lactoferrin and calprotectin fecal levels have beendemonstrated to correlate with colorectal and intestinalinflammation in several studies [14–16]. We evaluate theclinical applicability of inflammatory enzymes in the feces fordiagnosis of IBD, to assess disease activity, to monitor theresponse to treatment and for diagnosis of recurrent disease aftersurgery.

2. Fecal markers in chronic inflammatory bowel disease

Fecal markers include a heterogeneous group of substancesthat either leak from or are generated by the inflamed intestinalmucosa. The inflamed hyper-permeable mucosa of patients withinflammatory bowel disease is associated with increased proteincytokines and markers of neutrophil activation in fecal samples[17].

The fecal excretion of Indium 111-labeled leukocytes isconsidered the gold standard fecal marker of inflammation sincestrict correlations with intestinal inflammation were evidencedat colonoscopy and histology [18]. A sensitivity of 97% for

fecal in labeled leukocytes excretion compared with 79% forradiology and 70% for rectal histology has been reported for thediagnosis of IBD [18]. Furthermore, studies with radio-labeledproteins have demonstrated that there is fecal protein loss inpatients with active CD and they may be used as useful markersof disease activity [19]. Unfortunately, all radio-labeledtechniques expose patients to radiation and imply a 4 dayfecal collection and dedicated facilities; thus their routine use,other then clinical studies, is discouraged. In fact, concernsabout costs, radiation, and the need for prolonged fecalcollections did not suggest the application of these techniquesin the routine use, although they remain very important forresearch studies. The consequent idea, that emerged, was that itmight be possible to assay for cell proteins that are specificallyassociated with a certain cell type and that would then provideinformation on a specific component of the inflammatorycascade.

Ferguson's Edinburgh group was instrumental in expandingthis idea [20]. Concerned about bacterial degradation of markersthey used a whole gut lavage method involving ingestion ofpolyethylene-based purgatives (Kleenprep or GoLitely) forobtaining clear liquid fecal samples for analysis. The analysistook to various markers, such as immunoglobulins, neutrophils-specific elastase, and hemoglobin. Separate studies showed thatCD could be easily identified, and that the method had a greatersensitivity for colorectal cancer than the conventional fecaloccult blood technique [21,22]. Ideally suited for research, themethod has yet not found wide application for routine screeningpurposes, possibly because of the drawback of patients needingto ingest large volumes of liquid.

Direct analysis of these markers in feces would be a majoradvance on this method but the problem is the bacterialdegradation of these markers necessitating swift samplehandling. Alpha1-antitrypsin is a protease inhibitor producedby the liver, macrophages and intestinal epithelium. It is stablein the feces and can be detected by a variety of methods [23].Some studies have shown that fecal α1-antitrypsin clearance isa useful indicator of protein-losing enteropathy [24] and that inpatients with inflammatory bowel disease, 72-h fecal clearanceof α1-antitrypsin is a useful method for quantization ofintestinal protein loss [24,25]. Random fecal α1-antitrypsinlevels have been shown to be as useful as more prolongedcollection in measuring CD activity and correlated with severalother laboratory measures already known as indicators of CDactivity [26]. Fecal α1-antitrypsin has been generally acceptedas a useful marker of IBD; however, the method is not routinelyavailable and some studies are conflicting about the efficacy ofα1-antitrypsin in diagnosis of IBD. Furthermore, data about theefficiency in the follow-up after medical or surgical therapy,such as in case of pouchitis, are lacking [17,27] and othermarkers were demonstrated more accurate or cost-effective thanalpha1-antitrypsin in the stools of such patients [28].

Alpha2-macroglobulin is a serum anti-proteinase and itsexcretion in the feces is increased in IBD patients. The levels ofthis protein in the feces seem to correlate with CDAI in CD butnot in subjects with UC. There are only a few reports in theliterature and the method is not routinely available [17,29].

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3. Neutrophil-derived proteins in the feces in IBD

Lysozyme is a polymorphonuclear neutrophil-derived en-zyme which catalyses the hydrolysis of Gram-positive bacterialcell walls. Fecal lysozyme was found significantly elevated inpatients with active and inactive CD, active UC and non-inflammatory bowel gastrointestinal diseases with diarrhea,compared to healthy controls [30]. Fecal lysozyme correlateswith excretion of Indium 111-labeled granulocytes in patientswith colonic disease but not in those with small bowel disease[31], therefore it is of limited value in CD patients. Some otherstudies found that this enzyme is less effective than othermarkers in diagnosis of IBD [32,33].

Myeloperoxidase is a constituent of neutrophil azuregranules. Fecal levels are elevated in active IBD comparedwith controls and correlate with laboratory parameters andendoscopic grade of inflammation [34]. Fecal lactoferrincorrelates with fecal myeloperoxidase but the ratio of fecallactoferrin and myeloperoxidase is different in IBD compared toinfective diarrhea [35]. Maybe because of its short lifetime,other enzymes showed a stronger correlation with theendoscopic classified severity of inflammation and it appearsmore efficient than myeloperoxidase in the routine use fordiagnosis in IBD patients [32].

Granulocytes elastase was shown to correlate with the CDAIin CD and an activity index in UC [36]. Nevertheless, a directcomparison of accuracy and cost effectiveness concluded thatlactoferrin was a better neutrophil-derived fecal marker ofinflammation than granulocytes elastase, fecal lysozyme andmyeloperoxidase [28,37].

Tumor necrosis factor-alpha (TNF-α) is a proinflammatorycytokine produced also by polymorphonuclear neutrophils.Fecal TNF-α has been shown to be a useful marker of diseaseactivity in children with IBD but it needs to be further assessedin adults [38]. The results in children are equivalent to otherfecal markers such as calprotectin and the need to keep the stoolfrozen for risk of degradation makes this technique not usefulfor routine application [39]. Several studies using fecal tumornecrosis factor alpha have shown promising results, largeinterindividual variations in both serum and fecal levels havelimited its widespread applicability [16].

Lactoferrin is a 76 kDa iron binding glycoprotein that is themajor component of the secondary granules of polymorphonu-clear neutrophils and is secreted by most mucosal membranes.Lactoferrin is found in many body fluids such as human milk,tears, synovial fluid and serum. The secondary granules arereleased by polymorphonuclear neutrophils when they degra-nulate during inflammatory process since lactoferrin has animportant role in the innate immunity as a bactericidal [40,41].Other hematopoietic cells such as monocytes and lymphocytesdo not produce lactoferrin [42]. During intestinal inflammation,polymorphonuclear neutrophils infiltrate the mucosa, resultingin an increase in the concentration of lactoferrin in the feces andits presence is proportional to neutrophil translocation to thegastrointestinal tract [43]. The protein is resistant to proteolysisand undamaged by multiple freeze thaws, providing a usefulmarker in feces as an indicator of intestinal inflammation.

Moreover, it is remarkably stable and resistant to degradationwhen left in room temperatures for extended periods of timerather easy to detect and measure in stool using ELISA [44].This property makes this marker attractive for clinical use andseveral studies have been performed to identify the sensitivityand the qualitative assay for lactoferrin patients with IBD.

Lactoferrin was shown to be significantly increased in activeUC and CD compared to controls [37]. Several studies on thedifferential diagnosis of intestinal inflammatory conditionspointed out that fecal lactoferrin is a reliable marker of bowelinflammation with sensitivity of 90% and specificity of 98%[15,40–45]. Buderus S. et al. observed that fecal lactoferrinquickly decreased after clinically successful treatment withinfliximab therapy in children with severe CD and theysuggested that fecal lactoferrin was a sensitive and specificmarker for intestinal inflammation that can be used formonitoring the therapeutic response to infliximab in pediatricsCrohn's diseased patients [46]. In pouchitis, levels of lactoferrincorrelate with the clinical score of disease activity; therefore,fecal lactoferrin was demonstrated to differentiate patients withirritable pouch syndrome from those with pouchitis, cuffitis orCD with a sensitivity of 100% and specificity of 85%. Fecallactoferrin can serve as a sensitive and non-invasive initialscreening test in an algorithm for evaluation of symptomaticpatients after restorative proctocolectomy for UC [27]. Thismarker of bowel inflammation may find a reasonableapplication on detecting postoperative recurrence of CD [47].In our cohort of 63 CD patients who had a complete resection ofall diseased bowel the mean lactoferrin fecal levels were stillsignificantly higher than normal values after a 3 year follow-up[47]. Lactoferrin showed a reliable threshold value for systemicinflammation and seemed to predict the presence of episodes ofclinical recurrence during the post-operative follow-up. In ourexperience the lactoferrin threshold value for being predictive ofsystemic inflammation (CRPN6 mg/l) is 11 μg/ml with asensitivity of 71% and a specificity of 90% [47].

Calprotectin is the most used fecal protein. This proteinaccounts for up to 50% of the neutrophil cytosolic protein and itis resistant to colonic bacterial degradation. The protein is aheterocomplex protein consisting of 2 heavy (L1H) chains and 1light (L1L) chain which are non-covalently linked [48,49].Calprotectin appears to play a regulatory role in the inflamma-tory process and functions in both an antimicrobial andantiproliferative capacity [50]. Interest in calprotectin as amarker for inflammation in the gut followed the realization thatIndium 111-labeled granulocyte scans could be used to bothvisualize and quantify the acute inflammation in the gut ofpatients with inflammatory bowel disease [51]. These findingsled to the idea that an increased translocation of granulocytesinto the intestinal mucosa in conditions of inflammation mightgive increased levels of proteins from such cells in feces. Someauthors have demonstrated that eosinophilic granulocytes arethe main cellular source of calprotectin in the normal gutmucosa [52]. However, relatively high levels of calprotectin arefound in the stools of normal individuals—about 6 times theplasma levels (which are about 0.5 mg/l). This is compatiblewith data suggesting that in normal individuals most circulating

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neutrophils migrate through the mucosal membrane of the gutwall and thereby terminate their circulating life [52,53].Subsequent lyses within the gut lumen and release of cytosoliccalprotectin thereby accounts for the median fecal levels of2.0 mg/l seen in healthy controls [54]. It is easily measured infeces by a commercially available ELISA. The diagnostic use offecal calprotectin in a broad spectrum of intestinal diseases hasbeen studied by a number of groups with remarkable agreementbetween the results.

Since the method requires only a single stool sample,extraction and an ELISA, it was used as a screening test todistinguish between patients with IBD and irritable bowelsyndrome (IBS) in an outpatient setting. Several studies showedthat a cut off of 30 mg/l had a 100% sensitivity and 94%specificity for this purpose [15,55]. Fecal calprotectin concen-trations correlate with endoscopic and histological assessmentof disease activity in adults and children with IBD [56]. Severalother studies collected in a recent review confirmed a closecorrelation between fecal calprotectin concentrations anddisease activity and both endoscopic and histological scores[16]. Some other studies showed that fecal calprotectin appearsto be a good predictor of relapse in patients with IBD, althoughits value may be higher in patients with UC than those in CD[55,57]. In clinical practice fecal calprotectin could be measuredat intervals during follow-up, which may allow early detectionrather than just prediction of relapses. In our data of 63 patientsoperated for CD highly significant correlations were evidencedbetween calprotectin fecal levels and serum CRP (direct), serumiron and serum albumin (inverse) and, as observed forlactoferrin, patients with diarrhea reported significantly higherfecal calprotectin levels compared to asymptomatic ones [47].The calprotectin threshold value for being predictive ofsystemic inflammation (CPRN6 mg/l) was 284 ng/ml with asensitivity of 70% and specificity of 92% [47].

Fecal S100A12 is another marker of inflammation that asbeen proposed recently. It is a Ca-binding protein thatpreviously was known as calgranulin C or EN-RAGE and itis expressed as a cytoplasmatic protein in activated neutrophilswith proinflammatory properties. The S100A12 activates thenuclear factor-kB signal transduction pathway and inducesproinflammatory cytokine release, including TNF-α. Theavailable data suggest that S100A12 could contribute to theprocess of gut inflammation in the setting of IBD andfurthermore that S100A12 levels may reflect the presence andseverity of gut inflammation [58].

4. Fecal enzymes in the clinical management of inflammatorybowel disease

Apart from screening and assessing response to treatment,the fecal calprotectin and lactoferrin have a further majoradvantage over the leukocytes labeling technique in predictingrelapse of IBD. It has been shown that, in patients withclinically quiescent IBD, fecal calprotectin values N50 mg/l may be used to predict clinical relapse of disease within a fewmonths with over 80% sensitivity [16]. Symptoms of IBD arethe direct consequence of the inflammatory process itself and

vary depending on the location of the inflammation. Mostpatients with quiescent IBD have subclinic inflammation[47,55,57] and it is possible that symptomatic relapse occursonly when the inflammatory process reaches a critical intensity.The direct assessment of the level of intestinal inflammatoryactivity may provide a quantitative pre-symptomatic measure ofimminent clinical relapse of the disease [47,55,57]. Theidentification of patients at high risk of relapse would improvethe design of therapeutic regimes designed to maintain patientsin remission. Stratification by risk group using fecal calprotectinor lactoferrin would be efficient in predicting relapse and inidentifying patients at risk.

The fecal calprotectin and lactoferrin methods are the firstline of techniques that allow non-invasive assessment of theintestinal inflammatory cascade. At present they can be used formultiple purposes, described above, but it is likely that it will bepossible to widen their application tasks. Furthermore, manyother cells of the inflammatory cascade are numericallyincreased in biopsy specimens from patients with a variety ofgastroenterological conditions. Some, such as mast cells andeosinophils, are thought to play a central role in mediatingintestinal allergic reactions. However, both types of cell arefound to be activated in a number of other gastrointestinalinflammatory diseases such as IBD, coeliac disease, eosino-philic gastroenteritis and collagenous colitis, suggesting thatboth cell types may be involved in the pathogenesis of chronicintestinal inflammation [59,60]. It may therefore be possible,similarly to neutrophils and calprotectin and lactoferrin, toidentify mast cell granule proteins, such as tryptase andchymase, in fecal samples and use them as markers of aspecific component of the intestinal inflammatory response. Along-term objective might be to fully automate a fecal sampleassay method that provides specific information on the activityof acute inflammation (neutrophils), chronic inflammation (T-cells) and allergy (mast cells).

5. Conclusions

Lactoferrin and calprotectin appear to be the most used anduseful fecal markers of intestinal inflammation. Many studieshave confirmed the accuracy of these markers in the diagnosisand surveillance of patients with IBD. They are inexpensive andeasily measured and, therefore, suitable for extensive use. Bothtests appear to be useful in detecting bowel inflammation insymptomatic patients, achieving a similar diagnostic accuracy.Different cut-off values are suggested for different patientcategories, higher for patients with known inflammatoryconditions while lower for screening purposes [14].

The potential clinical implications of detection of calprotectinand lactoferrin in feces are considerable: differential diagnosis atan early stage, with the possibility of early treatment with less sideeffects, as well as monitoring of new therapeutic strategies tomaintain symptomatic remission [16]. In fact, theoretically themonitoring of the treatment with such fecal markers can lead to adramatic reduction in the frequency and severity of clinicalrelapses with an improvement in the patient's quality of life withreduction of invasive tests. More studies are necessary to define

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the role of fecal calprotectin and lactoferrin and the specificapplication of each in different settings of IBD patients.

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