The assessment of inflammatory activity in intestinal disease in man can be done using a variety of different techniques. These range from the use of non-invasive acute phase inflammatory markers measured in plasma such as C reactive protein (CRP) and the erythrocyte sedimentation rate (ESR) (both of which give an indirect assessment of disease activity) to the direct assessment of disease activity by intestinal biopsy performed during endoscopy in association with endoscopic scoring systems. Both radiology and endoscopy are conventional for the diagnosis of inflammatory bowel disease (IBD). However these techniques have severe limitations when it comes to assessing functional components of the disease such as activity and prognosis. Here we briefly review the value of two emerging intestinal function tests. Intestinal permeability, although ideally suited for diagnostic screening for small bowel Crohn’s disease, appears to give reliable predictive data for imminent relapse of small bowel Crohn’s disease and it can be used to assess responses to treatment. More significantly it is now clear that single stool assay of neutrophil specific proteins (calprotectin, lactoferrin) give the same quantitative data on intestinal inflammation as the 4-day faecal excretion of 111Indium labelled white cells. Faecal calprotectin is shown to be increased in over 95% of patients with IBD and correlates with clinical disease activity. It reliably differentiates between patients with IBD and irritable bowel syndrome. More importantly, at a given faecal calprotectin concentration in patients with quiescent IBD, the test has a specificity and sensitivity in excess of 85% in predicting clinical relapse of disease. This suggests that relapse of IBD is closely related to the degree of intestinal inflammation and suggests that targeted treatment at an asymptomatic stage of the disease may be indicated.

Permeability refers to that property of a membrane that enables passage of a solute by unmediated diffusion. The diffusion of a solute across a simple membrane is determined by the structure of the membrane (in terms of its composition, charge, thickness, etc.), the physicochemical properties of the solute (like molecular size, shape, charge and solubility) and its interaction with the media or solvent. Intestinal permeability is assessed non-invasively in vivo by measuring urinary excretion of orally administered substances. The ideal permeability probe is water-soluble, non-toxic, non-degradable and not metabolised before, during or after permeating the intestine[13]. The probes should preferably not be naturally present in urine, be completely excreted in the urine following intravenous administration and be easily and accurately measurable. Fordtran et al[14] were instrumental in the development of ideas for assessing intestinal permeability in man but it was Menzies who introduced oligosaccharides as test substances for the non-invasive assessment of intestinal permeability[15] in 1974, and later formulated the principle of differential urinary excretion of orally administered test substances[16]. The importance of the differential urinary excretion principle is that it overcomes most if not all the problems associated with the use of a single test substance, where urinary excretion is dependent on a number of pre-and post-mucosal factors as well as intestinal permeability. The differential principle advocates that a nonhydrolyzed disaccharide (i.e. lactulose) and a monosaccharide (L-rhamnose or mannitol) are ingested together. As the pre- and post-mucosal determinants of their excretion affects the two test substances equally and the differential 5 hour urinary excretion ratio (ratio of lactulose/L-rhamnose) is not affected by these variables the urinary excretion ratio becomes a specific measure of intestinal permeability.

Tests of intestinal permeability were initially designed to allow reliable non-invasive detection of patients with untreated coeliac disease[16]. The tests have since come to be viewed as synonymous with assessing intestinal barrier function. In clinically active small bowel Crohn’s disease the vast majority of patients (> 95%) have an increase in the differential urinary excretion of ingested di-/mono-saccharides (lactulose/L rhamnose or mannitol) and half of those with Crohn’s colitis are abnormal[13]. These figures are marginally improved with the use of 51CrEDTA, which requires a 24-hour, as opposed to a 5-hour urinary collection. The vast majority of patients with ulcerative colitis have normal small intestinal permeability when assessed by these methods. However, tests of intestinal permeability have not found widespread application as screening tests to discriminate between patients with Crohn’s disease and IBS. The reason for this is probably that the urinary sugar analysis is time consuming and demanding, and there may be some concern that the tests lack specificity being abnormal in a variety of small intestinal diseases (Table 1). At first sight the test appears to identify a number of “clinically irrelevant” diseases, which usually translates into disease for which no treatment is available, but in practice the tests seem often to identify small intestinal pathology where none was previously expected, thus expanding the number of identifiable small bowel pathologies.

There have been attempts to use intestinal permeability as an index of disease activity in Crohn’s disease. In general these have been disappointing because the degree of increase in the differential urinary excretion of lactulose/L-rhamnose or the excretion of ⁵¹CrEDTA is dependent on localisation and extent of disease within the small bowel as well as activity of the inflammation[13]. Abnormalities in intestinal permeability may, however, be used as a predictor of imminent relapse of quiescent Crohn’s disease. Three studies have now shown that, in patients with Crohn’s disease in clinical remission, an increased intestinal permeability can predict those at significant risk of relapse of disease in the next few months[17-19]. The strength of this association is difficult to assess from the published studies. Nevertheless, less than 20% of those with normal intestinal permeability appear to relapse over the ensuing 6 months. Interestingly, elevated levels of IL-6 in serum, which can be viewed as a surrogate marker of intestinal inflammation, also has a predictive value for relapse of Crohn’s disease[8], but receiver operating curve (ROC) analysis shows relatively low sensitivity and specificity (70 and 50%, respectively). The permeability ratio differs from such indices in that it is not based on concentrations of plasma proteins but rather represents functional changes in the intestinal mucosa, a direct consequence of intestinal inflammation.

The clinical implications of these findings are discussed later.

Intense neutrophil recruitment to the intestinal mucosa is a feature common to inflammatory bowel diseases[20]. When a patient’s own radiolabelled neutrophils are re-injected they migrate to sites of acute inflammation as well as to the liver, spleen and bone marrow[21]. Segal, Saverymuttu and Chadwick were instrumental in the introduction, validation and application of the 111Indium white cell technique for use in gastroenterology[20,22]. The technique visualises inflamed segments of bowel and quantitates the degree of inflammatory activity[20,23-26].

A number of studies have established that abdominal scans are abnormal in virtually all patients with active IBD; their accuracy in localisation of disease and distinguishing between actively inflamed and fibrous stricturing disease has implications for treatment. It was suggested that the technique could be used to discriminate, with an accuracy approaching 100%, between patients with IBD and IBS at the first outpatient visit. In practice this suggestion was not followed up with relevant research.

When combined with measurement of the 4 d faecal excretion of labelled white cells for quantitation of the inflammatory activity the technique becomes a formidable tool for research and investigation. The faecal excretion of the labelled white cells quantitate inflammation accurately and can be used to document therapeutic efficacy of various treatments in IBD[25,27]. It has also been used to define a number of enteropathies (NSAIDs, alcohol, chronic renal failure, hypogammaglobulinaemia, HIV-AIDS, etc.) where none were suspected or impossible to demonstrate by techniques other than perhaps the intestinal permeability tests (Table 1)[28]. The method is not disease specific, resembling that of the permeability tests, but it is specific for intestinal inflammation. This is not a drawback as it is a simple matter to distinguish between the inflammatory activity in patients with IBD and the above enteropathies, colonic cancer, diverticulitis, etc., since patients with active IBD have excretion values often an order of magnitude higher than the others.

Why has the white cell technique not been universally adapted for use as a diagnostic screen in IBD, and to assess disease activity? It requires expensive labeling facilities including labelling cabinets. The labeling procedure is time consuming, taking over 2 hours. The cost of isotope and material is in excess of £200 (US $300) and the radiation dose is not trivial if abdominal scans are carried out, being equivalent to that of a barium enema[29,30]. A complete 4-day faecal collection is also demanding and unpleasant for patients, occasionally requiring hospital admission.

Other methods have attempted to build on this success. One such is 99mTc labeling of white cells[31]. This is purported to give superior quality abdominal scintigraphy (which is not clinically important), but does not allow late (> 4 hours) scanning, because the label comes off and is excreted into the bowel independent of white cell excretion. Furthermore a faecal collection provides no quantitative information on intestinal inflammation (as the Technetium comes off the white cells and is excreted in faeces) and the labeling requires the same facilities as the white cells.

Newer techniques include E-selectin scanning[32]. This method is derived from the more conventional labelled white cell scintigraphy, but uses a labelled antibody to E-selectin, which is over-expressed in endothelial cells at sites of inflammation. It has the advantage of studying a more fixed entity that (unlike white cells) will not be shed at a variable rate into the bowel lumen and is applicable to the occasional patient with intestinal inflammation who is neutropenic.

In our opinion, the greatest impact that the white cell technique has had is that it ① emphasised that if a sensitive method is to be established for assessing intestinal function there are no shortcuts. Neurologists assess spinal fluid, respiratory physicians assess sputum, urologists urine and the gastroenterologist needs to come terms with the fact that faecal analysis is essential to obtain maximal information about the state of the intestine. ② emphasised that there is life beyond morphological assessment of the gut (x-ray and colonoscopic studies). ③ raised the possibility of dramatically changing our views on the treatment of IBD. Many patients with IBD in full clinical remission are shown to have significant intestinal inflammation[27,33]. At present treatment is non-specifically directed at maintaining remission (5-ASA, azathioprine, etc.). It seems highly probable that those patients with substantial inflammatory activity should be targeted for more aggressive therapy, in particular if they can be shown to be at significant risk of clinical relapse of disease. The analogy with the treatment of rheumatoid arthritis springs to mind. Here, first line treatment is directed to wards reducing the acute inflammatory component of the disease followed by a number of second line agents that can alter the natural history of the disease, reduce the frequency, duration and severity of relapses as well as reducing the joint damage.

Faecal analysis is unpleasant but has been with us for a long time. Measure of electrolytes and osmolality helped in the differential diagnosis of diarrhoea in children. Faecal fats were a widespread screening test for steatorrhoea for a while and faecal occult bloods have become the yardstick for colorectal screening with which other methods need to be compared. An improvement on these techniques was the introduction of radioisotopically labelled compounds (labelled red blood cells, proteins, white cells) which provided quantitative and functional data and which was event specific (blood loss, inflammation, protein losing enteropathy, etc.) but non-specific for disease.

The inflamed hyperpermeable mucosa of patients with inflammatory bowel disease is associated with increased protein loss into the bowel lumen[34]. Studies using radiolabelled proteins have demonstrated that there is faecal protein loss in patients with active Crohn’s disease and it may therefore be a useful marker of disease activity. Other studies have shown faecal α1 antitrypsin clearance to be a useful indicator of protein losing enteropathy[35] and that in patients with inflammatory bowel disease, 72 hour faecal clearance of α1 antitrypsin is a useful method for quantitating intestinal protein loss[36,37]. Faecal clearance of α1 antitrypsin correlates with that of ⁵¹Cr-albumin, and moderate rectal bleeding does not affect the α1 antitrypsin determination[36]. Random faecal α1 antitrypsin levels have been shown to be as useful as more prolonged collection in measuring Crohn’s disease activity[38] and correlated with several other laboratory measures that have been proposed as indicators of Crohn’s disease activity[39].

Concerns about costs, radiation, and the need for prolonged faecal collections all worked against these techniques for routine use, although many remain very important for research studies. The idea then emerged that it might be possible to assay for cell proteins or substances that are specifically associated with a certain cell type and which would then provide information on a specific component of the inflammatory cascade. Ferguson’s Edinburgh group was instrumental in expanding this idea[40]. Concerned about bacterial degradation of markers they used a whole gut lavage method involving ingestion of polyethylene-based purgatives (Kleenprep or GoLitely) for obtaining clear liquid faecal samples for analysis. The analysis took to various markers, such as immunoglobulins, neutrophils-specific elastase, andhaemoglobin. Separate studies showed that Crohn’s disease could be identified with ease, and that the method had a greater sensitivity for colorectal cancer than the conventional faecal occult blood technique. Ideally suited for research, the method has as yet not found wide application for routine screening purposes, possibly because of the drawback of patients needing to ingest large volumes of liquid.

Direct analysis of markers in faeces would be a major advance on this method. Here the problem is initially the bacterial degradation of the marker necessitating swift sample handling. One such marker, TNF, has been successfully used in children and in HIV infection in adults[41,42]. However, it is now clear that it is not necessary for the marker to be completely non-degraded, provided that the antibody (most of these assays are ELISA’s or radio immunoassay) is directed at an epitope of the molecule which resists degradation. One such assay is that for lactoferrin[43]. Lactoferrin is a relatively specific marker for neutrophils, in which it is present in cytoplasmic granules.