AIM: To study intestinal permeability (IP) and its relationship to the disease activity in patients with inflammatory bowel diseases (IBD) - Crohn’s disease (CD) and ulcerative colitis (UC).

METHODS: Fifty-eight patients with active IBD (32 with CD and 26 with UC) and 25 healthy controls consented to participate in the study. The clinical activity of CD was estimated using the Crohn’s Disease Activity Index (CDAI), and the endoscopic activity of UC using the Mayo scoring system. IP was assessed by the rise in levels of iohexol, which was administered orally (25 mL, 350 mg/mL) 2 h after breakfast. Three and six hours later serum (SIC mg/L) and urine (UIC g/mol) iohexol concentrations were determined by a validated HPLC-UV technique.

RESULTS: In the CD group, SIC values at 3 h (2.95 ± 2.11 mg/L) and at 6 h after ingestion (2.63 ± 2.18 mg/L) were significantly higher compared to those of healthy subjects (1.25 ± 1.40 mg/L and 1.11 ± 1.10 mg/L, respectively, P < 0.05). UIC (g/mol) values were also higher in patients, but the differences were significant only for UIC at 6 h. Significant positive correlation (P < 0.05) was found between the CDAI and IP, assessed by SIC at 3 h (r = 0.60) and 6 h (r = 0.74) after the ingestion. In comparison to controls, SIC and UIC of UC patients were higher in the two studied periods, but the differences were significant at 6 h only. Significantly higher values of SIC (P < 0.05) were found in patients with severe endoscopic activity of UC compared to those of patients with mild and moderate activity (3.68 ± 3.18 vs 0.92 ± 0.69 mg/L).

CONCLUSION: Serum levels of iohexol at 3 h and 6 h after its ingestion reflect increased IP, which is related to the disease activity in patients with IBD.

Crohn’s disease (CD) and ulcerative colitis (UC), the two principal forms of inflammatory bowel disease (IBD), are characterized by a chronic inflammation of the gastrointestinal tract. Over the past decade there has been increasing recognition of the importance of both epithelial barrier function and innate immunity in the genesis of intestinal inflammation[1-5].

Mucosal barrier function in IBD has been investigated in numerous studies and many of them, but not all[6], have demonstrated increased intestinal permeability (IP) in some of the patients[7-11]. Alterations of IP can be evaluated using several different probes such as disaccharides (lactulose, cellobiose), monosaccharides (mannitol), polyethylenglycols of different molecular mass and ⁵¹Cr-ethylenediaminetetraacetate (⁵¹Cr-EDTA). Studies have utilized different methods for the assessment of permeability, both in regard to administration procedures of the different probes and to outcome measures[12,13].

So far, IP assessment in patients has been performed by measuring urine recovery of ingested permeability substrates. The urine test can be relatively uncomfortable and can lead to potential inaccuracy[14,15] due to incomplete urine collection, renal dysfunction and variable hydration status of the tested subjects. In addition, some analytical difficulties in quantifying carbohydrates in urine have limited the widespread use of those permeability markers. Furthermore, urinary tract infections may compromise the recovery of sugar permeability markers[16]. Measurement of the permeability substrates in plasma or serum could eventually reduce the problems with urine recovery and could potentially have a valuable role, particularly in pediatric patients.

The aim of the present study was to evaluate IP in patients with active IBD (CD and UC) by measuring serum and urine levels of water-soluble contrast medium iohexol, to assess the relationship of IP to the disease activity, and to compare the reliability of serum vs urine levels of iohexol as an IP disease marker.

All permeability tests were well tolerated and no side effects were reported. Based on the results obtained from healthy subjects, values of SIC over 4.0 and 3.3 mg/L at 3 and 6 h respectively, as well as values of UIC over 35.5 and 31.4 g/mol at 3 and 6 h after ingestion, were accepted as abnormal (cut-offs). One control subject had slightly increased IP at 3 h, and two control subjects at 6 h, post-iohexol ingestion.

The results from applied Kruskal-Wallis test showed that the factor “group” is significant (P < 0.05). Mann-Whitney U test results indicated that in the group of CD patients the mean values of SIC at 3 h (2.95 ± 2.11 mg/L) and at 6 h (2.63 ± 2.18 mg/L) post-ingestion were significantly higher than those in the control group (1.25 ± 1.40 mg/L and 1.11 ± 1.10 mg/L, respectively). Abnormal IP was found in 10 patients at 3 h (31%) and in 16 patients at 6 h (50%) after iohexol intake. Urine recovery of iohexol was also higher in patients 3 h (18.09 ± 13.13 g/mol) and 6 h (36.92 ± 27.68 g/mol) post-ingestion compared to the control group (14.64 ± 10.44 g/mol and 14.18 ± 7.78 g/mol, respectively), but the difference was significant only for the mean UIC values at 6 h.

In the UC group, the mean serum levels (1.57 ± 1.55 mg/L and 2.49 ± 2.80 mg/L) and urine recovery of iohexol (9.86 ± 9.26 g/mol and 27.76 ± 25.18 g/mol) at 3 and 6 h after its ingestion, respectively, were higher than those for healthy controls, but the differences were significant only at 6 h for both parameters. IP was established as abnormal in 3 (11%) and 8 (31%) patients with UC at 3 and 6 h post-iohexol ingestion, respectively.

There was a significant positive correlation between the parameters SIC and CDAI in the group of CD patients at 3 h (r = 0.60) and at 6 h (r = 0.74) post-ingestion, while the correlation between CDAI and UIC was insignificant both at 3 h (r = 0.48) and at 6 h (r = 0.32) post-ingestion of iohexol.

In the subgroup “ЕА 3” of patients with severe endoscopic activity of UC (n=15), the mean values of SIC at 6 h were significantly higher (3.68 ± 3.18 mg/L), compared to those of the control group (1.11 ± 1.10 mg/L), while the values (0.92 ± 0.69 mg/L) for the patient subgroup “ЕА 1+2” (n = 11) with mild and moderate endoscopic activity did not differ from the controls (Figure 1A). The mean UIC values for patient subgroup “ЕА 3” at 6 h were also significantly higher (33.60 ± 28.42 g/mol) compared to those of the control group (14.18 ± 7.78 g/mol), and mean UIC values of the subgroup “EA 1+2” (19.78 ± 18.25 g/mol) were slightly but insignificantly higher from the control values (Figure 1B).

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Figure 1 Serum Iohexol concentrations (A) and urine Iohexol concentrations (B) (mean ± SE) at 6 h post-ingestion according to Endoscopic Activity Mayo score (EA) of patients with ulcerative colitis. EA3: Severe endoscopic activity; EA 1+2 : Mild and moderate endoscopic activity. ^aP < 0.05, SIC and UIC vs control group; ^bP < 0.05, EA 3 vs EA 1+2 group. SIC: Serum Iohexol concentrations; UIC:Urine Iohexol concentrations.

The permeability disturbances were five-fold more frequent in the subgroup of patients with severe activity (47%) in comparison to the subgroup of patients with mild and moderate activity (9%).

Altered barrier function in IBD is documented in experimental and clinical studies, but it is difficult to compare the results of IP due to the existing variability in the chemical nature of the chosen candidate markers in the experimental design and methodology. It is considered that the larger molecular weight markers reflect the changes predominantly in paracellular permeability, while smaller ones register transcellular transfer changes. Paracellular permeability across the epithelial cell monolayers is regulated primarily by the tight junctions (TJs) that encircle the apical poles of the epithelial cells[1,7]. The epithelium in the inflamed intestinal segments of patients with CD is characterized by a reduction of the TJ strands, strand breaks, and alterations of the TJ proteins. In patients with UC the epithelial leaks appear early due to micro-erosions resulting from upregulated epithelial apoptosis and in addition to a prominent increase of claudin-2[3]. Immune regulation of the epithelial functions by cytokines may cause a barrier dysfunction not only by the TJ impairments but also by apoptotic leaks, transcytotic mechanisms and mucosal gross lesions[4].

In our study, IP in patients with IBD was examined by measuring of the serum levels and urine recovery of iohexol, following oral administration. Andersen et al[19] demonstrated that water-soluble radiographic contrast media could be of use for evaluation of the altered intestinal barrier function. The contrast agent iohexol is a moderately large molecule (molecular weight 821 Dalton) with a low absorption under normal conditions, and enhanced absorption through the inflamed intestinal mucosa. It does not bind to serum proteins and is filtered through the glomerulus without indications of tubular secretion or reabsorption. Until now, to the best of our knowledge, serum levels of iohexol have not been used for the assessment of IP. The results of this study demonstrate that iohexol permeation through the intestinal mucosa is significantly increased in both CD and UC patients. Using different probes, similar findings have also been reported in numerous studies both in adult and pediatric populations with IBD[7-11,20-24]. The findings of this study support the understanding that patients with active IBD (including both CD and UC) have a mucosal barrier dysfunction, which can be assessed by measurement of IP. The permeability alterations were more frequent in CD than in UC patients: increased iohexol absorption as a marker for the abnormal IP was established in 50% of CD and in 31% of UC patients, figures which were significantly more frequent in comparison to the healthy subjects. Taking into account the fact that 3 h after ingestion iohexol is located in the large intestine[7], the higher SIC and UIC at 6 h after ingestion in patients with UC in our research suggest that this test can be used for evaluation of the colon permeability also.

The relationship between increased IP and disease activity in IBD has been established in some studies[9,11,20,21]. IP in CD patients is increased proportionally to the disease activity; it can be used to predict the clinical relapse of the disease (due to subclinical mucosal inflammation) and to assess prognosis[25,26]. However, the data in the literature are contradictory, most probably due to the usage of different permeability probes as mentioned above. Our study demonstrates that the permeation of iohexol through the intestinal mucosa, evaluated by serum concentrations, correlates positively with the disease activity in CD patients. These results are in agreement with data reported by Halme et al[7,20] for increased IP of iohexol (measured by its urinary recovery) and its correlation with the clinical disease activity indices. Furthermore, Halme et al[27] concluded that the iohexol test is a superior activity marker compared to the lactulose-mannitol test in patients with IBD. We established a relationship between serum levels of iohexol and the endoscopic activity score for UC patients. Using different permeability markers, Miki et al[11],

Arslan et al[23] and Casellas et al[24] found a relationship between IP and disease activity in IBD patients. However, other investigators did not establish such a correlation[8,28,29]. Our data for significant positive correlation between iohexol penetration through the intestinal mucosa and disease activity support the hypothesis of the important role of the impaired intestinal barrier in the pathogenesis of active IBD[2,3,5].

In conclusion, the water-soluble contrast medium iohexol is a suitable marker for assessing the gut barrier function as its penetration through the intestinal mucosa is increased in patients with active IBD (both CD and UC) and is related to the disease activity. In our study, serum iohexol concentration appears to be a superior marker of altered IP, compared to urinary iohexol level. Measurement of a single serum sample of iohexol 6 h following its oral administration makes the proposed permeability test more convenient and provides a possibility for the assessment of altered barrier function in both small and large intestine.