Characterisation of faecal protease activity in irritable bowel syndrome with diarrhoea: origin and effect of gut transit

Abstract

Objectives: Faecal serine proteases (FSPs) may play a role in irritable bowel syndrome with diarrhoea (IBS-D), but their origin is unclear. We aimed to structurally characterise them and define the impact of colonic cleansing and transit time.

Design: Faecal samples were obtained from 30 healthy volunteers (HV) and 79 patients with IBS-D participating in a trial of ondansetron versus placebo. Colonic transit was measured using radio-opaque markers. Samples were also obtained from 24 HV before and after colonic cleansing with the osmotic laxative MoviPrep. FSPs were purified from faecal extracts using benzamidine-Sepharose affinity chromatography. SDS-PAGE profiled components were identified using trypsinolysis and tandem mass spectrometry. Functional protease activity in faecal extracts was measured using a colorimetric assay based on the proteolysis of azo-casein.

Results: Protein analysis identified the most abundant FSPs as being of human origin and probably derived from pancreatic juice. Functional assays showed increased faecal protease (FP) and amylase in patients with IBS-D compared with HV. Those with higher amylase had significantly higher FP and greater anxiety. FP activity correlated negatively with whole gut transit in patients with IBS-D (Spearman r=-0.32, p=0.005) and HV (r=-0.55, p=0.014). Colon cleansing caused a significant rise in FP activity in HV from a baseline of median (IQR) 253 (140-426) to 1031 (435-2296), levels similar to those seen in patients with IBS-D. FSP activity correlated positively with days/week with urgency.

Conclusions: The most abundant FSPs are of human origin. Rapid transit through the colon and/or decreased (possibly bacterial) proteolytic degradation increases their faecal concentration and could contribute to visceral hypersensitivity in patients with IBS-D.

Clinicaltrialsgov: NCT00745004.

Keywords: DIARRHOEA; IRRITABLE BOWEL SYNDROME.

Figure 1

Methods and workflow.

Figure 2

SDS-PAGE of benzamidine-Sepharose enriched fraction of faecal extract samples. SDS-PAGE of chromatographic fractions of faecal extracts from patients with irritable bowel syndrome with diarrhoea (IBS-D) and healthy volunteers. Individual reference numbers are shown and red boxes denote representative regions excised for component identification. Note that similar profiles of components were recovered for all diseased individuals and that little or no protein was detected in healthy volunteers except for one individual who showed an SDS-PAGE profile comparable with IBS-D patients. Retrospectively it was apparent she did experience pain and some urgency though not enough to meet Rome criteria. The average transit time of these nine IBS patients was 21 h, stool frequency 3.0/day and urgency score 1.3 on a 0–3 scale, which is typical for the group whose average values were 19 h for transit, stool frequency 2.6/day and urgency score 1.6.

Figure 3

Faecal levels of amylase in patients with irritable bowel syndrome with diarrhoea (IBS-D) versus controls showing a biphasic distribution with one group of 22 showing a significant increase while a subgroup of 14 were not different from normal.

Figure 4

Faecal protease (FP) activity expressed in trypsin units/mg protein after colonic cleansing with MoviPrep at day −1 and days 2, 14 and 28 showing a significant rise in FSP activity after purging of colonic bacteria. One-way ANOVA for effect of time (p=0.001; day −1 vs day 2, p=0.0007, n=23).

Figure 5

Faecal protease activity expressed in trypsin units/mg protein showing that patients with irritable bowel syndrome with diarrhoea had significantly higher values than controls, but with wide variability in both groups. **p=0.003 (Mann–Whitney U test).

Figure 6

Inverse correlation between faecal serine protease (FSP) activity and whole gut transit in patients with irritable bowel syndrome with diarrhoea (Spearman r=−0.32, p=0.005, n=79).

Figure 7

Positive correlation between faecal protease (FP) activity and days/week with urgency in patients with irritable bowel syndrome with diarrhoea (Spearman r=0.26, p=0.02, n=73).