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

ABSTRACT: 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.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.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.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.NCT00745004.

SUBMITTER: Tooth D

PROVIDER: S-EPMC3995279 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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

Tooth David D Garsed Klara K Singh Gulzar G Marciani Luca L Lam Ching C Fordham Imogen I Fields Annie A Banwait Rawinder R Lingaya Melanie M Layfield Robert R Hastings Maggie M Whorwell Peter P Spiller Robin R

Gut 20130802 5

<h4>Objectives</h4>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.<h4>Design</h4>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 ...[more]

PMID: 23911555

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Project description:AIM:To study whether selected bacterial 16S ribosomal RNA (rRNA) gene phylotypes are capable of distinguishing irritable bowel syndrome (IBS). METHODS:The faecal microbiota of twenty volunteers with IBS, subdivided into eight diarrhoea-predominant (IBS-D), eight constipation-predominant (IBS-C) and four mixed symptom-subtype (IBS-M) IBS patients, and fifteen control subjects, were analysed at three time-points with a set of fourteen quantitative real-time polymerase chain reaction assays. All assays targeted 16S rRNA gene phylotypes putatively associated with IBS, based on 16S rRNA gene library sequence analysis. The target phylotypes were affiliated with Actinobacteria, Bacteroidetes and Firmicutes. Eight of the target phylotypes had less than 95% similarity to cultured bacterial species according to their 16S rRNA gene sequence. The data analyses were made with repeated-measures ANCOVA-type modelling of the data and principle component analysis (PCA) with linear mixed-effects models applied to the principal component scores. RESULTS:Bacterial phylotypes Clostridium cocleatum 88%, Clostridium thermosuccinogenes 85%, Coprobacillus catenaformis 91%, Ruminococcus bromii-like, Ruminococcus torques 91%, and R. torques 93% were detected from all samples analysed. A multivariate analysis of the relative quantities of all 14 bacterial 16S rRNA gene phylotypes suggested that the intestinal microbiota of the IBS-D patients differed from other sample groups. The PCA on the first principal component (PC1), explaining 30.36% of the observed variation in the IBS-D patient group, was significantly altered from all other sample groups (IBS-D vs control, P = 0.01; IBS-D vs IBS-M, P = 0.00; IBS-D vs IBS-C, P = 0.05). Significant differences were also observed in the levels of distinct phylotypes using relative values in proportion to the total amount of bacteria. A phylotype with 85% similarity to C. thermosuccinogenes was quantified in significantly different quantities among the IBS-D and control subjects (-4.08 +/- 0.90 vs -3.33 +/- 1.16, P = 0.04) and IBS-D and IBS-M subjects (-4.08 +/- 0.90 vs -3.08 +/- 1.38, P = 0.05). Furthermore, a phylotype with 94% similarity to R. torques was more prevalent in IBS-D patients' intestinal microbiota than in that of control subjects (-2.43 +/- 1.49 vs -4.02 +/- 1.63, P = 0.01). A phylotype with 93% similarity to R. torques was associated with control samples when compared with IBS-M (-2.41 +/- 0.53 vs -2.92 +/- 0.56, P = 0.00). Additionally, a R. bromii-like phylotype was associated with IBS-C patients in comparison to control subjects (-1.61 +/- 1.83 vs -3.69 +/- 2.42, P = 0.01). All of the above mentioned phylotype specific alterations were independent of the effect of time. CONCLUSION:Significant phylotype level alterations in the intestinal microbiotas of IBS patients were observed, further emphasizing the possible contribution of the gastrointestinal microbiota in IBS.

Project description:BACKGROUND: Increased concentrations of 5-hydroxytryptamine (5-HT) can be detected in the systemic circulation after a meal and may be involved in the physiological control of gastrointestinal motility. Abnormalities of 5-HT release after a meal might explain some of the postprandial symptoms associated with the irritable bowel syndrome (IBS). AIM: To investigate the effect of a standard meal on plasma 5-HT and urinary 5-hydroxyindole acetic acid (5-HIAA) concentrations in patients with diarrhoea predominant IBS and in healthy volunteers. METHODS: After an overnight fast, six volunteers and five patients with IBS were given a carbohydrate-rich meal. Blood and urine samples were taken before and for four hours after the meal. Platelet-poor plasma 5-HT and urinary 5-HIAA were analysed by reversed phase high performance liquid chromatography with fluorometric detection. 5-HIAA was expressed as a ratio with urinary creatinine concentration, which was measured by spectrophotometry. RESULTS: During the four hour postprandial period, 5-HT concentrations were significantly higher in patients with IBS than in healthy volunteers at 0.5 hours (p < 0.05), 2 hours (p < 0.05) and 2.5 hours (p < 0.05). 5-HT was not detected in the plasma in the fasting state in patients or volunteers. Median peak 5-HT in patients with IBS (359 (198-796) nmol/l) was significantly greater than volunteers (83 (7-190)) (p < 0.05). "Area under the curve" for 5-HT detection was greater for patients with IBS (317 (138-771)) than for healthy volunteers (51 (4-129); p < 0.05). The duration of the 5-HT peak was significantly longer in patients with IBS (3 (1-3) hours) than in the healthy volunteers (1 (1-1) hours; p < 0.01). Postprandial urinary median 5-HIAA values in controls (5.6 (5.5-5.8) mumol/mmol creatinine) and patients with IBS (3.0 (2.5-6.8) mumol/mmol creatinine) were not significantly different from preprandial values (controls: 5.9 (5.5-6.6) mumol/mmol creatinine; patients with IBS: (6.2 (2.4-9.3) mumol/mmol creatinine). CONCLUSION: These findings indicate that there may be a difference in the way that 5-HT is released in patients with diarrhoea predominant IBS, and could suggest a possible role for 5-HT in the postprandial symptoms of these patients.

Dataset Information

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

Publications

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

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