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

David Tooth, Klara Garsed, Gulzar Singh, Luca Marciani, Ching Lam, Imogen Fordham, Annie Fields, Rawinder Banwait, Melanie Lingaya, Robert Layfield, Maggie Hastings, Peter Whorwell, Robin Spiller, David Tooth, Klara Garsed, Gulzar Singh, Luca Marciani, Ching Lam, Imogen Fordham, Annie Fields, Rawinder Banwait, Melanie Lingaya, Robert Layfield, Maggie Hastings, Peter Whorwell, Robin Spiller

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.

Figures

Figure 1
Figure 1
Methods and workflow.
Figure 2
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
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
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
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
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
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).

References

    1. Spiller R, Aziz Q, Creed F, et al. Guidelines on the irritable bowel syndrome: mechanisms and practical management. Gut 2007;56:1770–98
    1. Gecse K, Roka R, Ferrier L, et al. Increased faecal serine protease activity in diarrhoeic IBS patients: a colonic lumenal factor impairing colonic permeability and sensitivity. Gut 2008;57:591–9
    1. Mertz H, Naliboff B, Munakata J, et al. Altered rectal perception is a biological marker of patients with irritable bowel syndrome. Gastroenterology 1995;109: 40–52
    1. Roka R, Rosztoczy A, Leveque M, et al. A pilot study of fecal serine-protease activity: a pathophysiologic factor in diarrhea-predominant irritable bowel syndrome. Clin Gastroenterol Hepatol 2007;5:550–5
    1. Otten CM, Kok L, Witteman BJ, et al. Diagnostic performance of rapid tests for detection of fecal calprotectin and lactoferrin and their ability to discriminate inflammatory from irritable bowel syndrome. Clin Chem Lab Med 2008;46:1275–80
    1. Garsed K, Singh G, Tooth D, et al. Origin of increased fecal serine protease in patients with irritable bowel syndrome and diarrhoea (IBS-D). Gut 2011;60:A25
    1. MacFarlane GT, Allison C, Gibson SA, et al. Contribution of the microflora to proteolysis in the human large intestine. J Appl Bacteriol 1988;64:37–46
    1. Garsed K, Hastings M, Marciani L, et al. Ondansetron is an effective treatment for patients with diarrhoea predominant irritable bowel syndrome. Gut 2011;60(Suppl 3):A40
    1. Garsed KC, Marciani L, Fields A, et al. Mode of action of a macrogol formulation on distribution of intestinal fluid: a MRI study. Gastroenterology 2012;142:S814
    1. Metcalf AM, Phillips SF, Zinsmeister AR, et al. Simplified assessment of segmental colonic transit. Gastroenterology 1987;92:40–7
    1. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54
    1. Tooth DJ, Krishna VG, Layfield R. An economical high-throughput protocol for multidimensional fractionation of proteins. International Journal of Proteomics 2012;2012:735132.
    1. Hellman U, Wernstedt C, Gonez J, et al. Improvement of an “In-Gel” digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal Biochem 1995;224:451–5
    1. Koshikawa N, Hasegawa S, Nagashima Y, et al. Expression of trypsin by epithelial cells of various tissues, leukocytes, and neurons in human and mouse. Am J Pathol 1998;153:937–44
    1. Cenac N, Andrews CN, Holzhausen M, et al. Role for protease activity in visceral pain in irritable bowel syndrome. J Clin Invest 2007;117:636–47
    1. Cottrell GS, Amadesi S, Grady EF, et al. Trypsin IV, a novel agonist of protease-activated receptors 2 and 4. J Biol Chem 2004;279:13532–9
    1. Kerckhoffs AP, Ter Linde JJ, Akkermans LM, et al. Trypsinogen IV, serotonin transporter transcript levels and serotonin content are increased in small intestine of irritable bowel syndrome patients. Neurogastroenterol Motil 2008;20:900–7
    1. Barbara G, Wang B, Stanghellini V, et al. Mast cell-dependent excitation of visceral-nociceptive sensory neurons in irritable bowel syndrome. Gastroenterology 2007;132:26–37
    1. Buhner S, Li Q, Vignali S, et al. Activation of human enteric neurons by supernatants of colonic biopsy specimens from patients with irritable bowel syndrome. Gastroenterology 2009;137:1425–34
    1. Klooker TK, Braak B, Koopman KE, et al. The mast cell stabiliser ketotifen decreases visceral hypersensitivity and improves intestinal symptoms in patients with irritable bowel syndrome. Gut 2010;59:1213–21
    1. Bohe M, Borgstrom A, Genell S, et al. Determination of immunoreactive trypsin, pancreatic elastase and chymotrypsin in extracts of human feces and ileostomy drainage. Digestion 1983;27:8–15
    1. Genell S, Gustafsson BE. Impaired enteric degradation of pancreatic endopeptidases in antibiotic-treated rats. Scand J Gastroenterol 1977;12:801–9
    1. Borgstrom A, Genell S, Ohlsson K. Elevated fecal levels of endogenous pancreatic endopeptidases after antibiotic treatment. Scand J Gastroenterol 1977;12:525–9
    1. Goldberg DM. Proteases in the evaluation of pancreatic function and pancreatic disease. Clin Chim Acta 2000;291:201–21
    1. Cenac N, Coelho AM, Nguyen C, et al. Induction of intestinal inflammation in mouse by activation of proteinase-activated receptor-2. Am J Pathol 2002;161:1903–15
    1. It-Belgnaoui A, Bradesi S, Fioramonti J, et al. Acute stress-induced hypersensitivity to colonic distension depends upon increase in paracellular permeability: role of myosin light chain kinase. Pain 2005;113:141–7
    1. Gareau MG, Jury J, Perdue MH. Neonatal maternal separation of rat pups results in abnormal cholinergic regulation of epithelial permeability. Am J Physiol Gastrointest Liver Physiol 2007;293:G198–203
    1. Zhou Q, Zhang B, Verne GN. Intestinal membrane permeability and hypersensitivity in the irritable bowel syndrome. Pain 2009;146:41–6

Source: PubMed

スポンサーと協力者

医学的状態

薬物療法

3
購読する