Modulation of protein fermentation does not affect fecal water toxicity: a randomized cross-over study in healthy subjects

PLoS One. 2012;7(12):e52387. doi: 10.1371/journal.pone.0052387. Epub 2012 Dec 20.


Objective: Protein fermentation results in production of metabolites such as ammonia, amines and indolic, phenolic and sulfur-containing compounds. In vitro studies suggest that these metabolites might be toxic. However, human and animal studies do not consistently support these findings. We modified protein fermentation in healthy subjects to assess the effects on colonic metabolism and parameters of gut health, and to identify metabolites associated with toxicity.

Design: After a 2-week run-in period with normal protein intake (NP), 20 healthy subjects followed an isocaloric high protein (HP) and low protein (LP) diet for 2 weeks in a cross-over design. Protein fermentation was estimated from urinary p-cresol excretion. Fecal metabolite profiles were analyzed using GC-MS and compared using cluster analysis. DGGE was used to analyze microbiota composition. Fecal water genotoxicity and cytotoxicity were determined using the Comet assay and the WST-1-assay, respectively, and were related to the metabolite profiles.

Results: Dietary protein intake was significantly higher during the HP diet compared to the NP and LP diet. Urinary p-cresol excretion correlated positively with protein intake. Fecal water cytotoxicity correlated negatively with protein fermentation, while fecal water genotoxicity was not correlated with protein fermentation. Heptanal, 3-methyl-2-butanone, dimethyl disulfide and 2-propenyl ester of acetic acid are associated with genotoxicity and indole, 1-octanol, heptanal, 2,4-dithiapentane, allyl-isothiocyanate, 1-methyl-4-(1-methylethenyl)-benzene, propionic acid, octanoic acid, nonanoic acid and decanoic acid with cytotoxicity.

Conclusion: This study does not support a role of protein fermentation in gut toxicity. The identified metabolites can provide new insight into colonic health.

Trial registration: NCT01280513.

Publication types

  • Randomized Controlled Trial
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Calorimetry
  • Cell Death / drug effects
  • Cluster Analysis
  • Colon / drug effects
  • Colon / metabolism
  • Cross-Over Studies
  • Diet
  • Discriminant Analysis
  • Energy Intake / drug effects
  • Feces / chemistry*
  • Feces / microbiology
  • Female
  • Fermentation*
  • HT29 Cells
  • Health*
  • Humans
  • Inhibitory Concentration 50
  • Least-Squares Analysis
  • Male
  • Metagenome
  • Mutagens / toxicity*
  • Proteins / metabolism*
  • Water / chemistry*
  • Young Adult


  • Mutagens
  • Proteins
  • Water

Associated data


Grants and funding

Sources of support are Fund for Scientific Research-Flanders (FWO Vlaanderen) Belgium (FWO project G.0674.10). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.