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

Karen Windey, Vicky De Preter, Thierry Louat, Frans Schuit, Jean Herman, Greet Vansant, Kristin Verbeke, Karen Windey, Vicky De Preter, Thierry Louat, Frans Schuit, Jean Herman, Greet Vansant, Kristin Verbeke

Abstract

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: ClinicalTrial.gov NCT01280513.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Enrollment of the volunteers.
Figure 1. Enrollment of the volunteers.
Figure 2. Bland-Altman plot comparing energy intake…
Figure 2. Bland-Altman plot comparing energy intake measured using indirect calorimetry (kcal/d) and energy intake calculated from the dietary records (kcal/d).
Mean energy intake measured by calorimetry and dietary record are plotted against the difference between energy intake measured by calorimetry and by dietary record.
Figure 3. Scatter plot of the comparison…
Figure 3. Scatter plot of the comparison between urinary p-cresol excretion (mg/24 h) and absolute protein intake (g/24 h).
Urinary p-cresol excretion correlated positively with absolute protein intake (Spearman’s r = 0.371, p = 0.007).
Figure 4. Scatter plot in which cytotoxicity…
Figure 4. Scatter plot in which cytotoxicity (IC50) is plotted against urinary p-cresol excretion (mg/24 h).
The plot shows a negative correlation between both parameters (Spearman’s r = −0.435, p = 0.001).
Figure 5. Score plots showing clustering of…
Figure 5. Score plots showing clustering of the metabolite profiles analyzed using PLS-DA according to genotoxicity (A) and cytotoxicity (B).
(A) High genotoxicity samples are located on the right side of the score plot, while low genotoxicity samples are present on the left side, indicating a difference in VOC profile between high and low genotoxicity samples. (B) High cytotoxicity samples are present on the upper right side of the score plot and low cytotoxicity samples on the lower left side, indicating a difference in VOC profile between high and low cytotoxicity samples.
Figure 6. Main metabolic pathways for the…
Figure 6. Main metabolic pathways for the production of microbial metabolites.
VOC are shown in bold .

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