Lise Deroover

Systemic availability and metabolism of colonic-derived short-chain fatty acids in healthy subjects: a stable isotope study

The short‐chain fatty acids (SCFAs) are bacterial metabolites produced during the colonic fermentation of undigested carbohydrates, such as dietary fibre and prebiotics, and can mediate the interaction between the diet, the microbiota and the host. We quantified the fraction of colonic administered SCFAs that could be recovered in the systemic circulation, the fraction that was excreted via the breath and urine, and the fraction that was used as a precursor for glucose, cholesterol and fatty acids. This information is essential for understanding the molecular mechanisms by which SCFAs beneficially affect physiological functions such as glucose and lipid metabolism and immune function.

Quantification of in Vivo Colonic Short Chain Fatty Acid Production from Inulin

Short chain fatty acids (SCFA), including acetate, propionate, and butyrate, are produced during bacterial fermentation of undigested carbohydrates in the human colon. In this study, we applied a stable-isotope dilution method to quantify the in vivo colonic production of SCFA in healthy humans after consumption of inulin. Twelve healthy subjects performed a test day during which a primed continuous intravenous infusion with [1-13C]acetate, [1-13C]propionate and [1-13C]butyrate (12, 1.2 and 0.6 μmol·kg−1·min−1, respectively) was applied. They consumed 15 g of inulin with a standard breakfast. Breath and blood samples were collected at regular times during the day over a 12 h period. The endogenous rate of appearance of acetate, propionate, and butyrate was 13.3 ± 4.8, 0.27 ± 0.09, and 0.28 ± 0.12 μmol·kg−1·min−1, respectively. Colonic inulin fermentation was estimated to be 137 ± 75 mmol acetate, 11 ± 9 mmol propionate, and 20 ± 17 mmol butyrate over 12 h, assuming that 40%, 10%, and 5% of colonic derived acetate, propionate, and butyrate enter the systemic circulation. In conclusion, inulin is mainly fermented into acetate and, to lesser extents, into butyrate and propionate. Stable isotope technology allows quantifying the production of the three main SCFA in vivo and proved to be a practical tool to investigate the extent and pattern of SCFA production.

Contribution of Colonic Fermentation and Fecal Water Toxicity to the Pathophysiology of Lactose-Intolerance

Whether or not abdominal symptoms occur in subjects with small intestinal lactose malabsorption might depend on differences in colonic fermentation. To evaluate this hypothesis, we collected fecal samples from subjects with lactose malabsorption with abdominal complaints (LM-IT, n = 11) and without abdominal complaints (LM-T, n = 8) and subjects with normal lactose digestion (NLD, n = 15). Lactose malabsorption was diagnosed using a 13C-lactose breath test. Colonic fermentation was characterized in fecal samples at baseline and after incubation with lactose for 3 h, 6 h and 24 h through a metabolomics approach using gas chromatography-mass spectrometry (GC-MS). Fecal water cytotoxicity was analyzed using a colorimetric assay. Fecal water cytotoxicity was not different between the three groups (Kruskall-Wallis p = 0.164). Cluster analysis of the metabolite patterns revealed separate clusters for NLD, LM-T and LM-IT samples at baseline and after 24 h incubation with lactose. Levels of 5-methyl-2-furancarboxaldehyde were significantly higher in LM-IT and LM-T compared to NLD whereas those of an unidentified aldehyde were significantly higher in LM-IT compared to LM-T and NLD. Incubation with lactose increased short chain fatty acid (SCFA) concentrations more in LM-IT and LM-T compared to NLD. In conclusion, fermentation patterns were clearly different in NLD, LM-IT and LM-T, but not related to differences in fecal water cytotoxicity.

Wheat Bran Does Not Affect Postprandial Plasma Short-Chain Fatty Acids from (13)C-inulin Fermentation in Healthy Subjects

Wheat bran (WB) is a constituent of whole grain products with beneficial effects for human health. Within the human colon, such insoluble particles may be colonized by specific microbial teams which can stimulate cross-feeding, leading to a more efficient carbohydrate fermentation and an increased butyrate production. We investigated the extent to which WB fractions with different properties affect the fermentation of other carbohydrates in the colon. Ten healthy subjects performed four test days, during which they consumed a standard breakfast supplemented with 10 g 13C-inulin. A total of 20 g of a WB fraction (unmodified WB, wheat bran with a reduced particle size (WB RPS), or de-starched pericarp-enriched wheat bran (PE WB)) was also added to the breakfast, except for one test day, which served as a control. Blood samples were collected at regular time points for 14 h, in order to measure 13C-labeled short-chain fatty acid (SCFA; acetate, propionate and butyrate) concentrations. Fermentation of 13C-inulin resulted in increased plasma SCFA for about 8 h, suggesting that a sustained increase in plasma SCFA can be achieved by administering a moderate dose of carbohydrates, three times per day. However, the addition of a single dose of a WB fraction did not further increase the 13C-SCFA concentrations in plasma, nor did it stimulate cross-feeding (Wilcoxon signed ranks test).

Mo1799 Increased Fecal Water Cytotoxicity in Patients With Ulcerative Colitis Is Associated With Low Levels of Short Chain Fatty Acids

Background: Many studies have focused on the fecal microbiota however it is now widely accepted that the gastrointestinal (GI) mucosa associated microbiota, rather than luminal content, is critically important in host-microbe interactions linked to health and disease. Mucosal biopsy is the most common sampling technique used to assess the mucosa associated microbiota. However, normal biopsy devices are designed to take samples for histologic assessment. Thus when biopsies are taken via working channels that are also used to aspirate luminal content it is highly likely that cross contamination occurs, that ultimately questions the validity of these samples. In view of this difficulty we have developed a novel device that allows targeted biopsies to be taken in any segment of the GI tract without cross contamination in an aseptic manner. Methods: Six patients undergoing upper GI endoscopy for iron deficiency were recruited with consent and ethical approval. In pilot experiments various prototypes of a sampling device were tested in vivo. Two final configurations of a sheath that covered miniature biopsy forceps were tested and compared with the normal single-use biopsy forceps (18 samples). Matched duodenal biopsy samples were collected from each patient, transferred individually into RNA later, and subject to gDNA extraction. Amplicon libraries spanning the V6-V8 region of the 16S rRNA gene were constructed, sequenced using the Illumina MiSeq platform, and analysed via the QIIME pipeline. Results: Microbial DNA, representing a diverse community, was observed in all duodenal samples obtained using the aseptic device, indicating this device is effective in sampling mucosa associated organisms present in the duodenum (Chao 1 estimated richness = 386, Goods coverage 99.8%). Assessment of the duodenal microbiota revealed a community dominated by the genera Streptococcus, Prevotella, Veillonella, Neisseria and Porphyromonas. There were substantial differences in the microbiota of samples obtained using the aseptic device and samples obtained with standard biopsy forceps. Only a low level of correlation (Pearsons r<0.6) between the matched biopsy pairs was seen across all bacterial phyla observed. Phylogenetic based (UniFrac) evaluation of beta-diversity revealed the aseptic samples clustered together and showed reduced inter-individual variability compared to those obtained with standard forceps. Conclusions: Sampling of duodenal biopsies with routine biopsy forceps resulted in greater microbial diversity as compared to samples acquired under aseptic conditions. This suggests cross contamination from other sites of the upper GI tract when utilising standard forceps. Based upon our data, aseptic techniques with a specific sampling device should be used to collect samples for studies that aim to define the mucosa associated microbiota.

Mo1800 Colonic Luminal Compounds Do Not Affect Butyrate Metabolism in Ulcerative Colitis

Background: Many studies have focused on the fecal microbiota however it is now widely accepted that the gastrointestinal (GI) mucosa associated microbiota, rather than luminal content, is critically important in host-microbe interactions linked to health and disease. Mucosal biopsy is the most common sampling technique used to assess the mucosa associated microbiota. However, normal biopsy devices are designed to take samples for histologic assessment. Thus when biopsies are taken via working channels that are also used to aspirate luminal content it is highly likely that cross contamination occurs, that ultimately questions the validity of these samples. In view of this difficulty we have developed a novel device that allows targeted biopsies to be taken in any segment of the GI tract without cross contamination in an aseptic manner. Methods: Six patients undergoing upper GI endoscopy for iron deficiency were recruited with consent and ethical approval. In pilot experiments various prototypes of a sampling device were tested in vivo. Two final configurations of a sheath that covered miniature biopsy forceps were tested and compared with the normal single-use biopsy forceps (18 samples). Matched duodenal biopsy samples were collected from each patient, transferred individually into RNA later, and subject to gDNA extraction. Amplicon libraries spanning the V6-V8 region of the 16S rRNA gene were constructed, sequenced using the Illumina MiSeq platform, and analysed via the QIIME pipeline. Results: Microbial DNA, representing a diverse community, was observed in all duodenal samples obtained using the aseptic device, indicating this device is effective in sampling mucosa associated organisms present in the duodenum (Chao 1 estimated richness = 386, Goods coverage 99.8%). Assessment of the duodenal microbiota revealed a community dominated by the genera Streptococcus, Prevotella, Veillonella, Neisseria and Porphyromonas. There were substantial differences in the microbiota of samples obtained using the aseptic device and samples obtained with standard biopsy forceps. Only a low level of correlation (Pearsons r<0.6) between the matched biopsy pairs was seen across all bacterial phyla observed. Phylogenetic based (UniFrac) evaluation of beta-diversity revealed the aseptic samples clustered together and showed reduced inter-individual variability compared to those obtained with standard forceps. Conclusions: Sampling of duodenal biopsies with routine biopsy forceps resulted in greater microbial diversity as compared to samples acquired under aseptic conditions. This suggests cross contamination from other sites of the upper GI tract when utilising standard forceps. Based upon our data, aseptic techniques with a specific sampling device should be used to collect samples for studies that aim to define the mucosa associated microbiota.