Colonic Absorption of Low-Molecular-Weight Metabolites Influenced by the Intestinal Microbiome: A Pilot Study

<div><p>Low-molecular-weight metabolites produced by the intestinal microbiome play a direct role in health and disease. However, little is known about the ability of the colon to absorb these metabolites. It is also unclear whether these metabolites are bioavailable. Here, metabolomics techniques (capillary electrophoresis with time-of-flight mass spectrometry, CE-TOFMS), germ-free (GF) mice, and colonized (Ex-GF) mice were used to identify the colonic luminal metabolites transported to colonic tissue and/or blood. We focused on the differences in each metabolite between GF and Ex-GF mice to determine the identities of metabolites that are transported to the colon and/or blood. CE-TOFMS identified 170, 246, 166, and 193 metabolites in the colonic feces, colonic tissue, portal plasma, and cardiac plasma, respectively. We classified the metabolites according to the following influencing factors: (i) the membrane transport system of the colonocytes, (ii) metabolism during transcellular transport, and (iii) hepatic metabolism based on the similarity in the ratio of each metabolite between GF and Ex-GF mice and found 62 and 22 metabolites that appeared to be absorbed from the colonic lumen to colonocytes and blood, respectively. For example, 11 basic amino acids were transported to the systemic circulation from the colonic lumen. Furthermore, many low-molecular-weight metabolites influenced by the intestinal microbiome are bioavailable. The present study is the first to report the transportation of metabolites from the colonic lumen to colonocytes and somatic blood <i>in vivo</i>, and the present findings are critical for clarifying host-intestinal bacterial interactions.</p></div

Metabolites detected in colonic feces but not in colonic tissue.

<p>Metabolites detected in colonic feces but not in colonic tissue.</p

Metabolomic snapshots of the known phenomenon of intestinal microbiome-dependent production of pro-atherogenic trimethylamine <i>N</i>-oxide by degradation of carnitine/choline in the intestinal tract.

<p>FMO: flavin-containing monooxygenase.</p

Estimation of metabolites to transport to body from colonic lumen using the difference of concentration between GF and Ex-GF mice (Ex-GF/GF ratio).

<p>(A) Metabolites with similar Ex-GF/GF ratios in colonic feces, colonic tissue, portal plasma, and cardiac plasma were considered to be transported from the colonic lumen to cardiac blood. (B) Metabolites with different Ex-GF/GF ratios between colonic feces and colonic tissue are controlled by (i) membrane transport system (C) Metabolites with different Ex-GF/GF ratios between colonic tissue and portal plasma are controlled by (ii) metabolism during transcellular transport. (D) Metabolites with different Ex-GF/GF ratios between portal plasma and portal.</p

Differences in the metabolomes of GF and Ex-GF mice.

<p>(A) Number of metabolites that were significantly (<i>p</i> < 0.05) or not significantly different between GF and Ex-GF mice.(B) PCA of metabolome profiles. Fec-Ex, colonic feces of Ex-GF mice; Fec-GF, colonic feces of GF mice; Col-Ex, colon of Ex-GF mice; Col-GF, colon of GF mice; PP-Ex, portal plasma of Ex-GF mice; PP-GF, portal plasma of GF mice; CP-Ex, cardiac plasma of Ex-GF mice; CP-GF, cardiac plasma of GF mice.(C) PCA of metabolome profiles in each specimen.</p

The ExGF/GF ratio and the influence of 3 gateways on each metabolite detected in the colonic feces, colon, portal plasma, and cardiac plasma.

<p>The ExGF/GF ratio and the influence of 3 gateways on each metabolite detected in the colonic feces, colon, portal plasma, and cardiac plasma.</p

Luminal and tissue 5-HT concentrations in the gastrointestinal tract of germ-free (GF) mice and GF mice reconstituted with SPF mouse feces.

<p>(A) GF and EX-GF mice were sacrificed at 10 weeks of age. Luminal (n = 6–9) and (B) tissue (n = 6–8) 5-HT levels in GF (open circles) and EX-GF (closed circles) mice. *** P < 0.001 relative to the corresponding GF value.</p

Dynamics of luminal 5-HT concentration in the gastrointestinal tract of GF mice after colonization with SPF feces.

<p>Male GF mice were sacrificed at 10 weeks of age for 5-HT measurements 3 (P3), 7 (P7), or 21 (P21) days after receiving SPF feces. GF mice without conventionalization were used as a control (P0). ***P < 0.001 and *P < 0.05 compared to the corresponding GF value.</p

Levels of free and conjugated 5-HT in the colonic lumen of GF and EX-GF mice^a.

<p>Levels of free and conjugated 5-HT in the colonic lumen of GF and EX-GF mice<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180745#t002fn001" target="_blank">^a</a>.</p

Relative expression levels of 5-HT-related genes determined with RT-qPCR.

<p>Relative expression levels of 5-HT-related genes determined with RT-qPCR.</p