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Am J Physiol Gastrointest Liver Physiol. 2012 January; 302(1): G1G9.

    Functional analysis of colonic bacterial metabolism: relevant to health?

    Henrike M. Hamer, Vicky De Preter, Karen Windey, and Kristin Verbeke

    Translational Research Center for Gastrointestinal Disorders and Leuven Food Science and Nutrition Research Center, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium

    Corresponding author.

    Address for reprint requests and other correspondence: K. Verbeke, TARGID, Univ. Hospital Leuven, Herestraat 49, 3000 Leuven, Belgium (e-mail: kristin.verbeke@med.kuleuven.be).

    Received February 7, 2011; Accepted October 16, 2011.

    Copyright ? 2012 the American Physiological Society

    Abstract

    With the use of molecular techniques, numerous studies have evaluated the composition of the intestinal microbiota in health and disease. However, it is of major interest to supplement this with a functional analysis of the microbiota. In this review, the different approaches that have been used to characterize microbial metabolites, yielding information on the functional end products of microbial metabolism, have been summarized. To analyze colonic microbial metabolites, the most conventional way is by application of a hypothesis-driven targeted approach, through quantification of selected metabolites from carbohydrate (e.g., short-chain fatty acids) and protein fermentation (e.g., p-cresol, phenol, ammonia, or HS), secondary bile 2

    acids, or colonic enzymes. The application of stable isotope-labeled substrates can provide an elegant solution to study these metabolic pathways in vivo. On the other hand, a top-down 1approach can be followed by applying metabolite fingerprinting techniques based on H-NMR or

    mass spectrometric analysis. Quantification of known metabolites and characterization of metabolite patterns in urine, breath, plasma, and fecal samples can reveal new pathways and give insight into physiological regulatory processes of the colonic microbiota. In addition, specific metabolic profiles can function as a diagnostic tool for the identification of several gastrointestinal diseases, such as ulcerative colitis and Crohn's disease. Nevertheless, future research will have to evaluate the relevance of associations between metabolites and different disease states.

    Keywords: fermentation, metabolites, metabolomics, microbiota, short-chain fatty acids THE HUMAN INTESTINAL MICROBIOTA complements our physiology with functions that we have

    not had to develop on our own. In fact, the intestinal microbiota have a metabolic capacity that is comparable to that of the liver (83). The human colon contains an extremely complex and 11dynamic microbial ecosystem with high densities of living bacteria in concentrations of 10-1210 cells/g of luminal contents belonging to more than 1,000 different species. In healthy adults, 80% of the identified fecal microbiota can be classified into three dominant phyla: Firmicutes, Bacteroidetes, and Actinobacteria, but there is substantial variation in the species composition between individuals (30, 101).

    The intestinal microbiota plays an important role in human physiology. For example, the intestinal microbiota is responsible for the further metabolism and energy harvest from nondigested nutrients, is involved in the synthesis of vitamins such as B and K and metabolism

    of polyphenols, provides colonization resistance toward potential pathogens, is involved in the metabolism of bile acids, and stimulates the immune function of the host (74, 83).

    Molecular approaches, mainly based on the 16S ribosomal RNA gene, have revolutionized the field of gut microbial ecology. Nowadays, the uncultured and complex microbial communities can be characterized with greater sensitivity by using high-throughput technologies, such as pyrosequencing (5) and phylogenetic microarrays (79), compared with former molecular

    fingerprinting methods, such as PCR-denaturing gradient gel electrophoresis (DGGE). Complementary quantitative technologies, such as fluorescence in situ hybridization (FISH) and real-time quantitative PCR, can be used to confirm shifts in particular groups or species (113).

    In recent years an increasing amount of literature has demonstrated that several diseases are related to alterations in the intestinal microbiota (known as dysbiosis), such as irritable bowel syndrome (54), inflammatory bowel disease (93), diabetes (55), atopic diseases (76), cancer (85),

    and obesity (7). For example, a reduction in the abundance and diversity of Firmicutes is frequently associated with inflammatory bowel disease and irritable bowel syndrome (105, 113).

    These studies have mainly shown that differences in the composition of the intestinal microbiota are associated with disease.

    Functional Capacity of the Microbiota

    The human microbiota is characterized by a significant degree of functional redundancy, meaning that different bacteria can perform similar functions and metabolize the same substrate (60, 64). Therefore, not only the composition but also the functional capacity of the intestinal microbiota is highly important regarding the clinical end points.

    Next-generation pyrosequencing can be applied to further evaluate the functional capacity of the colonic microbiota by creating a catalog of the genetic potential of the microbiota. However, it has to be realized that the detection of genes in a metagenomic library does not necessarily mean that these are functionally important (113). To gain better insight into the activity and

    functionality of the intestinal microbiota, other meta-“omics” approaches can be applied that use

    RNA, proteins, and metabolites as targets. In this review, we summarize the different approaches that have been used to quantify and characterize the metabolites produced by the microbiota, which yield information on the actual end products of metabolism. Colonic microbial fermentation results in the production of large amounts of different end products. The type and amount of these fermentation-derived metabolites largely depend on the composition of the microbiota, transit time, and the substrates available for fermentation (95). Some of these end

    products have been shown to be protective to the colonic epithelium, and others have proved to be proinflammatory or procarcinogenic metabolites (3, 48). By using knowledge of these specific

    metabolites, a hypothesis-driven targeted approach can be applied to evaluate changes in colonic metabolism following dietary interventions or during different disease states, for example, through quantification of selected metabolites from carbohydrate and protein fermentation, secondary bile acids, or colonic enzymes. On the other hand, a top-down approach can be 1followed by applying metabolite fingerprinting techniques based on H-nuclear magnetic

    resonance (NMR) or mass spectrometric analysis. By following this approach, novel metabolites and mechanisms can be identified that are involved in health and disease. This is, however, not an easy task, since the signals first have to be identified and their metabolic roles elucidated. For analyses of metabolites as end products of intestinal metabolism in humans, we mainly rely on fecal samples or on breath (for example, hydrogen, methane, and carbon dioxide), urine, and plasma samples due to the relative inaccessibility of the colon to sample at different locations

    (Fig. 1) (50). In these human studies, an elegant solution to study metabolic pathways in vivo is the application of stable isotope tracers.

    Types of Fermentation

    The colonic microbiota ferment endogenous host-derived substrates such as mucus, pancreatic enzymes, and exfoliated epithelial cells, as well as dietary components that escape digestion in the upper gastrointestinal tract. Two main types of colonic microbial fermentation can be distinguished, including saccharolytic fermentation of carbohydrate and proteolytic fermentation of protein (Fig. 2). In the proximal part of the colon, mainly saccharolytic fermentation takes place, since most microorganisms preferentially ferment carbohydrates and switch to protein fermentation when carbohydrate sources are depleted (75). The main products of carbohydrate

    metabolism are short-chain fatty acids (SCFA), mainly acetate, propionate, and butyrate, which have been shown to contribute to colonic health. SCFA provide energy to the colonic epithelial cells, decrease luminal pH, and improve mineral absorption. Furthermore, butyrate has been shown to possess an anti-inflammatory and anticarcinogenic potential (46, 63). Besides their

    contribution to gut health and maintenance, SCFA may provide further benefits for the systemic metabolism. For example, it has been shown that acetate and propionate affect hepatic lipid metabolism. Acetate is the primary substrate for cholesterol synthesis, whereas propionate can inhibit cholesterol synthesis (27, 110). Since the different SCFA may show distinct effects, not

    only the amount of SCFA produced but also their ratio is of importance with regard to these health effects. Furthermore, SCFA stimulate increased plasma levels of satiety hormones such as peptide YY (PYY), leptin, and glucagon-like peptide-1 (GLP-1) and may attenuate insulin resistance (1, 34). These effects may occur due to the fact that SCFA are ligands for G protein-coupled receptors (GPR) 41 and 43, expressed on adipocytes, enteroendocrine L-cells, and immune cells (88). In addition, recent studies have linked SCFA activation of GPR 43 to the suppression of colon cancer (94, 96). Proteolytic fermentation also leads not only to the

    production of SCFA (lower amounts than produced from carbohydrates) and branched-chain fatty acids but also to potentially toxic metabolites such as phenolic compounds, sulfur-containing compounds, amines, and ammonia (Fig. 2) (48). The toxicity of these protein

    fermentation metabolites has mainly been established in in vitro studies (6, 11, 58) and animal

    studies (4, 97). However, human epidemiological studies do not consistently support an association between protein intake and colorectal cancer (2) and inflammatory bowel disease

    (47). Unfortunately, actual protein fermentation metabolites were not determined in these studies. The different metabolites of protein fermentation and their potential involvement in colorectal cancer have previously been reviewed (48).

    Targeted Approach

    Quantification of carbohydrate fermentation.

    The beneficial effects of saccharolytic fermentation have mainly been ascribed to the production of SCFA (68, 91). Several studies have evaluated SCFA concentrations and profiles in patients with different diseases. For example, a lower butyrate-to-acetate ratio has been found in colonic luminal content of patients with adenomatous polyps or colon cancer compared with healthy controls (107), and increased amounts of SCFA were found in fecal samples from obese compared with lean individuals (89). Dietary interventions with prebiotics, such as inulin and

    oligofructose, defined as “nondigestible food ingredients that stimulate the growth and/or activity of bacteria in the digestive system which are beneficial to the health of the host,” aim to increase and prolong the saccharolytic fermentation toward the distal colon and thereby aim to reduce proteolytic fermentation (38) (Fig. 3). Increased SCFA production after addition of different

    prebiotics to the diet has previously been demonstrated by measuring fecal or plasma concentrations of SCFA (52, 73). However, the in situ production of SCFA is difficult to

    determine, since more than 95% of the produced SCFA are absorbed and metabolized by the host depending on the gastrointestinal transit time (59). To gain more insight into the actual SCFA

    production over time, the use of stable isotope tracers can be considered. A recent study 13with C-labeled barley has evaluated the kinetics of SCFA appearance in the systemic circulation in healthy volunteers. In this study, the pattern of SCFA appearing in the systemic circulation was different after consumption of a meal with dietary fiber (nonstarch polysaccharides) combined with resistant starch compared to a meal with dietary fiber alone (102). Further studies are necessary to address the significance of these different SCFA profiles with regard to health benefits. In another study, stable isotope tracers were applied to determine the production rate of acetate during colonic fermentation of lactulose in humans. Healthy 13volunteers received a primed continuous infusion of [1-C]acetate followed by the ingestion of

    lactulose. The colonic acetate production was calculated from the reduction in the plasma 13[C]acetate enrichment as a result of the colonic fermentation of lactulose (77). With the use of 13a dynamic in vitro model of the large intestine, the fermentation of C-labeled starch was

    evaluated by determination of the label incorporation into the microbial biomass and metabolites using stable isotope probing and NMR analysis (53). From the labeling pattern of microbial

    metabolites, it was concluded that cross-feeding between Ruminoccus bromii and Eubacterium

    rectale occurred, wherein R. bromiiproduced acetate, which was subsequently converted to

    butyrate by E. rectale.

    Quantification of protein fermentation.

    The degree of proteolytic fermentation can be determined by quantification of typical end products of protein fermentation (Fig. 2) (48). Some of these metabolites are reused as nitrogen

    source for bacterial growth, whereas others will be taken up by colonocytes and transported into the bloodstream. For instance, phenols and indoles are breakdown products of the aromatic acids tyrosine and tryptophan, respectively. Generally, once these compounds are produced, they enter the hepatic circulation to be detoxified in the liver and eventually excreted in urine. Since p-

    cresol and phenol are unique bacterial metabolites from protein fermentation that are not produced by human enzymes, these metabolites have been frequently used to assess the degree of proteolytic fermentation (25, 37). Urinary levels of p-cresol and phenol have shown to be

    increased during high protein intake (37) and decreased after oral supplementation with

    oligofructose-enriched inulin (OF-IN) (25).

    In hemodialysis patients, p-cresol generation and p-cresyl sulfate serum concentrations were

    lowered after oral OF-IN administration (69). As renal function declines in these patients,

    substances that are either excreted or metabolized by the kidney accumulate in the circulation, resulting in increased levels of numerous molecules in blood (33). A number of these retained solutes originate from colonic bacterial protein metabolism (33). In addition, small intestinal assimilation of proteins is impaired in renal failure (10), resulting in an increased availability of

    proteins for fermentation in the colon (31). Accumulation of the protein fermentation metabolites

    in serum has been suggested to alter endothelial function (32, 70) and has been associated with

    increased mortality in hemodialysis patients (9). As a consequence, a dietary strategy with OF-

    IN that contributes to a lower generation of protein fermentation metabolites might constitute a significant improvement in the treatment of these patients. In addition to p-cresol and phenol,

    other protein fermentation metabolites, such as sulfur-containing metabolites, were decreased in fecal samples after incubation with OF-IN in vitro (22). Increased concentrations of sulfides have

been associated with the pathogenesis of ulcerative colitis (UC). Hydrogen sulfide (HS) has 2

    been found to provoke genomic DNA damage in colonic cancer cells (HT-29 cells) in concentrations similar to those found in the human colon (6). In addition to inducing DNA

    damage, sulfide impairs the oxidation of butyrate, the major energy substrate in colonocytes, by