Gut Microbial Activity, Implications for Health and Disease: The

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Gut Microbial Activity, Implications for Health and Disease: The Potential Role of Metabolite Analysis Edna. P. Nyangale,*,† Donald. S. Mottram,† and Glenn. R. Gibson‡ †

The University of Reading, Food and Nutritional Sciences, Whiteknights, PO Box 226, Reading RG6 6AP, United Kingdom The University of Reading, Food Microbial Sciences Unit, Whiteknights, PO Box 226, Reading RG6 6AP, United Kingdom

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ABSTRACT: Microbial metabolism of proteins and amino acids by human gut bacteria generates a variety of compounds including phenol, indole, and sulfur compounds and branched chain fatty acids, many of which have been shown to elicit a toxic effect on the lumen. Bacterial fermentation of amino acids and proteins occurs mainly in the distal colon, a site that is often fraught with symptoms from disorders including ulcerative colitis (UC) and colorectal cancer (CRC). In contrast to carbohydrate metabolism by the gut microbiota, proteolysis is less extensively researched. Many metabolites are low molecular weight, volatile compounds. This review will summarize the use of analytical methods to detect and identify compounds in order to elucidate the relationship between specific dietary proteinaceous substrates, their corresponding metabolites, and implications for gastrointestinal health. KEYWORDS: protein fermentation, phenol, indole, p-cresol, CRC, volatile analysis

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INTRODUCTION The gut of an unborn child is considered sterile and bacterial colonisation occurs during the birth process via inoculation from maternal microbiota, the environment and diet subsequent to this. Following birth, when the infant gut becomes inoculated, growth of bacteria begins with facultative anaerobes being the initial colonizers. These consume available oxygen creating ideal conditions for the growth of more obligate anaerobes like Bifidobacterium spp. and Bacteroides spp.1 Diet also plays an important part in the initial development of the microbiota as colonization of breastfed infants occurs predominantly by Bif idobacterium spp.2 and that of formulafed infants occurs by anaerobes like Clostridium spp.3,4 The presence of complex oligosaccharides in human breast milk is stimulatory to bifidobacteria. By the age of two years, the intestinal flora begins to resemble that of an adult. Many different microorganisms exist along the gastrointestinal tract (GIT); populations consist of commensal and transient bacteria, which can have a significant impact upon host health via modulation of immunity, intestinal development, protection against pathogens and fermentation of substrates largely provided by the diet. In recent years, dysbacteriosis of the microbiome is associated with an expanding list of chronic diseases including obesity, inflammatory bowel disease (IBD), colorectal cancer (CRC) and type 2 diabetes. As the colonic microbiota plays host to some 1011− 1012 cfu/g content of bacteria with more than 1000 distinct species, it is not difficult to deduce that these bacteria play an important role in health, metabolism and nutrition. This occurs mainly by fermenting substrates that have escaped digestion in the stomach and small intestine, with the end products having different implications for host health.5 Indigenous bacteria also © XXXX American Chemical Society

influence colonization of pathogens by creating conditions adverse to their growth and by competitive exclusion. Factors that affect the microbial composition in the gut include host genetics,6 geography,7 mode of birth,2 diet,8,9 antibiotic use10,11 and the use of pre- or probiotics.11,12 The main functions of the gut microbiota can be divided into three distinct aspects: metabolic, trophic and protective.13 Trophic functions include control of cell proliferation and stability of the immune system. Metabolic functions of the microbiota are based around fermentation of available substrates which have escaped digestion in the upper GIT and, to a lesser extent, sloughed off intestinal epithelial cells. Fermentation of carbohydrates is often focused upon because of the production of beneficial short chain fatty acids (SCFA). Amino acids and proteins also escape digestion in the upper GIT and become available for colonic fermentation,14 however production of metabolites including ammonia, phenolic and indolic compounds is less preferential as several are known to exert toxic effects on the lumen.15−17 Proteolysis occurs along the entire colon but increases in the distal colon, where metabolites formed alter pH conditions to around 6.6−6.9.18 The relationship between diet and health is a long-standing one; it is now, however, moving from the general association between increased intake of certain micro/macro molecules and modulation of health toward elucidation of mechanisms involved. The relationship between increased meat consumption and wellbeing is complex due to a parallel increase in saturated fats, haem iron and heterocyclic amines, all of which are associated with a reduction in health status. A further factor Received: July 13, 2012

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dx.doi.org/10.1021/pr300637d | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

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then ready to be transported to the necessary tissues and organs. Early analysis of human volunteers undergoing gastric bypass revealed differences in their microbiota, this constitutes some of the earliest work linking changes in the gut environment to host adiposity.26 Using more robust techniques that have since been developed, the shift in the microbiota based on host adiposity has been shown with the use of the leptin-dificient ob/ob mouse which is predisposed to obesity. Culture independent techniques show higher Firmicutes and lower Bacteroidetes than the wild-type counterpart.27 It was also shown that adopting the “western diet” resulted in a shift within Firmicutes which has also been detected in obese humans, thus linking a shift in the microbiota to obesity.28 The balance of Firmicutes and Bacteroidetes also appears to change as obese individuals adopt a weight reducing diet.29 Analysis of conventionally raised (CONV-R) mice and germ free mice (GF) has shown that GF mice have decreased adiposity and hepatic triglycerides in comparison to CONV-R mice. They also appear to be resistant to diet-induced obesity, raising the interest into the effect of the microbiota on diet related obesity and the serum metabolome and lipidome.30 The gut microbiota has also been suggested to increase energy harvest from nutrients that escape digestion in the upper gastrointestinal tract and are available for fermentation in the gut. The production of SCFA occurs in the gut as a consequence of saccharolytic activity; these SCFA have been shown to reach the portal circulation after absorption in the gut.31 The remaining fatty acids which are not absorbed or excreted are used by the colonocytes as energy or are used by the liver for glucogenesis. Obese mice have also been shown to have much higher cecal SCFA suggesting the microbiota may affect lipid metabolism. The microbiota is suggested to affect lipid metabolism in several ways both in serum and via the liver. Lipoprotein lipase (LPL) is a fatty acid release regulator and facilitates the release from chylomicromns, the form in which fatty acids and very low-density lipoproteins are transported from the liver to adipose tissues. An increase in adipose LPL increased cellular uptake of fatty acids; the microbiota is suggested to downregulate the expression of an LPL inhibitor, which results in increased uptake of fatty acids via increased LPL activity. Another way in which the microbiota is suggested to affect lipid absorption is via increasing the expression of several genes including carbohydrate response element binding protein (CREBP), sterol response element binging protein 1 (SREBP1), acetyl CoA carboxylase (Acc1) and fatty acid synthase (FAS), the latter two of which are rate limiting enzymes in lipogenesis, which results in increased fatty acid levels as shown when GF mice are colonized.27,32

is increased protein consumption and the subsequent metabolism by colonic microbiota. Faecal water contains many bioactive compounds which are the result of bacterial transformation of dietary components, including secondary bile acids, phenolics and sulfur compounds alongside some beneficial compounds including SCFA and polyphenols. It is the presence of these compounds, retained in the non-solid phase of faeces that has led to the use of faecal water to determine the potentially toxic environment to which the lumen is exposed. This review intends to summarize compounds produced during fermentation by the microbiota, their relationship to the onset of gut disorders and their detection.

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SACCHAROLYTIC ACTIVITY Saccharolytic activity in the gut concerns the breakdown of carbohydrates by bacteria including species from the genera Bacteroides spp., Lactobacillus spp. and Bifidobacterium spp.18 The principal products of this degradation are SCFA (mainly butyrate, propionate, and acetate), lactate and other products including C02, H2, methane and ethanol. Production of SCFA mainly occurs via the Embden-Meyerhof-Parnas pathway which breaks down glucose molecules to pyruvate which is then transformed into SCFA and other organic acids. Colonocytes gain energy from SCFA, largely from butyrate.19 A large proportion of SCFA are absorbed in the lumen and less than 5% is excreted in faecal matter.20 Substrates involved in the production of SCFA include carbohydrate fibres, resistant starch, oligosaccharides, proteins and amino acids. In most cases, acetate is the most abundant SCFA followed by propionate then butyrate.18 Production of SCFA is one main benefits associated with the use of prebiotics. Prebiotics are nondigestible carbohydrates which surpass the upper GIT and reach the large gut intact and are then selectively fermented by bacteria that elicit beneficial effects.21 Impairment of the ability of the colonocytes to use SCFA as a source of energy can contribute to forms of IBD such as UC. Consequently, infusion of butyrate to UC sufferers leads to a reduction in symptoms of distal colitis.19,22 Rats fed a high wheat bran diet had significantly less malignant tumors than those on a high fiber diet; this correlated with higher levels of major SCFA in faecal samples of rats on a wheat bran diet.23 As the majority of saccharolytic activity occurs in the ascending colon, concentrations of SCFA are higher in this region, decreasing throughout the hindgut.24 An increase in luminal SCFA can be achieved by introducing nonstarch polysaccharides (NSP) and resistant starch to the diet. SCFA have been linked to a decrease in cell proliferation and apoptosis of colon carcinoma cell lines.25 However, data are lacking directly linking the increase in dietary fiber to reduced risk of colorectal cancer. Depletion in SCFA concentrations along the colon correlated with an increased prevalence of benign and malignant tumors in the distal colon, leading to the conclusion that beneficial effects seen by increased dietary consumption of resistant starches and NSP was due to their fermentation.20,23

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PROTEOLYTIC ACTIVITY Proteins from sources thought to be highly digestible can escape assimilation in the upper GIT and enter the colon following ingestion. For example, around 6% and 35% of cooked and raw egg proteins respectively escape digestion in the small intestine and reach the colon in healthy volunteers.33 Nitrogenous material in the large intestine exists mainly from proteins and amino acids, roughly 6−18 g/day reaches the colon and is available for bacterial fermentation.34 Although an increase in dietary protein greatly affects that which reaches the colon, endogenous secretions in the form of mucus and pancreatic secretions also have an impact.35 However, protein

LIPID METABOLISM IN THE GUT

On reaching the small intestine, dietary fats, which are in the form of triglycerides, are emulsified by bile; this allows pancreatic lipase to release free fatty acids and monoglycerides which can then be absorbed by the gut. Once absorbed they are packaged up into chylomicrometers and liposomes which are B



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Figure 1. Fate of dietary and nondietary proteins in the human large gut.

that reaches the colon is not restricted to those derived from animal sources, legumes like soya bean (G. max), peanuts (A. hypogaea) and chickpeas (C. arietinum) also contribute to dietary protein load, specifically in those who do not consume animal derived proteins. These ingredients constitute the major source of essential amino acids for those on vegetarian and vegan diets; however this does not imply a reduced dietary protein intake. Dietary macronutrient load of four diets including vegan, lacto-vegetarian, whole food omnivore and average omnivore were carried out by Lockie et al.36 This showed that dietary protein intake did not differ by a large amount, with those following a vegan diet consuming slightly less. This indicates that proteolytic activity may not only be influenced by dietary protein load but also its source. The dominant proteolytic bacteria in the human colon include Bacteroides spp. and Clostridium spp.37 In vitro studies using human faecal samples and pure cultures have identified several species of Clostridium spp. with proteolytic ability, via identification of known metabolites.38,39 Metabolites formed are dependent on the type and availability of amino acid precursors. Figure 1 shows the main metabolites formed. Several primary and secondary metabolites of amino acid fermentation are suggested to have a toxic effect on the colonic lumen and are implicated in the etiology of several gut disorders.

and produce sulfur compounds like H2S by reduction of sulfate. Bacterial fermentation of sulfur containing amino acids like cysteine also occurs and adds to the pool of colonic H2S. Cysteine desulfhydrase also converts cysteine to pyruvate, H2S and NH3. This ability occurs in bacteria like E. coli and Clostridium spp.40 An increase in dietary protein has been shown to positively correlate with an increase in urinary sulfide, showing that a dose-dependent relationship may exist.41 The majority of sulfide in the lumen is in the form of H2S but a smaller proportion, roughly 60 μmol/L (8%), is suggested to be free in the form of the sulfide ion.42 Diet contributes roughly 1.5−16 mmol per day of inorganic sulfate and 3.8 mmol per day of protein derived sulfate.35 Endogenous detoxification of free sulfide in the lumen has been suggested to occur via methylation of hydrogen sulfide to aliphatic sulfur compound methanethiol and then to dimethyl sulfide. However, the rate at which this occurs is much less than the suggested rate of hydrogen sulfide production as demonstrated in the rat cecum (10−7 mol/min),43,44 leaving a net increase of reactive, toxic hydrogen sulfide in the colonic lumen. There are also several other sulfur containing compounds including thiophenes and several thiazole compounds and several other aliphatic sulfur compounds that have been detected in animal derived proteins and during volatile analysis of stool samples. Indicating that although hydrogen sulphide appears to have more evidence to its link with a reduction in gut health, other sulfur containing compounds also make a significant contribution toward luminal

Sulfur Compounds

Sulfate-reducing bacteria like Desulfomonas spp. and Desulfovibrio spp. possess the ability to oxidize organic substrates to CO2 C



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Scheme 1. Reaction Mechanism for the Formation of N-Nitrosodimethylamine via Nitrosation of Dimethylamine by Dinitrogen Trioxide

cells in which concentrations as low as 250 μmol/L caused significant DNA damage to CHO at levels below that detected in faecal samples. Although HT-29 cells appeared to be less sensitive to damage, slight genomic damage was observed. This shows that increased luminal sulfide concentrations can affect barrier function of cells, leading to increased passage of compounds. Then once inside the cell it can go on to confer more damage by alteration of genes.52,53 On the contrary, studies on stool samples of a cohort of asymptomatic donors and patients with various types of IBD found no statistical difference in the detectable levels of volatile sulfur-containing compounds in the headspace of faecal samples.54 However, there is a distinct lack of recent data to supplement this. It has been suggested that volatiles detected from faecal samples may have use in a noninvasive diagnosis of gut disorders.55

sulfur concentration and this should possibly be an area of investigation for further studies into the overal contribution of each.45,46,55 However, contributions to the dietary load are made not only from protein sources but also from sulphates contained naturally in many fruits and vegetables and those included as additives during processing. Alongside is the possibility that increased levels of taurine in energy drinks may also constitute a reasonable contribution. An in vitro investigation looked at fermentation of green alga sea lettuce on metabolism and production of sulphates and concluded, partially as expected, that the bulk of the plant material was not broken down in the semi-continuous culture that was inoculated with human faecal microbiota.47 Less than 10% was transformed into short chain fatty acids, but 40% of sulphates contained were dissimilated by sulfate reducing bacteria to sulphides, demonstrating that dietary sulfate sources are not solely constricted to protein intake. Therefore, the link between high protein diets, specifically processed protein and increased luminal sulphide, therefore encompasses an overall load from many dietary sources where those on a vegetarian or low protein intake only have to contend with those derived from plant material, which appears to be less bioavailable due to the decreased capacity to dissimilate plant materials. This does however leave the possibility that increased consumption of taurine-based energy drinks may also play a role in elevated colonic load of sulphates. Taurine can also be assimilated by the body from cystine and so is naturally present in the body due to this as well as intake from meat and fish. However, the European Food Standards Agency (EFSA) recognizes that the amount of taurine consumed from energy drinks is more than that consumed on a normal omnivore diet, indicating that this may constitute a substantial load on the lumen as increased dietary intake of protein and amino acids results in increased colonic load of the amino acids and any metabolites formed that may also pose toxic effects. However, the current levels in available drinks is not seen to be excessive although many consulted studies concentrated only on cardiovascular and neurological effects; therefore, the effect on metabolism in the gut remains to be investigated.48 Hydrogen sulfide has been linked to incidences of inflammation of the colonic lumen, specifically UC.15 This is thought to be due to its ability to reduce barrier function of colonocytes thereby affecting their ability to mediate passage of solutes, a characteristic shared by known carcinogens and other protein metabolites.49 Luminal concentrations of sulfide have been documented at around 1.0−2.4 mmol/L50 and faecal concentrations of 0.22−3.38 mmol/kg.41,51 Genotoxicity of sulfide has also been investigated via the use of Chinese hamster ovaries (CHO) and human HT-29 colonic epithelial

N-Nitroso Compounds

N-Nitroso compounds (NOC) are endogenously formed via nitrosation of organic compounds, like amines, by nitrite sources. Endogenous N-nitrosation of primary and secondary amines form unstable and stable nitrosamines, respectively.56 Several secondary NOC are known to be carcinogenic and alkylation of DNA in GI tissues can cause base pair transitions seen in some cases of colorectal cancer.57−59 An increase in total N-nitroso compounds has been detected in faecal contents of individuals on high red meat diets compared to low red meat and vegetarian diets.60 This was suggested to be due to the presence of high levels of haem in red meat, more specifically iron. Beef steak and lamb contain 7.5 and 2.7 mg/g of haem as hemoglobin equivalent.56 However, levels observed in chicken and tuna are considerably low, at 0.3 and 0.6 mg/g, respectively, and white meat has been shown to cause fewer DNA double strand breaks in rats. Thus, increased consumption of white meat does not significantly correlate with colorectal cancer risk.61−63 Organic iron, mainly in the form of haem, has been suggested as one cause for elevated levels of NOC, often measured as apparent total N-nitroso compounds (ATNC). However, studies have reported analytical difficulties in measuring total NOC; methods developed to detect specific nitrosamines in biological fluids have been prone to false positives due to the abundance of nitrogen containing compounds.15,64 The ability of haem to catalyze endogenous formation of Nnitrosamines and free radicals begins with its own nitrosylation in the GIT, forming nitrosylhaem which then goes on to donate a nitrosonium ion to available amines and amino acids in the lumen.56 Other nitrosating agents include nitrous acid, nitric oxide, nitrosothiols and nitrite, which yield nitrous acid under D



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components derived from protein sources, to form compounds linked with a reduction in health status.

acidic conditions. Compounds, such as phenols, found in biological fluids of individuals on a high protein diet can also effect the formation of NOC. Nitric oxide is a free radical with a half-life of a few seconds due to its high reactivity; it is formed from amino acid Larginine, oxygen and nicotinamide adenine dinucleotide phosphate (NADPH) via different nitric oxide synthase enzymes.35,65 Although nitric oxide is an important cell signaling molecule and plays a significant role in vasodilatation, it is also a reactive nitrogen species and thought to have limited effect on organic compounds. However, nitric oxide aids in the production of a variety of nitrogen containing compounds which can go on to behave as nitrosating agents.66 Reactive nitrogen species like nitrogen III oxide (dinitrogen trioxide) nitrosate amines to form N-nitrosamines (Scheme 1). Natural biotransformation creates diazonium compounds which are potent alkylating agents creating DNA adducts and causing DNA base pair transitions,66 eventually leading to replication of altered DNA. The mutagenic properties of alkylated DNA have been confirmed in patients with various types of IBD.58 Although diets high in red meat have shown an increase in NOC in biological fluids, these compounds are also formed in the environment and in cured meats. While not discussed here, their presence should not be completely excluded from a possible impact on toxicity in vivo. Levels of NOC excreted in faecal contents of healthy individuals on a high red meat diet were comparable to those consuming the same concentration of organic iron as haem in blood sausage and liver pate.59 Levels were significantly higher than those on vegetarian, low red meat and inorganic iron diets. However, there was also a reported lack of difference in the genotoxicity of faecal water obtained from individuals on high red meat and oily fish diets.67 A decrease in NOC was detected, although this is not a surprising result due to the marked difference in organic iron, as haem, to catalyze the formation; however, this did not appear to lead to a significantly lower number of strand breaks in CaCo2 cells, indicating that a reduction in dietary meat may not be the solution. This study may have benefited from the use of a pre-, probiotic or dietary fiber, which may not necessarily have reduced the NOC but would have decreased transit time (in the case of fiber or prebiotics), reducing putrefaction, and increased levels of SCFA to aid detoxification and, as previously seen, reduce the effect of telomere shortening thereby protecting the genome against instability and the progression of cellular senescence. Another role played by trimethylamine (TMA) is in formation of trimethylamine N-Oxide (TMAO), a compound that has been associated with cardiovascular disease via increased atherosclerosis68 and non-alcoholic fatty liver disease in mice.69 Formation of TMAO occurs due to dietary supplementation of phosphodicholine (PC) which liberates choline during lipolysis, choline is then available for bacterial transformation to TMA,70 which is followed by hepatic formation of TMAO.68 An altered microbiota affects plasma concentrations of choline and TMAO as observed by feeding colonized mice a dose of deuterated PC before and after treatment with broad spectrum antibiotics. This lead to a complete reduction labeled metabolites, which increased after mice were conventionalized. Similar results were detected with GF mice.68 This demonstrates the role of the microbiota in transformation of dietary

Ammonia

An increase in dietary protein results in a marked increase in colonic total ammonia (NH+4 and NH3). Concentrations detected in the lumen are reported to be between 2 and 44 mM in healthy human subjects.71 Ammonia is formed via bacterial deamination of amino acids in the colon which produces the majority of ammonia in the body. Higher concentrations of ammonia are detected in the distal colon, the major site of amino acid fermentation.18 Ammonia concentrations are well regulated in the body with the majority of ammonia being absorbed and converted to urea in the liver, consequently only a small amount reaches circulation in a healthy individual.72 Concentrations of ammonia as low as 5−10 mM have been shown to alter the metabolism of cells in the intestine, affecting DNA synthesis and decreasing the lifespan of cells, therefore promoting the multiplication of damaged cells.73 Increased concentrations of ammonia are suggested to have an impact on hepatic coma syndrome, which is a loss of consciousness resulting from advanced cirrhosis and poisoning of the liver leading to liver failure. Around 4 g of ammonia are thought to be absorbed from the gut over a 24 h period. Although the majority of ammonia is absorbed and synthesized to urea, in those with impaired liver function, increased plasma ammonia concentrations can lead to various health issues.72,74 Excreted ammonia can be reduced by the addition of dietary fiber and resistant starch; intake of resistant starch has an inverse relationship with the risk of colorectal cancer (P < 0.001 in males and p < 0.01 in females) whereas protein and fat consumption have a positive association.75 Heterocyclic Amines

Heterocyclic amines (HCA) are compounds formed by the Maillard reaction, specifically the interaction between free amino acids, reducing sugars, creatine and creatinine at temperatures of around 150−200 °C.76,77 Formation of HCA often occurs on the surface of foods and is dependent on high temperature and low moisture levels. Studies carried out in several countries in the world on cooking practices identified the two most abundant HCA as 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) and 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx).77−79 Model reactions to simulate the formation of HCAs have been developed which found that temperatures as low as 37 °C are warm enough to form PhIP from phenylalanine, creatinine and glucose, although this did not occur at room temperature.80 It was also shown that PhIP was most abundant in chicken breast compared to beef and cod fish after dry heating for 30 min at 225 °C. This was consistent with data showing higher levels of phenylalanine, tyrosine and isoleucine in chicken breast which are all precursors of PhIP.81−83 HCA are activated by microsomal cytochrome P450 enzymes to form hydroxyl derivatives (i.e., arylhydroxylamines) which are esterified to more reactive compounds which have the ability to form DNA adducts.84,85 DNA adducts of PhIP have been detected in certain types of colorectal cancer cases. As with N-nitrosamines, alkylation of DNA bases by N-hydroxyl HCA which occurs either on the exocyclic amino group or carbon eight on guanine resulting in altered bases.85 Oral administration of PhIP at 400 mg/kg body weight to mice for 579 days resulted in 31 and 68% of male and female treated mice testing positive for lymphomas, respectively, in comparE



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Figure 2. Transformation of glucose and amino acids into SCFA and some species involved.

Figure 3. Degradation pathways for tryptophan and tyrosine metabolites.

ison to a control group.86 Sixty six week old Fischer 344 rats were fed 400 ppm of PhIP which produced 38 detectable colon

adenocarcinomas in over half of the treated male rats and 4 in 7% of treated female rats, showing a dose related relationship.87 F



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ascending,15 which confirms that the distal colon is the major site for proteolysis. Bacterial groups involved in the digestion of aromatic amino acids include Clostridium spp.95 and Bacteroides spp.96,97 Phenol is excreted as p-cresol in urine following detoxification in the liver, but phenol is also detected in high concentrations in the lumen of sudden death victims and in faecal samples of healthy individuals and those suffering from IBD.15,55,98 Increased levels of phenol and p-cresol have also been detected in individuals on a high protein diet.99,100 Less information has been documented on indole although it has been identified in faecal samples of healthy individuals and those who suffer from IBD.55 Phenol in the gut can be detoxified by the liver but colonocytes also have the ability to do so by sulfation.101 UC sufferers appear to have a diminished capacity to detoxify phenol which leads to its accumulation in the lumen and possible agitation of existing disease.102 Toxicity of phenol to the lumen has been demonstrated by showing increased permeability and reduced barrier function using a CaCo-2 cell line at concentrations detected in faecal samples, a characteristic shared with other carcinogens.103 However, Pedersen et al.104 found using HT-29 cells that cell viability did not diminish until phenol concentrations exceeded 20 mM, almost 20 times the minimum seen to affect cell permeability although exposure times were 24 and 12 h, respectively. This may suggest that an increase in cell permeability begins to occur at lower concentrations leading to a decrease in cell viability which could be a consequence of prolonged, increased passage of solutes into cells. Phenol has also been implicated as a cocarcinogen based on its ability to enhance N-nitrosation of secondary amines to form nitrosamines, many of which are known carcinogens.15,105 The nitrosation of phenol by nitrite in acidic conditions occurred faster than nitrosation of amines,106 which results in the formation of p-nitrosophenol and diazoquinone, a mutagen shown to behave as a bacteriocide in higher doses.107 Indole is the most abundant product of bacterial metabolism of tryptophan in comparison to other known metabolites.108 Bacterial groups involved in formation include Clostridium spp. and Bacteroides spp.108 Indole formation occurs via action of the enzyme tryptophanase, which has been isolated from several microbial species.109−111 Indole suppressed the growth of lactic acid bacteria at concentrations of 100 μg/mL after 24 h incubation, whereas p-cresol had an effect at much lower concentrations of 2, 20, and 100 μg/mL after 120 h of incubation, suggesting that other mechanisms of toxicity may exist.112 p-Cresol, another major metabolite detected in faecal and urine samples has also been shown to be toxic to cells. p-cresol is often the form in which phenol is transformed prior to excretion, however it is also present in faecal samples and therefore in the lumen. p-Cresol decreases the endothelial proliferation and wound repair of human umbilical vein endothelial cells (HUVEC) and decreases cell viability at concentrations of 10, 25, and 50 μg/mL. Functionality of the endothelium is important as vasodilatation helps movement of plasma around the body and dysfunction of the endothelium plays a role in cardiovascular disease.113 It is thought that bacterial metabolites like phenol, indole and p-cresol have an impact on renal failure. As mentioned, p-cresol has an impact on endothelial function, a marker of cardio vascular disease, a leading cause of mortality in patients with chronic renal failure.114 A suggested strategy for reduction in

The interest into HCAs centers around the idea that it is not only protein intake and source that may affect health status but also the way in which it is prepared prior to consumption. Therefore, with increased evidence showing the link between HCAs and alkylation of DNA bases, this cannot be seen as irrelevant. However, its impact will only be emphasized when mode of food preparation can be isolated as a factor in determining the risk of increased protein intake on health by reviews of existing clinical studies. Organic Acids

The principal degradation products in the large intestine are SCFA from carbohydrate fermentation but SCFA are also produced via degradation of proteins and amino acids. However, branched chain fatty acids (BCFA) are formed solely from proteins and amino acids. BCFA such as isobutyrate can be used as a precursor for the formation of longer chain fatty acids and aldehydes.88 With the use of a colon cell line, isobutyrate showed an effect on ionic movement through cells by regulating sodium absorption or activating sodium/hydrogen channels causing an increase of luminal crypts by swelling of cells in rat distal colon.89,90 BCFA are also important nitrogen donors for other important amino acids like glutamine and alanine which are both energy sources for the gastrointestinal tract. They are formed from branched chain amino acids valine, leucine and isoleucine. Effects of SCFAs are predominantly beneficial as they, specifically butyrate, constitute the main energy source for colonocytes. Several genera are responsible for the production of SCFA, although normally considered detrimental to colonic health clostridia clusters XIV a, b and cluster IV, XVI produce a large amount of butyrate from carbohydrate dissimilation.91 Other SCFA including acetate and propionate are produced by Bacteroides-prevotella group and Bif idobacterium spp. and Proteobacteria (Figure 2). Absorption of SCFA is quite efficient with less than 5% being excreted in faeces; production of SCFA in the gut is a complex system which benefits from cross feeding between species. Both F. prausnitzii and Roseburia spp. were shown to derive 85−90% of butyrate from external acetate when grown in the presence of 60 mM acetate and 10 mM of glucose; this was more than that produced by a Coprococcus related strain.92 Propionate is also absorbed from the colonic lumen where it is suggested along with butyrate to play a role in inducing gut hormones that reduce food intake which thereby may play a role in dietinduced obesity.93 However, in comparison to acetate and propionate, more than 70% of energy utilized by the colonocytes is preferentially derived from luminal butyrate.94 However, the formation of branched chain fatty acids is poorly covered in literature; although they are known to be derived from branched chain amino acids, it is yet to be stipulated which species may be involved in their formation. This may be done via analysis of enzyme involvement and intermediate compounds to help elucidate which bacterial groups maybe involved. Phenolic and Indolic Compounds

Phenolic (phenol and p-Cresol) and indolic (indole and skatole) compounds are products of catabolism of aromatic amino acids (Figure 3). As carbohydrate levels deplete along the colon the primary substrate becomes protein and amino acids. Fermentation products from amino acids are at higher concentrations in the descending colon compared to G



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are of great importance in the links between diet and cancer.127 Diets in most developing countries are higher in starch and carbohydrates in comparison to western countries which are higher in animal fats and proteins.7 Differences in dietary habits between countries have provided some useful information as to what the main factors in the association with CRC may be. Westernisation has had a large part to play in the increase in the incidence of CRC in nonwestern countries. In the 50s and 60s in comparison with the UK, Japan had a high intake of cereals and lower of meat products by almost 60 g/person per day. In the early 1990s this began to change in parallel to cereal intake which fell as meat consumption increased, this occurred alongside an increase in the incidence of CRC which rose by almost 30% in 30 years.128 However, epidemiological studies linking diet and cancer often lack a specific marker which is strongly associated with both cancer and diet modulation separately. More recently, the links between dietary metabolites of certain food components have shown an association with incidence of cancer. Those factors found to positively correlate include fat and protein, specifically from animals and processed meats. When fat is removed as a factor, the positive correlation with cancer risk did not decrease but did when animal proteins were removed, suggesting consumption of animal-derived proteins as a key driver in the link to CRC incidence.129 Three reviews of both epidemiology and prospective studies have found associations between increased consumption of animal-derived proteins, specifically red and processed meat and CRC.130−132 The main difference between red and white meat are the levels of hemoglobin, and more specifically the iron contained therein, which many studies have focused on as a risk factor for CRC via its catalytic involvement in the formation of NOC.133−135

accumulation of molecules known to affect renal failure aims at decreasing bacterial fermentation of their precursors via preand probiotic therapy.115

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DIETARY METABOLITES AND LARGE BOWEL DISORDERS Interactions between the microbiota and host immunity are important in maintaining host health status.116,117 Several members of the microbiota have been linked with diseases mainly affecting the gut, like inflammatory bowel disease (IBD) such as ulcerative colitis (UC) and Chrons Disease (CD); colorectal cancer (CRC) and Irritable Bowel Syndrome (IBS) although mechanisms involved are still not yet fully understood. Available energy affects the microbial community, an increase in carbohydrates reaching the gut increases biomass thereby increasing production of SCFA which are beneficial to the lumen.118,119 Diets in Uganda, India and Japan are considered primarily vegetarian compared to western diets, the main differences often being animal fats and proteins. Comparison of these individuals gut microbiota compared to those in the UK and USA, showed that those on mostly vegetarian diets possessed fewer Bacteroides spp., and more Enterococcus spp. and Eubacterium spp.7 However, much of these data were achieved prior to the development of molecular based identification techniques for the microbiota and therefore may not necessarily reflect outcomes achievable via such methods. Inflammatory Bowel Disease

UC is inflammation in the colon mucosa which can cause ulcers or open sores, this varies in severity from ulceration to hemorrhage and can occur along the full length of the colon. Involvement of the microbiota has been investigated by using ‘germ free’ mice which show a lack of inflammation in the mucosa.120 UC affects the mucosa and on analysis of the colonic wall it appears to be thickened with elongated crypts that contain abscesses.121 Impaired action of the mucosa can occur as a result of sulfides formed by sulfate reducing bacteria acting upon available sources of sulfur which are elevated with increased dietary protein intake. These are highly toxic and also interfere with butyrate oxidation by colonocytes.15,49 Analysis of bacteria in different areas of the colon of individuals with UC, compared to controls, showed that in both UC and intermediate colitis, concentrations of total facultative anaerobes, Gram negative and positives are significantly higher than the control in both the ileum and cecum.122 However, this study was based on a small subset of patients and although patients with CD were also assessed, larger numbers of assessments would provide a statistically better view. UC only affects the colon and rectum, CD can occur in any part of the GIT from the oral cavity to the anus. The most common site for the onset of CD is the ileocecal area.121,123 Although many studies report no significant correlation in the etiology of the disease, one suggestion is that it is an autoimmune disease due to up regulation of pro-inflammatory cytokines including TNF and IL-1β, both of which are also involved in apoptosis.124 Involvement of intestinal bacteria has also been linked to inflammation in affected areas.125,126

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METABOLITE ANALYSIS The human metabolomes encompasses both intrinsic and extrinsic factors, analysis of which can be carried out in several different ways. Metabolomics or metabonomics is the targeted and nontargeted study of the chemical aspect of the metabolome analyzed in biological samples as a result of metabolic activities. Metabolomics utilizes several analytical techniques in order to detect, identify and quantify a variety of chemical changes in biological samples as a result of modulation of extrinsic factors like diet and drug intake, alongside intrinsic factors like advancing age, health status and body composition.136 Volatile Metabolite Analysis

Volatile compounds are those with enough vapor pressure enabling them to change state and move from a liquid or solid state to a gaseous phase. Compounds with this ability are carbon based and include hydrocarbons, alcohols, aldehydes, ketones, esters and organic acids.137 Volatile organic compounds (VOC) can be detected from several mediums including human breath, urine, skin, sweat, blood and faeces. Many detectable compounds are produced as a consequence of endogenous metabolic activities or absorption of environmental chemicals. Around 1000 volatiles have been identified from human breath of healthy individuals, of which a few are common (e.g., acetone, ethane and methanol) and possibly arise from optimal functioning metabolic process.138 Therefore, those that fluctuate may be used as an indication of suboptimal cellular metabolism leading to, or indicative of, a diseased state.

Colorectal Cancer

Cancers of malignant neoplasm is a growth of a tumor often in the epithelium resulting from uncontrolled replication of damaged cells which have the ability to destroy adjacent cells or metastasize spreading to other tissues. Environmental factors H



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its ability to provide a large variety of information on the metabolites present but also provide structural information for compounds, which is beneficial when unknown compounds are detected. For this reason, NMR is widely used in both animal and human studies investigating host-microbiome interactions, both with dietary nutrients.146,147 It has also been used to document the metabolic profile along the colon of a rat; this in turn has helped to identify the different conditions present along the colon based on metabolites detected and remaining substrates.148 Many clinical studies are using NMR for analysis of several samples obtained; one such is that carried out by Schicho et al., where serum, plasma and urine were analyzed by 1H NMR spectroscopy and with the aid of discriminate analysis allowed for the division of metabolites that were more prevalent in patients with inflammatory bowel disease.149 It was found that several metabolites including urea, valine, tyrosine and citrate were all much higher in patients suffering from Crohn’s disease and ulcerative colitis than in healthy individuals. With the use of discriminant analysis, they were able to clearly distinguish between populations based on analysis of a defined subset of metabolites which appear significantly higher in specific groups. Studies like this are often fraught with the inter and intra person variation which occurs with a large metabolic data set; therefore, they lean toward the use of much larger data sets in order to overcome this. It is therefore easy to understand why this method, specifically with the use of 1H NMR is preferentially used for metabolite analysis over other nuclei like carbon 13 as it possess a higher sensitivity and shorter experimental time. However, one drawback to the use of NMR alone is the lack of sensitivity to metabolites that are low in abundance, but due to its nondestructive nature, more sensitive methods can always be applied in order to gain information from a different perspective; this is often the reason for combinations of methods which together aid in developing the metabolic profile. Liquid chromatography with mass spectrometry (LC−MS) is another method that is readily used in metabolomics for the detection of organic compounds with quantitative or semiquantitative analysis. The polar nature of many metabolites in human biological samples is sometimes a problem from chromatographic methods, in terms of separation; however, this is seemingly overcome with the use of hydrophilic interaction chromatography in combination with LC−MS, which enables better detections of compounds such as amino acids and nucleotides. This is of particular interest in metabolite analysis as LC−MS allows for the collection of relative peak abundances and m/z of even unknown compounds, allowing for relative concentrations to be calculated before elucidation of a possible chemical structure.136,150,151 A combination of methods will always be preferential in terms of gaining a much broader view of the metabolic profile; however, the acquisition of a large amount of data is an easy trap to fall into, and this is also fraught with the variations in response by the host requiring a larger data set in order for this to be reduced. However, the choice in which tools to use is one that needs careful consideration based on the metabolic response one is interested in, but there will always be an interest in the information that is not being gained via use of other methods.

Early applications of volatile analysis were based around detection of compounds from bacterial fermentation in human flatus;139 subsequent studies have examined metabolites in both urine and faecal samples. Several compounds produced during fermentation of substrates, including phenols, nitrosamines and indoles have been positively identified using headspace techniques,55,140,141 several of which are carcinogenic and cocarcinogenic. The successful use of these techniques have allowed for volatile patterns from faecal samples of volunteers suffering from gut disorders including UC and Campylobacter jejuni (CJ) to be elucidated.55 Twenty-eight compounds were used for a 2-dimentional discriminant analysis; these compounds showed a clear divide in clusters of the different groups analyzed. Of the compounds used, those with supporting evidence to their interaction with various gut disorders included sulfur containing compounds and some aromatic compounds derived from protein catabolism. This type of research has gone toward development of the “OdoReader” at The University of West of England (UWE) with the ability to analyze small liquid samples and make a diagnosis based on volatiles detected. Analysis of volatiles from faecal samples as an indication of gastrointestinal problems may increase in the future, although of course breath tests for 13C urea breakdown have long been used as a measure of Helicobacter pylori gastric infection.142 Implementation of laboratory expertise into clinical diagnostic tools has developed over recent years and may enable the use of such tools at the point of care. This work alongside that looking at detection of detection of ketones in expelled breath of diabetics constitutes some very early work linking volatile metabolites to disease states. However both the understanding and methodologies available have advanced a great deal with more complex techniques now being used in order to detect both volatile and non-volatile metabolites using liquid chromatography and mass spectrometry (LC−MS), high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). Non-volatile Metabolite Analysis

Metabolic profiling allows for investigations to into biomarkers of metabolic process which maybe a consequence of underlying pathological processes indicative of different health and disease states. The use of biological samples such as serum, urine and faecal water is increasing, the latter two as they can be provided in a non-invasive manner. Faecal water is often used in analysis of gut-based disorders as this aqueous phase contains a large number of bioactive compounds, many of which have been discussed earlier. Several studies have concentrated on pure metabolic profiling of faecal water in order to find any observable difference in profiles;143 however, some have chosen to use samples to look specifically at genotoxicity via use of a comet assay. The two methods would provide a much larger breadth of information beneficial to each other as the specific metabolites responsible for toxicity can then be investigated as to degree to which they contribute to the toxicity of samples, therefore allowing for identification of specific metabolites that drive the link between diet and modulation in gut health and the mechanisms by which specific metabolites alter this.144,145 One of the most commonly used tools in metabolomics is NMR spectroscopy, which is seen to provide somewhat of a holistic view of metabolites gained under specific conditions. Its wide use is based on nondestructive analysis of samples and its ability to quantify compounds at micromolar concentrations. NMR is used for the analysis of many biological fluids based on

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CONCLUSIONS This review provides an overview of the effects major dietary components on the microbiota species involved in catabolism I



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(12) Ziemer, C. J.; Gibson, G. R. An overview of probiotics, prebiotics and synbiotics in the functional food concept: Perspectives and future strategies. Int. Dairy J. 1998, 8 (5−6), 473−479. (13) Guarner, F.; Malagelada, J. Gut flora in health and disease. Lancet 2003, 361, 512−519. (14) Evenepoel, P.; Geypens, B.; Luypaerts, A.; Hiele, M.; Ghoos, Y.; Rutgeerts, P. Digestability of cooked and raw egg protein in humans as assessed by stable isotope techniques. J. Nutr. 1998, 128, 1716−1722. (15) Hughes, R.; Magee, E. A. M.; Bingham, S. Protein degredation in the large intestine: Relevance to colorectal cancer. Curr. Iss. Intest. Microbiol. 2000, 1 (2), 51−58. (16) Corpet, D. E.; Yin, Y.; Zhang, X. M.; Remesy, C.; Stamp, D.; Medline, A.; Thompson, L.; Bruce, W. R.; Archer, M. C. Colonic protein fermentation and promotion of colon carcinogenesis by thermolyzed casein Nutr. Cancer J. 1995, 23 (3), 271−281. (17) Gratz, S.; Duncan, S. H.; Richardson, A. J.; Johnstone, A. M.; Lobley, G. E.; Flint, H. J.; Wallace, R. J. High protein diets impact on microbial metabolites and toxicity in the human large intestine. Microb. Ecol. 2009, 57 (3), 572−573. (18) Cummings, J. H.; Macfarlane, G. T. The control and consequences of bacterial fermentation in the human colon. J. Appl. Bacteriol. 1991, 70 (6), 443−459. (19) Walker, A. W.; Duncan, S. H.; Leitch, E. C. M.; Child, M. W.; Flint, H. J. pH and peptide supply can radically alter bacterial populations and short-chain fatty acid ratios within microbial communities from the human colon. Appl. Environ. Microbiol. 2005, 71 (7), 3692−3700. (20) Topping, D. L.; Clifton, P. M. Short-chain fatty acids and human colonic function: Roles of resistant starch and nonstarch polysaccharides. Physiol. Rev. 2001, 81 (3), 1031−1064. (21) Gibson, G.; Roberfroid, M. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr. 1995, 125, 1401−1412. (22) Scheppach, W. Effects of short chain fatty acids on gut morphology and function. Gut 1994, 35 (1 Suppl), S35−S38. (23) McIntyre, A.; Gibson, P. R.; Young, G. P. Butyrate production from dietary fibre and protection against large bowel cancer in a rat model. Gut 1993, 34 (3), 386−391. (24) Cummings, J. H. Short chain fatty-acids in the human-colon. Gut 1981, 22 (9), 763−779. (25) Heerdt, B. G.; Houston, M. A.; Augenlicht, L. H. Potentiation by specific short chain fatty acids of differentiation and apoptosis in human colonic-carcinoma cell lines. Cancer Res. 1994, 54 (12), 3288− 3294. (26) Bjorneklett, A.; Viddal, K. O.; Midtvedt, T.; Nygaard, K. Intestinal and gastric bypass - Changes in intestinal microecology after surgical-treatment of morbid-obesity in man. Scand. J. Gastroenterol. 1981, 16 (5), 681−687. (27) Backhed, F.; Crawford, P. A. Coordinated regulation of the metabolome and lipidome at the host-microbial interface. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2010, 1801 (3), 240−245. (28) Backhed, F.; Ding, H.; Wang, T.; Hooper, L. V.; Koh, G. Y.; Nagy, A.; Semenkovich, C. F.; Gordon, J. I. The gut microbiota as an environmental factor that regulates fat storage. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (44), 15718−15723. (29) Ley, R. E.; Turnbaugh, P. J.; Klein, S.; Gordon, J. I. Microbial ecology: Human gut microbes associated with obesity. Nature 2006, 444 (7122), 1022−1023. (30) Velagapudi, V. R.; Hezaveh, R.; Reigstad, C. S.; Gopalacharyulu, P.; Yetukuri, L.; Islam, S.; Felin, J.; Perkins, R.; Boren, J.; Oresic, M.; Backhed, F. The gut microbiota modulates host energy and lipid metabolism in mice. J. Lipid Res. 2010, 51 (5), 1101−1112. (31) Fava, F.; Lovegrove, J. A.; Gitau, R.; Jackson, K. G.; Tuohy, K. M. The gut microbiota and lipid metabolism: Implications for human health and coronary heart disease. Curr. Med. Chem. 2006, 13 (25), 3005−3021. (32) Delzenne, N. M.; Williams, C. M. Prebiotics and lipid metabolism. Curr. Opin. Lipidol. 2002, 13 (1), 61−67.

and modulation in host health due to consumption. The interest in diet−host interactions is fuelled by the increase in clinical evidence showing attenuation in health based on specific dietary choices and decline in health status based on the dietary decisions of a population. The use of several techniques in metabolomics is based on the impact on metabolite activity based on interactions with not only host tissues but also other existing metabolites and creates a possibility for symbiosis between the use of metabolomics with biological models.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +44 1189 378 5360. Fax: +44 1189 31 0080. E-mail: e.p. [email protected]. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS CRC, colorectal cancer; UC, ulcerative colitis; IBD, inflammatory bowel disease; IBS, irritable bowel syndrome; CD, Crohn’s disease; CJ, Compylobacter jejuni; GCMS, gas chromatography and mass spectrometry; GIT, gastrointestinal tract; SPME, solid phase micro extraction; SCFA, short chain fatty acids; BCFA, branched chain fatty acids; VOC, volatile organic compounds; FISH, fluorescent in situ hybridization; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; TNF, tumour necrosis factor; IL, interleukin

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