Dissimilatory Amino Acid Metabolism in Human Colonic Bacteria

Anaerobe (1997) 3, 327–337

ECOLOGY

Dissimilatory Amino Acid Metabolism in Human Colonic Bacteria E. A. Smith and G. T. Macfarlane Medical Research Council Dunn Clinical Nutrition Centre, Hills Road, Cambridge CB2 2DH, UK (Received 28 May 1997, accepted in revised form 4 August 1997) Key Words: amino acid metabolism, large intestine, colonic bacteria

The abilities of slurries of human faecal bacteria to ferment 20 different amino acids were investigated in batch culture incubations. Ammonia, short chain fatty acids, and in some cases, amines, were the principal products of dissimilatory metabolism. The types of SCFA produced were dependent on the chemical compositions of the test substrates. Thus, acetate and butyrate were formed from the acidic amino acid glutamate, while acetate and propionate predominated in aspartate fermentations. Breakdown of the basic amino acids lysine and arginine was rapid, and yielded butyrate and acetate, and ornithine and citrulline, respectively. The major products of histidine deamination were also acetate and butyrate. However, fermentation of sulphur-containing amino acids was slow and incomplete. Acetate, propionate and butyrate were formed from cysteine, whereas the main products of methionine metabolism were propionate and butyrate. The simple aliphatic amino acids alanine and glycine were fermented to acetate, propionate and butyrate, and acetate and methylamine, respectively. Branched-chain amino acids were slowly fermented by colonic bacteria, with the main acidic products being branched-chain fatty acids one carbon atom shorter than the parent amino acid. Low concentrations of amines were also detected in these fermentations. Aliphatic-hydroxy amino acids were rapidly deaminated by large intestinal microorganisms. Serine was primarily fermented to acetate and butyrate, while threonine was mainly metabolised to propionate. Proline was poorly utilized by intestinal bacteria, but hydroxyproline was efficiently fermented to acetate and propionate. The aromatic amino acids tyrosine, phenylalanine and tryptophan were broken down to a range of phenolic and indolic compounds. © 1997 Academic Press

Introduction Peptides and amino acids are used as carbon, nitrogen and energy sources by both saccharolytic and asacAddress correspondence to: E.A. Smith, E-mail: [email protected]

1075-9964/97/050327 + 11 $25.00/0/an970121

charolytic bacteria. Some saccharolytic species such as Prevotella ruminicola are able to derive small amounts of energy from the carbon skeletons of peptides and amino acids, though not enough for these nutrients to serve as energy sources by themselves [1], while others including enterococci and enterobacteria ferment carbohydrates and amino acids. In contrast, other bacteria are obligate amino acid fermenters, and © 1997 Academic Press

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are unable to obtain energy from the metabolism of carbohydrates. The physiology and biochemistry of amino acid degradation by obligately anaerobic bacteria has been investigated most comprehensively in the clostridia [2–4], although a wide variety of nonsporing anaerobes are also known to ferment amino acids including species belonging to the genera Peptostreptococcus [5–7], Campylobacter [8] Acidaminococcus [9], Acidaminobacter [10], Fusobacterium [11] and Eubacterium [12]. Some sulphate-reducing bacteria can also grow at the expense of amino acids [13], while other organisms such as Fusobacterium nucleatum are able to ferment amino acids and sugars concurrently [14], with sugar transport being driven by energy derived from amino acid metabolism [15]. Bacterial amino acid catabolism in the human large intestine occurs via a number of mechanisms involving either deamination or decarboxylation reactions. Many different types of electron donors and acceptors are potentially available to participate in these reactions, including other amino acids, keto acids, molecular H2 and unsaturated fatty acids. There are four known direct pathways used for deaminating single amino acids, and a fifth, the Stickland reaction, in which pairs of amino acids are used. Deamination can be achieved by oxidation, with formation of an α-keto acid, by reduction in which a saturated fatty acid is produced, by hydrolysis with production of an α-hydroxy fatty acid, or alternatively, the elements of ammonia may be removed to leave an unsaturated product, as in the deamination of aspartate to fumarate by aspartase, and urocanic acid production from histidine [16]. Dissimilatory metabolism in amino acid fermenting clostridia is largely achieved by the Stickland reaction [17]. This involves coupled oxidationreduction reactions between suitable pairs of amino acids, where one substrate is oxidatively deaminated and decarboxylated whilst the other is reductively deaminated. Decarboxylation of many amino acids yields an amine and CO2, and a number of large intestinal bacteria are known to produce decarboxylases [18]. However, previous work in our laboratory [19] demonstrated that saturated fatty acids (acetate, propionate, butyrate) and ammonia were the main products of protein fermentation in mixed cultures of colonic bacteria in vitro indicating that amino acid metabolism in the large gut was primarily effected through reductive deamination. In addition to acetate, propionate and butyrate, branched chain-fatty acids (BCFA) and aromatic compounds, namely phenol, indole and a range of phenolic and indolic substituted fatty acids derived from phenylalanine, tyrosine and tryptophan may be formed. The branched chain amino acids valine, leucine and isoleucine have been shown to be oxidised to BCFA with one less carbon atom (isobutyrate,

isovalerate and 2-methylbutyrate, respectively) [17]. Isocaproate is a relatively minor BCFA in the large intestine which is formed by reduction of leucine [20]. Many metabolites produced by amino acid fermentation in the large intestine are harmful to the host. For example, high concentrations of ammonia in the colonic lumen may select for neoplastic growth [21] and in patients with liver disease, ammonia accumulates in body fluids, contributing to the onset of portalsystemic encephalopathy or hepatic coma [22]. Butyrate and valerate have also been implicated in the causation of hepatic coma where patients with cirrhosis have raised blood and cerebro-spinal fluid levels [23]. Production of BCFA by intestinal bacteria is not usually associated with toxicity, however, in children with isovaleric acidaemia, the onset of confusion and stupor is accompanied by a dramatic rise in this metabolite [24]. Amines, phenols and indoles have been implicated in schizophrenia, migraine and the onset of hypertensive symptoms [25–27]. Phenolic and indolic compounds are also thought to act as co-carcinogens [28], while amines serve as precursors of nitrosamine production [29]. Despite the known toxicological characteristics of many of the end products of nitrogen metabolism, our knowledge of the ecology and physiology of amino acid fermentation in the colon is limited. Although one or more dissimilatory pathways have been described for most amino acids [17,30], the degradation routes in bacterial communities is largely unknown. The objectives of this study, therefore, were to quantitate rates of amino acid breakdown in the colonic microbiota, and to determine the fermentation products formed from individual amino acids.

Materials and Methods Fermentation of individual amino acids by faecal bacteria Fresh stools were obtained from two healthy donors and slurries [33%, (w/v)] were prepared by homogenising the faeces in 0.1 mol/L potassium phosphate buffer (pH 6.8), saturated with argon gas. Particulate matter was removed as described by Allison and Macfarlane [31]. Samples (15 mL) were dispensed into serum bottles (70 mL capacity) containing 25 mL of a basal medium consisting of the following (g/L): CaCl2.2H2O, 0.02; MgSO4.7H2O, 0.02; K2HPO4, 0.08; KH2PO4, 0.08; NaHCO3, 0.8; NaCl, 0.2; haemin, 0.01; Tween 80, 2.0 Eight millilitres of a trace element solution [32] and 20 mL of a vitamin solution [33] were added per litre. The vitamin and trace element solutions were filter sterilised, all other components were autoclaved. Individual amino acid substrates

Amino Acid Fermentation were added to this medium in the form of a stock solution made in de-ionised water (10 mL), to give final concentrations in the serum bottles of 10 mmol/ L, or 5 mmol/L for aromatic amino acids (phenylalanine, tryptophan, tyrosine). Other individual amino acid substrates used were the L-forms of alanine, asparate, arginine, glutamate, glycine, histidine, leucine, isoleucine, lysine, methionine, proline, serine, threonine, valine, ornithine, hydroxyproline and cysteine. Bottles with no added amino acids were used as controls. Each bottle was capped with a butyl rubber stopper and crimped with an aluminium cap. These manipulations were done in a Whitley Mark 3 Anaerobic Cabinet, with an atmosphere consisting of 10%, H2, 10% CO2 and 80% N2. All bottles were subsequently incubated at 37°C in a rotary shaking incubator. Samples of headspace gas (2 mL) and liquid samples (4 mL) were taken at 0, 12 and 24 h, using gas tight 5 mL and 10 mL plastic syringes fitted with luer locks.

Chemical analyses Amines, phenols and indolic compounds were quantitated by gas chromatography or HPLC as previously described [18,34]. SCFA were measured by GC using a Pye Model 204 Gas Chromatograph fitted with a flame ionisation detector. SCFA were extracted using procedures outlined by Holdeman et al. [35], with the addition of an internal standard (t – butylacetate) at a concentration of 30 mmol/L. The SCFA were separated using 10% FFAP, 100/120 mesh Chromosorb WAW-DMCS in a 1.8 m 3 2 mm (i.d.) glass column (Unicam). Injector, detector and column temperatures were 200, 300 and 155°C, respectively. Flow rates of the N2 carrier gas, H2 and air were set at 50, 30 and 370 mL/min. These column conditions did not discriminate between the BCFA 2-methylbutyrate and isovalerate. All samples were quantitated by comparison of sample peak heights, with those of authentic standards. Ammonia was determined using the phenol-hypochlorite method of Solorzano [36] and by an LKB amino acid analyser.

Amino acid analysis These analyses were undertaken using a standard LKB 4151 Alpha Plus Amino Acid Analyser, equipped with a potentiometric recorder and a Hewlett Packard 3392A integrator. The analyser was fitted with a 200 mm 3 4.6 mm stainless steel column filled with Ultropac 8 cation exchange resin, lithium form (LKB Biochrom). Detection with ninhydrin was at 570 and 440 nm.

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For amino acid measurements, an internal standard of norleucine (10 mmol/L) was added to samples before deproteinisation with four parts 10% (w/v) trichloroacetic acid for 1 h at 4°C. The mixture was centrifuged at 13 000 3 g for 15 min and the supernatant filtered through a 0.22 µm membrane (Whatman). Samples (20 µL) were adjusted to pH 2.2 using 0.3 mol/L LiOH. Amino acids were quantitated by automatic integration by comparison to an amino acid physiological standard solution (Sigma).

Gas analysis Gases were measured by GC using a Pye Series 104 Gas Chromatograph, equipped with a 1 mL injection loop. The gases were separated on a 3 ft glass column (4 mm i.d.) containing Poropack Q (Waters). Column and detector temperatures were 40 and 110°C respectively. The carrier gas was argon, set at a pressure of 2 p.s.i. Detection was by a thermal conductivity detector connected to a Pye Unicam CDPI Computing Integrator. Results were quantitated by comparison with known gas standards.

Dry weight measurements Dry weights of bacterial cultures were determined as described by Degnan and Macfarlane [37].

Chemicals Bacteriological culture media were obtained from Oxoid (Basingstoke, England). Unless otherwise stated, all other chemicals were purchased from Sigma (Poole, England).

Results Metabolism of individual amino acids by faecal bacteria Figures 1–7 show fermentation products formed from the metabolism of single amino acids by faecal suspensions incubated anaerobically for 24 h. Results are mean concentrations from two samples and are corrected for simultaneously incubated control suspensions to which amino acids had not been added.

Fermentation of acidic amino acids Data in Figures 1a and b shows results of fermentation of acidic amino acids by faecal microorganisms.

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Fermentation rates were approximately 0.055 and 0.082 mmol/h/g dry wt. bacteria for glutamate and aspartate. Acetate and butyrate were major acidic products formed from glutamate, with propionate being a minor metabolic product (3.7, 4.2 and 0.2 mmol/L, respectively). The main products of aspartate fermentation were propionate and acetate, with lower levels of butyrate accumulating (4.4, 1.7 and 0.6 mmol/L, respectively). In both cultures, acetate concentrations declined after 24 h, while ammonia accumulated to approximately 5.5 mmol/L.

Fermentation of sulphur-containing amino acids

Fermentation of basic amino acids Catabolism of arginine and lysine was rapid [Figures 2(a) and (b)] with respective initial rates of utilization being approximately 0.093 and 0.077 mmol/h/g dry wt. bacteria. The amino acids ornithine and citrulline were the main products of arginine breakdown, with accumulation of ammonia (16.8 mmol/L). The main SCFA products of lysine fermentation were butyrate (8.1 mmol/L), and acetate (5.9 mmol/L) after 24 h. Trace levels of cadaverine were detected. Ammonia accumulated to approximately 77% of the maximum

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theoretical yield after 12 h incubation, and was apparently further assimilated by these cultures. The major products of histidine deamination [Figure 2(c)] were acetate, (4.0 mmol/L) and butyrate (1.5 mmol/L). Ornithine was slowly metabolised over 24 h (0.025 mmol/h/g dry wt. bacteria) without production of SCFA. Pyrrolidine was detected after 12 h (1.48 mmol/L), with the ratio of ammonia to pyrrolidine very close to unity (1.4 mmol/L), as shown in Figure 2(d).

Initial rates of cysteine and methionine utilization were approximately 0.034 and 0.049 mmol/h/g dry wt. bacteria, however, as shown in Figures 3(a) and 3(b), after 24 h incubation, residual amino acid was detected in both cultures (ca. 68 and 48% respectively). Deamination of these amino acids with release of ammonia was the primary route of dissimilatory metabolism. SCFA detected after 24 h incubation with cysteine were acetate, butyrate and propionate (3.1, 2.1 and 0.6 mmol/L, respectively). Alanine also accumulated in low amounts (up to 0.23 mmol/L) in these cultures. Methionine fermentation yielded propionate (2.9 mmol/L), butyrate (2.0 mmol/L), and the amino acid α-aminobutyrate.

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Figure 1. Fermentation of acidic amino acids by intestinal bacteria in batch culture (a) glutamate, (b) aspartate. Amino acid substrate (s), ammonia ( ), carbon dioxide (m), acetate (d;), propionate ( ), butyrate (n).

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Fermentation of alanine (ca. 0.057 mmol/h/ dry wt. bacteria) yielded acetate (1.6 mmol/L), propionate (3.4 mmol/L), butyrate (1.2 mmol/L), CO2 and ammonia (4.4 mmol/L), as shown in Figure 4(a). Glycine was rapidly utilized (ca. 0.10 mmol/h/g dry wt. bacteria), with near total degradation within 12 h. Acetate was the main acidic product (2.15 mmol/L), with decarboxylation of glycine forming methylamine, which accounted for approximately 24% of glycine disappearance [Figure 4(b)]. The branched chain amino acid valine was poorly metabolised by faecal bacteria (0.035 mmol/h/g dry wt. bacteria), with isobutyrate being the major SCFA product (2.9 mmol/L) as shown in Figure 4(c). 2-Methylbutylamine also accumulated (0.3 mmol/L) with concomitant release of CO2. Other branched chain amino acids (leucine, isoleucine) were slowly degraded (0.009 and 0.054 mmol/h/g dry wt. bacteria, respectively), with isovalerate (1.2 mmol/L) and ammonia being the main end products of leucine fermentation [Figures 4(d) and (e)]. Fermentation of isoleucine produced 2-methylbutyrate, but valine and its decarboxylation

Amino Acid Fermentation product 2-methylbutylamine were also detected [Figure 4(e)].

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rapidly accumulating. Trace levels of δ-aminovalerate were observed in these incubations [Figure 6(b)]. Metabolism of aromatic amino acids

Fermentation of aliphatic-hydroxy amino acids Acetate and butyrate were formed by rapid deamination of serine ( > 0.310 mmol/h/g dry wt. bacteria), while propionate and the amino acid glutamate were minor products [Figure 5(a)]. Faecal bacteria metabolised threonine less rapidly (0.095 mmol/h/g dry wt. bacteria), but higher yields of SCFA were produced [Figure 5(b)]. Although acetate and butyrate were detected, propionate was the main SCFA formed from this amino acid. α-Aminobutyrate and glycine were formed in trace amounts.

Approximately 80% of available tyrosine was assimilated after 24 h by faecal bacteria. A number of phenolic compounds were detected in concentrations above control levels, with phenol being present in highest amounts (1.2 mmol/L). Ammonia and CO2 were also produced [Figure 7(a)]. Phenylalanine and tryptophan were slowly assimilated by faecal bacteria. Phenylacetate and indoleacetate were the predominant end products of these fermentations (1.2 and 0.6 mmol/L, respectively), together with ammonia and CO2 [Figure 7(b) and (c)].

Fermentation of imino acids Proline was slowly metabolised by faecal bacteria (10% utilization in 24 h), with ammonia accumulating at the same rate [Figure 6(a)]. Low levels of acetate, propionate and propylamine, a decarboxylation product of γ-aminobutyrate were detected in these cultures. However, hydroxyproline was efficiently metabolised, with complete degradation after 12 h. Acetate and propionate were the main acid products (8.3 and 3.1 mmol/L, respectively) with ammonia also (a)

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Generation of free amino acids from proteins in the large intestine probably has little nutritional significance to the host, due to bacterial competition for the substrate, and the fact that the colon lacks absorptive machinery needed for their uptake [38]. In studies on amino acid metabolism, disappearance of

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Figure 2. Fermentation of basic amino acids by intestinal bacteria in batch culture (a) arginine, (b) lysine, (c) histidine, (d) ornithine. Amino acid substrate (s), ammonia ( ), carbon dioxide (m), acetate (d), propionate ( ), butyrate (n), ornithine ( ), citrulline ( ) cadaverine ( ).

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an amino acid can result from direct assimilation by bacteria, deamination, decarboxylation, or by their binding to the cell. However, Ruchim et al. [39] observed that human intestinal bacteria mainly degraded amino acids to form SCFA. In this study, fermentation of glutamate and aspartate by faecal bacteria was rapid (Figure 1). Turnover rates of these amino acids are also high in the rumen [40] and pure cultures of rumen bacteria have been reported to utilize acidic and neutral amino acids to the greatest extent [41]. On the basis of these data it was proposed that the acidic amino acids could spare C normally used for glutamate and aspartate synthesis. Support for this hypothesis was obtained by Hungate [42] where 14C-labelled glutamate and asparate were shown to be directly assimilated by rumen bacteria. However, from SCFA analysis in the current investigation (Figure 1), it can be seen that these amino acids were dissimilated, instead of being directly incorporated into bacterial protein. The products of glutamate decarboxylation, γ-aminobutyric acid and propylamine were not detected under these cultural conditions, which in view of the high amounts of this amine previously found in faeces, and in intestinal contents, was surprising [18]. This could be due to adaptation of bacterial populations towards SCFA production under conditions of increased substrate availability.

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Figure 3. Fermentation of sulphur-containing amino acids by intestinal bacteria in batch culture (a) cysteine, (b) methionine. Amino acid substrate (s), ammonia ( ), acetate (d), propionate ( ), butyrate (n), alanine ( ) α-aminobutyrate ( ).

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Two main pathways of dissimilatory glutamate metabolism occur in anaerobic bacteria, both of which have been extensively studied [43,44]. They are the methylaspartate pathway, which is used by the majority of clostridia, and the hydroxyglutamate pathway in fusobacteria, peptostreptococci and acidaminococci. The principal end products of both pathways are acetate, butyrate and CO2. In fermentations of glutamate and aspartate undertaken in this investigation (Figure 1), production of acetate by colonic bacteria was followed by the apparent utilization of this metabolite. In experiments described by Barker et al. [45], labelled acetate was shown to be extensively incorporated into butyrate by the oral anaerobe Fusobacterium nucleatum, and it seems that this process also occurs in the large intestine. When present as the sole source of energy, the end products of aspartate fermentation by Clostridium tetani are ammonia, CO2, SCFA (acetate, butyrate), hydroxy acids (lactate, malate) and ethanol [46], while Clostridium saccharobutyricum was reported to form acetic and butyric acids from aspartate in the ratio of 2 : 1 [47]. Alternatively, Clostridium perfringens has been shown to catalyse the decarboxylation of aspartate to alanine, and the reaction sequence involved in the reductive conversion of aspartate to threonine is also a possible pathway of aspartate fermentation. However, this must be coupled with a suitable oxidative reaction, the final metabolite in this case would be propionate, and as demonstrated in experiments in this study, propionate was the major product of dissimilatory aspartate metabolism [Figure 1(b)]. The high ammonia values detected in arginine, lysine and histidine fermentations can be attributed to the two or more potential amino groups present in each of these amino acids (Figure 2). The first step in histidine breakdown by Peptostreptococcus asaccharolyticus is deamination to urocanate, by the enzyme histidase. Urocanate is further degraded to formiminoglutamate by an enzyme system collectively termed urocanase, yielding formamide and glutamate [5]. Formamide has not been shown to be further metabolised by anaerobic bacteria, however, glutamate would be expected to be a transient product in the colon. In these faecal incubations, glutamate was not detected and was presumably metabolised in this way [Figure 2(c)]. The rapidity of glutamate fermentation was evident when the amino acid was incubated as sole N-source [Figure 1(a)]. Arginine was not fermented to SCFA by intestinal bacteria, but was converted to citrulline and ornithine [Figure 2(a)]. This has previously been demonstrated in Clostridium perfringens, Enterococcus faecalis and in a variety of enterobacteria and lactobacilli, where multienzyme systems are involved [48,49], with the initial deamination of arginine to citrulline occurring through the action of arginine deiminase:

Amino Acid Fermentation arginine + H2O → citrulline + NH3 Ornithine production proceeds by the action of catabolic ornithine carbamoyltransferase, according to the equation: citrulline + Pi → ornithine + carbamyl phosphate with concurrent ATP production via carbamate kinase: carbamyl phosphate + ADP → ATP + CO2 + NH3 All arginine utilized in this experiment could be accounted for by production of citrulline and ornithine [Figure 2(a)]. The resulting ornithine was then slowly decarboxylated and deaminated to produce the heterocyclic amine pyrrolidine [Figure 2(d)]. This (a)

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indicates that bacterial metabolism of dietary ornithine and arginine reaching the large intestine could account for the presence of this potentially toxic metabolite [18]. Agmatine, a known product of arginine decarboxylation [50,51], was not detected in these incubations. The reported optimum pH for arginine decarboxylase is 4.0, while ornithine decarboxylase functions most efficiently from pH 5.0 to near neutral conditions in Escherichia coli [52]. This route of arginine degradation may therefore not be viable in conditions prevailing in the human large intestine, as indicated by its absence in colonic contents obtained from human sudden death victims [18]. When lysine was incubated with faecal bacteria, the substrate was mainly fermented to ammonia, butyrate

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Figure 4. Fermentation of aliphatic amino acids by intestinal bacteria in batch culture (a) alanine, (b) glycine, (c) valine, (d) leucine, (e) isoleucine. Amino acid substrate (s), ammonia ( ), carbon dioxide (m), isovalerate/2- methylbutyrate ( ), valine ( ), 2-methylbutylamine (– –n– –).

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Higher yields of butyrate were detected in our studies, possibly due to conversion of acetate into butyrate, under conditions of substrate limitation. Notably, lysine fermentation did not give rise to appreciable amounts of CO2 in this study [Figure 2(b)], suggesting that it was mainly degraded by pathways that did not involve decarboxylation reactions. This has been demonstrated in Fusobacterium nucleatum, which is known to cleave lysine between carbon atoms 2 and 3 and carbon atoms 4 and 5 to produce acetate and butyrate from either ends of the amino acid substrate

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Figure 5. Fermentation of aliphatic-hydroxy amino acids by intestinal bacteria in batch culture (a) serine, (b) threonine. Amino acid substrate (s), ammonia ( ), carbon dioxide (m), hydrogen ( ), acetate (d), propionate ( ), butyrate (n), glycine ( ), α-aminobutyrate ( ), glutamate ( ).

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Figure 6. Fermentation of imino acids by intestinal bacteria in batch culture (a) proline, (b) hydroxy-proline. Amino acid substrate (s), ammonia ( ), carbon dioxide (m), acetate (d), propionate ( ), propylamine (– –), δ-aminovalerate ( ).

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Figure 7. Fermentation of aromatic amino acids by intestinal bacteria in batch culture (a) proline, (b) hydroxy-proline. Amino acid substrate (s), ammonia ( ), carbon dioxide ( ), p-cresol ( ), phenol ( ), hydroxyphenylpropionate ( ), phenylacetate ( ), phenylpyruvate (d), phenylpropionate ( ), indoleacetate ( ), indole ( ), indolepyruvate ( ), indolepropionate ( ).

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Amino Acid Fermentation [14]. Only trace levels of the amine cadaverine were detected [Figure 2(b)], further demonstrating that decarboxylation was not a major route of dissimilation of this amino acid, under these environmental conditions. Sulphur-containing amino acids were slowly degraded by faecal bacteria (Figure 3). Some anaerobic organisms are known to possess a desulphydrase [54], which converts cysteine to pyruvate, ammonia and hydrogen sulphide according to the following equation: cysteine + H2O → H2S + ammonia + pyruvate On the basis of fermentation products observed in faecal incubations [Figure 3(a)], it can be postulated that the pyruvate formed is fermented by different pathways according to the enzymic constitutions of bacteria in the microbiota. The alanine detected in these incubations suggests a biosynthetic role for this amino acid, since pyruvate can be transaminated to yield alanine. Methionine is attacked slowly by a number of clostridia [55], and C. sporogenes is thought to convert this amino acid to α-ketobutyrate, ammonia, and possibly, methanethiol. A number of fermentation products were formed from methionine, possibly due to further transformations of α-ketobutyrate [Figure 3(b)]. Although thiol production was not followed in these experiments, many mercaptans, especially methanethiol, are toxic to mammalian cells. For example, orally administered methionine precipitates hepatic coma in patients with liver disease [56]. Since intravenous methionine is not toxic per se, it is bacterial metabolism in the large intestine that is mainly responsible for its toxic effects [57]. The aliphatic amino acids glycine and alanine were efficiently assimilated by faecal bacteria, while the branched chain aliphatic amino acids valine, leucine and isoleucine were less readily metabolised (Figure 4). Multi-enzyme systems are used by anaerobic bacteria in the breakdown of glycine, and two distinct pathways have been reported, both resulting in production of acetate and CO2 [7,58]. Different ATP yields may be achieved in colonic bacteria depending on which pathway is used. In the glycine reductase pathway, 4 mol ATP are formed from 4 mol glycine, compared to only 1 mol ATP per 4 mol glycine, by glycine-serine-pyruvate conversion [59]. On the basis of fermentation data shown in Figure 4(b), the principal route of production of acetate in faecal bacterial populations is equivocal. However, methylamine which is a toxic substance in the large intestine [29], was apparently a major product of glycine metabolism in faecal bacteria. Branched chain amino acids were metabolised relatively slowly by intestinal bacteria. However, these substrates may be involved in Stickland reactions, and concentrations of free amino acids that function as

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electron acceptors in the faecal suspensions may have been too low to allow complete fermentation of these amino acids [Figure 4(c–e)]. Nevertheless, addition of valine to the incubations resulted in a small increase in the amount of isobutyrate [Figure 4(c)]. These data also suggest that decarboxylation of this amino acid to 2-methylbutylamine is a potential route of dissimilation in the colonic microbiota. Leucine has been shown to be able to act as both an electron acceptor and donor in a number of proteolytic clostridia and peptostreptococci [60,61]. In incubations with faecal bacteria described here, the reductive branch of leucine catabolism could not be demonstrated, as evidenced by the absence of isocaproate [Figure 4(d)]. The main product of isoleucine oxidation (2-methylbutyrate) was detected and it would appear to have a biosynthetic role because valine and its amine were also found [Figure 4e]. Serine and threonine dissimilation in intestinal microorganisms resulted in formation of a variety of products being formed (Figure 5). An enzyme that converts serine to pyruvate and ammonia is frequently found in anaerobic bacteria [62,63], and while threonine metabolism in rumen microorganisms initially involves deamination and rearrangement to give α-ketobutyrate and ammonia [64], the ultimate products depend on the catabolic pathways used by bacteria in the ecosystem. For example, Peptostreptococcus asaccharolyticus catalyses a simple dehydrogenation of α-ketobutyrate according to the equation: α-ketobutyrate + H2O → propionate + CO2 + H2 while Clostridium propionicum converts threonine to butyrate, propionate, CO2 and ammonia: 3 threonine + 2 H2O → butyrate + 2 CO2 + 3 NH3 The overall process represents an oxidation of two moles of threonine (or α-ketobutyrate) to propionate and CO2, coupled with reduction of one mole to butyrate. Glycine was also detected in incubations in this study, and is produced by direct cleavage of threonine to acetaldehyde and glycine, which has also been demonstrated in a number of clostridia [65]. Proline was not fermented by faecal bacteria under these growth conditions (Figure 6). Transient accumulation of low levels of δ-aminovalerate suggest that it can be reduced by bacteria catalysing the Stickland reaction: proline + 2H → δ-aminovalerate δ-Aminovalerate may be further fermented, as demonstrated in an unspeciated clostridium isolated from sewage sludge [66], according to the reaction: 2 δ-aminovalerate + 2 H2O → 2 NH3 + valerate + propionate + acetate In this study, propylamine was also detected, which

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may have been formed through production and subsequent decarboxylation of glutamate, although this biochemical pathway has not been reported to occur in colonic bacteria. While the aromatic amino acids tyrosine, phenylalanine and tryptophan were metabolised comparatively slowly by human intestinal microorganisms, a range of phenolic and indolic end-products were detected in the faecal incubations (Figure 7). They were mainly derived from oxidative decarboxylations of the parent amino acids, following transamination. Results obtained in this study show that quantitatively, direct incorporation of amino acids into cellular material is a minor pathway in the nitrogen economy of the human large intestinal ecosystem, at least during bacterial growth under carbohydrate-limiting conditions. Dissimilatory metabolism of amino acids resulted in the majority of their carbon and nitrogen appearing as fermentation products in the culture medium. Moreover, a consistent feature of these data was the fact that expected yields of ammonia, based on amino acid depletion and the appearance of SCFA production, were rarely observed. This would suggest that ammonia formed in deamination reactions was subsequently assimilated and incorporated into cellular material during growth of the amino acid fermenting components of the microbiota.

References 1. Russell J.B. (1983) Fermentation of peptides by Bacteroides ruminicola B14. Appl Environ Microbiol 45: 1566–1574 2. Mead G.C. (1971) The amino acid fermenting clostridia. J Gen Microbiol 67: 47–56 3. Elsden S.R. and Hilton M.G. (1979) Amino acid utilization patterns in clostridial taxonomy. Arch Microbiol 123: 137–141 4. Giesel H., Machacek G., Bayerl J. and Simon H. (1981) On the formation of 3-phenylpropionate and the different stereochemical course of the reduction of cinnamate by Clostridium sporogenes and Peptococcus anaerobius. FEBS Lett 123: 107–110 5. Whiteley H.R. (1957) Fermentation of amino acids by Micrococcus aerogenes. J Bacteriol 74: 324–330 6. Cato E.P., Johnson J.L., Hash D.E. and Holdeman L.V. (1983) Synonymy of Peptococcus glycinophilus with Peptostreptococcus micros and electrophoretic differentiation of Peptostreptococcus micros from Peptococcus magnus. Inst J Syst Bacteriol 33: 207–210 7. Durre ¨ P., Spahr R. and Andreesen J.R. (1983) Glycine fermentation via glycine reductase in Peptococcus glycinophilus and Peptococcus magnus. Arch Microbiol 134: 127–135 8. Nanninga H.J., Drent W.J. and Gottschal J.C. (1986) Major differences between glutamate-fermenting species isolated from chemostat enrichments at different dilution rates. FEMS Microbiol Ecol 38: 321–329 9. Buckel W. (1986) Substrate stereochemistry of the biotindependent sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans. Eur J Biochem 156: 259–263 10. Stams A.J.M. and Hansen T.A. (1984) Fermentation of glutamate and other compounds by Acidaminobacter hydrogenoformans gen. nov. sp. nov., an obligate anaerobe isolated from black mud. Studies with pure cultures and mixed cultures with sulfate reducing and methanogenic bacteria. Arch Microbiol 137: 329–337

11. Rogers A.H., Gully N.J., Pfennig A.L. and Zlim P.S. (1992) The breakdown and utilization of peptides by strains of Fusobacterium nucleatum. Oral Microbiol Immunol 7: 299–303 12. Zindel U., Freudenberg W., Rieth M., Andreeseen J.R., Schnell J. and Widdel F. (1988) Eubacterium acidaminophilum sp. nov., a versatile amino acid-degrading anaerobe producing or utilizing H2 or formate. Description and enzymatic studies. Arch Microbiol 150: 254–266 13. Widdel F. (1988) Microbial ecology of sulfate- and sulfur reducing bacteria. In Zehnder A.J.B. (ed) Biology of Anaerobic Microorganisms, pp. 469–585. John Wiley and Sons, New York 14. Loesche W.J. and Gibbons R.J. (1968) Amino acid fermentation by Fusobacterium nucleatum. Arch Oral Biol 13: 191–201 15. Robrish S.A., Oliver C. and Thompson J. (1987) Amino aciddependent transport of sugars by Fusobacterium nucleatum ATTC 10953. J Bacteriol 169: 3891–3897 16. Drasar B.S. and Hill M.J. (1974) Human Intestinal Microflora. Academic Press, London 17. Barker H.A. (1961) Fermentation of nitrogenous organic compounds. In Gunsalus I.C. and Stannier R.Y. (eds) The Bacteria vol 2, pp. 151–207. Academic Press, London 18. Smith E.A. and Macfarlane G.T. (1996) Studies on amine production in the human colon: Enumeration of amine forming bacteria and physiological effects of carbohydrate and pH. Anaerobe 2: 285–297 19. Macfarlane G.T. and Allison C. (1986) Utilisation of protein by human gut bacteria. FEMS Microbiol Ecol 38: 19–24 20. Macfarlane G.T., Gibson G.R., Beatty E. and Cummings J.H. (1992) Estimation of short-chain fatty acid production from protein by human intestinal bacteria based on branched-chain fatty acid measurements. FEMS Microbiol Ecol 101: 81–88 21. Matsui T., Matsui T., Matsukawa Y., Sakai T., Nakamura K., Aoike A and Kawai K. (1995) Effect of ammonia on cell-cycle progression of human gastric cancer cells. Eur J Gastroenterol Hepatol 7: S79–S81 22. Weber F.L., Banwell J.G., Fresard K.M. and Cummings J.H. (1987) Nitrogen in fecal bacteria, fiber and soluble fractions of patients with cirrhosis: effects of lactulose and lactulose plus neomycin. J Lab Clin Med 110: 259–263 23. Vince A.J. (1986) Metabolism of ammonia, urea, and amino acids, and their significance in liver disease. In Hill M.J. (ed) Microbial Metabolism in the Digestive Tract, pp. 83–105. CRC Press, Boca Raton 24. Tanaka K., Budd M.A., Efron M.L. and Isselbacher K.J. (1976) Isovaleric acidemia: a new genetic defect of leucine metabolism. Proc Natl Acad Sci 56: 236–242 25. Dalgliesh C.E., Kelley W. and Horning E.C. (1958) Excretion of a sulphatoxyl derivative of skatole in pathological studies in man. Biochem J 70: 13P 26. Anon (1968) Headache, tyramine, serotonin and migraine. Nutr Rev 26: 40–44 27. Boulton A.A., Cookson B. and Paulton R. (1970) Hypertensive crisis in a patient on MAOI antidepressants following a meal of beef liver. CMAJ 102: 1395 28. Dunning W.F., Curtis M.R. and Maun M.E. (1950) The effect of added dietary tryptophane on the occurrence of 2-acetylaminfluorene-induced liver and bladder cancer in rats. Cancer Res 10: 454–459 29. Shephard S.E., Schlatter C. and Lutz W.K. (1987) N-Nitrosocompounds: relevance to human cancer. In Bartels H., O’Neill I.K. and Herman R.S. (eds) IARC Scientific Publications No 57, pp. 328–332. IARC Publications, Lyons 30. Barker H.A. (1981) Amino acid degradation by anaerobic bacteria. Ann Rev Biochem 50: 23–40 31. Allison C. and Macfarlane G.T. (1988) Effect of nitrate on methane production and fermentation by slurries of human faecal bacteria. J Gen Microbiol 134: 1397–1405 32. Evans C.G.T., Herbert D. and Tempest D.W. (1970) The continuous cultivation of microorganisms. II. The construction of a chemostat. Meth Microbiol 2: 277–327 33. Wolin E.A., Wolin M.J. and Wolfe R.S. (1963) Formation of methane by bacterial extracts. J Biol Chem 238: 2882–2886 34. Smith E.A. and Macfarlane G.T. (1996) Enumeration of human colonic bacteria producing phenolic and indolic compounds: Effects of pH, carbohydrate availability and retention time on

Amino Acid Fermentation

35. 36. 37.

38.

39. 40.

41. 42. 43. 44. 45. 46. 47. 48.

49.

dissimilatory aromatic amino acid metabolism. J Appl Bacteriol 81: 288–302 Holdeman L.V., Cato E.P. and Moore W.E.C. Eds (1977) Anaerobic Laboratory Manual. 4th Edn. Virginia Polytechnic Institute Anaerobe Laboratory, Blacksburg, Virginia Solorzano L. (1969) Determination of ammonia in natural waters by the phenolhypochlorite method. Limnol Oceanog 14: 799–801 Degnan B.A. and Macfarlane G.T. (1995) Arabinogalactan utilization in continuous cultures of Bifidobacterium longum. Effect of co-culture with Bacteroides thetaiotaomicron. Anaerobe 1: 103–112 Wrong O.M. (1988) Bacterial metabolism of protein and endogenous nitrogen compounds. In Rowland I.R. (ed) Role of the Gut Flora in Toxicity and Cancer, pp. 227–262. Academic Press, New York Ruchim M.A., Makino D. and Zarling E.J. (1984) Volatile fatty acid production from amino acid degradation by human faecal suspensions. Gastroenterology 86: 1225 Prins R.A., van Hal-van Gestel J.C. and Counotte G.H.M. (1979) Degradation of amino acids and peptides by mixed rumen micro-organisms. Z Tierphzsiol Tierer¨aenhr Futtermittelfd 42: 333–339 Scheifinger C., Russell N. and Chalupa W. (1976) Degradation of amino acids by pure cultures of rumen bacteria. J Anim Sci 43: 821–827 Hungate E.R. (1966) The Rumen and its Microbes. Academic Press, New York Horler D.F., Westlake D.W.S. and McConnell W.B. (1966) Conversion of glutamic acid to volatile acids by Micrococcus aerogenes. Can J Microbiol 12: 47–53 Buckel W. (1980) Analysis of the fermentation pathways of clostridia using double labelled glutamate. Arch Microbiol 127: 167–169 Barker H.A., Kahn J.M. and Hedrick L. (1982) Pathway of lysine degradation in Fusobacterium nucleatum. J Bacteriol 152: 201–207 Pickett M.J. (1943) Studies on the metabolism of Clostridium tetani. J Biol Chem 151: 203–209 Cohen G.N., Nisman B. and Cohen-Bazire G. (1948) Sort des amino-acides d´egrad´es par Cl. saccharobutyricum et Cl. sporogenes. Bull Soc Chim Biol 30: 109–111 Chen K.C.S., Culbertson N.J., Knapp J.S., Kenny G.E. and Holmes K.K. (1982) Rapid method for simultaneous detection of the arginine dihydrolase system and amino acid decarboxylases in microorganisms. J Clin Microbiol 16: 909–919 Liu S.Q., Pritchard G.G., Hardman M.J. and Pilone G.J. (1995) Occurrence of arginine deiminase pathway enzymes in arginine catabolism by wine lactic acid bacteria. Appl Environ Microbiol 61: 310–316

337

50. Møller V. (1955) Simplified tests for some amino acid decarboxylases and for the arginine dihydrolase system. Acta Path Microb Scand 36: 158–172 51. Goldschmidt M.C. and Lockhart B.M. (1971) Rapid methods for determining decarboxylase activity: arginine decarboxylase. Appl Microbiol 22: 350–357 52. Gale E.F. (1940) The production of amines by bacteria. Biochem J 34: 392–413 53. Dohner P.M. and Cardon B.P. (1954) Anaerobic fermentation of lysine. J Bacteriol 67: 608–611 54. Fromageot C. (1951) Desulfhydrases. In Summer J.B. and Myrback K. (eds) The Enzymes: Chemistry and Mechanism of Action vol 1, pp. 1237–1243. Academic Press, New York 55. Wiesendanger S. and Nisman S. (1953) LaL-m´ethionine d´emercaptod´esaminase: un nouvel enzyme a` pyridoxal-phosphate. Acad Sci 237: 764–765 56. McLain C.J., Zieve L., Doizaki W.M., Gilberstadt S. and Anstad G.R. (1980) Blood methanethiol in alcoholic liver disease with and without hepatic encephalopathy. Gut 21: 318–323 57. Roediger W.E.W. and Nance S. (1990) Selective reduction of fatty acid oxidation in colonocytes: correlation with ulcerative colitis. Lipids 25: 646–652 58. Lebertz H. and Andreesen J.R. (1988) Glycine fermentation by Clostridium histolyticum. Arch Microbiol 150: 11–14 59. Andreesen J.R. (1994) Glycine metabolism in anaerobes. Anton Van Leeuwen Int J Gen Mol Microbiol 66: 223–237 60. Elsden S.R. and Hilton M.G. (1978) Volatile acid production from threonine, valine, leucine and isoleucine by clostridia. Arch Microbiol 117: 165–172 61. Giesel H. and Simon H. (1983) On the occurrence of enoate reductase and 2-oxo-carboxylate reductase in clostridia and some observations on the amino acid fermentation by Peptostreptococcus anaerobius. Arch Microbiol 135: 51–57 62. Wood W.A. and Gunsalus I.C. (1949) Serine and threonine deaminases of Escherichia coli. activators for a cell-free enzyme. J Biol Chem 181: 171–182 63. Pardee A. and Prestidge L. (1955) Induced formation of serine and threonine deaminases by Escherichia coli. J Bacteriol 70: 667–674 64. Walker D.J. (1958) The purification and properties of the L-threonine deaminase of the rumen microorganism LCI. Biochem J 69: 524–530 65. Golovchenko N.P., Belokopytov B.F. and Akimenko V.K. (1982) Threonine catabolism in the bacterium Clostridium sticklandii. Bioch Engl Trans Biokhimiya 47: 969–974 66. Hardman J.K., Stadtman T.C. and Szulmajster J. (1958) Bacterial fermentation of n-aminovaleric acid. Bacteriol Proc (Soc Am Bacteriologists) P76: 120