Amino Acid Composition and Amino Acid-Metabolic Network in Supragingival Plaque

Biomedical Research (Tokyo) 37 (4) 251–257, 2016

Amino acid composition and amino acid-metabolic network in supragingival plaque

Jumpei WASHIO, Tamaki OGAWA, Keisuke SUZUKI, Yosuke TSUKIBOSHI, Motohiro WATANABE and Nobuhiro TAKAHASHI Division of Oral Ecology and Biochemistry, Tohoku University Graduate School of Dentistry, 4-1, Seiryo-machi, Aoba, Sendai, Miyagi 980-8575, Japan (Received 12 July 2016; and accepted 22 July 2016)

ABSTRACT Dental plaque metabolizes both carbohydrates and amino acids. The former can be degraded to acids mainly, while the latter can be degraded to various metabolites, including ammonia, acids and amines, and associated with acid-neutralization, oral malodor and tissue inflammation. How- ever, amino acid metabolism in dental plaque is still unclear. This study aimed to elucidate what kinds of amino acids are available as metabolic substrates and how the amino acids are metabolized in supragingival plaque, by a metabolome analysis. Amino acids and the related metabolites in supragingival plaque were extracted and quantified comprehensively by CE-TOFMS. Plaque samples were also incubated with amino acids, and the amounts of ammonia and amino acid-related metabolites were measured. The concentration of glutamate was the highest in supragingival plaque, while the ammonia-production was the highest from glutamine. The obtained metabolome profile revealed that amino acids are degraded through various metabolic pathways, including deamination, decarboxylation and transamination and that these metabolic systems may link each other, as well as with carbohydrate metabolic pathways in dental plaque ecosystem. Moreover, glutamine and glutamate might be the main source of ammonia production, as well as arginine, and contribute to pH-homeostasis and counteraction to acid-induced demineralization in supragingival plaque.

Dental plaque is known to metabolize both carbohy- ever, the amino acid metabolism of dental plaque is drates and amino acids. Carbohydrates can be de- still unclear, probably due to the wide range of met- graded mainly through glycolysis and converted into abolic substrates and metabolites, as compiled in a organic acids, such as lactic acid. This acid produc- recent review paper (35), although it is well known tion can demineralize the tooth surface and initiate that dental plaque contains abundant amounts of dental caries (20). Amino acids can be degraded by amino acids (7, 12). dental plaque bacteria to various metabolites, includ- It was reported that several amino acids can be ing ammonia, organic acids, and amines, which are decarboxylated by dental plaque and converted into associated with acid neutralization (32), oral mal- amine and carbon oxide (4, 11), thereby the acidic odor (2, 38), and tissue inflammation (2, 15). How- plaque environment can be neutralized (11). It was also suggested that the arginine deiminase system is one of the main metabolic pathways to counteract Address correspondence to: Jumpei Washio, DDS, PhD Division of Oral Ecology and Biochemistry, Tohoku Uni- tooth demineralization by neutralizing acids in dental versity Graduate School of Dentistry, 4-1, Seiryo-machi, plaque (18, 22, 25). Various oral bacteria including Aoba, Sendai, Miyagi 980-8575, Japan Streptococcus sanguinis, Streptococcus mitis, Strepto- Tel: +81-22-717-8295, Fax: +81-22-717-8297 coccus gordonii, Streptococcus parasanguis, and E-mail: [email protected] some Lactobacillus and Actinomyces species were re- 252 J. Washio et al. ported to possess this metabolic system (18), where Billerica, MA, USA), evaporated to dryness, sus- arginine is deiminated via citrulline with the produc- pended in Milli-Q water containing internal standards tion of ammonia, ornithine, carbon oxide, and ATP (Internal standard-3; Human Metabolome Technolo- (5). In addition, Porphyromonas gingivalis, Prevotella gies), and stored at −80°C until analysis. Internal intemedia, and Fusobacterium nucleatum are known standard-3 contains trimesic acid and 3-hydroxynaph- to utilize glutamate and/or aspartate or their peptides thalene-2,7-disulfonic acid for calibration of the re- as energy substrates (30, 31) with the production of tention time for CE. ammonia, organic acids, and carbon oxide (28, 29) In a separate experiment, plaque samples collect- and contribute to acid neutralization (32). In these ed from four volunteers were washed twice with metabolic systems, many enzymatic reactions, such 2 mM potassium phosphate buffer (pH 7.0) contain- as deamination and transamination, are involved. ing 0.15 M potassium chloride and 5 mM magne- Furthermore, Veillonella and Fusobacterium are sium chloride, and mixed and suspended at 20 mg thought to possess cysteine-β-lyase and/or cysteine- wet weight of plaque per mL in the same buffer. γ-lyase, which degrade cysteine into hydrogen sul- This suspension was used as plaque suspension for fide, pyruvate, and ammonia (38, 41). However, the following study of amino acid metabolism. there is no information available about which amino acids are mainly degraded and how those amino ac- Amino acid metabolism by supragingival plaque. ids are metabolized in dental plaque. The plaque suspension was mixed with pH-neutral- In recent years, we established techniques of me- ized alanine, arginine, asparagine, aspartate, cysteine, tabolome analysis using capillary electrophoresis and glutamate, glutamine, glycine, histidine, isoleucine, a time-of-flight mass spectrometer (CE-TOFMS) to leucine, lysine, methionine, proline, serine, threo- examine a small amount of dental plaque (33, 34). nine, tyrosine, tryptophan, and valine, at a final con- Therefore, this study aimed to clarify what kinds of centration of 50 mM at pH 7.0. Before and after a amino acids are contained, which amino acids are 15-min incubation at 37°C, 75 μL of plaque suspen- mostly utilized, and how these amino acids are me- sion (containing approximately 1.5 mg wet weight tabolized in dental plaque, based on metabolome of plaque) was taken and centrifuged, then the su- analysis using CE-TOFMS. pernatant were kept at −80°C until the measurement of ammonia. The residues were immediately washed twice with ice-cold buffer, mixed with 800 μL of MATERIALS AND METHODS methanol containing internal standards (Internal stan- Supragingival plaque sampling. After informed con- dard solution-1; Human Metabolome Technologies), sent was obtained, sixteen volunteers (age: 26.1 ± and sonicated for 30 s (55W, US-1R; AS ONE Cor- 4.5 yrs) were asked to refrain from toothbrushing poration) for the efficient extraction of metabolites. and allow dental plaque to accumulate overnight. The methanol extracts were mixed well with 800 μL The volunteers had not taken any antibiotics recent- of chloroform and 320 μL of Milli-Q water, vortexed ly. After confirming that the volunteers had not con- for 30 s, and centrifuged at 2,300 × g and 4°C for sumed any food for at least 2 h, we collected all the 5 min. The aqueous layer (250 μL) was treated as available supragingival plaque, using sterilized tooth- described above. picks, mainly from marginal and interproximal areas. Simultaneously, as a marker of amino acid metabo- Immediately, plaque samples were weighed, mixed lism, the amount of ammonia produced in the super- with 0.80 mL ice-cold methanol containing internal natant was measured with an ammonia meter (Ami- standards (Internal standard-1; Human Metabolome Chek meter, Arkley, Japan). Technologies, Tsuruoka, Japan), and sonicated for 30 s (55W, US-1R; AS ONE Corporation, Osaka, CE-MS conditions. CE-MS was carried out by CE Japan) for the extraction of intracellular metabolites (G1600AX; Agilent Technologies, Waldbronn, Ger- including amino acids. Internal standard-1 contains many) equipped with a time-of-flight mass spec- methionine sulfone and camphor-10-sulfonic acid trometer (TOFMS) (G1969A; Agilent Technologies). for calibration of the quantification of MS. Follow- Separations and detections of metabolites were per- ing methanol extraction, the extracts were re-extract- formed as described previously (26, 27). A fused ed with 0.80 mL chloroform and 0.32 mL Milli-Q silica capillary (H3305-2002; Human Metabolome water by being vortexed for 30 s and were then cen- Technologies), sheath liquid (H3301-1020; Human trifuged. The aqueous layer was ultrafiltrated (Ul- Metabolome Technologies), and electrolytes (H3302- trafree-MC 5000NMWL UFC3 LCCNB; Millipore, 1021; Human Metabolome Technologies) were used Amino acids metabolism in plaque 253 for analysis. The applied voltage was set at +30 kV when the electrospray ionization was operated in the negative ion mode or +27 kV in the positive ion mode, and the capillary voltage was set at 3.5 kV in the negative ion mode and 4 kV in the positive ion mode. The flow rate of heated dry nitrogen gas (300°C) was maintained at 7 L/min. All standard metabolites and chemicals used were of analytical or reagent grade. We analyzed the data with calculating software (MassHunter Workstation Software Quali- tative Analysis; Agilent Technologies), using data obtained from standard metabolite solutions. Amino acids and related compounds (listed in Fig. 1), the metabolites in the tri-carbonic acid cycle (TCA cycle in Fig. 4), acetyl-CoA, and pyruvate were targeted and quantified with this system.

Statistical analysis. Differences in the amounts of metabolites by amino acid addition were analyzed with the paired t-test.

RESULTS Amino acids and related compounds in supragingival plaque Fig. 1 Amino acids and related metabolites in supragingival In the resting dental plaque, most major amino acids plaque (nmol/mg wet weight of dental plaque). Values are were detected: glutamate (10.6 ± 8.1 nmol/mg wet the means with standard deviations obtained from sixteen weight of plaque) was the highest, followed by aspar- individuals. Cysteine was not detected due to the charac- tate (3.0 ± 3.1), proline (1.7 ± 1.3), alanine (1.4 ± 1.2), teristics of CE-TOFMS. glycine (1.2 ± 0.7), and glutamine (1.1 ± 0.7) (Fig. 1). Cysteine was not detected due to the limitations of CE-TOFMS method. Moreover, many compounds tration of glutamate (Fig. 3a), while the addition of related to amino acid metabolism, such as citrulline, glutamate increased the concentrations of alanine, ornithine, putrescine, γ-aminobutylic acid (GABA), aspartate, 2-oxoglutarate, and GABA (Fig. 3b). Sim- and β-alanine, were detected. ilarly, the addition of asparagine increased aspartate (Fig. 3c), while the addition of aspartate increased Ammonia production from amino acids by supragin- fumarate and glutamate, and decreased 2-oxoglutarate gival plaque (Fig. 3d). The addition of arginine increased the con- When incubated with amino acids, dental plaque pro- centrations of citrulline, ornithine, putrescine, spermi- duced ammonia from all amino acids analyzed in this dine, and agmatine (Fig. 3e). The addition of cysteine study. The highest production was observed from glu- increased the concentration of serine (Fig. 3f), but tamine, followed by asparagine, arginine, serine, glu- hydrogen sulfide was not measured in this study. The tamate, aspartate, and cysteine (Fig. 2). The amounts addition of serine increased pyruvate (Fig. 3g). The of ammonia production from the other amino acids other metabolites in the TCA cycle were not changed were less than that from cysteine. during metabolic reactions of these amino acids (data not shown). Metabolome analysis of intracellular metabolites during amino acid metabolism by supragingival plaque DISCUSSION Following the addition of 7 amino acids that produced abundant ammonia (Fig. 2), the intracellular In the present study, glutamate was present in high- metabolites of dental plaque were quantified with est concentration, followed by aspartate, proline, al- metabolome analysis using CE-TOFMS (Fig. 3). anine, glycine, and glutamine (Fig. 1). In previous The addition of glutamine increased the concen- studies (7, 12), although the same amino acids were 254 J. Washio et al. detected as major compounds, their proportions var- ied. This variation may be due to high inter-individ- ual variability, differences in sampling and analytical conditions. Nevertheless, the major amino acids were common between the studies. Some compounds related to amino acid metabolism were also detected (Fig. 1), suggesting that the amino acids were metabolized in supragingival plaque. A 15-min incubation of supragingival plaque with amino acids revealed that the highest ammonia production was observed from glutamine, followed by asparagine, arginine, serine, glutamate, aspartate, and cysteine (Fig. 2), indicating that these amino acids are preferable metabolic substrates as energy and carbon sources in supragingival plaque. The exclusive production of glutamate from glutamine (Fig. 3a) indicates that glutamine is deaminated to glutamate by glutaminase. The production of aspartate, alanine, and 2-oxoglutarate from glutamate (Fig. 3b) suggests that glutamate can be deaminated to 2-oxoglutarate directly by glutamate dehydrogenase, or transaminated to 2-oxoglutarate with the production of aspartate and alanine by aminotransferase. Oral streptococci and other plaque bacteria are known to possess glutaminase (37), glutamate dehydrogenase (10), and aminotransferase (40). The high concentration of glutamine and glutamate (Fig. 1) Fig. 2 Ammonia production from amino acids by supragingival plaque (nmol/mg wet weight of plaque). Values are the and the high ammonia-producing activity from glu- means with standard deviations obtained from three inde- tamine and glutamate (Fig. 2) suggest that these pendent experiments. Ammonia production from other ami- amino acids may be the main metabolic substrates no acids was less than that from lysine. in supragingival plaque. GABA also increased in dental plaque after the glutamate addition (Fig. 3b), indicating that the decarboxylation of glutamate also the main substrate for ammonia production through occurred. the arginine deiminase system in dental plaque (18, The production of aspartate from asparagine 22, 25), where arginine is deiminated via citrulline (Fig. 3c) indicates that asparagine was deaminated to with the production of ammonia, ornithine, carbon aspartate by asparaginase. The production of fuma- oxide, and ATP with acid neutralization (3, 5, 13). rate and glutamate from aspartate (Fig. 3d) suggests The present study supports that it occurs in suprag- that aspartate can be deaminated to fumarate by as- ingival plaque (Figs. 1, 2, and 3e). An exogenous partase, or transaminated to oxaloacetate with the supply of arginine might activate the bacterial argi- production of glutamate by aminotransferase. Oral nine deiminase system and counteract acid deminer- bacteria, such as Porphyromonas gingivalis, Pre- alization of tooth surface. In fact, a protective effect votella interemedia, and some Neisseria species, are against caries of toothpaste containing arginine is known to possess asparaginase (14, 37), aspartase, considered, and its efficacy is suggested (1, 16). and aminotransferase (28, 32). β-Alanine is known Moreover, agmatine was produced by the addition of to be produced by the carboxylation of aspartate (14), arginine (Fig. 3e), indicating that arginine was decar- however no production was observed under the ex- boxylated in supragingival plaque. Polyamines such perimental conditions in the present study. β-Alanine as putrescine and spermidine also increased. These was found in supragingival plaque (Fig. 1), suggest- compounds are known to be produced by the decar- ing that its production is low in supragingival plaque, boxylation of ornithine (Fig. 3e) (17, 18) and in- but the β-alanine produced is stable and not effi- volved in bacterial growth, etc. (39). ciently utilized. Ammonia production from cysteine and serine was Arginine has been considered to be taken up as also relatively high (Fig. 2). Oral bacteria such as Amino acids metabolism in plaque 255

Fig. 3 Proposed metabolic pathways of amino acids in supragingival plaque based on metabolome profiles. Graphs show the change in concentration of metabolites (nmol/mg wet weight of plaque) for 15-min incubation without amino acids (control: left bar) or with amino acids (right bar). The data were obtained from three independent experiments. Oxaloacetate was difficult to detect with CE-TOFMS. CO2 and H2S were not measured in the present study. Significant difference from control (*P < 0.05). GABA, γ-aminobutyric acid.

Veillonella and Fusobacterium are reported to have serine (Fig. 3f) indicates that cystathionine-β-synthase cysteine-γ-lyase and be able to degrade cysteine into is active in dental plaque. After the addition of ser- hydrogen sulfide, pyruvate, and ammonia (38, 42). ine, pyruvate increased significantly (Fig. 3f) with In addition, these bacteria also have cystathionine-β- an increase of ammonia, indicating that serine was synthase, which degrades cysteine into serine and deaminated by serine dehydratase. It was reported pyruvate (38, 43). Ammonia production from cyste- that Veillonella atypica and Fusobacterium nuclea- ine (Fig. 3f) suggests the cysteine-γ-lyase activity, but tum have this enzyme (38, 43). the absence of the detection of pyruvate necessitates It is assumed that various amino acids are utilized further study to clarify the pathway. The increase of for biosynthesis of the proteins that oral bacteria need 256 J. Washio et al.

The amine production from amino acids also releas- es carbon dioxide and can contribute to acid neutralization (11, 35), however amines and polyamines, as well as ammonia, are known to be cytotoxic (22, 23, 24) and thus may induce gingival inflammation. These compounds are also known to be oral malodorous (9), together with sulfur compounds (hydrogen sulfide and methyl mercaptan) (9, 36) and short-chain fatty acids (butyric acid, valeric acid, etc.) (19), which were not examined in the present study.

Acknowledgement This study was supported by Grants-in-Aid for Young Scientific Research B (No.23792498), Scientific Re- search B (No. 26293439), and Scientific Research C (No. 26463154) from JSPS, Japan, and by Research and Education Funding for the Inter-University Re- search Project (2012–2017) from Ministry of Educa- tion, Culture, Sports, Science and Technology, Japan.

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