- Email: [email protected]
Monitoring ammonia to assess halitosis
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Vol. 94 No. 6 December 2002
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY ORAL MEDICINE
Editor: Martin S. Greenberg
Monitoring ammonia to assess halitosis Akiko Amano, DDS,a Yasuo Yoshida, DDS, PhD,b Takahiko Oho, DDS, PhD,c and Toshihiko Koga, DDS, PhD,de Fukuoka, Japan KYUSHU UNIVERSITY
Objective. This study examined the applicability of ammonia monitoring for assessing halitosis. Study design. The actual degree of halitosis was determined by using an organoleptic test in 61 subjects aged 28 ⫾ 10 years (mean ⫾ SD). Levels of volatile sulfur compounds and ammonia were determined by using gas chromatography and ammonia monitoring, respectively. Levels of ammonia and methyl mercaptan produced by bacteria in dental plaque and tongue-coating samples obtained from 25 subjects were quantified. In addition, changes in ammonia levels were measured before and after removing tongue coating or dental plaque. Results. There was no significant correlation between the organoleptic score and the ammonia level measured with ammonia monitoring, whereas there was a significant correlation between ammonia level and the total level of volatile sulfur compounds measured with gas chromatography. Significant correlations were also observed between ammonia level and levels of methyl mercaptan produced by bacteria in dental plaque and tongue coating. Furthermore, the ammonia level decreased after the removal of tongue coating and dental plaque. Conclusion. These results indicate that measuring ammonia levels is useful for assessing halitosis, specifically for halitosis arising from a lack of oral hygiene. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:692-6)
Halitosis affects a large section of the population1 and may cause significant social and psychologic problems.2 Halitosis originates from microbial putrefaction within the oral cavity, and volatile sulfur compounds (VSCs), such as hydrogen sulfide, methyl mercaptan, and dimethyl sulfide, are considered the major gases associated with halitosis.3-5 In addition to their obvious
Supported in part by Grant-in-Aid for Developmental Scientific Research (B)(2)(13557184) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. a Resident, Department of Preventive Dentistry, Faculty of Dental Science, Kyushu University. b Research Associate, Department of Preventive Dentistry, Faculty of Dental Science, Kyushu University. c Assistant Professor, Department of Preventive Dentistry, Faculty of Dental Science, Kyushu University. d Professor, Department of Preventive Dentistry, Faculty of Dental Science, Kyushu University. e Deceased, October 14, 2001. This article is in recognition of his fine work and is dedicated to his memory. Received for publication Jan 15, 2002; accepted for publication May 13, 2002. © 2002, Mosby, Inc. 1079-2104/2002/$35.00 ⫹ 0 7/13/126911 doi:10.1067/moe.2002.126911
692
odor, VSCs have pathologic consequences as a result of their penetration and subsequent degradation of oral tissues by affecting cell metabolism and collagen synthesis.6-8 The volatile fraction above putrefied saliva contains ammonia.1,9 On the basis of the hypothesis that ammonia produced by oral bacteria reflects halitosis, a portable monitor to measure the ammonia produced by oral bacteria after rinsing with urea solution was recently developed in Japan.10 However, few studies have examined the correlation between ammonia production by oral bacteria and the actual degree of halitosis. This study examined the applicability of ammonia monitoring to the assessment of halitosis. The level of oral malodor in subjects was determined by several methods, including an organoleptic test, gas chromatography, and ammonia monitoring, and correlation analyses of the results were performed. Dental plaque and tongue-coating samples were obtained from the subjects, and the ability of bacteria in these samples to produce ammonia and methyl mercaptan was examined in vitro. In addition, the effects of removing tongue coating and dental plaque on the production of ammonia in the oral cavity were examined.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 94, Number 6
SUBJECTS AND METHODS Subjects Sixty-one volunteers (27 men and 34 women, 28 ⫾ 10 years of age [mean ⫾ SD]) were examined; all the subjects were Japanese. Written informed consent was obtained from all subjects, and the experimental protocol was approved by the ethics committee of the Faculty of Dental Science, Kyushu University, Fukuoka, Japan. None of the subjects had a history of diseases such as sinusitis or diabetes mellitus that are thought to be nonoral causes of malodor.11 The volunteers were asked to refrain from oral activities, including eating, drinking, chewing, sucking, brushing, and mouth rinsing, for at least 2 hours before testing. Organoleptic test The degree of halitosis was estimated by using the organoleptic test of Oho et al.12 Briefly, the organoleptic panel consisted of 3 dentists who were trained to perform an examination standardized by the Japanese Bureau of Environmental Health. The results of the organoleptic test were recorded as a score of 0 to 3 (0, no appreciable odor; 1, slight odor [not unpleasant]; 2, clearly noticeable malodor; and 3, strong malodor). The percentage of agreement in the organoleptic scores among the 3 examiners always exceeded 85% ( ⫽ 0.67).13 Measurement of VSCs The level of VSCs was analyzed with a Shimadzu GC-14B gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a flame photometric detector system. Ten milliliters of mouth air was applied to a glass column packed with 25% , ⬘-oxydipropionitrile on a 60- to 80-mesh Chromosorb W AW-DMCS-ST support system (Shimadzu) at 70°C.12 Measurement of ammonia produced from urea in the oral cavity The ammonia produced by oral bacteria was measured with a portable ammonia-monitoring device (ATTAIN; Taiyo, Osaka, Japan), according to the manufacturer’s instructions. Briefly, volunteers rinsed with 20 mL of 0.17 mol/L urea solution for 30 seconds and then refrained from opening their mouth for 5 minutes. This instrument contained a pump that drew 50 mL of air through an ammonia gas detector tube connected to the disposable mouthpiece placed inside the subject’s mouth. The concentration of ammonia produced by oral bacteria was read directly from the scale on the detector tube.
Amano et al 693
Measurement of methyl mercaptan produced by oral bacteria Dental plaque and tongue-coating samples were collected by using a microspoon and suspended in ice-cold sterile phosphate-buffered saline (PBS; 10 mmol/L Na2HPO4, 5 mmol/L KH2PO4, 0.12 mol/L NaCl, [pH, 7.5]). Each sample was mixed well and then centrifuged at 100 g for 1 minute at 4°C to remove masses that were not suspended in PBS. The supernatant including bacterial cells was diluted with sterile PBS to an optical density at 550 nm of 0.2, and then 700 L of GAM broth (Nissui Medical Co, Tokyo, Japan) was added to 300 L of the cell suspension. A reaction mixture consisting of the cell suspension and GAM broth was incubated anaerobically (10% CO2, 10% H2, 80% N2) in a sterile 15-mL polypropylene tube at 37°C. After a 60-minute incubation, the tube was sealed with a silicon plug and then incubated for an additional 90 minutes. To determine methyl mercaptan level, 1 mL of the vapor above the reaction mixture in the tube was collected with a syringe and analyzed by using gas chromatography, as described above. Measurement of ammonia produced by oral bacteria A cell suspension that was diluted with sterile PBS to an optical density at 550 nm of 0.2 was prepared as described above. One hundred microliters of the cell suspension was then mixed with the same volume of the reaction mixture (50 mmol/L urea, 50 mmol/L K2HPO4-KH2PO4, pH 7.0).14 After the mixture was incubated at 37°C for 60 minutes, the amount of ammonia produced from urea was determined by using Ammonia-testwako (Wako Pure Chemical Industries Ltd, Osaka, Japan). Effects of oral prophylaxis on the ammonia production Twelve volunteers were asked to refrain from brushing their teeth or removing their tongue coating for at least 15 hours before the measurement. The ammonia level of each subject was measured as described previously. Precleaning measurement was carried out after rinsing with distilled water. The tongue coating was removed as thoroughly as possible with a tongue scraper, and another measurement was carried out immediately after tongue cleaning. Subsequently, the subjects underwent professional mechanical tooth cleaning to remove dental plaque that was disclosed with erythrosine, and then a final measurement was made. Thirty seconds before each measurement to remove residual urea, the volunteers rinsed with distilled water (20 mL) for 30 seconds 5 times.
694 Amano et al
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY December 2002
Table I. Correlations between halitosis levels as determined with 3 methods
Organoleptic test Gas chromatography*
Gas chromatography*
Ammonia monitoring
0.47† —
0.16 0.39‡
*Common logarithms of measured values were used for analysis. † Spearman rank correlation coefficient, P ⬍ .01. ‡ Pearson product moment correlation coefficient, P ⬍ .01.
Statistical analyses The common logarithms of the measured values were used to statistically analyze the results obtained with gas chromatography. Correlation analyses were performed with either the Spearman rank correlation or the Pearson product moment correlation. The ammonia levels among treatments were compared with the Wilcoxon signed rank test. RESULTS The actual degree of halitosis and levels of VSCs and ammonia in 61 subjects were determined by using the organoleptic method, gas chromatography, and ammonia monitoring, respectively. The correlations between the halitosis levels obtained by these methods were then analyzed (Table I). There was a significant correlation between the halitosis level obtained with the organoleptic test and the total level of VSCs determined by gas chromatography. There was, however, no significant correlation between the halitosis level obtained with the organoleptic test and the ammonia level determined by ammonia monitoring, whereas there was a significant correlation between the ammonia level measured by ammonia monitoring and the total level of VSCs measured by gas chromatography. Dental plaque and tongue-coating samples were obtained from 5 volunteers to examine the ability of oral bacteria to produce ammonia. Bacteria in dental plaque and tongue coating produced ammonia in a concentration-dependent manner (Fig 1). The bacteria in dental plaque produced more ammonia than those in tongue coating. There was a significant correlation between the ammonia and the methyl mercaptan levels produced by bacteria in dental plaque and tongue coating (Table II). In addition, there was a significant correlation between the ammonia levels produced by bacteria in dental plaque and those in tongue coating. There was also a significant correlation between the methyl mercaptan levels produced by bacteria in dental plaque and those in tongue coating. To examine whether ammonia monitoring is useful for assessing oral hygiene, the ammonia levels in the
Fig 1. Ammonia levels produced by bacteria in dental plaque and tongue-coating samples obtained from 5 subjects. A, Dental plaque samples; B, tongue-coating samples. CFU, Colony-forming unit.
oral cavity were compared before and after the removal of tongue coating and dental plaque (Fig 2) by using the means ⫾ SD of the ammonia levels expressed as a percentage of the baseline ammonia level. There was no significant decrease in ammonia level after water rinsing compared with the baseline, whereas there was a significant difference between the baseline value and the level measured after removing the tongue coating.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 94, Number 6
Table II. Correlations between ammonia and methyl mercaptan produced from dental plaque and tongue coating
NH3 from dental plaque NH3 from tongue coating MESH from dental plaque
NH3 from tongue coating
MESH from dental plaque
MESH from tongue coating
0.44* — —
0.45* 0.16 —
0.43 0.55† 0.50*
*Pearson product moment correlation coefficient, P ⬍ .05. † Pearson product moment correlation coefficient, P ⬍ .01. NH3, Ammonia; MESH, methyl mercaptan.
Fig 2. Effects of removing tongue coating and dental plaque on ammonia production in the oral cavity. The values are the mean ⫾ SD (bars) of ammonia levels expressed as a percentage of the baseline ammonia level. TC, Tongue coating; DP, dental plaque; n.s., not significant. *P ⬍ .05; **P ⬍ .01.
A further significant decrease in the ammonia level was observed after also removing the dental plaque. DISCUSSION Oral malodor results from by-products arising from bacterial amino acid metabolism of materials such as food debris, desquamated cells from oral mucosa, and leukocytes that accumulate in the mouth. These metabolites include VSCs, indole, skatole, amines, and ammonia.15,16 Because VSCs are the main cause of oral malodor,15 recent investigations have focused on VSCs-measuring techniques such as gas chromatography and sulfide monitoring. Consequently, little attention has been paid to gases other than VSCs, and there are no portable instruments to measure gases other than VSCs for assessing halitosis. Although fresh saliva contains volatile ammonia, its
Amano et al 695
odor is considered sweet and pleasant.1 This may explain why it is considered meaningless to detect ammonia directly from the mouth. In addition, the natural concentration of ammonia in the oral cavity is too low to detect. The ammonia monitoring used in this study was supposed to detect the ammonia produced from urea by bacterial metabolism in the oral cavity. The applicability of ammonia monitoring was examined through correlation between the ammonia and the methyl mercaptan levels produced by oral bacteria. There was no significant correlation between the halitosis level determined with the organoleptic test and the measured ammonia level. There was, however, a significant correlation between the ammonia level and total VSC levels measured by gas chromatography. On the basis of these findings, ammonia monitoring can be used as an alternative to gas chromatography. The relationship between the number of oral bacteria and the amount of ammonia they produce was investigated; bacterial samples obtained from both dental plaque and tongue coating produced ammonia from urea in a concentration-dependent manner. Therefore, we examined the effect of removing tongue coating and dental plaque on ammonia production in the oral cavity (Fig 2). Ammonia production decreased after the removal of tongue coating and dental plaque; this result coincides well with those in Fig 1, suggesting that ammonia was produced from bacteria in both dental plaque and tongue coating. These findings suggest that ammonia monitoring can be used to assess the halitosis caused by poor oral hygiene. On the basis of information from the National Institute for Occupational Safety and Health (http://www. cdc.gov/niosh/ipcs/iscpgdx6.html), the VSC with the lowest odor threshold value is methyl mercaptan, as detected by gas chromatography. In addition, methyl mercaptan has pathologic significance for oral tissues.6,7,15,17 Hence, the correlation between ammonia and methyl mercaptan levels produced by oral bacteria was analyzed. A significant correlation between the ammonia and the methyl mercaptan levels produced by bacteria derived from dental plaque and tongue coating was observed. These results suggest that the determination of ammonia production from urea in the oral cavity is useful for assessing halitosis. In conclusion, the degree of halitosis determined by ammonia monitoring was correlated with the results of VSC measurement by gas chromatography. A significant correlation was also observed between the ammonia and the methyl mercaptan levels produced by bacteria in dental plaque and tongue coating. The ammonia production level decreased after the removal of tongue coating or dental plaque in the oral cavity. Therefore,
696 Amano et al
ammonia monitoring is a useful tool for the assessment of halitosis and the evaluation of oral hygiene. REFERENCES 1. Tonzetich J. Production and origin of oral malodor: a review of mechanisms and methods of analysis. J Periodontol 1977;48:1320. 2. Hine M. Halitosis. J Am Dent Res 1956;55:37-46. 3. Tonzetich J. Evaluation of volatile odoriferous components of saliva. Arch Oral Biol 1964;9:39-45. 4. Tonzetich J. Direct gas chromatographic analysis of sulphur compounds in mouth air in man. Arch Oral Biol 1971;16:587-97. 5. Schmidt NF, Missan SR, Tarbet WJ. The correlation between organoleptic mouth-odor ratings and levels of volatile sulfur compounds. Oral Surg Oral Med Oral Pathol 1978;45:560-7. 6. Johnson PW, Ng W, Tonzetich J. Modulation of human gingival fibroblast cell metabolism by methyl mercaptan. J Periodontal Res 1992;27:476-83. 7. Johnson PW, Yaegaki K, Tonzetich J. Effect of volatile thiol compounds on protein metabolism by human gingival fibroblasts. J Periodontal Res 1992;27:553-61. 8. Johnson P, Yaegaki K, Tonzetich J. Effect of methyl mercaptan on synthesis and degradation of collagen. J Periodontal Res 1996;31:323-9. 9. Goldberg SK, Kozalovsky A, Rosenberg M. In: Rosenberg M, editor. Bad breath: research perspectives. 2nd ed. Tel Aviv: Ramot publishing; 1997. p. 71-85. 10. Ueda H, Sato Y, Nakayama T, Hashimoto M, Honda S. Clinical significance of the oral ammonia analyzer ATTAIN for halitosis treatment. In: Abstracts of the 5th International Conference on Breath Odor. Tel Aviv: International Society for Breath Odor Research; 2001. p. 44.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY December 2002 11. Scully C, el-Maaytah M, Porter SR, Greenman J. Breath odor: etiopathogenesis, assessment and management. Eur J Oral Sci 1997;105:287-93. 12. Oho T, Yoshida Y, Shimazaki Y, Yamashita Y, Koga T. Characteristics of patients complaining of halitosis and the usefulness of gas chromatography for diagnosing halitosis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:531-4. 13. Aickin M. Maximum likelihood estimation of agreement in the constant predictive probability model, and its relation to Cohen’s kappa. Biometrics 1990;46:293-302. 14. Chen YY, Clancy KA, Burne RA. Streptococcus salivarius urease: genetic and biochemical characterization and expression in a dental plaque streptococcus. Infect Immun 1996;64:585-92. 15. Ratcliff PA, Johnson PW. The relationship between oral malodor, gingivitis, and periodontitis: a review. J Periodontol 1999; 70:485-9. 16. van Steenberghe D. Breath malodor. Curr Opin Periodontol 1997;4:137-43. 17. Yaegaki K, Sanada K. Biochemical and clinical factors influencing oral malodor in periodontal patients. J Periodontol 1992;63: 783-9. Reprint requests: Takahiko Oho, DDS, PhD Department of Preventive Dentistry Kyushu University Faculty of Dental Science 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 Japan [email protected]
Vol. 94 No. 6 December 2002
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY ORAL MEDICINE
Editor: Martin S. Greenberg
Monitoring ammonia to assess halitosis Akiko Amano, DDS,a Yasuo Yoshida, DDS, PhD,b Takahiko Oho, DDS, PhD,c and Toshihiko Koga, DDS, PhD,de Fukuoka, Japan KYUSHU UNIVERSITY
Objective. This study examined the applicability of ammonia monitoring for assessing halitosis. Study design. The actual degree of halitosis was determined by using an organoleptic test in 61 subjects aged 28 ⫾ 10 years (mean ⫾ SD). Levels of volatile sulfur compounds and ammonia were determined by using gas chromatography and ammonia monitoring, respectively. Levels of ammonia and methyl mercaptan produced by bacteria in dental plaque and tongue-coating samples obtained from 25 subjects were quantified. In addition, changes in ammonia levels were measured before and after removing tongue coating or dental plaque. Results. There was no significant correlation between the organoleptic score and the ammonia level measured with ammonia monitoring, whereas there was a significant correlation between ammonia level and the total level of volatile sulfur compounds measured with gas chromatography. Significant correlations were also observed between ammonia level and levels of methyl mercaptan produced by bacteria in dental plaque and tongue coating. Furthermore, the ammonia level decreased after the removal of tongue coating and dental plaque. Conclusion. These results indicate that measuring ammonia levels is useful for assessing halitosis, specifically for halitosis arising from a lack of oral hygiene. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:692-6)
Halitosis affects a large section of the population1 and may cause significant social and psychologic problems.2 Halitosis originates from microbial putrefaction within the oral cavity, and volatile sulfur compounds (VSCs), such as hydrogen sulfide, methyl mercaptan, and dimethyl sulfide, are considered the major gases associated with halitosis.3-5 In addition to their obvious
Supported in part by Grant-in-Aid for Developmental Scientific Research (B)(2)(13557184) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. a Resident, Department of Preventive Dentistry, Faculty of Dental Science, Kyushu University. b Research Associate, Department of Preventive Dentistry, Faculty of Dental Science, Kyushu University. c Assistant Professor, Department of Preventive Dentistry, Faculty of Dental Science, Kyushu University. d Professor, Department of Preventive Dentistry, Faculty of Dental Science, Kyushu University. e Deceased, October 14, 2001. This article is in recognition of his fine work and is dedicated to his memory. Received for publication Jan 15, 2002; accepted for publication May 13, 2002. © 2002, Mosby, Inc. 1079-2104/2002/$35.00 ⫹ 0 7/13/126911 doi:10.1067/moe.2002.126911
692
odor, VSCs have pathologic consequences as a result of their penetration and subsequent degradation of oral tissues by affecting cell metabolism and collagen synthesis.6-8 The volatile fraction above putrefied saliva contains ammonia.1,9 On the basis of the hypothesis that ammonia produced by oral bacteria reflects halitosis, a portable monitor to measure the ammonia produced by oral bacteria after rinsing with urea solution was recently developed in Japan.10 However, few studies have examined the correlation between ammonia production by oral bacteria and the actual degree of halitosis. This study examined the applicability of ammonia monitoring to the assessment of halitosis. The level of oral malodor in subjects was determined by several methods, including an organoleptic test, gas chromatography, and ammonia monitoring, and correlation analyses of the results were performed. Dental plaque and tongue-coating samples were obtained from the subjects, and the ability of bacteria in these samples to produce ammonia and methyl mercaptan was examined in vitro. In addition, the effects of removing tongue coating and dental plaque on the production of ammonia in the oral cavity were examined.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 94, Number 6
SUBJECTS AND METHODS Subjects Sixty-one volunteers (27 men and 34 women, 28 ⫾ 10 years of age [mean ⫾ SD]) were examined; all the subjects were Japanese. Written informed consent was obtained from all subjects, and the experimental protocol was approved by the ethics committee of the Faculty of Dental Science, Kyushu University, Fukuoka, Japan. None of the subjects had a history of diseases such as sinusitis or diabetes mellitus that are thought to be nonoral causes of malodor.11 The volunteers were asked to refrain from oral activities, including eating, drinking, chewing, sucking, brushing, and mouth rinsing, for at least 2 hours before testing. Organoleptic test The degree of halitosis was estimated by using the organoleptic test of Oho et al.12 Briefly, the organoleptic panel consisted of 3 dentists who were trained to perform an examination standardized by the Japanese Bureau of Environmental Health. The results of the organoleptic test were recorded as a score of 0 to 3 (0, no appreciable odor; 1, slight odor [not unpleasant]; 2, clearly noticeable malodor; and 3, strong malodor). The percentage of agreement in the organoleptic scores among the 3 examiners always exceeded 85% ( ⫽ 0.67).13 Measurement of VSCs The level of VSCs was analyzed with a Shimadzu GC-14B gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a flame photometric detector system. Ten milliliters of mouth air was applied to a glass column packed with 25% , ⬘-oxydipropionitrile on a 60- to 80-mesh Chromosorb W AW-DMCS-ST support system (Shimadzu) at 70°C.12 Measurement of ammonia produced from urea in the oral cavity The ammonia produced by oral bacteria was measured with a portable ammonia-monitoring device (ATTAIN; Taiyo, Osaka, Japan), according to the manufacturer’s instructions. Briefly, volunteers rinsed with 20 mL of 0.17 mol/L urea solution for 30 seconds and then refrained from opening their mouth for 5 minutes. This instrument contained a pump that drew 50 mL of air through an ammonia gas detector tube connected to the disposable mouthpiece placed inside the subject’s mouth. The concentration of ammonia produced by oral bacteria was read directly from the scale on the detector tube.
Amano et al 693
Measurement of methyl mercaptan produced by oral bacteria Dental plaque and tongue-coating samples were collected by using a microspoon and suspended in ice-cold sterile phosphate-buffered saline (PBS; 10 mmol/L Na2HPO4, 5 mmol/L KH2PO4, 0.12 mol/L NaCl, [pH, 7.5]). Each sample was mixed well and then centrifuged at 100 g for 1 minute at 4°C to remove masses that were not suspended in PBS. The supernatant including bacterial cells was diluted with sterile PBS to an optical density at 550 nm of 0.2, and then 700 L of GAM broth (Nissui Medical Co, Tokyo, Japan) was added to 300 L of the cell suspension. A reaction mixture consisting of the cell suspension and GAM broth was incubated anaerobically (10% CO2, 10% H2, 80% N2) in a sterile 15-mL polypropylene tube at 37°C. After a 60-minute incubation, the tube was sealed with a silicon plug and then incubated for an additional 90 minutes. To determine methyl mercaptan level, 1 mL of the vapor above the reaction mixture in the tube was collected with a syringe and analyzed by using gas chromatography, as described above. Measurement of ammonia produced by oral bacteria A cell suspension that was diluted with sterile PBS to an optical density at 550 nm of 0.2 was prepared as described above. One hundred microliters of the cell suspension was then mixed with the same volume of the reaction mixture (50 mmol/L urea, 50 mmol/L K2HPO4-KH2PO4, pH 7.0).14 After the mixture was incubated at 37°C for 60 minutes, the amount of ammonia produced from urea was determined by using Ammonia-testwako (Wako Pure Chemical Industries Ltd, Osaka, Japan). Effects of oral prophylaxis on the ammonia production Twelve volunteers were asked to refrain from brushing their teeth or removing their tongue coating for at least 15 hours before the measurement. The ammonia level of each subject was measured as described previously. Precleaning measurement was carried out after rinsing with distilled water. The tongue coating was removed as thoroughly as possible with a tongue scraper, and another measurement was carried out immediately after tongue cleaning. Subsequently, the subjects underwent professional mechanical tooth cleaning to remove dental plaque that was disclosed with erythrosine, and then a final measurement was made. Thirty seconds before each measurement to remove residual urea, the volunteers rinsed with distilled water (20 mL) for 30 seconds 5 times.
694 Amano et al
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY December 2002
Table I. Correlations between halitosis levels as determined with 3 methods
Organoleptic test Gas chromatography*
Gas chromatography*
Ammonia monitoring
0.47† —
0.16 0.39‡
*Common logarithms of measured values were used for analysis. † Spearman rank correlation coefficient, P ⬍ .01. ‡ Pearson product moment correlation coefficient, P ⬍ .01.
Statistical analyses The common logarithms of the measured values were used to statistically analyze the results obtained with gas chromatography. Correlation analyses were performed with either the Spearman rank correlation or the Pearson product moment correlation. The ammonia levels among treatments were compared with the Wilcoxon signed rank test. RESULTS The actual degree of halitosis and levels of VSCs and ammonia in 61 subjects were determined by using the organoleptic method, gas chromatography, and ammonia monitoring, respectively. The correlations between the halitosis levels obtained by these methods were then analyzed (Table I). There was a significant correlation between the halitosis level obtained with the organoleptic test and the total level of VSCs determined by gas chromatography. There was, however, no significant correlation between the halitosis level obtained with the organoleptic test and the ammonia level determined by ammonia monitoring, whereas there was a significant correlation between the ammonia level measured by ammonia monitoring and the total level of VSCs measured by gas chromatography. Dental plaque and tongue-coating samples were obtained from 5 volunteers to examine the ability of oral bacteria to produce ammonia. Bacteria in dental plaque and tongue coating produced ammonia in a concentration-dependent manner (Fig 1). The bacteria in dental plaque produced more ammonia than those in tongue coating. There was a significant correlation between the ammonia and the methyl mercaptan levels produced by bacteria in dental plaque and tongue coating (Table II). In addition, there was a significant correlation between the ammonia levels produced by bacteria in dental plaque and those in tongue coating. There was also a significant correlation between the methyl mercaptan levels produced by bacteria in dental plaque and those in tongue coating. To examine whether ammonia monitoring is useful for assessing oral hygiene, the ammonia levels in the
Fig 1. Ammonia levels produced by bacteria in dental plaque and tongue-coating samples obtained from 5 subjects. A, Dental plaque samples; B, tongue-coating samples. CFU, Colony-forming unit.
oral cavity were compared before and after the removal of tongue coating and dental plaque (Fig 2) by using the means ⫾ SD of the ammonia levels expressed as a percentage of the baseline ammonia level. There was no significant decrease in ammonia level after water rinsing compared with the baseline, whereas there was a significant difference between the baseline value and the level measured after removing the tongue coating.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 94, Number 6
Table II. Correlations between ammonia and methyl mercaptan produced from dental plaque and tongue coating
NH3 from dental plaque NH3 from tongue coating MESH from dental plaque
NH3 from tongue coating
MESH from dental plaque
MESH from tongue coating
0.44* — —
0.45* 0.16 —
0.43 0.55† 0.50*
*Pearson product moment correlation coefficient, P ⬍ .05. † Pearson product moment correlation coefficient, P ⬍ .01. NH3, Ammonia; MESH, methyl mercaptan.
Fig 2. Effects of removing tongue coating and dental plaque on ammonia production in the oral cavity. The values are the mean ⫾ SD (bars) of ammonia levels expressed as a percentage of the baseline ammonia level. TC, Tongue coating; DP, dental plaque; n.s., not significant. *P ⬍ .05; **P ⬍ .01.
A further significant decrease in the ammonia level was observed after also removing the dental plaque. DISCUSSION Oral malodor results from by-products arising from bacterial amino acid metabolism of materials such as food debris, desquamated cells from oral mucosa, and leukocytes that accumulate in the mouth. These metabolites include VSCs, indole, skatole, amines, and ammonia.15,16 Because VSCs are the main cause of oral malodor,15 recent investigations have focused on VSCs-measuring techniques such as gas chromatography and sulfide monitoring. Consequently, little attention has been paid to gases other than VSCs, and there are no portable instruments to measure gases other than VSCs for assessing halitosis. Although fresh saliva contains volatile ammonia, its
Amano et al 695
odor is considered sweet and pleasant.1 This may explain why it is considered meaningless to detect ammonia directly from the mouth. In addition, the natural concentration of ammonia in the oral cavity is too low to detect. The ammonia monitoring used in this study was supposed to detect the ammonia produced from urea by bacterial metabolism in the oral cavity. The applicability of ammonia monitoring was examined through correlation between the ammonia and the methyl mercaptan levels produced by oral bacteria. There was no significant correlation between the halitosis level determined with the organoleptic test and the measured ammonia level. There was, however, a significant correlation between the ammonia level and total VSC levels measured by gas chromatography. On the basis of these findings, ammonia monitoring can be used as an alternative to gas chromatography. The relationship between the number of oral bacteria and the amount of ammonia they produce was investigated; bacterial samples obtained from both dental plaque and tongue coating produced ammonia from urea in a concentration-dependent manner. Therefore, we examined the effect of removing tongue coating and dental plaque on ammonia production in the oral cavity (Fig 2). Ammonia production decreased after the removal of tongue coating and dental plaque; this result coincides well with those in Fig 1, suggesting that ammonia was produced from bacteria in both dental plaque and tongue coating. These findings suggest that ammonia monitoring can be used to assess the halitosis caused by poor oral hygiene. On the basis of information from the National Institute for Occupational Safety and Health (http://www. cdc.gov/niosh/ipcs/iscpgdx6.html), the VSC with the lowest odor threshold value is methyl mercaptan, as detected by gas chromatography. In addition, methyl mercaptan has pathologic significance for oral tissues.6,7,15,17 Hence, the correlation between ammonia and methyl mercaptan levels produced by oral bacteria was analyzed. A significant correlation between the ammonia and the methyl mercaptan levels produced by bacteria derived from dental plaque and tongue coating was observed. These results suggest that the determination of ammonia production from urea in the oral cavity is useful for assessing halitosis. In conclusion, the degree of halitosis determined by ammonia monitoring was correlated with the results of VSC measurement by gas chromatography. A significant correlation was also observed between the ammonia and the methyl mercaptan levels produced by bacteria in dental plaque and tongue coating. The ammonia production level decreased after the removal of tongue coating or dental plaque in the oral cavity. Therefore,
696 Amano et al
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