768
Salivary and Metabolic Factors
Involved in Oral Malodor Formation /.
Kleinberg and G. Westbay
Saliva plays a central role in the formation of oral malodor. Such formation has as its basis bacterial putrefaction, the degradation of proteins, and the resulting amino acids by microorganisms. Saliva provides substrates that are readily oxidized and in the process facilitates oxygen depletion. This favors the reduced conditions conducive to production of odoriferous volatiles. At the same time, saliva is a major source of oxygen for the oral bacteria which generally is inhibitory of their formation. The pH is also critical to malodor development; acidity inhibits, whereas neutrality and alkalinity favor malodor production. Since the pH on oral mucosal surfaces where odor formation occurs is largely determined by the fermentative and putrefactive activities of the adhering bacteria, these acid-base processes are necessarily of major regulatory importance. Because oral malodor and Periodontitis both involve excessive oral putrefaction, a better understanding of putrefaction could lead to more substantive methods of oral malodor treatment than exists today, as well as identifying new approaches to amelioration of the bacterial attack on the soft tissues leading to the destruction associated with periodontal disease. / Periodonts 1992; 63:768-775.
Key Words: Halitosis/etiology; saliva/analysis.
A considerable amount of fundamental information on the key metabolic elements involved in oral malodor formation has come from studies using incubated whole saliva obtained from humans by chewing an inert material such as paraffin wax. Two things happen using such a collection method. One is that the chewing facilitates dislodgement of epithelial cells and bacteria; the other is that a saliva milieu is provided containing ingredients much like those bathing the bacteria within the oral cavity. Many of the bacteria are attached to epithelial cells1 which are easily shed from the outer layers of the oral mucosa (Fig. 1). Bacteria are also dislodged from the outer layers of dental plaque covering the teeth. Incubation and extensive examination of this simple in vitro system by Fosdick and his colleagues,2-6 Tonzetich and his co-workers,7-14 McNamara et al.,15 and others has led to identification of several of the essential elements involved in oral malodor
production.
A dramatic difference in malodor formation is observed when a fermentable sugar, such as glucose, is added to or omitted from the whole saliva before it is incubated. In the absence of glucose, intense malodor is produced whereas with the addition, less or none occurs, depending on the amount of glucose added. Since the pH falls below neutrality when sugar is added and remains near or above neu-
'Department of Oral Biology and Pathology, School of Dental Medicine, University of New York, Stony Brook, NY.
State
Figure 1. Oral epithelial cells permission).
with attached bacteria
(from Nolte' with
trality when the sugar is omitted, it is clear that pH is a key malodor variable.15 McNamara et al.15 concluded that the cut-off for malodor cessation is below about pH 6.5. In saliva incubation experiments, it was also noted that
Volume 63 Number 9
shift of the initial resident microflora to a more Gramnegative bacterial composition15 acco' ">anied odor formainvolvement and tion. Additional studies implicated t imino acids, cysthe degradation of the sulfur-contair central role.8'10'13 teine, cystine, and methionine, as play Low levels of these, and for that matte, other amino acids, are characteristic of most freshly collected salivas.16 In contrast, amino acids are abundantly present as parts of peptides and proteins. These become readily available after bacterial hydrolysis, another essential process in oral malodor formation.4 6 A major source of protein with the sulfur-containing amino acids is the epithelial cells7'11-13 to which many of the bacteria are attached. To relate the incubated salivary system to events within the oral cavity, comparison of the odoriferous compounds produced in the two systems during odor formation showed them to be similar.8'9 This led Tonzetich and others7,9'17'18 to conclude that similar malodor producing metabolic processes are involved. Modification of the salivary system to more closely and quantitatively parallel the metabolic behavior of the bacterial deposits on oral surfaces in vivo was accomplished a number of years ago in our laboratory.19-22 The most important modification that had to be made was to increase and control the concentration of bacteria in the system. In freshly collected whole saliva, the concentration of sediment and bacteria generally varies between about 0.5 and 2%. Such low bacterial cell concentrations produce metabolic reactions that are much slower than those seen on oral tissue surfaces in vivo. However, by centrifuging the whole saliva and by discarding excess supernatant, the sediment in the whole saliva samples can be concentrated and the more rapid metabolic changes seen on the oral surfaces simulated. This modified salivary system, named the suspended salivary sediment (SSS) system,19 was then used in parallel with in vitro21-25 and in vivo26-31 plaque studies to develop an understanding of the acid-base32 and oxygen metabolisms33'34 of the oral mixed bacteria. A most important observation arising out of these investigations was that salivary supernatant had a major regulatory effect on the metabolism of the sediment bacteria.35 This suggested that such might also be true for oral malodor, especially since Tonzetich7,11 had found some enhancement of odor production by saliva. Accordingly, the main focus of the present paper is description of new and fundamental information on the effect of salivary supernatant on oral malodor formation. At the same time, the influence of the oxygen and acid-base metabolisms of the oral mixed flora on this relationship has been assessed.
KLEINBERG, WESTBAY
a
Demonstration of the Enhancing Effect of Salivary Supernatant on Oral Malodor Formation The substantial effect of salivary supernatant on oral malodor was demonstrated in the SSS system in our laboratory by 2 methods. One was by organoleptic assessment of mal-
3.0i
4h
24 h
0 h
4h
769
»
OmmHg Figure 6. A diagram illustrating effects of salivary oxygen and dental or mucosa! plaque metabolism on plaque aerobicIanaerobic composition.46 In thinner bacterial deposits on the teeth and oral soft tissues, the anaerA anaerobic zone-no oxygen B aerobic zone-high oxygen =
=
obic zone would be less
or
higher bacterial numbers, oral soft and hard tisharboring thicker plaque can also be expected to harbor higher proportions of the Gram-negative anaerobic bacteria15 that are key elements in the odor-producing process. In plaque accumulation and experimental gingivitis studies,47"49 it was clearly shown that upon cessation of oral hygiene, plaque accumulates spontaneously and as the plaque thickens, the flora shifts to one containing a greater number of Gram-negative anaerobic bacteria. These plaque bacteria are those best able to engage in proteolytic and aminolytic processes and hence in the production of odoriferous volatiles. Gram-positive bacteria are poor contributors to these different aspects of nitrogen degradative metabolism but, as indicated above, are major players in plaque fermentation and thus, in acid production processes. Intra-oral location can be expected to influence plaque Besides
Salivo
or
=
non-existent.
It is interesting that proline and glutamate comprise an extremely high percentage of the amino acids in salivary peptides and proteins.45 Hence, it is possible that their availability and rapidity of oxidation33 are important elements in oxygen depletion and thus, in the oral malodor
formation process.
oxygen levels because saliva access to intra-oral sites varies. Sites with least salivary access would be exposed to less oxygen, which should make them more prone to anaerobiosis, all other factors being equal. Since these sites are also those where plaque build-up readily occurs, then the oxygen depletion and associated sequelae would be conducive to development of malodor producing plaque. Accordingly, it is not surprising that interproximal sites, where plaque is generally thickest and salivary access is poor, are those where malodor readily shows when sensed
organoleptically.50 Experimental gingivitis studies48'49 have also shown that
rise in gingival inflammation occurs as plaque thickens and microflora compositions change. Again, the initial predominantly Gram-positive coccal and bacillary flora change from a microflora more or less harmless to the soft tissues to one richer in Gram-negatives51,52 and not as benign. The latter bacteria are well suited to degrade protein and the resulting amino acids. The end-products produced, which include ammonia, amines, propionate, butyrate, hydrogen sulfide, and methyl mercaptan, can be damaging53"57 to the gingival and periodontal tissues while at least some contribute simultaneously to oral malodor.58,59 a
Relation of Plaque Oxygen Levels to Plaque Thickness and Intra-Oral Location Plaque in different oral locations varies in its thickness and microbial composition. The tongue dorsum and dentition embrasure sites are locations where bacterial deposits can be particularly thick. In a thicker plaque, it is easier for the bacteria in the innermost layers to be in an oxygen depleted micro-environment (Fig. 6). Stralfors46 pointed out that the innermost plaque layers are prone to a depleted oxygen condition because the more aerobic bacteria in the outermost layers utilize the oxygen that enters the plaque mainly from saliva. Stralfors46 estimated that the plaque thickness critical to development of inner layer anaerobiosis would be about 0.32 mm. This was based on ambient oxygen being that of atmospheric air and hence an oxygen level of 150 mm Hg. However, ambient oxygen may be lower since the oxygen in saliva when measured was about 65 mm Hg.27 At this ambience, plaque anaerobiosis would be favored at a thickness of only 0.20 mm. In either case, it would not require many days of oral hygiene cessation for the plaque on the teeth and adjacent gingiva to acquire oxygen depleted zones even on readily accessible dentition sites. The same principles are likely to apply to the oral soft tissues. For example, the tongue, because of its many indentations, is likely to be covered with bacterial deposits that are thicker and more anaerobic than those on the
smoother, easily-abraded cheek mucosa.
Relation of Plaque Acid-Base Metabolism to Oral Malodor Formation Saliva plays a central role in oral bacterial acid-base metabolism and in turn, this metabolism largely determines the pH.20,32 As indicated above, pH plays a central regulatory role in oral malodor formation;15 an acidic pH reduces or inhibits odor formation whereas a pH near neutrality and above favors it. Displacement of the pH in an acidic direction rapidly occurs when sugars and other fermentable carbohydrates are provided to the plaque bacteria.60-63,30 These come mainly from the diet with small amounts from salivary glycoprotein.64 The high cell concentrations that characterize plaque accumulations on the oral surfaces together with the physical constraints of being essentially in surface deposits ensure a dynamic that easily results in end-product accumu-
Volume 63 Number 9
KLEINBERG, WESTBAY
lation.63 Hence, the rapid drop in the pH as a result of sugar fermentation60 is to be expected. Comparable displacement
rosine.70 This leads to formation of amines and carbon diox-
ide and results in pressure for the pH to shift towards neutrality. The other is de-amination of amino acids32 at alkaline pH, mainly serine, glutamine, and asparagine.36 This leads to formation of acids and ammonia, the former stronger than the latter, and hence, pressure for the pH to shift downwards towards neutrality. Additional means for returning the pH to neutrality have been identified in certain oral bacteria. The most important is arginine degradation via the arginine deiminase pathway. Even though this shows a pH optimum at and slightly above neutrality,71 it is very effective in elevating the pH from acidity. Under the right circumstances, it can also counter and reduce the acidification72 that occurs when sugar is introduced into the mouth. Another means of raising the pH, about which there is little known, is the oxidation of amino acids which occurs above about pH 5.5.34 This appears to result in ammonia and carbon dioxide generation (unpublished data) which would lead to pH elevation. Collectively, these various metabolic reactions carried out by the mix of bacteria in plaque go a long way toward determining the acid-base levels and thus whether the malodor process would be operative or not. In a sense, the pH might be like a thermostat, in that malodor occurs near and above neutrality but is turned off when the pH becomes sufficiently acidic.
Types of Acid-Base Plaques Extensive pH measurements in vivo have indicated that the acid-base metabolism of plaque in different oral sites results in a characteristic pH pattern28 which has been attributed mainly to variations in saliva availability.28 These include variations in salivary flow, location of gland duct openings, compartmentalization of the oral cavity by the teeth, gravity, and flow of salivary films evidently present on oral tissue surfaces.28,67,68,74 They also include variations in oral mucosa and dentition morphologies, since these affect not
protein amino acids samino acids NH3 +R-C00H (organic acids) v2 —
—
Urea
of the
pH in an alkaline direction occurs when urea is the challenging substrate29 but urea is usually provided continuously at quite low levels from saliva.65 Some may be provided at somewhat higher levels from gingival crevicular fluid when there is gingival inflammation.66 Sugar substrates favoring acidic pH displacement and urea substrate favoring alkaline pH displacement are opposed by certain physical and biochemical mechanisms that favor return of the pH to neutrality. The physical means of pH return consists of the removal of end-products whether acidic or alakaline by diffusion out of the plaque63 and clearance by repetitive salivary washings.67,68 The biochemical mechanisms include salivary buffering and the metabolic processes discovered by Gale69 many years ago. One is the decarboxylation of certain amino acids at acidic pH, such as Ornithine, lysine, histidine, glutamate, and ty-
773
—NH3 + C02 pH Glucose acetic
—
+
C02,
lactic, propionic, formic
/arginine —C02 + Putrescine + NH3
'amino
acids—C02+R-NH2 (amines)
protein— amino acids Time-
Figure 7. Acid-base metabolism diagram illustrating pH vectors resulting from plaque fermentation and putrefaction processes.
PH
jpr
Time
Figure 8. Acid-base metabolism diagrams for different types ofplaques. From left to right, the first two sets of vectors would be those for plaques with balanced fermentative and putrefactive metabolisms. The first would presumably be for a thicker plaque and the second for a thinner plaque. In the third set, putrefaction and hence, malodor and Periodontitis would be favored whereas, in the fourth set fermentation would dominate and favor caries development.
only the amounts of plaque that accumulate in micro-sites but also their clearance by saliva.74,75 Although each microbial community possesses the same fundamental acid-base processes indicated by the metabolic vectors shown in Figure 7, variation in magnitude and location on the pH scale (Fig. 8) would enable the diverse types of plaque pH changes seen in vivo30,31 to be accommodated. Location of acid-base vectors on the pH scale appears to be influenced more by the submandibular-sublingual than by the parotid secretions.28 Very little attention has been paid to the acid-base changes on mucosal surfaces in situ. Interestingly, at the same time that Stephan61 showed the now famous Stephan curve in plaque on the teeth following a sugar rinse, he also showed a similar large and rapid decrease in pH on the dorsum of the tongue. It is probable that, like plaque on dento-gingival surfaces, bacterial deposits on the soft tissues also show acid-base vectors60 and intra-oral pH variation. Conclusions Saliva evidently plays a central role in malodor formation. On the one hand, it provides oxygen which, as indicated
774
above, favors inhibition of odor formation. On the other hand, saliva contains oxidizable substrates that can cause oxygen depletion33 and as a result, conditions favorable to
malodor formation. How this might apply in the mouth and which effect would dominate might depend, among other things, upon the salivary flow rate. Rapid flow, greater availability of oxygen, and less opportunity for salivary peptides and proteins to be degraded by the oral bacteria could mean dominance of the inhibitory property of saliva. On the other hand, when conditions are reversed, as for example during sleep when flow rates and oxygen availability are at their lowest,73 malador stimulation by saliva ought to be highly favored. This would explain why malodor is generally severest upon arising in the morning.2'7 The pH and hence the acid-base metabolism of the oral bacteria play a central role in malodor formation. Near and above neutrality are conditions favorable to the production of the putrefactive end-products that are malodorous and harmful to the oral soft tissues. Central to oral malodor formation is oral putrefaction.3-5 Such excessive formation is associated with Periodontitis3-6 and is the basis of the suggestion that oral malodor might be used as a Periodontitis indicator.76-77 Hence, a better understanding of oral bacterial putrefaction could lead to more substantive methods of malodor treatment than exist, and at the same time, might identify new approaches to the amelioration of the bacterial attack on the soft tissues leading to the destruction associated with periodontal disease.
Acknowledgments The secretarial assistance of Mrs. Patricia Calia and the diagram preparations by Mr. James Skillman are gratefully
acknowledged.
REFERENCES 1. Nolte WA. Defense mechanisms of the mouth. In: Nolte WA, ed. Oral Microbiology with Basic Microbiology and Immunology, 4th ed. St. Louis: CV Mosby Company; 1982;245-260. 2. Sulser GF, Brening RH, Fosdick LS. Some conditions that affect the odor concentration of breath. J Dent Res 1939;18:355-359. 3. Berg M, Fosdick LS. Studies in periodontal disease. II. Putrefactive 4.
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ORAL MALODOR FORMATION
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10. Tonzetich J, Richter VJ. Evaluation of volatile odoriferous components of saliva. Arch Oral Biol 1964;9:39-45. 11. Tonzetich J, Kestenbaum RC. Odor production by human salivary fractions and plaque. Arch Oral Biol 1969;14:815-827. 12. Tonzetich J, Eigen E, King WJ, Weiss S. Volatility as a factor in the inability of certain amines and indole to increase the odor of saliva. Arch Oral Biol 1967;12:1167-1175. 13. Tonzetich J, Carpenter PAW. Production of volatile sulphur compounds from cysteine, cystine and methionine by human dental plaque. Arch Oral Biol 1971;16:599-607. 14. Tonzetich J, Johnson PW. Chemical analysis of thiol, di-sulphide and total sulphur content of human saliva. Arch Oral Biol 1977;22:125131. 15. McNamara TF, Alexander JF, Lee M. The role of microorganisms in 'the production of oral malador. Oral Surg Oral Med Oral Pathol 16.
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I. Effect of varying sediment and glucose concentrations and acid production in human salivary sediment mixtures. Arch Oral Biol 1967;12:1457-1473. 20. Kleinberg I. Biochemistry of the dental plaque. In: Staple PH, ed. Advances in Oral Biology, Vol. 4. Orlando, FL: Academic Press; 1970:43-90. 21. Singer DL, Chatterjee R, Denepitiya L, Kleinberg I. A comparison of the acid-base metabolisms of pooled human dental plaque and salivary sediment. Arch Oral Biol 1983;28:29-35. 22. Denepitiya L, Kleinberg I. A comparison of the microbial compositions of pooled dental plaque and salivary sediment. Arch Oral Biol 23.
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Effect of urea concentration on human plaque pH levels in situ. Arch Oral Biol 1967;12:175-1484. 24. Wijeyeweera RL, Kleinberg I. Arginolytic and ureolytic activities of pure cultures of human oral bacteria and their effects on the pH response of salivary sediment and dental plaque in vitro. Arch Oral
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Berg M, Burrill DY, Fosdick LS. Chemical studies in periodontal disease. IV. Putrefaction rate as index of periodontal disease. J Dent Res 1947;26:67-71. Fosdick LS, Piez KA. Chemical studies in periodontal disease. X. Paper Chromatograph investigation of the putrefaction associated with Periodontitis. / Dent Res 1953;32:87-100. Tonzetich J. Production and origin of oral malodor: A review of mechanisms and methods of analysis. / Periodontol 1977;48:13-20. Richter VJ, Tonzetich J. The application of instrumental technique for the evaluation of odoriferous volatiles from saliva and breath. Arch Oral Biol 1964;9:47-53. Tonzetich J. Direct gas Chromatographie analysis of sulphur compounds in mouth air in man. Arch Oral Biol 1971;16:587-597.
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1972;34:41^t8. Kleinberg I, Kanapka JA, Chatterjee R, Craw D, D'Angelo , Sandham HJ. Metabolism of nitrogen by the oral mixed bacteria. In: Kleinberg I, Ellison SA, Mandel ID, eds. Saliva and Dental Caries. Washington, DC: Information Retrieval; 1979;357-377. Solis-Gaffar MC, Niles HP, Rainieri WC, Kestenbaum RC. Instrumental evaluation of mouth odor in a human clinical study. / Dent Res 1975;54:351-357.
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I. Acid-base pH curves in vitro with mixtures of pure cultures of human oral microorganisms. Arch Oral Biol 1989;34:55-64. Singer DL, Kleinberg I. Quantitative assessment of urea, glucose and ammonia changes in human dental plaque and saliva following rinsing with urea and glucose. Arch Oral Biol 1983;28:923-929. Globerman DY, Kleinberg I. Intra-oral P02 and its relation to bacterial accumulation on the oral tissues. In: Kleinberg I, Ellison SA, Mandel ID, eds. Saliva and Dental Caries. New York: Information Retrieval; 1979:275-291. Kleinberg I, Jenkins GN. The pH of dental plaques in the different areas of the mouth before and after meals and their relationship to the pH and rate of flow of resting saliva. Arch Oral Biol 1964;9:493516. Kleinberg I. Effect of urea concentration on human plaque in situ. Arch Oral Biol 1967;12:1475-1484. Kleinberg I, Jenkins GN, Denepitiya L, Chatterjee R. Diet and dental plaque. In: Ferguson DB, ed. The Environment of the Teeth. Basel, Switzerland: Karger; 1981:88-107. Kleinberg I, Jenkins GN, Chatterjee R, Wijeyeweera RL. The antimony pH electrode and its role in the assessment and interpretation of dental plaque pH. / Dent Res 1982;61:1139-1147.
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Kleinberg I. Regulation of the gingival plaque and its relation ease.
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KLEINBERG, WESTBAY
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acid-base metabolism of the dentoperiodontal dis-
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Korayem M, Westbay G, Kleinberg I. Constituents of salivary supernatant responsible for stimulation of oxygen uptake by the bacteria in human salivary sediment. Arch Oral Biol 1990;35:145-152. Korayem M, Traudt M, Kleinberg I. Oxygen uptake and its relation to pH in a human salivary sediment during fermentation of glucose.
Arch Oral Biol 1990;35:759-764. Kleinberg I, Craw D, Komiyama K. Effect of salivary supernatant on the glycolytic activity of the bacteria in salivary sediment. Arch Oral Biol 1973;18:787-798. 36. Westbay G. Studies on the biochemical basis of oral malodor formation (Thesis). Stony Brook, NY: State University of New York; 1990. 226 p. 37. Kenney EB, Ash MM. Oxidation-reduction potential of developing plaque, periodontal pockets, and gingival sulci. / Periodontol 35.
1969;40:630-633.
38. Traudt M, Kleinberg I. Bacteria in human dental plaque responsible for its oxygen uptake activity. / Dent Res 1988;67(Spec.
Issue):204(Abstr.732). Kleinberg I. Comparison of the acidogenicities glucose. / Dent Res 1988;67(Spec. Issue):203(Abstr.726). Eisenbrandt LL. Initial oxidation reduction potentials of saliva. JDent
39. Salako NO, Ryan C, of galactose and 40.
Res 1943;22:293-294.
41. Molan P, Hartles RL. The nature of the intrinsic salivary substrate used by the human oral flora. Arch Oral Biol 1971;16:1449-1462. 42. Chatterjee R, D'Angelo , Kleinberg I. Degradation of salivary proteins by the oral mixed bacteria. / Dent Res 1980;59(Spec.
Issue):686(Abstr.686).
43. Converso C, Kleinberg I. Degradation of salivary cultures of oral bacteria. / Dent Res
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proteins by pure 1988;67(Spec.
44. Shiota T, Kunkel Jr MF. In vitro chemical and bacterial changes in saliva. / Dent Res 1958;37:780-787. 45. Jenkins GN. The Physiology and Biochemistry of the Mouth, 4th ed. Oxford: Blackwell Scientific; 1978:292. 46. Stralfors A. An investigation of the respiratory activities of oral bacteria. Acta Odontol Scand 1956;14:153-186. 47. Ritz HL. Microbial population shifts in developing human dental plaque. Arch Oral Biol 1967;12:1561-1568. 48. Löe , Theilade E, Borglum-Jensen SB. Experimental gingivitis in man. / Periodontol 1965;36:177-187. 49. Lindhe H, Hamp SE, Löe . Plaque induced periodontal disease in beagle dogs: A 4-year clinical roentgenographical and histometrical study. / Periodontal Res 1975;10:243-255. 50. Rosenberg M, Kalkarni GV, Bosy A, McCulloch CAG. Reproducibility and sensitivity of oral malodor measurements with a portable sulphide monitor. / Dent Res 1991;70:1436-1440. 51. Dzink JL, Tanner ACR, Haffajee AD, Socransky SS. Gram-negative species associated with active destructive periodontal lesions. J Clin Periodontol 1985;12:648. 52. Katayama T. Suzuki T, Okada S. Clinical observation of dental plaque maturation. Application of oxidation-reduction indicator dyes. / Per-
iodontol 1975;46:610. 53. Van Steenbergen JM, Van der Mispel LMS, DeGraff J. Effects of ammonia and volatile fatty acids produced by oral bacteria on tissue culture cells. / Dent Res 198.6;65:909-912. 54. Singer RE, Buckner BA. Butyrate and proprionate: Important components of toxic dental plaque extracts. Infect Immun 1981;32:458-463.
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55. Levine M. The role for butyrate and propionate in mediating Hela cells growth inhibition by human dental plaque fluid from adult periodontal disease. Arch Oral Biol 1985;30:155-159. 56. Tonzetich J, McBride BC. Characterization of volatile sulphur production by pathogenic and non-pathogenic strains of oral bacteroides. Arch Oral Biol 1981;26:963-969. 57. Dean RT, Jessup W, Roberts CR. Effects of exogenous amines on mammalian cells with particular reference to membrane flow. Biochem J 1984;217:27-40. 58. Slots J. The predominant cultivable microflora of advanced Periodontitis. Scand J Dent Res 1977;85:114-121. 59. Pianotti R, Pitts G. Effects of an antiseptic mouthwash on odorigenic microbes in the human gingival crevice. J Dent Res 1978;57:175179. 60. Imfeld T. Identification of Low Caries Risk Dietary Components. Basel, Switzerland: Karger; 1983;1-198. 61. Stephan RM. Changes in hydrogen ion concentration on tooth surfaces and in carious lesions. J Am Dent Assoc 1940;27:718-723. 62. Neff D. Acid production from different carbohydrate sources in plaque in situ. Caries Res 1967;1:78-87. 63. Stralfors A. Investigation into the bacterial chemistry of dental plaques. Odont Tidsker 1950;58:153-341. 64. Kleinberg I. Formation and accumulation of acid on the tooth surface. JDent Res 1970;49:1300-1316. 65. Jenkins GN. The Physiology and Biochemistry of the Mouth, 4th ed. Oxford: Blackwell Scientific; 1978;286-287. 66. Golub LM, Borden SM, Kleinberg I. Urea content of gingival crevicular fluid and its relation to periodontal disease in humans. / Periodont Res 1971;6:243-251. 67. Dawes C. A mathematical model of salivary clearance of sugar from the oral cavity. Caries Res 1983;17:321-334. 68. Dawes C. Physiological factors affecting salivary flow rate, oral sugar clearance, and the sensation of dry mouth in man. / Dent Res
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69. Gale EF. The bacterial amino acid
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1946;6:1-32. 70. Hayes ML, Hyatt AT. The decarboxylation of amino acids by bacteria derived from human dental plaque. Arch Oral Biol 1974;19:361-369. 71. Kanapka JA, Kleinberg I. Catabolism of arginine by the mixed bac72.
teria in human salivary sediment under conditions of low and high glucose concentration. Arch Oral Biol 1983;28:1007-1015. Mandel ID, Kleinberg I. Methods for plaque biochemistry alteration.
State-of-the-science review. In: Löe H, Kleinman DY, eds. Dental
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Schneyer LS, Levin LK. Rate of secretion by individual salivary gland pairs of man under conditions of reduced exogenous stimulation. / Appi Physiol 1955;7:508-609. Besic FC. Molecular transport in micro-volumes simulating salivary
retention areas. J Dent Res 1966;45:1499-1510. 75. Nevin RB, Walsh JP. Some physiochemical factors in relation to the causation of interproximal caries. J Dent Res 1951;30:235-250. 76. Tonzetich J. Oral malador: An indicator of health status and oral cleanliness. Int Dent J 1978;28:309-319. 77. Kostelc JG, Preti G, Zelson PR, Brauner L, Baehni P. Oral odors in early experimental gingivitis. J Periodont Res 1984;19:303-312.
Send reprint requests to: Dr. I. Kleinberg, Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York, Stony Brook, NY 11794-8702.
Salivary and Metabolic Factors
Involved in Oral Malodor Formation /.
Kleinberg and G. Westbay
Saliva plays a central role in the formation of oral malodor. Such formation has as its basis bacterial putrefaction, the degradation of proteins, and the resulting amino acids by microorganisms. Saliva provides substrates that are readily oxidized and in the process facilitates oxygen depletion. This favors the reduced conditions conducive to production of odoriferous volatiles. At the same time, saliva is a major source of oxygen for the oral bacteria which generally is inhibitory of their formation. The pH is also critical to malodor development; acidity inhibits, whereas neutrality and alkalinity favor malodor production. Since the pH on oral mucosal surfaces where odor formation occurs is largely determined by the fermentative and putrefactive activities of the adhering bacteria, these acid-base processes are necessarily of major regulatory importance. Because oral malodor and Periodontitis both involve excessive oral putrefaction, a better understanding of putrefaction could lead to more substantive methods of oral malodor treatment than exists today, as well as identifying new approaches to amelioration of the bacterial attack on the soft tissues leading to the destruction associated with periodontal disease. / Periodonts 1992; 63:768-775.
Key Words: Halitosis/etiology; saliva/analysis.
A considerable amount of fundamental information on the key metabolic elements involved in oral malodor formation has come from studies using incubated whole saliva obtained from humans by chewing an inert material such as paraffin wax. Two things happen using such a collection method. One is that the chewing facilitates dislodgement of epithelial cells and bacteria; the other is that a saliva milieu is provided containing ingredients much like those bathing the bacteria within the oral cavity. Many of the bacteria are attached to epithelial cells1 which are easily shed from the outer layers of the oral mucosa (Fig. 1). Bacteria are also dislodged from the outer layers of dental plaque covering the teeth. Incubation and extensive examination of this simple in vitro system by Fosdick and his colleagues,2-6 Tonzetich and his co-workers,7-14 McNamara et al.,15 and others has led to identification of several of the essential elements involved in oral malodor
production.
A dramatic difference in malodor formation is observed when a fermentable sugar, such as glucose, is added to or omitted from the whole saliva before it is incubated. In the absence of glucose, intense malodor is produced whereas with the addition, less or none occurs, depending on the amount of glucose added. Since the pH falls below neutrality when sugar is added and remains near or above neu-
'Department of Oral Biology and Pathology, School of Dental Medicine, University of New York, Stony Brook, NY.
State
Figure 1. Oral epithelial cells permission).
with attached bacteria
(from Nolte' with
trality when the sugar is omitted, it is clear that pH is a key malodor variable.15 McNamara et al.15 concluded that the cut-off for malodor cessation is below about pH 6.5. In saliva incubation experiments, it was also noted that
Volume 63 Number 9
shift of the initial resident microflora to a more Gramnegative bacterial composition15 acco' ">anied odor formainvolvement and tion. Additional studies implicated t imino acids, cysthe degradation of the sulfur-contair central role.8'10'13 teine, cystine, and methionine, as play Low levels of these, and for that matte, other amino acids, are characteristic of most freshly collected salivas.16 In contrast, amino acids are abundantly present as parts of peptides and proteins. These become readily available after bacterial hydrolysis, another essential process in oral malodor formation.4 6 A major source of protein with the sulfur-containing amino acids is the epithelial cells7'11-13 to which many of the bacteria are attached. To relate the incubated salivary system to events within the oral cavity, comparison of the odoriferous compounds produced in the two systems during odor formation showed them to be similar.8'9 This led Tonzetich and others7,9'17'18 to conclude that similar malodor producing metabolic processes are involved. Modification of the salivary system to more closely and quantitatively parallel the metabolic behavior of the bacterial deposits on oral surfaces in vivo was accomplished a number of years ago in our laboratory.19-22 The most important modification that had to be made was to increase and control the concentration of bacteria in the system. In freshly collected whole saliva, the concentration of sediment and bacteria generally varies between about 0.5 and 2%. Such low bacterial cell concentrations produce metabolic reactions that are much slower than those seen on oral tissue surfaces in vivo. However, by centrifuging the whole saliva and by discarding excess supernatant, the sediment in the whole saliva samples can be concentrated and the more rapid metabolic changes seen on the oral surfaces simulated. This modified salivary system, named the suspended salivary sediment (SSS) system,19 was then used in parallel with in vitro21-25 and in vivo26-31 plaque studies to develop an understanding of the acid-base32 and oxygen metabolisms33'34 of the oral mixed bacteria. A most important observation arising out of these investigations was that salivary supernatant had a major regulatory effect on the metabolism of the sediment bacteria.35 This suggested that such might also be true for oral malodor, especially since Tonzetich7,11 had found some enhancement of odor production by saliva. Accordingly, the main focus of the present paper is description of new and fundamental information on the effect of salivary supernatant on oral malodor formation. At the same time, the influence of the oxygen and acid-base metabolisms of the oral mixed flora on this relationship has been assessed.
KLEINBERG, WESTBAY
a
Demonstration of the Enhancing Effect of Salivary Supernatant on Oral Malodor Formation The substantial effect of salivary supernatant on oral malodor was demonstrated in the SSS system in our laboratory by 2 methods. One was by organoleptic assessment of mal-
3.0i
4h
24 h
0 h
4h
769
»
OmmHg Figure 6. A diagram illustrating effects of salivary oxygen and dental or mucosa! plaque metabolism on plaque aerobicIanaerobic composition.46 In thinner bacterial deposits on the teeth and oral soft tissues, the anaerA anaerobic zone-no oxygen B aerobic zone-high oxygen =
=
obic zone would be less
or
higher bacterial numbers, oral soft and hard tisharboring thicker plaque can also be expected to harbor higher proportions of the Gram-negative anaerobic bacteria15 that are key elements in the odor-producing process. In plaque accumulation and experimental gingivitis studies,47"49 it was clearly shown that upon cessation of oral hygiene, plaque accumulates spontaneously and as the plaque thickens, the flora shifts to one containing a greater number of Gram-negative anaerobic bacteria. These plaque bacteria are those best able to engage in proteolytic and aminolytic processes and hence in the production of odoriferous volatiles. Gram-positive bacteria are poor contributors to these different aspects of nitrogen degradative metabolism but, as indicated above, are major players in plaque fermentation and thus, in acid production processes. Intra-oral location can be expected to influence plaque Besides
Salivo
or
=
non-existent.
It is interesting that proline and glutamate comprise an extremely high percentage of the amino acids in salivary peptides and proteins.45 Hence, it is possible that their availability and rapidity of oxidation33 are important elements in oxygen depletion and thus, in the oral malodor
formation process.
oxygen levels because saliva access to intra-oral sites varies. Sites with least salivary access would be exposed to less oxygen, which should make them more prone to anaerobiosis, all other factors being equal. Since these sites are also those where plaque build-up readily occurs, then the oxygen depletion and associated sequelae would be conducive to development of malodor producing plaque. Accordingly, it is not surprising that interproximal sites, where plaque is generally thickest and salivary access is poor, are those where malodor readily shows when sensed
organoleptically.50 Experimental gingivitis studies48'49 have also shown that
rise in gingival inflammation occurs as plaque thickens and microflora compositions change. Again, the initial predominantly Gram-positive coccal and bacillary flora change from a microflora more or less harmless to the soft tissues to one richer in Gram-negatives51,52 and not as benign. The latter bacteria are well suited to degrade protein and the resulting amino acids. The end-products produced, which include ammonia, amines, propionate, butyrate, hydrogen sulfide, and methyl mercaptan, can be damaging53"57 to the gingival and periodontal tissues while at least some contribute simultaneously to oral malodor.58,59 a
Relation of Plaque Oxygen Levels to Plaque Thickness and Intra-Oral Location Plaque in different oral locations varies in its thickness and microbial composition. The tongue dorsum and dentition embrasure sites are locations where bacterial deposits can be particularly thick. In a thicker plaque, it is easier for the bacteria in the innermost layers to be in an oxygen depleted micro-environment (Fig. 6). Stralfors46 pointed out that the innermost plaque layers are prone to a depleted oxygen condition because the more aerobic bacteria in the outermost layers utilize the oxygen that enters the plaque mainly from saliva. Stralfors46 estimated that the plaque thickness critical to development of inner layer anaerobiosis would be about 0.32 mm. This was based on ambient oxygen being that of atmospheric air and hence an oxygen level of 150 mm Hg. However, ambient oxygen may be lower since the oxygen in saliva when measured was about 65 mm Hg.27 At this ambience, plaque anaerobiosis would be favored at a thickness of only 0.20 mm. In either case, it would not require many days of oral hygiene cessation for the plaque on the teeth and adjacent gingiva to acquire oxygen depleted zones even on readily accessible dentition sites. The same principles are likely to apply to the oral soft tissues. For example, the tongue, because of its many indentations, is likely to be covered with bacterial deposits that are thicker and more anaerobic than those on the
smoother, easily-abraded cheek mucosa.
Relation of Plaque Acid-Base Metabolism to Oral Malodor Formation Saliva plays a central role in oral bacterial acid-base metabolism and in turn, this metabolism largely determines the pH.20,32 As indicated above, pH plays a central regulatory role in oral malodor formation;15 an acidic pH reduces or inhibits odor formation whereas a pH near neutrality and above favors it. Displacement of the pH in an acidic direction rapidly occurs when sugars and other fermentable carbohydrates are provided to the plaque bacteria.60-63,30 These come mainly from the diet with small amounts from salivary glycoprotein.64 The high cell concentrations that characterize plaque accumulations on the oral surfaces together with the physical constraints of being essentially in surface deposits ensure a dynamic that easily results in end-product accumu-
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KLEINBERG, WESTBAY
lation.63 Hence, the rapid drop in the pH as a result of sugar fermentation60 is to be expected. Comparable displacement
rosine.70 This leads to formation of amines and carbon diox-
ide and results in pressure for the pH to shift towards neutrality. The other is de-amination of amino acids32 at alkaline pH, mainly serine, glutamine, and asparagine.36 This leads to formation of acids and ammonia, the former stronger than the latter, and hence, pressure for the pH to shift downwards towards neutrality. Additional means for returning the pH to neutrality have been identified in certain oral bacteria. The most important is arginine degradation via the arginine deiminase pathway. Even though this shows a pH optimum at and slightly above neutrality,71 it is very effective in elevating the pH from acidity. Under the right circumstances, it can also counter and reduce the acidification72 that occurs when sugar is introduced into the mouth. Another means of raising the pH, about which there is little known, is the oxidation of amino acids which occurs above about pH 5.5.34 This appears to result in ammonia and carbon dioxide generation (unpublished data) which would lead to pH elevation. Collectively, these various metabolic reactions carried out by the mix of bacteria in plaque go a long way toward determining the acid-base levels and thus whether the malodor process would be operative or not. In a sense, the pH might be like a thermostat, in that malodor occurs near and above neutrality but is turned off when the pH becomes sufficiently acidic.
Types of Acid-Base Plaques Extensive pH measurements in vivo have indicated that the acid-base metabolism of plaque in different oral sites results in a characteristic pH pattern28 which has been attributed mainly to variations in saliva availability.28 These include variations in salivary flow, location of gland duct openings, compartmentalization of the oral cavity by the teeth, gravity, and flow of salivary films evidently present on oral tissue surfaces.28,67,68,74 They also include variations in oral mucosa and dentition morphologies, since these affect not
protein amino acids samino acids NH3 +R-C00H (organic acids) v2 —
—
Urea
of the
pH in an alkaline direction occurs when urea is the challenging substrate29 but urea is usually provided continuously at quite low levels from saliva.65 Some may be provided at somewhat higher levels from gingival crevicular fluid when there is gingival inflammation.66 Sugar substrates favoring acidic pH displacement and urea substrate favoring alkaline pH displacement are opposed by certain physical and biochemical mechanisms that favor return of the pH to neutrality. The physical means of pH return consists of the removal of end-products whether acidic or alakaline by diffusion out of the plaque63 and clearance by repetitive salivary washings.67,68 The biochemical mechanisms include salivary buffering and the metabolic processes discovered by Gale69 many years ago. One is the decarboxylation of certain amino acids at acidic pH, such as Ornithine, lysine, histidine, glutamate, and ty-
773
—NH3 + C02 pH Glucose acetic
—
+
C02,
lactic, propionic, formic
/arginine —C02 + Putrescine + NH3
'amino
acids—C02+R-NH2 (amines)
protein— amino acids Time-
Figure 7. Acid-base metabolism diagram illustrating pH vectors resulting from plaque fermentation and putrefaction processes.
PH
jpr
Time
Figure 8. Acid-base metabolism diagrams for different types ofplaques. From left to right, the first two sets of vectors would be those for plaques with balanced fermentative and putrefactive metabolisms. The first would presumably be for a thicker plaque and the second for a thinner plaque. In the third set, putrefaction and hence, malodor and Periodontitis would be favored whereas, in the fourth set fermentation would dominate and favor caries development.
only the amounts of plaque that accumulate in micro-sites but also their clearance by saliva.74,75 Although each microbial community possesses the same fundamental acid-base processes indicated by the metabolic vectors shown in Figure 7, variation in magnitude and location on the pH scale (Fig. 8) would enable the diverse types of plaque pH changes seen in vivo30,31 to be accommodated. Location of acid-base vectors on the pH scale appears to be influenced more by the submandibular-sublingual than by the parotid secretions.28 Very little attention has been paid to the acid-base changes on mucosal surfaces in situ. Interestingly, at the same time that Stephan61 showed the now famous Stephan curve in plaque on the teeth following a sugar rinse, he also showed a similar large and rapid decrease in pH on the dorsum of the tongue. It is probable that, like plaque on dento-gingival surfaces, bacterial deposits on the soft tissues also show acid-base vectors60 and intra-oral pH variation. Conclusions Saliva evidently plays a central role in malodor formation. On the one hand, it provides oxygen which, as indicated
774
above, favors inhibition of odor formation. On the other hand, saliva contains oxidizable substrates that can cause oxygen depletion33 and as a result, conditions favorable to
malodor formation. How this might apply in the mouth and which effect would dominate might depend, among other things, upon the salivary flow rate. Rapid flow, greater availability of oxygen, and less opportunity for salivary peptides and proteins to be degraded by the oral bacteria could mean dominance of the inhibitory property of saliva. On the other hand, when conditions are reversed, as for example during sleep when flow rates and oxygen availability are at their lowest,73 malador stimulation by saliva ought to be highly favored. This would explain why malodor is generally severest upon arising in the morning.2'7 The pH and hence the acid-base metabolism of the oral bacteria play a central role in malodor formation. Near and above neutrality are conditions favorable to the production of the putrefactive end-products that are malodorous and harmful to the oral soft tissues. Central to oral malodor formation is oral putrefaction.3-5 Such excessive formation is associated with Periodontitis3-6 and is the basis of the suggestion that oral malodor might be used as a Periodontitis indicator.76-77 Hence, a better understanding of oral bacterial putrefaction could lead to more substantive methods of malodor treatment than exist, and at the same time, might identify new approaches to the amelioration of the bacterial attack on the soft tissues leading to the destruction associated with periodontal disease.
Acknowledgments The secretarial assistance of Mrs. Patricia Calia and the diagram preparations by Mr. James Skillman are gratefully
acknowledged.
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Send reprint requests to: Dr. I. Kleinberg, Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York, Stony Brook, NY 11794-8702.
