Formation and Accumulation of Acid

on

the Tooth Surface

1. KLEINBERG Department of Oral Biology, The University of Manitoba, Winnipeg, Canada

Dental plaque, the bacterial deposit found in regions of the dentition difficult to reach with oral hygiene procedures, is essential for the formation and accumulation of acid on the surface of the tooth' (Fig 1). The high bacterial concentration in plaque enables it to convert dietary carbohydrate to acid at a rapid rate. Because diffusion of substances out of the plaque is a comparatively slow process, the acids thus formed tend to accumulate and cause the pH to fall. In most instances, the dental plaque is open to its environment and therefore consists of entry, metabolic, and exit components. The metabolic component encompasses those biochemical processes within the plaque whereby the plaque obtains, mainly from the degradation of carbohydrate and nitrogenous substrates, the energy necessary for maintaining its integrity.2 At the same time that the plaque derives energy from these substrates, it forms acidic and basic end products, the former from carbohydrate and the latter from nitrogenous substrates, particularly urea. The entry and exit components of the system relate the plaque to its environment and, based on the properties of open systems, are important regulators of the overall metabolism of the plaque.3 Because saliva is normally the major fluid phase of the plaque system, the physical and chemical properties of saliva are important determinants of the overall metabolism of the plaque and of whether a particular plaque is capable of producing sufficient acid to produce a carious lesion. The tendency of many investigators has been to consider acid formation in the caries process in isolation. In this study the formation and accumulation of acid within the plaque and on the tooth surface have been examined in relation to total plaque function, particularly the acid-base metabolism and its regulation. 1300

Acid Formation in Plaque In Situ Information of any consequence on the formation of acid in the dental plaque was not obtained until Stephan4 measured the pH of plaque in situ with microelectrodes constructed from antimony. Stephan4 showed that rinsing with a 10% glucose or sucrose solution caused an immediate, rapid fall in pH, which reached a minimum within about ten minutes and then slowly rose over a period of approximately one or more hours. Repetition of the rinse resulted in a pH curve of similar shape (Fig 1). Besides showing that the formation of acid by the plaque is a rapid process, his experiments indicated that acid formation, at least on the more accessible surfaces of the teeth, is an intermittent process. Stephan5 also showed that plaques on the labial surfaces of the maxillary and mandibular anterior teeth differed in pH minimums and, because of the inverse relation of the pH minimums to the incidence of caries in these regions of the dentition, he postulated that a certain pH level must be reached before caries can occur. These findings led to the concept of a critical pH. support for which was provided when it was found that individuals who showed higher caries activities also showed lower plaque pH minimums. Using the lactobacillus count as an index of caries susceptibility, Strilfors68 confirmed that those individuals who showed higher counts were those who showed lower pH minimums after a rinse with glucose. Using pH as a master variable,7 Kleinberg8 9 examined the system characteristics of the dental plaque and found that although the plaque is morphologically complex, with regard to its acid-base changes. the plaque functions as a synchronized unit. Using glucose in one study, and urea in a second, the relation between substrate availability and pH response was estab-

Vol 49 1970
First glucose
Lef t
teeth brushed

ACID ON THE TOOTH SURFACE

Second glucose
rinse

1301

rinse

I.

TIME IN MINUTES FIG L-.pH on the tooth surface after rinsing with 10% glucose both before and after plaque removal. Plaques on labial cervical surfaces of right and left maxillary incisors were measured before and for one hour after rinsing. Thereafter, plaques on left side were removed with toothbrush and rinsing and pH measurements were repeated. Note that both curves on the right side and the first on left are almost the same. On the other hand, the second curve on left, the one obtained after plaque removal, shows an initial small rise in pH (attributable to the slight stimulation of saliva that occurs during and immediately after rinsing) and no subsequent pH fall (adapted from Stephan
and Miller').

wished. It was shown clearly that limited availability of substrate resulted in a fallrise type of curve with glucose and a risefall type of curve with urea. On the other hand, when availability of substrate to the plaque was not limited, the pH response was to steady state levels that were above base line with urea and below base line with glucose (Fig 2, Kleinberg'0). The difference between the steady state level and that of base line was a function of the substrate concentration. Measurement of the pH of plaques located in different regions of the human dentition showed the existence of regional patterns, the basis of which was attributed to variation in the flow of saliva to the different regions of the dentition." Plaques in locations favored by more saliva showed higher plaque pH levels than those with less saliva and individuals with a more rapid resting salivary flow rate showed higher plaque pH levels than those with slower flow

rates."'

Macromolecular Structure of the Dental Plaque and Its Relation to Acid Formation and Accumulation Most investigators have assumed that plaque has a gel-like structure characterized by little or no space among the plaque cells and intercellular matrix material. A gel-like structure would enable one to explain why acids formed within the plaque can accumulate and why, as the acids subsequently slowly diffuse out of the plaque, the pH slowly rises. However, one point that has been overlooked in this hypothesis is the fact that a gel-like structure would also retard the rate of entry of substrate into the plaque. As a result, a slow entry would be followed by a rapid conversion process that kinetically could not lead to the rapid build-up of acid necessary for the pH to fall rapidly when sugar enters the mouth. That plaque is not a simple gel is suggested by recent findings'2 which show that rinsing with either a glucose or urea solution results in their almost instantaneous uptake and plaque levels of glucose and urea that are much higher than the levels in saliva. This could not have occurred unless uptake were faster than the brief time it takes for the solution to equilibrate with the saliva and suggests that, at least for fasting plaque, a floc structure (a macromolecular arrangement with spaces between the aggregating substances1) is more likely to be the correct macromolecular arrangement of the constituents of the dental plaque.'6 Characteristics of a floc structure are such that sugars could penetrate through the plaque much easier than if the structure was a gel.13 Also a large internal surface area, like a sponge, would favor uptake of a larger volume of solution. Because exit of fluids from narrow spaces is difficult5 diffusion of acid out of a floclike plaque would be difficult, but probably not as difficult as exit from a gel. Should the intracellular spaces become filled with extracellular polysaccharide that was synthesized during uptake of sugar by the plaque, the plaque might become less floclike and more gellike, as envisaged by earlier investigators.6 For a gel-like plaque, and with the salivary flow rate and sugar concentration outside the plaque at constant levels (conditions one might expect when the plaque pH is at its minimum after ingestion of dietary carbo-

1302

KLEINBERG
5ml 1%
urea

J Dent

JRes Supplement
I-

to

No. 6

0.05ml 1%

urea

0.05ml 167%

ursa

0.05ml 0.17% ure/a min

intervals

9.0-

pH
8.0
I

1\
*-.

I
I

i''. \ *.-., N IsI_

V
*

A/ a_--r \_
\.

9.0.,.

IA!

pH

f;i ?~~~~~~\eA
b
40
6 400 80

'%

i'
a
100 120

8.0

7n] ..-

20

40

60

80

(.V.4 7

.lml 1% 8.0

glucose

Olml 1% glucose

O.Iml 50% glucose

8.0I K
pH
7.0
s

0.06ml of 0.5% glucose/2 min

intervals

pIlHA

7.01
6.0

~~~~~~~~
\

6.0

C
0

I
0

vsfifs~AV
20

/1/

d
60

-2

20

40

60

80

100

120

40

50

Time in
hydrate), Stralfors6 has derived the following relationship:
QH2

Minutes

FIG 2.-Effect of varying availability of glucose and urea on pH of dental plaque in situ. The availability of urea and glucose were varied in a and c by varying the concentration and fixing the time period over which aliquots of substrate were added to plaque. In b and d, availability was varied by varying time and keeping concentration constant (adapted from Kleinberg10).

UO =

2D,

where U0 is the concentration of acid at the enamel surface; Q, the rate of sugar consumption by the bacteria; H, the thickness of the plaque; and D1, the coefficient for acid diffusion through the plaque. From this equation, the acid concentration in the innermost layer rises linearly with increase in Q and geometrically with increase in H. The decided effect of an increase in H, was demonstrated when StrAlfors6 showed in experiments in vitro that a bacterial gel only 0.6 mm thick could sustain a pH differential between inner and outer surfaces of approximately 1.5 pH units. Because plaque has the necessary enzymes to remove extracellular polysaccharides, including dextran,'6"17 it is probable that the porosity of the plaque, and as a result the

ability of the plaque to take up sugar from the environment, is variable. If these arguments are correct, uptake would be most efficient when the environmental supply of sugar is low and the plaque is most porous, whereas uptake would be least efficient when the environmental supply is high and the plaque contains its highest levels of extracellular polysaccharide. Under the first conditions, the deeper layers could receive substrate before the surface layers have used it up; under the second conditions, the deeper layers should still be able to receive substrate, because substrate is likely to be in excess at this time and, because of the low pH, its rate of utilization is likely to be

low.6,18
Saliva as a Determinant of the Fasting Steady State pH Before breakfast, the plaque pH is at its highest level and immediately decreases on eating a meal, unless meticulous precautions

Vol 49 1970

ACID ON THE TOOTH SURFACE

pH
,

1303

are taken to ensure that the meal does not contain fermentable carbohydrate.1 The high pH at this time, has been attributed partly to a low level of acid formation and a somewhat higher level of base formation. Absence of dietary carbohydrate and availability of only- low levels of carbohydrate from the constituents of saliva and tissue debris would account for low acid formation (Fig 3), whereas the higher base formation can be accounted for from the urea present in the saliva.9 The urea content of the saliva is sufficient under these conditions of low acid formation to produce enough base to account for the plaque pH being not only at an alkaline level on arising, but also for the pH of plaque usually being considerably above the pH of the saliva.1' On the other hand, diets usually contain large amounts of carbohydrate, which on entry into the oral cavity results in a decrease in the pH. The nitrogenous substrates it contains, unlike urea, cannot be catabolized rapidly to form base and cause the pH to rise (Fig 3). Because eating is an intermittent process and the dietary constituents favor acid formation in the plaque, the effect of eating is to displace the plaque pH from its steady state level1' (Fig 4). The magnitude and duration of the response is dependent on the availability of carbohydrate. The longer carbohydrate is available, the longer the pH is displaced from the base-line level.8

8.0
Mandibular Interproximal
_X|
7.0

, Z Mean for

4.---

AII Areas-.0

/a' / t / '-/

Maxillary Interproximal

6.0
I ~~~~~

Time in Hours

FIG 4.-Relationship between pH of dental plaque and time of eating. Prebreakfast (zero time) plaque pH levels are above pH of resting saliva, which has an average value of approximately 6.7; after eating, pH falls below that of saliva and with increase in time after eating, pH slowly rises toward base-line levels (adapted from Kleinberg'9).

Availability of dietary carbohydrate differs from that of salivary substrates such as urea, in that the former occurs in large concentrations and is available for short periods of time, whereas the latter occurs in

pH
Meat Distilled 9.0- Homogenate Water

pH
Gingival
9.0 Homogenate

Distilled

I
7.0

I'

Water

7.0-

5.01
0 30

a
60

5.0

b
0o

30I

60

90

Time in Minutes
FIG 3.-Effect of (a) meat and (b) gingival tissue homogenates on pH of plaques located on the labial surfaces of the maxillary incisors. Unlike urea, they do not cause plaque pH to rise.

1304

KLEINBERG

J Dent Res Supplement to No. 6

low concentration and is available more or less continuously. Saliva and diets that contain carbohydrate have opposite effects on the plaque pH. This is seen particularly at the time dietary carbohydrate enters the mouth when acid formation and an increased flow of saliva are stimulated simultaneously.20 Depending on the relative magnitudes of the two effects, the plaque pH can fall, but remain at a higher pH than if there were no stimulation of saliva, or rise, if the pH1 at the time of ingestion is so low because of previous carbohydrate ingestion that further carbohydrate cannot cause the pH to fall further (Fig 5).

Dentition Morphology and Its Effect on Substrate Availability Retention of dietary carbohydrate occurs most frequently in the pits and fissures, in

regions where teeth are in contact, and on the smooth surfaces of the teeth that are adjacent to the gingiva. Retention in these sites, particularly in the first two, which the saliva does not easily reach, would prolong the time of carbohydrate availability and result in prolongation of acid formation and the duration of the plaque pH below its base-line level. Since carbohydrate foodstuffs are easily crammed into the pits and fissures of the occlusal surfaces of the posterior teeth during eating, the time that substrate could be available to acid-producing bacteria in these regions is exceedingly long. In fact, with frequent eating, it would not be surprising if carbohydrate were available to the bacteria in these sites virtually uninterruptedly. This may also occur at or near areas of contact of approximating teeth and within carious lesions, particularly those that have

pH

pH

7.01
fig cookie
(
cola

6.0 A_

beverage
NA 1

-I%A
5.00

Vln-~
15

-glIucose solution

30

15

30

Time in Minutes
FIG 5.-Effect of carbohydrate foodstuffs on pH of (a) plaques initially at neutral and (b) curious lesions initially at acidic pH. Rinsing with 10% glucose stimulates salivary flow only slightly, whereas foodstuffs, such as fig cookie and cola beverage stimulate flow much more.21 Consequently, pH with glucose falls in both a and b, but less in b because the pH is near its minimum. Qn the other hand, greater stimulation of saliva with the fig cookie and cola beverage would account for the reduction in extent of pH fall in a and rise in pH in b. (The data for the construction of a were taken from Ludwig and Bibby22 and the data for b were taken from Caldwell and Bibby.23

Vol 49 1970

ACID ON THE TOOTH SURFACE

1305

CL~~~~~~~~~~~~~~~~~~w

5.0 is~Tyne

10

15 Time,

20

25

30

mrin

FIG 6.-Effect of a 10% glucose rinse on the pH of carious lesions having different size apertures. Lesions with the narrowest openings (type 1 ) show lower pHI levels and are less affected by saliva than those with wider openings (types 2 and 3). Type 3 lesions are those with the widest openings (adapted from Dirksen et a124).

narrow openings24 (Fig 6). Soluble carbohydrates, such as the sugars, may not enter these regions as rapidly as they might reach the smooth surfaces, but once they have entered they could remain there for long periods of time (Fig 7). Regions of the dentition that favor increased retention of dietary carbohydrate also have poor access to saliva. The dentition morphology has the effect of altering the relative supply of substrate to the plaque microorganisms that are engaged in base formation and the microorganisms that are
pH
0.5 ml 30% sucrose approx every 40 sec added to the interdental area

involved in the formation of acid. The more a region of the dentition favors such an imbalance, ie, more retention and less saliva access, the more likely acid formation, a cariogenic flora, and caries would occur. This hypothesis, that alterations in dentition morphology can decidedly affect the relative supply of substrate to the plaque and alter its flora and cariogenicity, can be applied to a variety of oral situations with considerable success. For example, it is a common clinical observation that caries in approximating teeth proceed at different rates. If one of the adjacent teeth is extracted, caries arrest usually occurs in the tooth that remains, provided that the lesion is in a relatively early stage. Extraction exposes a previously inaccessible tooth surface to saliva and at the same time destroys the previous ability to retain dietary carbohydrate in the immediate vicinity. According to our hypothesis, this alteration would favor increased base formation, because of the increased access to saliva, and reduced acid formation, because of reduced retention of carbohydrate resulting in a higher plaque pH, caries arrest, and remineralization.26 With time, the lesion would feel hard when probed with a dental explorer. The opposite effect on the relative supply of substrates occurs when erupting teeth, not yet in contact, move into apposition with their neighbors. Hemmens et a127 have shown that as a tooth erupts, the incidence of lactobacilli in proximal plaques is low
9-day- old plaque

6 5

4 _

m...;._..
-T

20

40

60

80

min

FIG 7.-pH before, during, and after continuous addition of 0.5 ml of 50% sucrose solution to intradental 9-day-old plaque. The pH of plaque in the intradental region was measured radiotelemetrically with a pH glass electrode and radio transmitter built into a partial denture.25 (Reprinted with permission of authors and publisher.)

1306

KLEINBERG

J Dent Res Supplement to No. 6

but rises decidedly once contact is made. Numerous studies have established that a rise in the incidence of lactobacilli is a clear signal of increased acid formation and that caries is likely to follow.28 The observation that proximal surfaces of teeth that are not in contact (diastema) show a much lower caries incidence than similar surfaces that are in contact29 supports these observations. A carious lesion, once it has progressed through the enamel, shows considerable lateral enlargement when it enters the dentin. As a result, any carbohydrate entering such a lesion would, because the lesion is almost inaccessible to saliva, result in low pH levels for a long period of time. Absence of saliva and retention of carbohydrate could account for the observation of Stephan30 and that of Caldwell and Bibby23 that many hours after ingestion of carbohydrate, lesions of this type show low pH levels. On occasion, the enamel of such lesions breaks away, which results in exposure to saliva and a more favorable condition for carbohydrate substrate to be cleared. Thus reduction of acid formation and an increase in base formation would be favored, and, as for a diastema or a diastema produced by extraction, favor a higher pH level and caries arrest.' Properties of Foodstuffs Affecting Substrate Availability The effect of a wide variety of carbohydrate foodstuffs on acid formation in the plaque has been investigated. Some carbohydrates cause a fall in plaque pH, whereas others have no effect or cause the pH to rise. Although there are numerous types of dietary carbohydrate, there seem to be three properties that are mainly responsible for their variable effects on acid formation in the plaque: fermentability, retentiveness, and ability to stimulate salivary flow. With regard to fermentability, Stephan and Hemmens32 examined individually the rapidity with which 40 different strains of microorganisms isolated from the dental plaque formed acid from each of several sugars (glucose, fructose, galactose, maltose, lactose, and sucrose) and from starch. The experiments were carried out at a cell concentration of 33% (v/v) to ensure that catabolism occurred as rapidly as in the dental plaque and the rate of acid formation was measured as the drop in pH during

the first ten minutes. Monosaccharides produced the most rapid changes, sucrose and maltose the next, and the slowest changes in pH occurred with lactose and soluble starch (Fig 8). When mixed bacterial populations are used, the differences in acid formation between the various carbohydrates decrease. Miller, Muntz, and Bradel33 found that dental plaque produced acid rapidly and at comparable rates from glucose, fructose, maltose, and sucrose, somewhat less from starch, and least from lactose (Fig 9). The more rapid rate of breakdown of disaccharide and polysaccharides by a mixed popution of microorganisms can be accounted for by some of the microorganisms in the population contributing the enzymes needed to carry out the initial hydrolysis to monosaccharides, so that those microorganisms, which are only able to catabolize monosacharides, can produce acid. When both saliva and mixed populations are used, except for galactose and lactose, there are even less differences in acid formation between the various sugars and polysaccharides than when the saliva is not included (Fig 10, 11). Most probably the saliva contributes large amounts of amylase to facilitate hydrolysis of the starchy foodstuffs. It should be noted that these studies with pure cultures could easily lead to the conclusions that there is bacterial specificity and
pH units /min

0.2i
a)
I-.

a)

c)
~0
10

ix,
v4

u w

No GI Fr Ga Su Ma La Starch Substrate Monosacch Disacch FIG 8.-Comparison of rates of fermentation of several sugars and starch by pure cultures of microorganisms isolated from dental plaque. Shown are mean rates and standard errors for acid formation by 17 different microorganisms shown in Stephan and Hemmens.32 Gl, glucose; Fr, fructose; Ga, galactose; Su, sucrose; Ma, maltose; La, lactose,

|
1 _a

M W.64

Vol 49 1970

-f

22.4 2.4
CD

0 a) 0

0 0 A-0
0

0 *0

FIG 9.-Comparison of rates of fermentation of several sugars and starch by mixed microbial flora of dental plaque in absence of saliva. Shown are mean values and standard errors for lactic acid produced during a 30 minute incubation of plaque-carbohydrate mixtures in five experiments shown in Miller, Muntz, and Bradel33 in which there was complete data for all the substrates tested. GI, glucose; Fr, fructose; Su, sucrose; Ma, maltose; La, lactose.

a)fw
c

ACID ON THE TOOTH SURFACE

1307

pH units
/min
U,

1.6
.0

an

a)

0.8
C.
t-r1

o In
a0

a) CO)
,

I11

w9
.'

a,

M- GIFr SuMacLa Starch lonosacch Disocch

Monosacch Disacch
Dextrin

that sugars are more easily fermented than starchy foodstuffs. However, the mixed flora experiments, particularly those with saliva present, indicate that the opposite is carbohydrate availability and as a result, true and that work with pure cultures can increase in the duration of the pH response. be misleading. There is also some indication that starchy In vivo, starchy foodstuffs such as bread foodstuffs adhere more readily to the dentiand sweets are equally fermentable.20 Con- tion when introduced into the mouth at a sequently any variability between these two time that saliva is in an unstimulated rather classes of dietary carbohydrate as substrates than in a stimulated state.35 This suggests for acid formation by the plaque bacteria that ingestion of carbohydrate at the end would have to arise from differences in the of a meal may not lead to as much retenother two properties mentioned, namely, tion as compared to ingestion between meals their retentiveness and their effects on saliva when saliva is essentially unstimulated. during ingestion. These effects can be attributed to saliva If high substrate concentrations are used, facilitating clearance of acid from the more extracellular polysaccharide can be plaque, facilitating clearance of carbohyformed from sucrose than from the other drate from both plaque and saliva, and prosugars and may have the effect of slowing viding buffers and the constituents of saliva the rate of the pH return either by slowing involved in its base-forming capacity for the diffusion of acid out of the plaque or by removing and neutralizing the acids formed providing substrate for the plaque micro- during carbohydrate breakdown. flora. Effect of Bacterial Composition on With regard to retentiveness, such factors Acid Formation as particle size and adhesiveness should be Considerable evidence, both direct and because formation in acid simply important these factors can lead to prolongation of indirect, has accumulated which indicates

FIG 1O.-Comparison of rates of fermentation of several sugars, starch, and dextrin by the mixed microbial flora in unstimulated whole saliva in the presence of salivary supernatant. Shown are values for the rate of pH decrease for first 30 minutes of four hour incubation at 37 C. Titratable acidity measured at the end of four hours showed the same relationship as pH decrease during first 30 minutes, namely, starch showed most acid, glucose, sucrose, and fructose showed less but were about equal, and dextrin showed the least (data taken from Volker and Pinkerton34).

1308

KLEINBERG

J Dent Res Supplement to No. 6

pH
8.0
6.0

pH
8.0

gal

gluE
10

6.0 4.0

fructose
glucose
0

4.0

10 20 30

pH
8,0
enI fb.U

pH
8501
41(

lac

6.0d glucose

Iglut
4.0
0

sucrose

10

4.C

10

20 30

Sugar Concentration (gm%)

FIG 11.-Comparison of rates of fermentation of several sugars by the microbial flora in salivary sediment in the presence of salivary supernatant. Shown are pH values in salivary sediment-sugar mixtures after three hours of incubation at 37 C. Because of their limited solubility, galactose and lactose were tested at concentrations between 0 and 10% (W/V); the other sugars were tested between 0 and 30% (W/V). The sediment concentration was 25% (v/v).

that the microflora of caries-active and caries-inactive individuals differ. For example, lactobacilli increase with increase in the incidence of caries28 or in response to those conditions such as high carbohydrate
diet that leads to caries.36 Also the incidence of carbohydrate-storing microorganisms is higher in the plaques of caries-active subjects than in caries inactive subjects.37 In an attempt to determine the bacterial factors responsible for the differences in the pH minimums of plaques of subjects with varying caries activity, Stephan and Hemmens32 examined the effect of cell concentration and bacterial type; they used a bacterial system that consisted of pure cultures of plaque microorganisms suspended in an artificial medium designed to resemble the buffering properties of saliva as their model. They showed that a high cell conmentration

is necessary to produce the rapid fall in pH seen in plaque in situ and that the microorganisms could, to varying degrees, not only produce acid and a pH fall but also reduce the extent of the fall and favor a pH rise. This could only be observed at low glucose levels because at higher concentrations, the pH-rise phase of the pH curve does not appear. Some of the microorganisms produced their lowest pH level at high cell concentrations, others at intermediate cell concentrations, and one out of the 17 strains of microorganisms studied, a diphtheroid, produced a pH response that was directly proportional to the cell concentration. The diphtheroid was different from the other organisms examined in that it showed no acid consumption phase. The variability of effects with the dif-

Vol 49 1970

ACID ON THE TOOTH SURFACE

1309

ferent microorganisms was attributed by these authors to differences in the relative rates of acid production and acid consumption by the different microorganisms. The general effects of cell and glucose concentration on the pH of mixtures that contained pure cultures have since been confirmed for salivary sediment and dental plaque.38'39 However, the behavior of plaque is much closer to that of sediment than to that of pure cultures. To study the pH-rise or acid consumption phase of the fall-rise type of pH curve, Stephan and Hemmens32 replaced glucose in their bacterial mixtures with lactic acid. In this way, its use could be examined separately from its formation (Fig 12). They found that a large number of the plaque microorganisms used lactic acid. Of two types of lactobacilli tested, one type of lactobacillus produced no rise in pH with lactic acid, whereas a second produced an initial rapid rise and no change thereafter. The authors suggested that for the first type of lactobacillus, no change occurs because lactic acid is the final end product of its glucose catabolism, whereas for the second type, lactic acid is converted to the weaker acetic acid.

Hu and Sandham41 have shown that of the streptococci isolated from plaque that have been tested for cariogenicity in animal experiments, most form lactic acid as their final end product but some, such as the cariogenic strain AHT, produce acetic acid. In mixed populations, such as salivary sediment or dental plaque, lactic acid is an intermediate and not an end product of glucose catabolism, as shown by the fact that lactic acid is rapidly used in both these bacterial systems (Fig 12, 13). Experiments carried out by Stephan and Hemmens32 under conditions of high cell concentration and limited substrate in which different types of oral microorganisms were mixed with lactobacilli in varying proportion showed that the pH fall is considerably reduced and usually in proportion to the extent of dilution of the lactobacilli with the added microorganisms. Experiments in progress in our laboratory suggest that microorganisms isolated as groups from the mixed population in sediment on the basis of function can be combined to produce pH curves the shape of which, as in the experiments of Stephan and Hemmens32 with pure cultures, is dependent on the proportions of the groups combined.

'
pH

Lactic aci( d

I Glucose

Time in Hours
FIG 12.-Glucose and lactic acid utilization in (a) bacterial system containing pure culture of a sarcina isolated from dental plaque and (b) suspended salivary sediment system in which sediment concentration was 16.7% (v/v). Cell concentration in a was 33% (v/v). (a was adapted from Stephan and Hemmens32 and b from Sandham and Kleinberg.40)

1310

KLEINBERG

J Dent Res

Supplement

to No. 6

ypEq/yil

x102

0
4-

.-

aI)
c

.5
0_

Time in Hours
FIG 13.-Acid formation during glucose catabolism (a) in suspension of dental plaque and (b) in the SSS system (a adapted from Muntz42 and b from Sandham and Kleinberg43). Cell concentration in sediment system was 16.7% (v/v) and glucose concentration was 0.2%. Exact cell concentration of plaque suspension was not given, but was obviously quite high and probably above that of the sediment in b; glucose concentration was 0.4%. Higher glucose concentration and probable higher cell concentration in a would account for sharper rise and fall in lactic acid observed in a.

Removal of Acid from the Plaque The removal of acid can be accomplished either by physicochemical or by metabolic means. StrAlfors6 suggested that the acid that accumulates in plaque in situ during glucose breakdown, is removed mainly by diffusion because of the difference in concentration between the acid in plaque and that in saliva. Stephan and Hemmens32 recognized that saliva could facilitate removal of acid from the plaque by diffusion, but their experiments showed that removal could occur by metabolism of acids such as lactic to less acidic end products, or neutralization with base formed from urea or by buffers, or both. Since clearance of end products from plaque on surfaces readily accessible to saliva is a comparatively slow process,'2 in regions where saliva access is poor, one

would expect clearance to be slow and metabolic removal to be proportionately more. However, recent studies suggest that metabolic removal under these circumstances may not be proportionately as much as one might have expected, because saliva is also essential for metabolic removal to
occur.

Acid formed in the plaque could be neutralized by buffers in both saliva and in plaque. Buffering by saliva appears to be quite significant and rapid; the latter effect is consistent with plaque being porous and allowing easy entry of the saliva. Bicarbonate is the main buffer of saliva and phosphate contributes to a much lesser extent.45 Protein and calcium phosphate appear to be the most important buffering substances in plaque. Calcium phosphate buffers particularly well when the pH approaches ap-

Vol 49 1970

ACID ON THE TOOTH SURFACE

1311

proximately 5.0.46 This pH is also critical for enamel dissolution and for the inhibition and activation of a number of metabolic processes within the plaque. For example, fluoride inhibits acid formations in this region of the pH scale, whereas base formation is stimulated.18
Metabolic Pathways The metabolic pathways used during the catabolism of glucose and urea by the cells in salivary sediment have been outlined by techniques (chromatography and radioactive tracer compounds) that have been found to be successful in establishing pathways in other tissues and cells (Fig 14). These studies were carried out under conditions that led to acid-base changes similar to those that occur in dental plaque, and

showed that lactic, acetic, and propionic acids are the main acids formed, although others are present in smaller amounts.42'43'49 Once the roles of glucose and urea as substrate regulators of the acid-base metabolism of both salivary sediment and dental plaque had been established, experiments were initiated to determine the nonsubstrate regulators of this metabolism. The nonsubstrate regulators that have been identified to date include oxygen, fluoride, and a constituent of saliva, named pH-rise factor. The last of the three regulators stimulates both the uptake of glucose and the uptake of oxygen. Studies to date indicate that in the suspended salivary sediment (SSS) system, oxygen is involved in both the oxidation of carbohydrate and nitrogenous substrates. With regard to the oxidation of

C02

synthesizing
pathways

Aspartate
Urea-NH2
Oxaloacetate

'--ketoglutarate
Glutamate
--

Alanine

glucose
Pyruvate f

FIG 14.-Metabolic pathways involved in carbohydrate and nitrogen metabolism of dental plaque responsible for acid-base changes. According to this scheme, a source of amino group, such as urea, favors formation of alanine from glucose and as a result, some reduction of acid formation. How alanine formation could occur is shown at the bottom.48 (Reprinted with permission of the publisher.)

1311.7

KLEINBERG

J Dent Res

Supplement

to No. 6

carbohydrate, oxygen appears to be essential for the formation of acetic acid and CO, and as a result, more rapid removal of lactic acid. Oxygen also appears to be essential for the oxidation of amino acids that arise from the diversion of carbohydrate intermediates into amino acids during glucose catabolism and from amino acids formed during protein hydrolysis. Fluoride has been shown to inhibit the uptake of glucose. In spite of such inhibition, at low glucose levels, the fall in pH is stimulated slightly. However, at higher levels of glucose, the pH may fall below 5.0, where fluoride inhibition of glucose uptake results in decided inhibition of the pH fall.50 The pH-rise factor in saliva, in contrast to fluoride, stimulates the uptake of glucose. Its effect on the pH is opposite to that of fluoride. At low glucose concentrations, the pH fall is decidedly inhibited, whereas at higher glucose levels, the pH-rise factor stimulates the pH fall slightly.51 Salivary protein also has a role in acidbase metabolism by providing amino acids that can be decarboxylated at low pH and others that can be deaminated at high pH. These effects facilitate the return of the pH to neutrality when it has been displaced to a low level with glucose or a high level with urea.52 One result of these studies is that knowledge of the overall metabolism and its regulation should enable fractionation of the flora in plaque and sediment to proceed on a functional rather than a morphologic basis. The task of isolating individual microorganisms and reassembling them without some idea of how the overall flora functions would appear hopeless, as is evident from the confusion that now exists in the studies using animal models and the cariogenic streptococci to study the caries process.53 The futility of such an approach arises from a fundamental point that is unfortunately overlooked by many investigators, namely that in organized biologic systems, seldom do the parts function like the whole.2 Role of Oxygen in Acid Formation Saliva from caries-resistant subjects shows a higher rate of oxygen uptake during the catabolism of glucose than the saliva of caries-active individuals54 (Fig 15). If the glucose were metabolized, as has been as-

s- caries resistant

200
150-

oz- caries active

, ,

100

e-I-

50-

+ glucose

['1

/7

[I

- glucose

FIG 15.-Uptake of oxygen in caries-active and caries-resistant salivas with and without glucose present. Note that oxygen uptake is higher in caries-resistant saliva both in presence and absence of glucose. Glucose concentration in these experiments was 250 mg% and experiments were carried out for 30 minutes at 37 C. The rate of oxygen consumption is expressed in cu mm/ml of salivary mixture/hour. The mean and standard errors for the nine subjects in each group are shown (data taken from Eggers-Lura54).

sued in the past, by an intact tricarboxylic acid cycle, then a more rapid rate of oxygen uptake should be associated with a reduced rate of acid formation. Eggers-Lura54 has shown that the opposite is true (Fig 16). Eggers-Lura54 also showed that saliva from caries-resistant subjects uses oxygen and glucose several times faster, and forms lactic and pyruvic acids almost twice as rapidly, as does saliva from caries-active subjects (Fig 16). In his experiments, he showed that the lactic acid concentration rises and falls, whereas the pyruvic acid concentration rises asymptotically in both types of saliva (Fig 17). Under anaerobic conditions, caries-resistant saliva formed approximately three times the amount of lactic acid formed in caries-active saliva (Fig 18). Because he found that glucose use and acid formation were each more extensive

M - caries

resistant
200,

o - caries

active

jiEq /mi/hr

200O

M - caries resistant
-caries active
E

100

100

UIL

_0
C-)

0-

Oxygen uptake

Glucose Lactic Pyruvic utilized acid acid for med formed

FIG 16.-Oxygen uptake, glucose utilization, and lactic and pyruvic acid formation during incubation of mixed salivas of caries-active and caries-resistant individuals. Glucose concentration in these experiments was 250 mg% and the experiments were carried out for 30 minutes at 37 C. Rate of oxygen consumption is expressed in cu mm/ml of salivary mixture/hour and the glucose and acid concentrations are in mg%. Mean and standard errors for eight subjects in each group are shown (data taken from EggersLura54).

Anaerobic

Aerobic

FIG 18.-Lactic acid formation in cariesresistant and caries-active salivas incubated under aerobic and anaerobic conditions at 37 C. Initial glucose concentration in these experiments was 250 mg% and volume of saliva incubated was 1 ml. Incubation was carried out for 30 minutes at a pH of 7.2 (data taken from Eggers-Lura54).

*--caries resistant
mg%

o---o

caries

active

20.01
0

Lactic

mg% 20.0
5.0-

Pyruvic

15.0
10.0
I/
% ,1

-q

C) 0
-

So
_0

100
-

5.0
d

5).O

I2
Time in

0
Hours

FIG 17.-Changes in lactic and pyruvic acid concentrations in cariesresistant and caries-active salivas incubated at 37 C for two hours. Initial glucose concentration in these experiments was 100 mg% and the volume of saliva incubated was 1 ml (data taken from Eggers-Lura54).

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in caries-resistant saliva than in caries- and the selection out of plaques of differing active saliva, Eggers-Lura54 concluded that microbial composition. Miller's55 acid decalcification theory may Saliva and oxygen do not have ready acnot be valid for the initiation of caries. cess to the fissures and proximal contacting This paradox can, however, be accounted surfaces of the dentition or to the deepest for from information obtained in our studies layers of mature dental plaque. Conseon the regulation of the acid-base metab- quently, these regions would be deprived olism of the mixed microbial flora that is not only of oxygen but also of salivary pHcontained in salivary sediment. These studies rise factor. This should favor the selection showed that the oxygen uptake activity of of those microorganisms that have an ansaliva resides in the sediment fraction and aerobic type of metabolism and that use that the supernatant portions contain a carbohydrate at a comparatively slow rate. factor (the pH-rise factor previously men- On the other hand, surface layers of the tioned), which stimulates the oxygen uptake plaque have access to saliva and oxygen activity of the sediment both in the presence and should therefore favor those microand absence of added glucose56 (cf Hartles organisms that have a metabolism that inand Wasdell57). The same factor stimulates cludes not only oxidative steps but also the uptake of glucose and more rapid for- more rapid use and "metabolic" clearance of mation and subsequent use of lactic acid, the ingested carbohydrate, as outlined in the effects by which salivas from caries-resistant previous section. and caries-active subjects differ. Dentition morphology that leads to poor The experiments with the SSS system also saliva access would, for the reasons given showed that stimulation of glucose uptake earlier, also affect the ratio of salivary to and acid formation is accompanied by in- dietary substrates and as a result favor more creased base formation, a process essential acid than base formation. At the same for reducing the extent of the pH decrease time, dentition morphology that leads to and facilitating the return of the pH to start- poor saliva access would reduce the rate ing levels. If, as our studies suggest, the of removal of acid by saliva and possibly saliva from the caries-active subjects studied reduce the proportion of the removal proby Eggers-Lura54 contains less pH-rise fac- cess that is by diffusion rather than by tor than saliva from caries-resistant subjects, metabolism. then the microflora of caries-resistant indiConclusions viduals may use and "clear" glucose (and In summary, by altering availability of other sugars) more rapidly than that of caries-active subjects without the harmful saliva and retention of carbohydrate, dentition morphology would affect the entry and decrease in pH. The fact that the saliva of caries-resistant exit functions of the plaque system, the individuals can catabolize glucose at an availability of regulators of plaque metaaccelerated rate suggests that any procedure bolism and, as a result, both the microbial or chemotherapy that reduces base forma- flora and the formation and accumulation tion could leave acid formation unchecked of acid by the dental plaque. and lead to formation of caries under cirReferences cumstances where it might not normally 1. and MILLER, B.F.: A quanSTEPHAN, R.M., occur. The animal models that have been titative Method for Evaluating Physical used to study the cariogenicity of specific and Chemical Agents which Modify Promicroorganisms appear to fall into this duction of Acids in Bacterial Plaques on category. Human Teeth, J Dent Res 22:45-51, 1943. 2. VON BERTALANFFY, L.: General System Environmental Niches Resulting from Theory: Foundations, Development, AppliVariation of Dentition Morphology cations, New York: George Braziller Inc., 1968. Enough has been learned from the studies 3. BRAY, H.G., and WHITE, K.: Kinetics and on the metabolism of the mixed microbial Thermodynamics in Biochemistry, 2nd ed, floras in sediment and plaque to suggest, in New York: Academic Press, 1966, pp 197a general way, how dentition morphology 204. can lead to varying environmental niches 4, STEPHAN, R.M.: Changes in Hydrogen-Ion

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Concentration on Tooth Surfaces and in Carious Lesions, JADA 27:718-723, 1940. 5. STEPHAN, R.M.: Intra-oral Hydrogen-Ion Concentrations Associated with Dental Caries Activity, J Dent Res 23:257-266, 1944. 6. STRALFORS, A.: Investigations into the Bacterial Chemistry of Dental Plaques, Odont T 58:153-341, 1950. 7. SILLEN, L.G.: Treatise on Analytical Chemistry. Part I. Theory and Practice, Kolthoff, I.M. and Elving, P.J., (eds), New York: Interscience, 1959, p 277. 8. KLEINBERG, I.: Studies on Dental Plaque. I. The Effect of Different Concentrations of Glucose on the pH of Dental Plaque In Vivo, J Dent Res 40:1087-1111, 1961. 9. KLEINBERG, I.: Effect of Urea Concentration on Human Plaque pH Levels In Situ, Arch Oral Biol 12:1475-1484, 1967. 10. KLEINBERG, I.: Biochemistry of the Dental Plaque, in Staple, P.H. (ed): Advances Oral Biol, Vol 4, New York: Academic Press, 1970, p 43. 11. KLEINBERG, I., and JENKINS, G.N.: 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 9:493-516, 1964. 12. SINGER, D.L., and KLEINBERG, I.: NH3 Formation in Plaque In Situ, abstracted IADR, Program and Abstracts of Papers, No. 637, 1969. 13. LAMER, V.K., and HEALY, T.W.: Adsorption-Flocculation Reactions of Macromolecules at the Solid-Liquid Interface, Rev Pure Appl Chem 13:112-133, 1963. 14. SILVERMAN, G., and KLEINBERG, I.: Studies on Factors Affecting the Aggregation of the Microorganisms in Human Dental Plaque, Arch Oral Biol 12:1407-1416, 1967. 15. NEVIN, R.B.: The Diet and Mastication; Their Effects on Diffusion and on the Inception of Dental Caries, Dunedin: Progress Printing Co., Ltd. 1954. 16. HALHOUL, M.N., and KLEINBERG, I.: Catabolism of Glucose, Fructose, Sucrose, Dextrans and Levans in a Salivary Sediment System, abstracted IADR Program and Abstracts of Papers, No. 383, 1969. 17. WOOD, J.M.: The State of Hexose Sugar in Human Dental Plaque and its Metabolism by the Plaque Bacteria, Arch Oral Biol 14:161-168, 1969. 18. KORAYEM, M., and KLEINBERG, I.: Effect of pH on Glucose Utilization and Acid Formation by Salivary Sediment, abstracted IADR Program and Abstracts of Papers, No. 586, 1970. 19. KLEINBERG, I.: Dental Caries, in Nolte,

W.A. (ed): Oral Microbiology St. Louis,

C.V. Mosby, Chap 8, 1968. 20. KLEINBERG, I., and JENKINS, G.N.: Further Studies on the Effect of Carbohydrate Substrates on Plaque pH In Vivo, J Dent Res 38:704-705, 1959. 21. KLEINBERG, I.: Studies on the Influence of Diet on the Dental Plaque with Special Reference to the Initiation of Caries, PhD thesis, University of Durham, England, 1958. 22. LUDWIG, T.G., and BIBBY, B.G.: Acid Production from Different Carbohydrate Foods in Plaque and Saliva, J Dent Res 36:5660, 1957. 23. CALDWELL, R.C., and BIBBY, B.G.: The Effect of Foodstuffs on the pH of Dental Cavities, JADA 57:685-692, 1958. 24. DIRKSEN, T.R.; LITTLE, M.F.; BIBBY, B.G.; and CRUMP, S.L.: The pH of Carious Cavities. I. The Effect of Glucose and Phosphate Buffer on Cavity pH, Arch Oral Biol 7:49-58, 1962. 25. DEBOEvER, J., and MUHLEMANN, H.R.: pH of Interproximal Plaque with Regard to Continuous Sucrose Application, Helv Odont Acta 13:97-99, 1969. 26. KOULOURIDES, T.; FEAGIN, F.; and PIGMAN, W. Remineralization of Dental Enamel by Saliva In Vitro, Ann NY Acad Sci 131: 751-757, 1965. 27. HEMMENS, E.S.; BLAYNEY, J.R.; BRADEL, S.F.; and HARRISON, R.W. The Microt ic Flora of the Dental Plaque in Relation to the Beginning of Caries, J Dent Res 25: 195-205, 1946. 28. BECKS, H.; JENSEN, A.L.; and MILLARR, C.B.: Rampant Dental Caries: Prevention and Prognosis. A Five Year Clinical Survey, JADA 31:1189-1200, 1944. 29. WELANDER, E.: The Occurrence of Dental Caries in the Permanent Dentition, Uppsala: Almqvist and Wiksells, 1955, pp 94117. 30. STEPHAN, R.M.: Hydrogen Ion Concentration of the Dental Plaque, J Dent Res 17: 251-256, 1938. 31. ANDERSON, B.G.: Clinical Study of Arresting Dental Caries, J Dent Res 17:443-452, 1938. 32. STEPHAN, R.M., and HEMMENS, E.S.: Studies of Changes in pH Produced by Pure Cultures of Oral Micro-organisms, J Dent Res 26:15-41, 1947. 33. MILLER, B.F.; MUNTZ, J.A.; and BRADEL, S. Decomposition of Carbohydrate Substrates by Dental Plaque Material. J Dent Res 19:473-478, 1940. 34. VOLKER, J.F., and PINKERTON, D.M.: Acid Production in Saliva-Carbohydrate Mixtures, J Dent Res 26:229-232, 1947. 35. ERICHSEN, A., and SCHULERUD, A.: Forsk-

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36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

jellige brodsorters klebrighet under tyggningen, Odont T 50:221, 1942, cited by Lanke, L.S.: Influence on Salivary Sugar of Certain Properties of Foodstuffs and Individual Oral Conditions, Acta Odont Scand 15 (suppl 23):1957. ONISI, M., and KONDO, W.: Establishing an Environment for Growth of Aciduric Bacteria in the Oral Cavity, J Dent Res 35:596-602, 1956. GIBBONS, R.J., and SOCRANSKY, S.S.: Intracellular Polysaccharide Storage by Organisms in Dental Plaques. Its Relation to Dental Caries and Microbial Ecology of the Oral Cavity, Arc/i Oral Biol 7:73-80, 1962. KLEINBERG, I.: Effect of Varying Sediment and Glucose Concentrations on the pH and Acid Production in Human Salivary Sediment Mixtures, Arch Oral Biol 12:14571473, 1967. SINGER, D.L., and KLEINBERG, I.: Cell concentration and the pH of Suspended Plaque Mixtures In Vitro, abstracted, IADR Program and A bstracts of Papers, No. 494, 1970. SANDHAM, H.J., and KLEINBERG, I.: Utilization of Glucose and Lactic Acid by Salivary Sediment, Arch Oral Biol 14:597-602, 1969. Hu, G., and SANDHAM, H.J.: Lactic Acid Utilization by Streptococci, abstracted, IADR Programn and Abhstracts of Papers, No. 173. 1969. MUNTZ, J.A.: Production of Acids from Glucose by Dental Plaque Material, J Biol Chem 148:225-236, 1943. SANDHAM, H.J., and KLEINBERG, I.: Contribution of Lactic and Other Acids to the pH of a Salivary Sediment System during Glucose Catabolism, Arch Oral Biol. in press, 1970. KLEINBERG, 1.; CRAW, D.; and KAY. M.: The Effect of a Salivary pH-Rise Factor on Salivary Sediment Metabolism. abstracted IADR Program and A bstracts of Papers, No. 90, 1968. LILrENTHAL, B.: An Analysis of the Buffer Systems in Saliva, J Dent Res 34:516-530, 1955. HOLT, L.E., JR.; LA MER, V.K.; and CHOWN, H.B.: Studies in Calcification. I. The Solubility Product of Secondary and

47. 48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

Tertiary Calcium Phosphate under Various Conditions, J Bilo Chem 64:509-565, 1925. JENKINS, G.N.: The Effect of pH on the Fluoride Inhibition of Salivary Acid Production, Arch Oral Biol 1:33-41, 1959. KLEINBERG, I.: Regulation of the Acid-Base Metabolism of the Dento-Gingival Plaque and Its Relation to Dental Caries and Periodontal Disease. Ift Dent J, in press, 1970. GILMOUR, M.N., and POOLE, A.E.: The Fermentative Capabilities of Dental Plaque, Caries Res 1:247-260, 1967. SANDHAM, H.J., and KLEINBERG, I.: The Effect of Fluoride on the Interrelation Between Glucose Utilization, pH and Carbohydrate Storage in a Salivary Sediment System, Arch Oral Biol 14:619-628, 1969. CRAw, D., and KLEINBERG, I.: Effect of Salivary Supernatant and Fluoride on the Metabolism of Glucose in a Salivary Sediment System, abstracted IADR Programi Abstracts of Papers, No. 385, 1969. KLEINBERG, I.; KAY, M.; and CRAw, D.: Saliva in the Regulation of the pH of Salivary Sediment During Urea Utilization, abstracted IADR Program and A bstracts of Papers, No. 382, 1969. KRASSE, B., and CARLSSON, J.: Various Types of Streptococci and Experimental Caries in Hamsters, Arch Oral Biol 15:2532. 1970. EGGERS-LURA, H1.: Untersuchungen fiber den aeroben und anaeroben Abbau der Kohlehydrate im Speichel, Deutsch Zahn Mund Kieferheilk 21:97-111, 1954. MILLER, W.D.: The Micro-Organisms of the Human Mouth, Philadelphia: S.S. White Dental Mfg Co., 1890. KORAYEM, M., and KLEINBERG, I.: Studies on the Effect of Glucose and Salivary Supernatant on the Uptake of Oxygen by the Mixed Microbial Flora in a Suspended Salivary Sediment System Using the Clark Oxygen Electrode, Arch Oral Biol, submitted for publication, 1970. HARTLES, R.L., and WASDELL, M.: The Metabolism of the Oral Flora. 6. Preliminary Observations on a Water-Soluble Factor in Saliva which Enhances the Respiratory and Glycolytic Activity of the Salivary Flora, Brit Dent J 98:334-337, 1955.