Composition of Pooled Plaque Fluid from Caries-free and Caries-positive Individuals Following Sucrose Exposure H.C. MARGOLIS and E.C. MORENO Forsyth Dental Center, 140 The Fenway, Boston, Massachusetts 02115
The composition of pooled plaque fluid from five population samples was determined before and at selected times (7, 15, 30, and 60 min) after a 10% sucrose rinse. Subjects were grouped according to caries status (caries-free, CF, DMFS = 0; caries-positive, CP, DMFS > 10). Samples were also studied from white-spot surfaces and from sound surfaces of the same mouths of two additional CP groups. Plaque fluid was isolated by centrifugation and analyzed for organic acids, inorganic ions (ion chromatography), and pH (microelectrodes). Prior to sucrose exposure, plaque fluids from the CF subgroups and from sound surfaces of the CP subjects had higher pH values than samples from CP subgroups and from white-spot surfaces, respectively; the ionic compositions were otherwise similar. Starved plaque fluids were also found to be supersaturated with respect to enamel and to a significantly greater degree in the CF samples, suggesting that CF plaque fluid may have a greater remineralization potential than CP samples. Following sucrose exposure, a rapid decrease in plaque fluid pH was observed, which corresponded primarily to lactic acid production. For all times examined, mean pH and DS(En) values were lower and lactic acid concentrations were higher in the CP samples than in the CF samples; noted differences were statistically significant at 7 min for pH and DS(En), and at 7, 15, and 30 min for lactic acid. Lower values of DS(En) suggest that plaque fluid from CP subjects had a measurably greater cariogenic potential. Calcium concentrations also increased in plaque fluid, following sucrose exposure, and to similar levels in samples from CF and CP subjects, despite significantly lower acid production in the CF samples. These latter results appear to be associated with the further finding that whole plaque from the CF subgroups contained significantly more calcium than the samples from the CP subjects. The availability of mineral ions like calcium within plaque may, therefore, play an important role in controlling enamel demineralization. J Dent Res 71(11):1776-1784, November, 1992
Introduction. Fermentation of dietary carbohydrate by micro-organisms in dental plaque brings about changes in the ionic composition ofthe extracellular aqueous phase of plaque, referred to as plaque fluid. Such chemical changes can lead to the establishment of the necessary conditions for caries formation. These cariogenic conditions in plaque, however, have not been well-defined, although associations have been made between changes in plaque pH and caries experience (e.g., Stephan, 1944; Abelson and Mandel, 1981). It has also been suggested that changes in plaque pH values are primarily associated with lactic acid production (Muntz, 1943; Gilmour and Poole, 1967; Geddes, 1972; Cole et al., 1978). Results of in vitro enamel demineralization studies, however, show that in addition to pH and lactic acid concentration, the rate of enamel demineralization is greatly influenced by the degree of saturation, DS(En), ofthe demineralizing medium with respect to enamel (Moreno and Zahradnik, 1974; Theuns et al., 1983; Margolis et al., 1985). ReReceived for publication January 6, 1992 Accepted for publication May 26, 1992 This study was supported by grants (DE-07493, DE-07009, and DE03187) from the National Institute of Dental Research.
1776
cently, we have also shown that the kinetics of hydroxyapatite (HA) dissolution can be described by a function of the DS(HA) and the total activity of undissociated acid species (i.e., organic and phosphoric acids) present in solution (Margolis and Moreno, 1992). These same parameters also appear to influence enamel demineralization in vitro (Lee et al., 1990). Although several studies (Edgar et al., 1986; Higham and Edgar, 1989) have investigated changes in the organic acid composition of plaque fluid following carbohydrate exposure, no studies have measured simultaneously acid production and the saturation status of plaque fluid in vivo. The present study was undertaken to determine the effect of fermentable carbohydrate exposure on the composition and degree of saturation of plaque fluid and to associate these parameters with caries experience. Plaque samples were pooled from all surfaces of caries-free individuals and from all surfaces of caries-positive individuals, before and following exposure to an oral sucrose rinse. Comparisons were also made between samples from white-spot surfaces and from sound surfaces of the same mouths in cariespositive individuals. Materials and methods. Subjects.-Due to analytical limitations previously discussed (Margolis et al., 1988a), pooled plaque samples were required for completion of all the analyses. Whole-mouth plaque samples were obtained from all tooth surfaces offive groups of subjects. Group A subjects (ages 18-22).from Northeastern University (Boston, MA) and group B subjects (ages 10-15) from the Massachusetts Migrant Education Program (Taunton, MA) were divided into two subgroups (n = 4-6) of caries-free, CF (DMFS = 0), and caries-positive, CP (DMFS: group A, 23 ± 7; group B, 18 ± 6), individuals, the latter previously referred to as being caries-susceptible (Margolis et al., 1988a,b). An additional CF group (group C: n = 5) from Northeastern University was also studied. Subjects were scored for caries without the use of radiography. The selected subjects did not have sealants or orthodontic bands, nor did they exhibit fluorosis. CF and CP individuals, therefore, differed with regard to caries history and not necessarily in any measure of current caries activity. Essentially all surfaces recorded as decayed for the CP subjects of group B were unfilled, and each subject was advised to seek dental treatment. The complete study was carried out with use of both subgroups ofgroup A on two separate occasions; results from these two studies are referred to as Al and A2. Site-specific plaque samples were also collected and pooled separately from sound and white-spot surfaces in two additional groups ofCP (group 1: n = 3, DMFS =25 ± 13; group 2, n = 4, DMFS = 21 - 16) individuals from Northeastern University. The locations of sound and white-spot surfaces of CP subjects were carefully charted during preliminary screening sessions. Whole-mouth plaque samples.-Prior to plaque collection, all subjects refrained from oral hygiene for 48 h and fasted overnight. Plaque samples were removed from the mouth, as previously described (Rankine et al., 1985). Initially, for establishment of a baseline for the plaque fluid composition, four pooledplaque samples were obtained from each subgroup. Plaque samples were collected from each subgroup member in a fashion such that all quadrants of the dentition were equally sampled. Prior to plaque collection, charts were prepared, for each subgroup member, which noted from
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1777
CARIOGENIC POTENTIAL OF PLAQUE FLUID
Vol. 71 No. 11
TABLE 1 MEAN INORGANIC ION COMPOSITION OF WHOLE-MOUTH PLAQUE FLUID SAMPLES BEFORE AND AT 7, 15, 30, AND 60 MIN FOLLOWING A ONE-MINUTE 10% SUCROSE RINSEa
6.02
60 6.19
6.791
Caries-positive 15 5.58 5.382
0.28
-
0.12
0.09
Caries-free pH
ob 7.021
s.d.
0.05
7
15
30
5.632
5.80
0.08
0.36
ob
7
0.22
30 5.73
60 5.88
0.20
0.37
Na+
14.2
11.7
10.3
10.7
12.2
16.5
16.5
16.1
20.2
19.0
s.d.
3.5
6.1
4.4
5.2
4.7
5.4
1.8
0.7
3.6
6.6
NH4+
26.3
18.5
18.5
19.6
21.2
22.6
16.5
18.7
22.1
20.7
s.d.
3.9
2.6
3.7
2.6
2.0
3.9
2.4
2.5
4.3
2.0
K+
59.9
64.1
58.2
62.2
63.4
71.4
84.3
88.0
86.5
91.8
s.d.
4.9
14.6
11.1
8.8
4.9
11.3
6.8
11.3
1.3
5.0
Mg2+ s.d.
2.0
2.3
2.2
2.4
2.0
2.6
2.5
2.9
2.9
2.7
0.4
0.3
0.3
0.2
0.1
0.4
-
0.4
0.8
0.2
Cat~2.8
9.6
5.9
4.2
3.5
2.7
8.2
5.8
4.3
3.5
s.d.
0.2
1.5
0.5
1.2
1.0
0.5
1.9
1.2
0.4
0.4
Cl-
23.8
16.0
17.8
19.9
20.8
27.2
22.3
22.3
22.0
29.2
s.d.
3.6
3.1
4.6
5.0
3.7
2.9
7.3
2.2
4.0
11.0
Ptat
13.9
17.7
15.0
14.8
13.5
15.6
20.6
20.0
17.4
16.4
4.8 3.5 2.5 1.4 3.6 3.0 3.3 3.8 3.5 1.9 s.d. aAll concentrations expressed as mmol/L. bMean values calculated from baseline data (typically, 13 values for CF data and 12 values for CP data); for pH, the negative log ofthe mean of the hydrogen ion activity is given as the mean value; the s.d. of individual pH measurements from this mean pH is also given. 1CF and CP values significantly different at p < 0.001 (Student's t test). 2CF and CP values significantly different at p < 0.025 (Student's t test).
which teeth plaque samples would be taken. From each subgroup member, plaque was removed from lingual and buccal surfaces of two designated teeth of each quadrant and placed under watersaturated mineral oil in a 0.4-mL microcentrifuge tube maintained in an ice bath. Thus, for example, tube #1 contained plaque collected from eight teeth (e.g., from teeth nos. 17 & 16,21 & 22,37 & 36, and 41 & 42) of one subject ofthe subgroup. This procedure was repeated three times for the same subject, each time with plaque removed from eight "other" designated teeth (two from each quadrant) and samples pooled from the selected (eight) teeth in different microcentrifuge tubes. Thus, fourrepresentative whole-mouth plaque samples were collected from one subject, with each pooled sample placed in a separate microcentrifuge tube. This procedure was then repeated for each subject of a subgroup, with plaque samples being combined with the samples collected from the other subgroup members. Thus, in this fashion, four pooled plaque samples were collected for each subgroup. For maximum random sampling, sampling sites were staggered. For example, in tube #1 (referred to before), plaque samples from a second subject were collected from teeth 15 & 14,23 & 24,35 & 34, and 43 & 44. Since pooled samples of plaque were collected from all areas of the mouth, localized differences in plaque fluid composition cannot be assessed from the present data. Plaque fluid was then separated from the whole plaque and analyzed, as described below, to provide baseline data
(zero-time concentrations) for the subsequent rinse studies. Approximately one week later, plaque samples were collected at 7, 15, 30, and 60 min, following a one-minute rinse with 10% sucrose. These times denote the time at which plaque collection was begun. Exactly three minutes were allowed for the collection ofeach plaque sample. For each time point (i.e., at 7, 15, 30, or 60 min), plaque samples were collected and pooled from the same designated areas which were used for one of the four pooled samples obtained in the baseline studies. Plaque fluid was then isolated and analyzed as described below. In separate studies, it was determined that it was necessary and adequate for isolated plaque samples to be maintained at ice temperature, so that subsequent acid production, following sucrose exposure, would be minimized for the duration of the collection time (up to 90 min). CF and CP samples from each subgroup (Al, A2, or B) were collected and processed at the same time. For group C, a single whole-mouth sample of pooled plaque was collected, following overnight fasting; during subsequent sessions, this procedure was repeated following a one-minute, 10% sucrose rinse.
Site-specific plaque samples.-Prior to plaque collection, all subjects refrained from oral hygiene for 48 h and fasted overnight. Plaque samples from (1) white-spot surfaces of the CP subjects (groups 1 and 2) and (2) sound surfaces ofthe same CP subjects were collected, in the same general fashion as described above, before and
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1778
MARGOLIS & MORENO
J Dent Res November 1992
TABLE 2 MEAN ORGANIC ACID COMPOSITION OF WHOLE-MOUTH PLAQUE FLUID SAMPLES BEFORE AND AT 7,15,30, AND 60 MIN FOLLOWING A ONE-MINUTE 10% SUCROSE RINSEa
Succinate
Ob 4.4
7 3.2
s.d.
1.5
0.8
Lactate
1.8
s.d.
Caries-free 15 4.0
Caries-positive 30 5.2
60 5.6
1.4
1.7
36.01
21.91
0.7
8.6
19.9
s.d. Propionate
Acetate
Ob
4.6
7 3.0
2.4
1.0
12.51
6.8
1.9
2.0
17.6
18.7
3.5
4.0
5.8
8.0
15 2.8
30 6.0
60 7.5
0.7
0.1
0.4
1.8
2.6
51.11
39.91
19.1'
13.2
2.4
1.2
2.5
6.2
2.2
5.6
20.7
19.8
20.3
19.8
24.1
29.9
25.3
5.4
4.4
4.6
4.6
2.7
2.1
4.5
3.5
7.4
8.9
7.6
9.2
11.7
13.7
17.5
14.3
3.4 1.5 4.1 3.2 1.9 3.6 1.3 1.9 4.2 s.d. 4.0 aAll concentrations expressed in mmol/L. bMean values calculated from the baseline data (typically, 13 values for CF data and 12 values for CP data); formic and butyric acid concentrations were low (0-5 mmolJL) and not consistently detected in all samples. 'CF and CP values significantly different at p < 0.025 (Student's t test).
Calculation of ion activities and degrees of saturation.-By use ofthe analytical data obtained, individual ion activities and degrees of saturation (DS) with respect to enamel (En) and dicalcium phosphate dehydrate (DCPD) were calculated as previously described in detail (Moreno and Margolis, 1988). These calculations include calcium and magnesium binding by organic acid anions and phosphate species. The ionic concentrations, C, were then used in the following equation defining the condition of electrical neutrality in the plaque fluid: E = C AZ, with the valences, Z., taken with their sign. If the concentrations of all ionic species were determined, the value of E would be equal to zero. In the present study, a value ofE different from zero was considered as the concentration of a monosample and analyzed as described below. Plaquefluid isolation. -Plaque fluid was isolated by centrifuga- valent species not determined and was included in the calculation tion, as previously described (Margolis et al., 1988a), with the of ionic strength. modification that centrifugation was carried out at 2-4°C; 3-6 PL of Determination of total calcium and fluoride in plaque resifluid was obtained from each pooled plaque sample. Immediately dues. -Pooled plaque residues (stored at -20'C) collected duringthe following the separation of plaque fluid, samples were diluted for baseline studies (i.e., without sucrose exposure) were removed from subsequent analyses by the addition of approximately 2-AL aliquots the microcentrifuge tubes; microcentrifuge tubes were cut with a (Drummond microdispenser, Drummond Scientific Co., Broomall, scalpel just below the oil-plaque interface to facilitate plaque rePA) of plaque fluid to 75 pL of 3.0 mmol/L OSA (octane sulfonic acid) moval and avoid contamination with mineral oil. Plaque samples contained in a 1.5-mL polypropylene microcentrifuge tube. The from each tube (four tubes per subgroup) were then individually precise volume of plaque fluid used was determined by calibration freeze-dried to constant weight. Accurately weighed dried plaque ofthe microdispenser (Margolis et al., 1988a). Duplicate or triplicate samples (1.8-12 mg) were then digested in acid (0.2 mL of a 1:1 dilutions ofeach sample were made, depending on the volume of the mixture of 14.3 mol/L nitric acid and 11.6 molIL perchloric acid) at sample available. Diluted samples were then frozen at -20°C, and 60°C for 2 h, by means of a modification of a published procedure were stored at this temperature prior to analysis; analyses were (Birkeland, 1970). Concentrations of fluoride and calcium (extypically completed within 72 h after plaque sample collection. pressedper plaque dry weight) in the neutralized acid digests were Remaining plaque fluid, maintained on ice, was used for pH deter- determined with a fluoride ion-selective electrode (Orion, Cammination, as described below. The compressed plaque residues, bridge, MA) and an atomic absorption spectrophotometer, respecobtained following centrifugation (maintained under mineral oil), tively. were then stored in a freezer at -20°C and later analyzed for total calcium and fluoride, also as described below. Organic acid and inorganic ion analyses. -Diluted plaque fluid Results. samples were analyzed for mono- and divalent cations, inorganic The mean inorganic ion and organic acid compositions of wholeanions, and organic acids, as described previously (Margolis et al., mouth plaque fluid samples from the CF and CP subgroups, ob1988a). tained before and after exposure to sucrose, are given in Tables 1 The pH value for each plaque fluid sample (0.35-1-PL aliquots) and 2, respectively. was determined by use of micro-glass and reference electrodes Baseline composition.-As shown in Table 1, the mean pH value (Microelectrodes, Inc., Londonderry, NH), as previously described ofthe baseline CF samples (13 values, fourfor each ofgroups Al, A2, and B, and one for group C), 7.02 ± 0.05, was significantly (p < 0.001) (Margolis et al., 1988b). at selected times (7, 15, 30, and 60 min) following a 5% and/or 10% sucrose rinse. During a single session, two plaque samples were collected-one from white-spot surfaces (e.g. at 7 min) and one from sound surfaces (e.g. at 15 min)-and placed under oil in separate microcentrifuge tubes maintained at ice temperature. During four consecutive weekly sessions, samples were collected from the same subjects and the same sites at a different designated time following sucrose exposure (e.g. at 7 min from sound surfaces and 15 min from white-spot surfaces). In this fashion, pooled plaque samples were obtained from both surface types ofthe same individuals at various times after the sucrose rinse. Plaque fluid was isolated from each
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1779
CARIOGENIC POTENTIAL OF PLAQUE FLUID
Vol. 71 No. 11
TABLE 3 COMPOSITION OF PLAQUE FLUID SAMPLES FROM WHITE-SPOT AND SOUND SURFACES OF CARIES-POSITIVE SUBJECTS BEFORE AND AT 7,15,30, AND 60 MIN FOLLOWING A ONE-MINUTE 5% OR 10% SUCROSE RINSEa
Sound surfaces 7 15 30 5.66 5.87 5.99 5.79 5.92 5.96 5.90 5.99 5% 6.11 10% 12.3 13.3 10.3 14.9 21.1 11.2 10% 5% 12.7 12.8 16.5 6.9 10.2 8.0 NH4+1 10% 11.5 2 10% 10.8 11.1 2 5% 13.7 7.9 11.9 K+ 1 10% 46.2 75.6 63.4 2 10% 61.4 73.7 " 2 5% 66.1 68.4 70.9 5.4 4.7 3.5 2.1 Mg2+1 10% 1.7 2 10% 1.8 1.6 1.8 1.8 2 5% 1.9 2.2 4.4 5.3 2.2 2.1 Catot 1 10% 2 10% 3.1 2.5 2.5 1.8 3.4 3.0 2.3 2 5% 14.5 Cl- 1 10% 32.0 23.6 18.4 2 10% 22.1 33.4 29.0 21.5 28.1 27.0 2 5% 12.7 9.6 12.9 11.8 Ptot 1 10% 8.7 7.6 9.2 2 10% 9.0 10.5 8.9 10.1 2 5% 0.1 0.9 1.8 3.3 Suc 1 10% 1.9 2.3 2.9 2 10% 5.3 4.4 1.7 2.3 2 5% 31.4 34.5 11.4 1.7 Lac 1 10% 24.5 17.7 26.1 2 10% 3.9 14.4 2 5% 23.3 18.9 12.0 20.5 15.1 14.5 Ace 1 10% 12.5 13.0 15.2 2 10% 13.7 12.2 13.7 15.1 2 5% 8.2 5.4 6.1 3.4 Pro 1 10% 8.8 7.2 8.9 2 10% 7.3 9.7 2 5% 5.9 7.9 aAl concentrations expressed in mmol/L; - missing sample.
Group No. pH 1 2 2 Nay 1 2 2
Sucrose
conc. 10% 10%
0 6.92 6.99 " 18.2 19.4 " 12.7 12.2 a 56.2 68.1
60 6.66 -
6.67 8.8 -
13.2 9.7 -
17.8 41.9 -
66.4 3.5 -
1.9 2.1 -
2.3 16.2 -
25.6 9.7 -
11.1 2.3 -
4.8 5.7 -
6.8 11.3 -
13.6 4.1 -
9.1
0 6.73 6.72
16.4 24.0 " 13.4 14.9 66.6 69.5
4.9 2.0 2.1 2.2 37.7 29.5 " 14.4 9.2 5.1 6.4 2.3 4.0 18.2 12.4 6.0 5.7 "
7 5.76 5.42 5.53 7.9 18.7 11.3 4.6 10.4 7.8 55.3 -
71.5 4.1 1.3 2.0 2.5 1.8 3.5 14.6 -
27.7 8.7 7.8 9.5 0.9 1.8 1.6 33.7 31.6 30.7 11.9 11.8 11.5 5.3 6.9 8.8
White spots 15 5.68 6.01 5.94 10.6 12.8 10.7 7.2 11.8 9.0 52.3 66.5 66.9 3.8 1.6 2.0 2.8 2.2 2.0 18.1 22.2 20.5 10.2 9.0 8.7 1.0 1.8 2.0 29.7 17.1 16.5 14.3 13.2 12.7 5.1 10.5 7.8
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30 -
6.18 -
15.5 7.1 -
16.8 -
77.5 -
2.6 1.8 -
2.6 -
-
29.4 -
-
12.0 -
-
2.4 -
15.7 -
-
21.0 -
-
14.8
60 6.25 6.44 6.34 12.3 13.8 12.5 8.0 12.7 10.8 64.5 79.0 72.3 1.8 1.2 1.7 1.6 2.3 1.7 20.7 30.9 23.0 11.5 11.5 10.2 3.5 3.5 4.4 7.4 9.7 9.2 12.4 17.2 14.8 6.3 12.4 10.6
1780
MARGOLIS & MORENO
higher than that found for the CP samples (11 values), 6.79 ± 0.12. The pH values of plaque fluid from white-spot surfaces (Table 3) were in the range of the whole-mouth samples from the CP subgroups and were measurably lower (by 0.19-0.27 pH units) than those of samples taken from sound surfaces of the same CP mouths. Other than pH, no meaningful differences were observed in the mean baseline concentrations in the whole-mouth samples from CF and CP groups. With regard to organic acid composition (Table 2), in all samples, acetic acid was present in the highest concentration, whereas lactic acid was found in relatively low concentrations. Negligible concentrations of formic and butyric acids were found in the site-specific samples. In general, the baseline concentrations of ions in plaque fluids from white-spot and sound surfaces ofCP individuals (Table 3) were comparable with those found (Tables 1 and 2) in the whole-mouth samples, with the notable exception of calcium and ammonium concentrations, which were lower in the site-specific samples from the CP individuals. Other than pH, no apparent differences were observed between the ionic composition of plaque fluid from whitespot and sound surfaces within the same mouths of CP subjects, prior to sucrose exposure. Plaque fluid composition following sucrose exposure: pH.-As shown in Table 1, following exposure to sucrose, plaque fluid pH decreased rapidly to an apparent minimum at 7 min, and then increased slowly. In no case did the plaque fluid pH return to baseline values within the experimental time-frame of 60 min. The mean pH values were lower in the CP samples at all times examined and significantly so (p < 0.025) at 7 min. In group 2 (Table 3), pH values in samples from white-spot surfaces, at 7 min following exposure to both 5% and 10% sucrose solutions, were found to be lower (by 0.37 pH units) than those observed in samples from sound surfaces within the same mouths. The pH values in plaque fluids from white-spots and sound surfaces from CP subjects of group 1 were, however, comparable, except for samples obtained at 60 min. At 60 min, pH values in all samples from CP individuals and from white-spot surfaces were substantially lowerthan the pH in samples from corresponding CF individuals (Table 1) or from sound surfaces in CP individuals (Table 3). The limited amount of data presently available for site-specific samples, however, does not lend itself to statistical analysis. Organic acids.-A rapid increase in lactic acid concentration was observed (Tables 2 and 3), in all samples, concomitant with the decrease in pH. Mean lactic acid concentrations were significantly larger(p < 0.025)inthe CPsamples at 7,15, and30min aftersucrose exposure. In addition, in site-specific samples from group 2 (Table 3), the lactic acid concentration at 7 min was greater in the whitespot sample than in samples from the sound surfaces of the same mouths. Again, the limited amount of data presently available for site-specific samples does not lend itself to statistical analysis. The increase in lactic acid concentration (up to 51 mmol/L) in plaque fluid was the largest change in any constituent ion observed following sucrose exposure. Relatively little change (< 5 mmol/L) was observed in the concentration of the other organic acids in the whole-mouth samples from CF subgroups during the experimental time period. However, the mean acetic and propionic acid concentrations in the whole-mouth CP samples each increased slowly and were significantly (p < 0.05) larger (8-9 mmol/L) at 30 min, compared with baseline values. In the site-specific samples, observed changes in other organic acid concentrations were relatively small compared with the changes in lactic acid concentrations. Inorganic ions.-Calcium concentrations increased in wholemouth plaque fluid samples, following sucrose exposure (Table 1), reaching an apparent maximum concentration at 7 min. Calcium concentrations then sharply decreased and approached baseline values. Compared with baseline values, calcium concentrations in plaque fluids were significantly greater at 7 and 60 min for the CP samples (p < 0.05) and at 7 and 15 min forth CF samples (p < 0.005).
J Dent Res November 1992
TABLE 4 TOTAL CONCENTRATIONS OF CALCIUM AND FLUORIDE IN WHOLE PLAQUEa
Group A
CF
mg Ca/g 16.2 ± 5.2
jig F/g 62.4 ± 17.1
*
B
CP CF
6.9 ± 12.8 ±
0.4 1.5
47.5 ± 3.5 57.6 ± 14.1
**
CP C CF 1 sound white-spot 2 sound
7.9 + 3.0 24.7 ± 4.3 9.3 45.8 6.8 32.3 3.5 25.4 16.7 54.0 3.8 white-spot 32.0 aExpressedper gram dry weight of plaque. *Significantly different at p < 0.05. Despite the fact that significantly greater acid production was present in CP samples, the mean calcium concentrations of the CF and CP plaque fluid samples at 7 min were not significantly different. Increases in calcium concentrations in plaque fluids from specific sites were much less apparent and were clearly detected only in samples from sound surfaces of group 1. An increase in the mean phosphate concentrations in whole-mouth samples (Table 1) was also observed, but differences were not significant. Changes in the measured concentrations of other ions (Tables 13) were relatively small, following sucrose exposure. However, the concentration of ammonium ion sharply decreased in all wholemouth plaque fluid samples, following sucrose exposure, and then increased slowly and exceeded the initial values (by 2 mmol/L) at 60 min in only one subgroup (group B, CP). Mean concentrations of ammonium ion in samples from CF and CP subgroups were not significantly different either before or following sucrose exposure. In general, similar findings were obtained with the site-specific samples. Ca and F in plaque residues.-Table 4 shows the calcium and fluoride contents in whole plaque samples of the experimental groups. Significantly (p < 0.05) higher concentrations of calcium were found in the whole plaque samples (centrifuged residues) obtained from CF individuals than in samples from CP individuals. In addition, higher concentrations of fluoride were also present in the CF samples than in the CP samples; this difference in group B was highly significant (p < 0.05). Similarly, concentrations of calcium in samples from sound surfaces were measurably higher than those found in samples from the white-spot surfaces; the lowest calcium concentrations were in the white-spot samples. Degrees of saturation and electrical imbalance.-By use of the analytical data, the degrees of saturation (DS) in plaque fluid with respect to both enamel (En) and DCPD and the apparent electrical imbalance, E, were calculated for each time point examined, before and after sucrose exposure. As shown in Table 5, prior to sucrose exposure, plaque fluid was found to be supersaturated with respect to enamel mineral, and to a significantly (p < 0.001) greater degree in the whole-mouth CF samples, with a mean of 7.11 ± 0.66 (n = 12), than in comparable samples from the CP groups, with a mean value of 5.42 ± 0.68 (n = 11). DS(En) values in samples from sound surfaces ofCPindividuals were similar to those in whole-mouth samples from CP subgroups. The lowest DS(En) values were found for the samples from white-
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CARIOGENIC POTENTIAL OF PLAQUE FLUID
Vol. 71 No. 11
1781
TABLE 5 DEGREES OF SATURATION IN PLAQUE FLUID SAMPLES BEFORE AND AT 7,15,30, AND 60 MIN FOLLOWING A ONE-MINUTE SUCROSE RINSEa
DS(En) s.d.
ob 7.11' 0.66
7 1.762 0.28
Caries-free 15 2.29 1.24
30 2.48 1.08
60 3.30 1.80 sound: 1 2 2-5% S
ob
5.421 0.68 5.50 5.00
7 1.022 0.30
Caries-positive 15 30 1.35 1.52 0.81 0.54
1.06 1.04 1.40
1.68 1.07 1.46
1.41 1.29 1.60
0.88 0.39 0.72 1.24 0.32
0.87 1.30 1.09 1.42 0.61
-
1.94 1.62 0.00
0.98 0.89 1.13
2.12 0.82 1.09
1.11 0.99 1.13
60 1.87 1.08 3.80 -
3.89
white-spot:
DS(DCPD) s.d.
2.56 0.20
1.74 0.23
1.77 0.63
1.76 0.54
1 2 2-5% S 1.84 0.33
4.30 3.86 2.37 0.30
1.82 2.80 2.00 1.45 0.50
sound: 1 2 2-5% S
2.10 1.80
1.79 -
1.84
white-spot: 1 2.00 0.73 0.82 1.20 2 1.70 0.42 0.97 1.58 2-5% S 0.73 0.86 1.34 1.22 aAll data following a 10% sucrose rinse, except where indicated for a 5% sucrose rinse (5% S); whole-mouth samples were obtained for groups Al, A2, B, and C; for groups 1 and 2, samples were collected from sound surfaces and white-spot surfaces of CP individuals.
bMean values calculated from the baseline data. 'CF and CP values significantly different at p < 0.001 (Student's t test). 2CF and CP values significantly different at p < 0.05 (Student's t test). spots with values of 4.30 and 3.86. DS(DCPD) values were similar plaque fluid from sound surfaces ofCP individuals were lower than in the CF and CP whole-mouth samples. DS(DCPD) values for the those found for the whole-mouth samples from CF groups, and, in site-specific samples from CP individuals were on the low end of fact, they were in the range of values found for the whole-mouth values obtained for the whole-mouth samples. samples from CP groups. In this respect, it is noteworthy that Following sucrose exposure, the DS(En) in all plaque fluid samples from white-spot surfaces had DS(En) values which were samples rapidly decreased and, for all times examined, to a greater lower than those for samples from all other surface types studied. extent in samples from CP individuals. The mean DS(En) value for Supersaturated conditions in starved plaque fluid can also the whole-mouth samples from CP subgroups at 7 min was signifi- support the precipitation of calcium phosphate deposits within dental plaque. Consistent with this idea, it was shown (Table 4) that cantly lower (p < 0.05) than that for the CF samples. The electrical imbalance, E, was found to be positive in all significantly higher concentrations of calcium were present in samples. The mean value for CF samples (21.5 ± 7.1 mmol/L, plaque samples obtained from CF groups and from sound surfaces including sound surfaces from groups 1 and 2) was not differentfrom of CP individuals than in whole-mouth samples from CP subjects the mean (21.3 ± 8.7, including white-spot surfaces from groups 1 and in white-spot samples, respectively. This finding is consistent with results from several studies (Ashley, 1975; Shaw et al., 1983) and 2) for CP samples. which found inverse relationships between caries experience and calcium (and phosphate) levels in whole plaque. As found in this Discussion. study, calcium ions are released following acid production and In the present study, starved plaque fluid from CF subjects had a hence ameliorate the driving force for enamel demineralization, by higher pH and was more highly supersaturated with respect to preventing greater decreases in the DS(En) if no calcium ions enamel than was plaque fluid from CP subjects. Consequently, a entered the fluid. The availability of mineral ions like calcium (and greater driving force for enamel remineralization may be present possibly fluoride) within plaque thus may play an important role in under starved conditions in the CF plaque fluid than in the plaque controlling enamel demineralization and, therefore, deserves furfluid of CP subjects. This factor may contribute to the difference in ther study. In fact, effective caries-preventive procedures have been caries history of these subjects. Furthermore, it appears that even advanced (Pearce, 1982) based on the formation and subsequent the plaque from sound tooth surfaces in the CP subjects enhances dissolution of (fluoridated) mineral phases within dental plaque the risk for enamel demineralization. Thus, the DS(En) values for upon acid production. Downloaded from jdr.sagepub.com at Bobst Library, New York University on April 29, 2015 For personal use only. No other uses without permission.
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In this study, degrees of saturation were calculated from individual ion activities determined from total plaque fluid compositions, by use of computational procedures previously described (Moreno and Margolis, 1988). A limitation of this approach is that it does not take into account calcium binding by plaque fluid ligands other than phosphate and organic acid anions. The mean total calcium concentrations in the whole-mouth starved plaque fluid samples from CF and CP groups were 2.8 ± 0.2 and 2.7 ± 0.5 mmol/ L, respectively. The mean values calculated for the corresponding free calcium ion concentrations for the CF and CP groups were 1.88 and 1.87 mmol/L, respectively. Thus, approximately 32% ofthe total calcium in starved plaque fluid appears to be bound by organic acid and phosphate ions. The direct measurement ofcalcium ion activities by means of ion-selective electrodes in plaque fluid has previously presented some difficulties (Tatevossian and Gould, 1976; Moreno and Margolis, 1988). Recently, however, more consistent results have been obtained which suggest that the free calcium concentration in "single-site", fasted, plaque fluid samples is around 1 mmoVL (Carey et al., 1986) and around 1.2 mmol/L in pooled, fasted samples (Tatevossian, 1987). In the former study, total calcium concentrations were not given, but a comparison of the latter value with the total calcium measured in the same samples (2.6 mmol/L: similar to the values reported here) suggests that approximately 54% of the total calcium is bound. It should be pointed out that, in this latter study, a direct measurement was not made in plaque fluid but rather in a sample diluted (1:1) with a buffer to adjust the ionic strength. Nevertheless, these results suggest that in plaque fluid about 20% ofthe total calcium is bound to species other than phosphate and organic acid anions. In preliminary studies in our laboratory, using a neutral ion carrier calcium electrode (Mere-1, WPI Instruments, Sarasota, FL), we have found that about 15-20% of the total calcium is bound to other species in starved plaque fluid samples from both CF and CP groups, in good agreement with the above estimation. If 20% additional calcium binding is considered in our calculations, the DS with respect to enamel is reduced by approximately 17% to values of 6.13 and 4.43 for the CF and CP groups, respectively, and the DS(DCPD) for the CF group would be reduced to 2.27 and to a value of 2.02 for the CP group. Therefore, the inclusion of the additional calcium binding does not change the general conclusion that starved plaque fluid is highly supersaturated with respect to both enamel and other calcium phosphate phases, such as DCPD, nor does it change the relative difference between CF and CP groups. In another study (Carey et al., 1986), in which calcium activity was measured directly, plaque fluid was also found to be supersaturated with respect to calcium phosphate phases. No studies, however, have determined the extent of calcium binding in plaque fluid following exposure to fermentable carbohydrate. Following sucrose exposure, the DS(En) sharply decreased, and to a greater extent in the whole-mouth CP samples and in samples from white-spots. This observation is an important one, since several studies (Moreno and Zahradnik, 1974; Theuns et al., 1983; Margolis et al., 1985) have clearly shown that rates of in vitro enamel demineralization are greater in undersaturated solutions with lower values of DS(En). Plaque fluid from CF individuals is thus characterized by a lower demineralization potential, as well as a greater remineralization potential, than that in CP individuals. Previously, we reported (Margolis et al., 1988ab) concentrations of acetic acid, propionic acid, and ammonium ion in plaque fluid (in the absence of sucrose) as approximately 45, 30, and 56 mmolfL, respectively. In those samples, the average calcium concentrations were in the range of 4-6 mmol/L. Such values differ substantially from those reported here (Tables 1-3). In the previous studies, plaque fluid samples were isolated at room temperature, whereas in this study they were maintained in ice during plaque fluid isolation (collection times: up to 90 min). Similar concentrations of acetic and propionic acids (33-45 mmoV/L and ±L27 mmolfL, respectively) were
J Dent Res November 1992
also previously found (Moreno and Margolis, 1988) in plaque fluid samples isolated from plaque kept in ice during a lengthy collection period (6-8 h). These latter values are also substantially higher than those reported in this study. Therefore, such differences appear to be related to the isolation procedures used and may be attributed to a greater rate of endogenous metabolic activity in plaque samples maintained at room temperature and to the accumulation of endproducts in samples held in ice for relatively long periods of time. Higher calcium concentrations in these samples may result from higher acid concentrations, either through the dissolution of mineral deposits in plaque or through the release into the more acidic medium ofcalcium bound by plaque components. However, whether samples were isolated at room temperature (Margolis et al., 1988b) or at ice temperature (this study), no significant differences in either acetic or propionic acid concentrations were found in samples from CF and CP subgroups. Similarly, in these studies on starved plaque fluid samples, no consistent differences in ammonium ion concentration were observed. It appears, then, that general findings based upon results of comparative studies are consistent, regardless ofthe isolation procedures used. It should also be pointed out that the mean electrical imbalance found in plaque fluid was positive and around 20 mequiv/L for all sample types. This finding may suggest that a corresponding concentration of anions has gone undetected in the present analyses of plaque fluid. Although this imbalance is small, compared with the high concentrations of ions found in plaque fluid, it is not unreasonable, since the present protocol did not include carbonate and amino acid analyses. Carbonate was previously found in plaque fluid at levels of 5-7 mmol/L (Margolis et al., 1988b). Similar concentrations of acidic amino acids (aspartate plus glutamate) have also been found in plaque fluid (Edgaret al., 1986; Higham and Edgar, 1989). Thus, the combined levels of these anionic species (not determined) are consistent with the observed electrical imbalance. Dental plaque appears to contain a pool of calcium ions which is released following acid production (Tables 1 and 3). Increases in calcium concentrations in plaque fluids following sucrose exposure have been previously observed by us in samples from human subjects (Rankine et al., 1985), and by Edgar et al. (1981) in plaque fluid from monkeys. In the present study, as much or more calcium was released into plaque fluid from CF subjects than from CP subjects, in spite ofthe significantly lower acid production in the CF samples. As noted above, these findings are consistent with the observation that plaque from CF subjects contains a greater pool of calcium ions than that from CP subjects, as reported in Table 4. Corresponding increases in phosphate concentrations have been observed (Rankine et al., 1985), but appear to be less apparent (this study; Edgar et al., 1981). Dawes and Jenkins (1962) and Luoma (1964) reported a decrease in the amount of extractable inorganic phosphate in human plaque following carbohydrate exposure. In these latter studies, the phosphate concentration in the plaque fluid compartment was not determined. It is possible that the total extractable phosphate concentration may decrease while the plaque fluid concentration (in some cases) increases. Based on additional laboratory studies, Luoma (1964) suggested that the decrease in extractable plaque phosphate was mainly attributable to uptake of phosphate by plaque cells. This latter finding may explain why better correlations have not been observed between increases in calcium and phosphate concentrations in plaque fluid following acid production. The results obtained indicate that the changes in the pH value of plaque fluid and in DS(En), following sucrose exposure, are brought about primarily by the rapid production oflactic acid. This finding is in agreement with results from a numberof studies on acid production in dental plaque (Muntz, 1943; Gilmour and Poole, 1967; Geddes, 1972; Cole et al., 1978; Higham and Edgar, 1989). As lactic acid concentrations decrease in plaque fluid (presumably due to diffusion out of plaque), following maximum acid production at
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CARIOGENIC POTENTIAL OF PLAQUE FLUID
Vol. 71 No. 11
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about 7 min, concentrations of high pK acids (acetic and propionic) Carey C, Gregory T, Rupp W, Tatevossian A, Vogel GL (1986). The driving began to increase (by a total of about 18 mmol/L at 30 min, Table 2) forces in human dental plaque fluid for demineralisation and remineralisation of enamel mineral. In: Leach SA, editor. Factors in the CP samples; following this, these acid concentrations derelating to demineralisation and remineralisation of the teeth. Oxford creased. This observation apparently reflects a change from a homofermentive pathway to a heterofermentive metabolic pathway (UK): IRL Press Ltd., 163-173. as carbohydrate became limiting (Carlsson, 1984). Results from Carlsson J (1984). Regulation of sugar metabolism in relation to the feastand-famine existence in plaque. In: Guggenheim B, editor. Cariology several studies (Geddes, 1975, 1984; Edgaretal., 1981; Higham and Today. International Congress, Zurich, 1983. Basel (Switzerland): Karger, Edgar, 1989; Oliveby et al., 1990) have suggested that, initially, 205-211. following sucrose exposure, acetic acid concentrations in plaque decrease; this was also observed in some samples in this work Cole MF, Bowden GH, Korts DC, Bowen WH (1978). The effect ofpyridoxine, phytate, and invert sugar on production of plaque acids in situ in the (Tables 2 and 3). monkey (M. fascicularis). Caries Res 12:190-201. During the experimental time period (60 min), pH values remained quite low relative to the baseline values. This can, in part, Dawes C and Jenkins GN (1962). Some inorganic constituents of dental plaque and their relationship to early calculus formation and caries. be related to the relatively high total acid concentrations in plaque Arch Oral Biol 7:161-172. fluid which were 15 to 35 mmolfL greater than those found prior to sucrose exposure. During this 60-minute time period, however, no Edgar WM, Bowen WH, Cole MF (1981). Development of rampant dental caries, and composition of plaque fluid and saliva in irradiated primates. substantial production of ammonium ion was observed; in fact, its concentration initially decreased, following sucrose exposure. SimiJ Oral Pathol 10:284-295. larly, another study (Edgar et al., 1986) showed little change in Edgar WM, Dodds MWJ, Higham SM (1986). Changes in carboxylic acid and ammonia concentrations in plaque fluid within the first 23 min free amino acid profiles in human dental plaque after a carbohydrate challenge in situ. Biochem Soc Trans 14:977. following sucrose exposure. The importance of base (ammonia) production, however, may become evident at times greater than 60 Geddes DAM (1972). The production ofL(+) and D(-) lactic acid and volatile min following exposure to fermentable carbohydrate, as plaque acids by human dental plaque and the effect of plaque buffering and fluid pH and other constituents return to baseline values. In fact, we acidic strength on pH. Arch Oral Biol 17:537-545. previously found that resting plaque fluid samples from CF sub- Geddes DAM (1975). Acids produced by human dental plaque metabolism in jects, collected one to several hours after they had eaten, had situ. Caries Res 9:98-109. significantly higher ammonium concentrations than did similar Geddes DAM (1984). Current view ofplaque acidogenicity. In: Guggenheim samples from CP subjects (Margolis et al., 1988a). Further studies B, editor. Cariology Today. International Congress, Zurich, 1983. Basel in this area are required. (Switzerland): Karger, 199-204. Although only a limited number of site-specific samples have Gilmour MN, Poole AE (1967). The fermentative capabilities of dental plaque. Caries Res 1:247-260. been studied, it is clear from the present work that differences can be detected in the cariogenic potential of plaque fluid from white- Higham SM, Edgar WM (1989). Human dental plaque pH, and the organic acid and free amino acid profiles in plaque fluid, after sucrose rinsing. spots and sound surfaces in CP subjects. In fact, the differences in Arch Oral Biol 34:329-334. starved-plaque-fluid pH, calcium content of plaque residues, acid production, and DS(En) (before and following sucrose exposure) Lee CY, Margolis HC, Moreno EC (1990). Factors influencing the rate of enamel demineralization in vitro (abstract). JDentRes 69 (Spec Iss):302. between white-spot and sound surfaces pooled from CP individuals are similar to those between whole-mouth plaque fluids from CF Luoma H (1964). Lability ofinorganic phosphate in dental plaque and saliva. Its appearance in association with some other factors related to dental and CP groups. These differences may reflect different bacterial caries. Acta Odontol Scand 22 (Suppl 41):1-127. populations in the two sites considered. Thus, in similar samples, the in vitro pH-lowering potential and proportions of mutans strep- Margolis HC, Duckworth JH, Moreno EC (1988a). Composition of pooled resting plaque fluid from caries-free and caries-susceptible individuals. tococci were found to be greater in plaque samples from white spots JDent Res 67:1468-1475. than in samples from sound surfaces of CP individuals (Van Houte Margolis HC, Duckworth JH, Moreno EC (1988b). Composition and buffer etal., 1991). capacity of pooled starved plaque fluid from caries-free and cariesDespite the fact that the pooling of plaque samples may obscure some local differences, this investigation has shown that measursusceptible individuals. J Dent Res 67:1476-1482. able and significant differences exist between the plaque and plaque Margolis HC, Moreno EC (1992). Kinetics of hydroxyapatite dissolution in acetic, lactic, and phosphoric acid solutions. Calcif Tissue Int 50:137fluid compositions in samples from caries-free and caries-positive 143. human subjects. The identification and the measurement of such differences provide a unique insight into the etiology of caries Margolis HC, Murphy BJ, Moreno EC (1985). Development of carious-like lesions in partially saturated lactate buffers. Caries Res 19:36-45. formation.
Acknowledgment. WethankDr. P.M. Soparkar, Department ofClinical Trials, Forsyth Dental Center, for conducting the dental examinations. REFERENCES Abelson DC and Mandel ID (1981). The effect ofsaliva on plaque pH in vivo. JDent Res 60:1634-1638. Ashley FP (1975). Calcium and phosphorus concentrations ofdental plaque related to dental caries in 11- to 14-year-old male subjects. Caries Res 9:351-362. Birkeland JM (1970). Direct potentiometric determination offluoride in soft tooth deposits. Caries Res 4:243-255.
Moreno EC, Margolis HC (1988). Composition ofhuman plaque fluid. JDent Res 67:1181-1189. Moreno EC, ZahradnikRT (1974). Chemistryofenamel subsurface demineralization. J Dent Res 53:226-235. Muntz JA (1943). Production of acids from glucose by dental plaque material. JBiol Chem 148:225-236. Oliveby A, Weetman DA, Geddes DAM, Lagerlof F (1990). The effect of salivary clearance of sucrose and fluoride on human dental plaque acidogenicity. Arch Oral Biol 35:907-911. Pearce EIF (1982). Effect of plaque mineralization on experimental dental caries. Caries Res 16:460-471. Rankine CAN, Moreno EC, Vogel GL, Margolis HC (1985). Micro- analytical determination of pH, calcium, and phosphate in plaque fluid. JDentRes 64:1275-1280. Shaw L, Murray JJ, Burchell CK, Best JS (1983). Calcium and phosphorus content of plaque and saliva in relation to dental caries. Carries Res 17:543-548.
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Stephan RM (1944). Intra-oral hydrogen-ion concentration associated with dental caries activity. JDent Res 23:257-266. Tatevossian A (1987). Calcium and phosphate in human dental plaque and their concentrations after overnight fasting and after ingestion of a boiled sweet. Arch Oral Biol 32:201-205. Tatevossian A, Gould CT (1976). Methods for sampling and analysis of the aqueous phase of human dental plaque. Arch Oral Biol 21:313-317.
J Dent Res November 1992 Theuns HM, Van Dijk JWE, Driessens FCM, Groeneveld A (1983). Effect of time and degree of saturation of buffer solutions on artificial carious lesion formation in human tooth enamel. Caries Res 17:503-512. Van Houte J, Sansone C, Joshipura K, Kent R (1991). In vitro acidogenic potential and mutans streptococci of human smooth surface plaque associated with initial caries lesions and sound enamel. J Dent Res 70:1497-1502.
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The composition of pooled plaque fluid from five population samples was determined before and at selected times (7, 15, 30, and 60 min) after a 10% sucrose rinse. Subjects were grouped according to caries status (caries-free, CF, DMFS = 0; caries-positive, CP, DMFS > 10). Samples were also studied from white-spot surfaces and from sound surfaces of the same mouths of two additional CP groups. Plaque fluid was isolated by centrifugation and analyzed for organic acids, inorganic ions (ion chromatography), and pH (microelectrodes). Prior to sucrose exposure, plaque fluids from the CF subgroups and from sound surfaces of the CP subjects had higher pH values than samples from CP subgroups and from white-spot surfaces, respectively; the ionic compositions were otherwise similar. Starved plaque fluids were also found to be supersaturated with respect to enamel and to a significantly greater degree in the CF samples, suggesting that CF plaque fluid may have a greater remineralization potential than CP samples. Following sucrose exposure, a rapid decrease in plaque fluid pH was observed, which corresponded primarily to lactic acid production. For all times examined, mean pH and DS(En) values were lower and lactic acid concentrations were higher in the CP samples than in the CF samples; noted differences were statistically significant at 7 min for pH and DS(En), and at 7, 15, and 30 min for lactic acid. Lower values of DS(En) suggest that plaque fluid from CP subjects had a measurably greater cariogenic potential. Calcium concentrations also increased in plaque fluid, following sucrose exposure, and to similar levels in samples from CF and CP subjects, despite significantly lower acid production in the CF samples. These latter results appear to be associated with the further finding that whole plaque from the CF subgroups contained significantly more calcium than the samples from the CP subjects. The availability of mineral ions like calcium within plaque may, therefore, play an important role in controlling enamel demineralization. J Dent Res 71(11):1776-1784, November, 1992
Introduction. Fermentation of dietary carbohydrate by micro-organisms in dental plaque brings about changes in the ionic composition ofthe extracellular aqueous phase of plaque, referred to as plaque fluid. Such chemical changes can lead to the establishment of the necessary conditions for caries formation. These cariogenic conditions in plaque, however, have not been well-defined, although associations have been made between changes in plaque pH and caries experience (e.g., Stephan, 1944; Abelson and Mandel, 1981). It has also been suggested that changes in plaque pH values are primarily associated with lactic acid production (Muntz, 1943; Gilmour and Poole, 1967; Geddes, 1972; Cole et al., 1978). Results of in vitro enamel demineralization studies, however, show that in addition to pH and lactic acid concentration, the rate of enamel demineralization is greatly influenced by the degree of saturation, DS(En), ofthe demineralizing medium with respect to enamel (Moreno and Zahradnik, 1974; Theuns et al., 1983; Margolis et al., 1985). ReReceived for publication January 6, 1992 Accepted for publication May 26, 1992 This study was supported by grants (DE-07493, DE-07009, and DE03187) from the National Institute of Dental Research.
1776
cently, we have also shown that the kinetics of hydroxyapatite (HA) dissolution can be described by a function of the DS(HA) and the total activity of undissociated acid species (i.e., organic and phosphoric acids) present in solution (Margolis and Moreno, 1992). These same parameters also appear to influence enamel demineralization in vitro (Lee et al., 1990). Although several studies (Edgar et al., 1986; Higham and Edgar, 1989) have investigated changes in the organic acid composition of plaque fluid following carbohydrate exposure, no studies have measured simultaneously acid production and the saturation status of plaque fluid in vivo. The present study was undertaken to determine the effect of fermentable carbohydrate exposure on the composition and degree of saturation of plaque fluid and to associate these parameters with caries experience. Plaque samples were pooled from all surfaces of caries-free individuals and from all surfaces of caries-positive individuals, before and following exposure to an oral sucrose rinse. Comparisons were also made between samples from white-spot surfaces and from sound surfaces of the same mouths in cariespositive individuals. Materials and methods. Subjects.-Due to analytical limitations previously discussed (Margolis et al., 1988a), pooled plaque samples were required for completion of all the analyses. Whole-mouth plaque samples were obtained from all tooth surfaces offive groups of subjects. Group A subjects (ages 18-22).from Northeastern University (Boston, MA) and group B subjects (ages 10-15) from the Massachusetts Migrant Education Program (Taunton, MA) were divided into two subgroups (n = 4-6) of caries-free, CF (DMFS = 0), and caries-positive, CP (DMFS: group A, 23 ± 7; group B, 18 ± 6), individuals, the latter previously referred to as being caries-susceptible (Margolis et al., 1988a,b). An additional CF group (group C: n = 5) from Northeastern University was also studied. Subjects were scored for caries without the use of radiography. The selected subjects did not have sealants or orthodontic bands, nor did they exhibit fluorosis. CF and CP individuals, therefore, differed with regard to caries history and not necessarily in any measure of current caries activity. Essentially all surfaces recorded as decayed for the CP subjects of group B were unfilled, and each subject was advised to seek dental treatment. The complete study was carried out with use of both subgroups ofgroup A on two separate occasions; results from these two studies are referred to as Al and A2. Site-specific plaque samples were also collected and pooled separately from sound and white-spot surfaces in two additional groups ofCP (group 1: n = 3, DMFS =25 ± 13; group 2, n = 4, DMFS = 21 - 16) individuals from Northeastern University. The locations of sound and white-spot surfaces of CP subjects were carefully charted during preliminary screening sessions. Whole-mouth plaque samples.-Prior to plaque collection, all subjects refrained from oral hygiene for 48 h and fasted overnight. Plaque samples were removed from the mouth, as previously described (Rankine et al., 1985). Initially, for establishment of a baseline for the plaque fluid composition, four pooledplaque samples were obtained from each subgroup. Plaque samples were collected from each subgroup member in a fashion such that all quadrants of the dentition were equally sampled. Prior to plaque collection, charts were prepared, for each subgroup member, which noted from
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CARIOGENIC POTENTIAL OF PLAQUE FLUID
Vol. 71 No. 11
TABLE 1 MEAN INORGANIC ION COMPOSITION OF WHOLE-MOUTH PLAQUE FLUID SAMPLES BEFORE AND AT 7, 15, 30, AND 60 MIN FOLLOWING A ONE-MINUTE 10% SUCROSE RINSEa
6.02
60 6.19
6.791
Caries-positive 15 5.58 5.382
0.28
-
0.12
0.09
Caries-free pH
ob 7.021
s.d.
0.05
7
15
30
5.632
5.80
0.08
0.36
ob
7
0.22
30 5.73
60 5.88
0.20
0.37
Na+
14.2
11.7
10.3
10.7
12.2
16.5
16.5
16.1
20.2
19.0
s.d.
3.5
6.1
4.4
5.2
4.7
5.4
1.8
0.7
3.6
6.6
NH4+
26.3
18.5
18.5
19.6
21.2
22.6
16.5
18.7
22.1
20.7
s.d.
3.9
2.6
3.7
2.6
2.0
3.9
2.4
2.5
4.3
2.0
K+
59.9
64.1
58.2
62.2
63.4
71.4
84.3
88.0
86.5
91.8
s.d.
4.9
14.6
11.1
8.8
4.9
11.3
6.8
11.3
1.3
5.0
Mg2+ s.d.
2.0
2.3
2.2
2.4
2.0
2.6
2.5
2.9
2.9
2.7
0.4
0.3
0.3
0.2
0.1
0.4
-
0.4
0.8
0.2
Cat~2.8
9.6
5.9
4.2
3.5
2.7
8.2
5.8
4.3
3.5
s.d.
0.2
1.5
0.5
1.2
1.0
0.5
1.9
1.2
0.4
0.4
Cl-
23.8
16.0
17.8
19.9
20.8
27.2
22.3
22.3
22.0
29.2
s.d.
3.6
3.1
4.6
5.0
3.7
2.9
7.3
2.2
4.0
11.0
Ptat
13.9
17.7
15.0
14.8
13.5
15.6
20.6
20.0
17.4
16.4
4.8 3.5 2.5 1.4 3.6 3.0 3.3 3.8 3.5 1.9 s.d. aAll concentrations expressed as mmol/L. bMean values calculated from baseline data (typically, 13 values for CF data and 12 values for CP data); for pH, the negative log ofthe mean of the hydrogen ion activity is given as the mean value; the s.d. of individual pH measurements from this mean pH is also given. 1CF and CP values significantly different at p < 0.001 (Student's t test). 2CF and CP values significantly different at p < 0.025 (Student's t test).
which teeth plaque samples would be taken. From each subgroup member, plaque was removed from lingual and buccal surfaces of two designated teeth of each quadrant and placed under watersaturated mineral oil in a 0.4-mL microcentrifuge tube maintained in an ice bath. Thus, for example, tube #1 contained plaque collected from eight teeth (e.g., from teeth nos. 17 & 16,21 & 22,37 & 36, and 41 & 42) of one subject ofthe subgroup. This procedure was repeated three times for the same subject, each time with plaque removed from eight "other" designated teeth (two from each quadrant) and samples pooled from the selected (eight) teeth in different microcentrifuge tubes. Thus, fourrepresentative whole-mouth plaque samples were collected from one subject, with each pooled sample placed in a separate microcentrifuge tube. This procedure was then repeated for each subject of a subgroup, with plaque samples being combined with the samples collected from the other subgroup members. Thus, in this fashion, four pooled plaque samples were collected for each subgroup. For maximum random sampling, sampling sites were staggered. For example, in tube #1 (referred to before), plaque samples from a second subject were collected from teeth 15 & 14,23 & 24,35 & 34, and 43 & 44. Since pooled samples of plaque were collected from all areas of the mouth, localized differences in plaque fluid composition cannot be assessed from the present data. Plaque fluid was then separated from the whole plaque and analyzed, as described below, to provide baseline data
(zero-time concentrations) for the subsequent rinse studies. Approximately one week later, plaque samples were collected at 7, 15, 30, and 60 min, following a one-minute rinse with 10% sucrose. These times denote the time at which plaque collection was begun. Exactly three minutes were allowed for the collection ofeach plaque sample. For each time point (i.e., at 7, 15, 30, or 60 min), plaque samples were collected and pooled from the same designated areas which were used for one of the four pooled samples obtained in the baseline studies. Plaque fluid was then isolated and analyzed as described below. In separate studies, it was determined that it was necessary and adequate for isolated plaque samples to be maintained at ice temperature, so that subsequent acid production, following sucrose exposure, would be minimized for the duration of the collection time (up to 90 min). CF and CP samples from each subgroup (Al, A2, or B) were collected and processed at the same time. For group C, a single whole-mouth sample of pooled plaque was collected, following overnight fasting; during subsequent sessions, this procedure was repeated following a one-minute, 10% sucrose rinse.
Site-specific plaque samples.-Prior to plaque collection, all subjects refrained from oral hygiene for 48 h and fasted overnight. Plaque samples from (1) white-spot surfaces of the CP subjects (groups 1 and 2) and (2) sound surfaces ofthe same CP subjects were collected, in the same general fashion as described above, before and
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MARGOLIS & MORENO
J Dent Res November 1992
TABLE 2 MEAN ORGANIC ACID COMPOSITION OF WHOLE-MOUTH PLAQUE FLUID SAMPLES BEFORE AND AT 7,15,30, AND 60 MIN FOLLOWING A ONE-MINUTE 10% SUCROSE RINSEa
Succinate
Ob 4.4
7 3.2
s.d.
1.5
0.8
Lactate
1.8
s.d.
Caries-free 15 4.0
Caries-positive 30 5.2
60 5.6
1.4
1.7
36.01
21.91
0.7
8.6
19.9
s.d. Propionate
Acetate
Ob
4.6
7 3.0
2.4
1.0
12.51
6.8
1.9
2.0
17.6
18.7
3.5
4.0
5.8
8.0
15 2.8
30 6.0
60 7.5
0.7
0.1
0.4
1.8
2.6
51.11
39.91
19.1'
13.2
2.4
1.2
2.5
6.2
2.2
5.6
20.7
19.8
20.3
19.8
24.1
29.9
25.3
5.4
4.4
4.6
4.6
2.7
2.1
4.5
3.5
7.4
8.9
7.6
9.2
11.7
13.7
17.5
14.3
3.4 1.5 4.1 3.2 1.9 3.6 1.3 1.9 4.2 s.d. 4.0 aAll concentrations expressed in mmol/L. bMean values calculated from the baseline data (typically, 13 values for CF data and 12 values for CP data); formic and butyric acid concentrations were low (0-5 mmolJL) and not consistently detected in all samples. 'CF and CP values significantly different at p < 0.025 (Student's t test).
Calculation of ion activities and degrees of saturation.-By use ofthe analytical data obtained, individual ion activities and degrees of saturation (DS) with respect to enamel (En) and dicalcium phosphate dehydrate (DCPD) were calculated as previously described in detail (Moreno and Margolis, 1988). These calculations include calcium and magnesium binding by organic acid anions and phosphate species. The ionic concentrations, C, were then used in the following equation defining the condition of electrical neutrality in the plaque fluid: E = C AZ, with the valences, Z., taken with their sign. If the concentrations of all ionic species were determined, the value of E would be equal to zero. In the present study, a value ofE different from zero was considered as the concentration of a monosample and analyzed as described below. Plaquefluid isolation. -Plaque fluid was isolated by centrifuga- valent species not determined and was included in the calculation tion, as previously described (Margolis et al., 1988a), with the of ionic strength. modification that centrifugation was carried out at 2-4°C; 3-6 PL of Determination of total calcium and fluoride in plaque resifluid was obtained from each pooled plaque sample. Immediately dues. -Pooled plaque residues (stored at -20'C) collected duringthe following the separation of plaque fluid, samples were diluted for baseline studies (i.e., without sucrose exposure) were removed from subsequent analyses by the addition of approximately 2-AL aliquots the microcentrifuge tubes; microcentrifuge tubes were cut with a (Drummond microdispenser, Drummond Scientific Co., Broomall, scalpel just below the oil-plaque interface to facilitate plaque rePA) of plaque fluid to 75 pL of 3.0 mmol/L OSA (octane sulfonic acid) moval and avoid contamination with mineral oil. Plaque samples contained in a 1.5-mL polypropylene microcentrifuge tube. The from each tube (four tubes per subgroup) were then individually precise volume of plaque fluid used was determined by calibration freeze-dried to constant weight. Accurately weighed dried plaque ofthe microdispenser (Margolis et al., 1988a). Duplicate or triplicate samples (1.8-12 mg) were then digested in acid (0.2 mL of a 1:1 dilutions ofeach sample were made, depending on the volume of the mixture of 14.3 mol/L nitric acid and 11.6 molIL perchloric acid) at sample available. Diluted samples were then frozen at -20°C, and 60°C for 2 h, by means of a modification of a published procedure were stored at this temperature prior to analysis; analyses were (Birkeland, 1970). Concentrations of fluoride and calcium (extypically completed within 72 h after plaque sample collection. pressedper plaque dry weight) in the neutralized acid digests were Remaining plaque fluid, maintained on ice, was used for pH deter- determined with a fluoride ion-selective electrode (Orion, Cammination, as described below. The compressed plaque residues, bridge, MA) and an atomic absorption spectrophotometer, respecobtained following centrifugation (maintained under mineral oil), tively. were then stored in a freezer at -20°C and later analyzed for total calcium and fluoride, also as described below. Organic acid and inorganic ion analyses. -Diluted plaque fluid Results. samples were analyzed for mono- and divalent cations, inorganic The mean inorganic ion and organic acid compositions of wholeanions, and organic acids, as described previously (Margolis et al., mouth plaque fluid samples from the CF and CP subgroups, ob1988a). tained before and after exposure to sucrose, are given in Tables 1 The pH value for each plaque fluid sample (0.35-1-PL aliquots) and 2, respectively. was determined by use of micro-glass and reference electrodes Baseline composition.-As shown in Table 1, the mean pH value (Microelectrodes, Inc., Londonderry, NH), as previously described ofthe baseline CF samples (13 values, fourfor each ofgroups Al, A2, and B, and one for group C), 7.02 ± 0.05, was significantly (p < 0.001) (Margolis et al., 1988b). at selected times (7, 15, 30, and 60 min) following a 5% and/or 10% sucrose rinse. During a single session, two plaque samples were collected-one from white-spot surfaces (e.g. at 7 min) and one from sound surfaces (e.g. at 15 min)-and placed under oil in separate microcentrifuge tubes maintained at ice temperature. During four consecutive weekly sessions, samples were collected from the same subjects and the same sites at a different designated time following sucrose exposure (e.g. at 7 min from sound surfaces and 15 min from white-spot surfaces). In this fashion, pooled plaque samples were obtained from both surface types ofthe same individuals at various times after the sucrose rinse. Plaque fluid was isolated from each
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1779
CARIOGENIC POTENTIAL OF PLAQUE FLUID
Vol. 71 No. 11
TABLE 3 COMPOSITION OF PLAQUE FLUID SAMPLES FROM WHITE-SPOT AND SOUND SURFACES OF CARIES-POSITIVE SUBJECTS BEFORE AND AT 7,15,30, AND 60 MIN FOLLOWING A ONE-MINUTE 5% OR 10% SUCROSE RINSEa
Sound surfaces 7 15 30 5.66 5.87 5.99 5.79 5.92 5.96 5.90 5.99 5% 6.11 10% 12.3 13.3 10.3 14.9 21.1 11.2 10% 5% 12.7 12.8 16.5 6.9 10.2 8.0 NH4+1 10% 11.5 2 10% 10.8 11.1 2 5% 13.7 7.9 11.9 K+ 1 10% 46.2 75.6 63.4 2 10% 61.4 73.7 " 2 5% 66.1 68.4 70.9 5.4 4.7 3.5 2.1 Mg2+1 10% 1.7 2 10% 1.8 1.6 1.8 1.8 2 5% 1.9 2.2 4.4 5.3 2.2 2.1 Catot 1 10% 2 10% 3.1 2.5 2.5 1.8 3.4 3.0 2.3 2 5% 14.5 Cl- 1 10% 32.0 23.6 18.4 2 10% 22.1 33.4 29.0 21.5 28.1 27.0 2 5% 12.7 9.6 12.9 11.8 Ptot 1 10% 8.7 7.6 9.2 2 10% 9.0 10.5 8.9 10.1 2 5% 0.1 0.9 1.8 3.3 Suc 1 10% 1.9 2.3 2.9 2 10% 5.3 4.4 1.7 2.3 2 5% 31.4 34.5 11.4 1.7 Lac 1 10% 24.5 17.7 26.1 2 10% 3.9 14.4 2 5% 23.3 18.9 12.0 20.5 15.1 14.5 Ace 1 10% 12.5 13.0 15.2 2 10% 13.7 12.2 13.7 15.1 2 5% 8.2 5.4 6.1 3.4 Pro 1 10% 8.8 7.2 8.9 2 10% 7.3 9.7 2 5% 5.9 7.9 aAl concentrations expressed in mmol/L; - missing sample.
Group No. pH 1 2 2 Nay 1 2 2
Sucrose
conc. 10% 10%
0 6.92 6.99 " 18.2 19.4 " 12.7 12.2 a 56.2 68.1
60 6.66 -
6.67 8.8 -
13.2 9.7 -
17.8 41.9 -
66.4 3.5 -
1.9 2.1 -
2.3 16.2 -
25.6 9.7 -
11.1 2.3 -
4.8 5.7 -
6.8 11.3 -
13.6 4.1 -
9.1
0 6.73 6.72
16.4 24.0 " 13.4 14.9 66.6 69.5
4.9 2.0 2.1 2.2 37.7 29.5 " 14.4 9.2 5.1 6.4 2.3 4.0 18.2 12.4 6.0 5.7 "
7 5.76 5.42 5.53 7.9 18.7 11.3 4.6 10.4 7.8 55.3 -
71.5 4.1 1.3 2.0 2.5 1.8 3.5 14.6 -
27.7 8.7 7.8 9.5 0.9 1.8 1.6 33.7 31.6 30.7 11.9 11.8 11.5 5.3 6.9 8.8
White spots 15 5.68 6.01 5.94 10.6 12.8 10.7 7.2 11.8 9.0 52.3 66.5 66.9 3.8 1.6 2.0 2.8 2.2 2.0 18.1 22.2 20.5 10.2 9.0 8.7 1.0 1.8 2.0 29.7 17.1 16.5 14.3 13.2 12.7 5.1 10.5 7.8
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30 -
6.18 -
15.5 7.1 -
16.8 -
77.5 -
2.6 1.8 -
2.6 -
-
29.4 -
-
12.0 -
-
2.4 -
15.7 -
-
21.0 -
-
14.8
60 6.25 6.44 6.34 12.3 13.8 12.5 8.0 12.7 10.8 64.5 79.0 72.3 1.8 1.2 1.7 1.6 2.3 1.7 20.7 30.9 23.0 11.5 11.5 10.2 3.5 3.5 4.4 7.4 9.7 9.2 12.4 17.2 14.8 6.3 12.4 10.6
1780
MARGOLIS & MORENO
higher than that found for the CP samples (11 values), 6.79 ± 0.12. The pH values of plaque fluid from white-spot surfaces (Table 3) were in the range of the whole-mouth samples from the CP subgroups and were measurably lower (by 0.19-0.27 pH units) than those of samples taken from sound surfaces of the same CP mouths. Other than pH, no meaningful differences were observed in the mean baseline concentrations in the whole-mouth samples from CF and CP groups. With regard to organic acid composition (Table 2), in all samples, acetic acid was present in the highest concentration, whereas lactic acid was found in relatively low concentrations. Negligible concentrations of formic and butyric acids were found in the site-specific samples. In general, the baseline concentrations of ions in plaque fluids from white-spot and sound surfaces ofCP individuals (Table 3) were comparable with those found (Tables 1 and 2) in the whole-mouth samples, with the notable exception of calcium and ammonium concentrations, which were lower in the site-specific samples from the CP individuals. Other than pH, no apparent differences were observed between the ionic composition of plaque fluid from whitespot and sound surfaces within the same mouths of CP subjects, prior to sucrose exposure. Plaque fluid composition following sucrose exposure: pH.-As shown in Table 1, following exposure to sucrose, plaque fluid pH decreased rapidly to an apparent minimum at 7 min, and then increased slowly. In no case did the plaque fluid pH return to baseline values within the experimental time-frame of 60 min. The mean pH values were lower in the CP samples at all times examined and significantly so (p < 0.025) at 7 min. In group 2 (Table 3), pH values in samples from white-spot surfaces, at 7 min following exposure to both 5% and 10% sucrose solutions, were found to be lower (by 0.37 pH units) than those observed in samples from sound surfaces within the same mouths. The pH values in plaque fluids from white-spots and sound surfaces from CP subjects of group 1 were, however, comparable, except for samples obtained at 60 min. At 60 min, pH values in all samples from CP individuals and from white-spot surfaces were substantially lowerthan the pH in samples from corresponding CF individuals (Table 1) or from sound surfaces in CP individuals (Table 3). The limited amount of data presently available for site-specific samples, however, does not lend itself to statistical analysis. Organic acids.-A rapid increase in lactic acid concentration was observed (Tables 2 and 3), in all samples, concomitant with the decrease in pH. Mean lactic acid concentrations were significantly larger(p < 0.025)inthe CPsamples at 7,15, and30min aftersucrose exposure. In addition, in site-specific samples from group 2 (Table 3), the lactic acid concentration at 7 min was greater in the whitespot sample than in samples from the sound surfaces of the same mouths. Again, the limited amount of data presently available for site-specific samples does not lend itself to statistical analysis. The increase in lactic acid concentration (up to 51 mmol/L) in plaque fluid was the largest change in any constituent ion observed following sucrose exposure. Relatively little change (< 5 mmol/L) was observed in the concentration of the other organic acids in the whole-mouth samples from CF subgroups during the experimental time period. However, the mean acetic and propionic acid concentrations in the whole-mouth CP samples each increased slowly and were significantly (p < 0.05) larger (8-9 mmol/L) at 30 min, compared with baseline values. In the site-specific samples, observed changes in other organic acid concentrations were relatively small compared with the changes in lactic acid concentrations. Inorganic ions.-Calcium concentrations increased in wholemouth plaque fluid samples, following sucrose exposure (Table 1), reaching an apparent maximum concentration at 7 min. Calcium concentrations then sharply decreased and approached baseline values. Compared with baseline values, calcium concentrations in plaque fluids were significantly greater at 7 and 60 min for the CP samples (p < 0.05) and at 7 and 15 min forth CF samples (p < 0.005).
J Dent Res November 1992
TABLE 4 TOTAL CONCENTRATIONS OF CALCIUM AND FLUORIDE IN WHOLE PLAQUEa
Group A
CF
mg Ca/g 16.2 ± 5.2
jig F/g 62.4 ± 17.1
*
B
CP CF
6.9 ± 12.8 ±
0.4 1.5
47.5 ± 3.5 57.6 ± 14.1
**
CP C CF 1 sound white-spot 2 sound
7.9 + 3.0 24.7 ± 4.3 9.3 45.8 6.8 32.3 3.5 25.4 16.7 54.0 3.8 white-spot 32.0 aExpressedper gram dry weight of plaque. *Significantly different at p < 0.05. Despite the fact that significantly greater acid production was present in CP samples, the mean calcium concentrations of the CF and CP plaque fluid samples at 7 min were not significantly different. Increases in calcium concentrations in plaque fluids from specific sites were much less apparent and were clearly detected only in samples from sound surfaces of group 1. An increase in the mean phosphate concentrations in whole-mouth samples (Table 1) was also observed, but differences were not significant. Changes in the measured concentrations of other ions (Tables 13) were relatively small, following sucrose exposure. However, the concentration of ammonium ion sharply decreased in all wholemouth plaque fluid samples, following sucrose exposure, and then increased slowly and exceeded the initial values (by 2 mmol/L) at 60 min in only one subgroup (group B, CP). Mean concentrations of ammonium ion in samples from CF and CP subgroups were not significantly different either before or following sucrose exposure. In general, similar findings were obtained with the site-specific samples. Ca and F in plaque residues.-Table 4 shows the calcium and fluoride contents in whole plaque samples of the experimental groups. Significantly (p < 0.05) higher concentrations of calcium were found in the whole plaque samples (centrifuged residues) obtained from CF individuals than in samples from CP individuals. In addition, higher concentrations of fluoride were also present in the CF samples than in the CP samples; this difference in group B was highly significant (p < 0.05). Similarly, concentrations of calcium in samples from sound surfaces were measurably higher than those found in samples from the white-spot surfaces; the lowest calcium concentrations were in the white-spot samples. Degrees of saturation and electrical imbalance.-By use of the analytical data, the degrees of saturation (DS) in plaque fluid with respect to both enamel (En) and DCPD and the apparent electrical imbalance, E, were calculated for each time point examined, before and after sucrose exposure. As shown in Table 5, prior to sucrose exposure, plaque fluid was found to be supersaturated with respect to enamel mineral, and to a significantly (p < 0.001) greater degree in the whole-mouth CF samples, with a mean of 7.11 ± 0.66 (n = 12), than in comparable samples from the CP groups, with a mean value of 5.42 ± 0.68 (n = 11). DS(En) values in samples from sound surfaces ofCPindividuals were similar to those in whole-mouth samples from CP subgroups. The lowest DS(En) values were found for the samples from white-
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CARIOGENIC POTENTIAL OF PLAQUE FLUID
Vol. 71 No. 11
1781
TABLE 5 DEGREES OF SATURATION IN PLAQUE FLUID SAMPLES BEFORE AND AT 7,15,30, AND 60 MIN FOLLOWING A ONE-MINUTE SUCROSE RINSEa
DS(En) s.d.
ob 7.11' 0.66
7 1.762 0.28
Caries-free 15 2.29 1.24
30 2.48 1.08
60 3.30 1.80 sound: 1 2 2-5% S
ob
5.421 0.68 5.50 5.00
7 1.022 0.30
Caries-positive 15 30 1.35 1.52 0.81 0.54
1.06 1.04 1.40
1.68 1.07 1.46
1.41 1.29 1.60
0.88 0.39 0.72 1.24 0.32
0.87 1.30 1.09 1.42 0.61
-
1.94 1.62 0.00
0.98 0.89 1.13
2.12 0.82 1.09
1.11 0.99 1.13
60 1.87 1.08 3.80 -
3.89
white-spot:
DS(DCPD) s.d.
2.56 0.20
1.74 0.23
1.77 0.63
1.76 0.54
1 2 2-5% S 1.84 0.33
4.30 3.86 2.37 0.30
1.82 2.80 2.00 1.45 0.50
sound: 1 2 2-5% S
2.10 1.80
1.79 -
1.84
white-spot: 1 2.00 0.73 0.82 1.20 2 1.70 0.42 0.97 1.58 2-5% S 0.73 0.86 1.34 1.22 aAll data following a 10% sucrose rinse, except where indicated for a 5% sucrose rinse (5% S); whole-mouth samples were obtained for groups Al, A2, B, and C; for groups 1 and 2, samples were collected from sound surfaces and white-spot surfaces of CP individuals.
bMean values calculated from the baseline data. 'CF and CP values significantly different at p < 0.001 (Student's t test). 2CF and CP values significantly different at p < 0.05 (Student's t test). spots with values of 4.30 and 3.86. DS(DCPD) values were similar plaque fluid from sound surfaces ofCP individuals were lower than in the CF and CP whole-mouth samples. DS(DCPD) values for the those found for the whole-mouth samples from CF groups, and, in site-specific samples from CP individuals were on the low end of fact, they were in the range of values found for the whole-mouth values obtained for the whole-mouth samples. samples from CP groups. In this respect, it is noteworthy that Following sucrose exposure, the DS(En) in all plaque fluid samples from white-spot surfaces had DS(En) values which were samples rapidly decreased and, for all times examined, to a greater lower than those for samples from all other surface types studied. extent in samples from CP individuals. The mean DS(En) value for Supersaturated conditions in starved plaque fluid can also the whole-mouth samples from CP subgroups at 7 min was signifi- support the precipitation of calcium phosphate deposits within dental plaque. Consistent with this idea, it was shown (Table 4) that cantly lower (p < 0.05) than that for the CF samples. The electrical imbalance, E, was found to be positive in all significantly higher concentrations of calcium were present in samples. The mean value for CF samples (21.5 ± 7.1 mmol/L, plaque samples obtained from CF groups and from sound surfaces including sound surfaces from groups 1 and 2) was not differentfrom of CP individuals than in whole-mouth samples from CP subjects the mean (21.3 ± 8.7, including white-spot surfaces from groups 1 and in white-spot samples, respectively. This finding is consistent with results from several studies (Ashley, 1975; Shaw et al., 1983) and 2) for CP samples. which found inverse relationships between caries experience and calcium (and phosphate) levels in whole plaque. As found in this Discussion. study, calcium ions are released following acid production and In the present study, starved plaque fluid from CF subjects had a hence ameliorate the driving force for enamel demineralization, by higher pH and was more highly supersaturated with respect to preventing greater decreases in the DS(En) if no calcium ions enamel than was plaque fluid from CP subjects. Consequently, a entered the fluid. The availability of mineral ions like calcium (and greater driving force for enamel remineralization may be present possibly fluoride) within plaque thus may play an important role in under starved conditions in the CF plaque fluid than in the plaque controlling enamel demineralization and, therefore, deserves furfluid of CP subjects. This factor may contribute to the difference in ther study. In fact, effective caries-preventive procedures have been caries history of these subjects. Furthermore, it appears that even advanced (Pearce, 1982) based on the formation and subsequent the plaque from sound tooth surfaces in the CP subjects enhances dissolution of (fluoridated) mineral phases within dental plaque the risk for enamel demineralization. Thus, the DS(En) values for upon acid production. Downloaded from jdr.sagepub.com at Bobst Library, New York University on April 29, 2015 For personal use only. No other uses without permission.
1782
MARGOLIS & MORENO
In this study, degrees of saturation were calculated from individual ion activities determined from total plaque fluid compositions, by use of computational procedures previously described (Moreno and Margolis, 1988). A limitation of this approach is that it does not take into account calcium binding by plaque fluid ligands other than phosphate and organic acid anions. The mean total calcium concentrations in the whole-mouth starved plaque fluid samples from CF and CP groups were 2.8 ± 0.2 and 2.7 ± 0.5 mmol/ L, respectively. The mean values calculated for the corresponding free calcium ion concentrations for the CF and CP groups were 1.88 and 1.87 mmol/L, respectively. Thus, approximately 32% ofthe total calcium in starved plaque fluid appears to be bound by organic acid and phosphate ions. The direct measurement ofcalcium ion activities by means of ion-selective electrodes in plaque fluid has previously presented some difficulties (Tatevossian and Gould, 1976; Moreno and Margolis, 1988). Recently, however, more consistent results have been obtained which suggest that the free calcium concentration in "single-site", fasted, plaque fluid samples is around 1 mmoVL (Carey et al., 1986) and around 1.2 mmol/L in pooled, fasted samples (Tatevossian, 1987). In the former study, total calcium concentrations were not given, but a comparison of the latter value with the total calcium measured in the same samples (2.6 mmol/L: similar to the values reported here) suggests that approximately 54% of the total calcium is bound. It should be pointed out that, in this latter study, a direct measurement was not made in plaque fluid but rather in a sample diluted (1:1) with a buffer to adjust the ionic strength. Nevertheless, these results suggest that in plaque fluid about 20% ofthe total calcium is bound to species other than phosphate and organic acid anions. In preliminary studies in our laboratory, using a neutral ion carrier calcium electrode (Mere-1, WPI Instruments, Sarasota, FL), we have found that about 15-20% of the total calcium is bound to other species in starved plaque fluid samples from both CF and CP groups, in good agreement with the above estimation. If 20% additional calcium binding is considered in our calculations, the DS with respect to enamel is reduced by approximately 17% to values of 6.13 and 4.43 for the CF and CP groups, respectively, and the DS(DCPD) for the CF group would be reduced to 2.27 and to a value of 2.02 for the CP group. Therefore, the inclusion of the additional calcium binding does not change the general conclusion that starved plaque fluid is highly supersaturated with respect to both enamel and other calcium phosphate phases, such as DCPD, nor does it change the relative difference between CF and CP groups. In another study (Carey et al., 1986), in which calcium activity was measured directly, plaque fluid was also found to be supersaturated with respect to calcium phosphate phases. No studies, however, have determined the extent of calcium binding in plaque fluid following exposure to fermentable carbohydrate. Following sucrose exposure, the DS(En) sharply decreased, and to a greater extent in the whole-mouth CP samples and in samples from white-spots. This observation is an important one, since several studies (Moreno and Zahradnik, 1974; Theuns et al., 1983; Margolis et al., 1985) have clearly shown that rates of in vitro enamel demineralization are greater in undersaturated solutions with lower values of DS(En). Plaque fluid from CF individuals is thus characterized by a lower demineralization potential, as well as a greater remineralization potential, than that in CP individuals. Previously, we reported (Margolis et al., 1988ab) concentrations of acetic acid, propionic acid, and ammonium ion in plaque fluid (in the absence of sucrose) as approximately 45, 30, and 56 mmolfL, respectively. In those samples, the average calcium concentrations were in the range of 4-6 mmol/L. Such values differ substantially from those reported here (Tables 1-3). In the previous studies, plaque fluid samples were isolated at room temperature, whereas in this study they were maintained in ice during plaque fluid isolation (collection times: up to 90 min). Similar concentrations of acetic and propionic acids (33-45 mmoV/L and ±L27 mmolfL, respectively) were
J Dent Res November 1992
also previously found (Moreno and Margolis, 1988) in plaque fluid samples isolated from plaque kept in ice during a lengthy collection period (6-8 h). These latter values are also substantially higher than those reported in this study. Therefore, such differences appear to be related to the isolation procedures used and may be attributed to a greater rate of endogenous metabolic activity in plaque samples maintained at room temperature and to the accumulation of endproducts in samples held in ice for relatively long periods of time. Higher calcium concentrations in these samples may result from higher acid concentrations, either through the dissolution of mineral deposits in plaque or through the release into the more acidic medium ofcalcium bound by plaque components. However, whether samples were isolated at room temperature (Margolis et al., 1988b) or at ice temperature (this study), no significant differences in either acetic or propionic acid concentrations were found in samples from CF and CP subgroups. Similarly, in these studies on starved plaque fluid samples, no consistent differences in ammonium ion concentration were observed. It appears, then, that general findings based upon results of comparative studies are consistent, regardless ofthe isolation procedures used. It should also be pointed out that the mean electrical imbalance found in plaque fluid was positive and around 20 mequiv/L for all sample types. This finding may suggest that a corresponding concentration of anions has gone undetected in the present analyses of plaque fluid. Although this imbalance is small, compared with the high concentrations of ions found in plaque fluid, it is not unreasonable, since the present protocol did not include carbonate and amino acid analyses. Carbonate was previously found in plaque fluid at levels of 5-7 mmol/L (Margolis et al., 1988b). Similar concentrations of acidic amino acids (aspartate plus glutamate) have also been found in plaque fluid (Edgaret al., 1986; Higham and Edgar, 1989). Thus, the combined levels of these anionic species (not determined) are consistent with the observed electrical imbalance. Dental plaque appears to contain a pool of calcium ions which is released following acid production (Tables 1 and 3). Increases in calcium concentrations in plaque fluids following sucrose exposure have been previously observed by us in samples from human subjects (Rankine et al., 1985), and by Edgar et al. (1981) in plaque fluid from monkeys. In the present study, as much or more calcium was released into plaque fluid from CF subjects than from CP subjects, in spite ofthe significantly lower acid production in the CF samples. As noted above, these findings are consistent with the observation that plaque from CF subjects contains a greater pool of calcium ions than that from CP subjects, as reported in Table 4. Corresponding increases in phosphate concentrations have been observed (Rankine et al., 1985), but appear to be less apparent (this study; Edgar et al., 1981). Dawes and Jenkins (1962) and Luoma (1964) reported a decrease in the amount of extractable inorganic phosphate in human plaque following carbohydrate exposure. In these latter studies, the phosphate concentration in the plaque fluid compartment was not determined. It is possible that the total extractable phosphate concentration may decrease while the plaque fluid concentration (in some cases) increases. Based on additional laboratory studies, Luoma (1964) suggested that the decrease in extractable plaque phosphate was mainly attributable to uptake of phosphate by plaque cells. This latter finding may explain why better correlations have not been observed between increases in calcium and phosphate concentrations in plaque fluid following acid production. The results obtained indicate that the changes in the pH value of plaque fluid and in DS(En), following sucrose exposure, are brought about primarily by the rapid production oflactic acid. This finding is in agreement with results from a numberof studies on acid production in dental plaque (Muntz, 1943; Gilmour and Poole, 1967; Geddes, 1972; Cole et al., 1978; Higham and Edgar, 1989). As lactic acid concentrations decrease in plaque fluid (presumably due to diffusion out of plaque), following maximum acid production at
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CARIOGENIC POTENTIAL OF PLAQUE FLUID
Vol. 71 No. 11
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about 7 min, concentrations of high pK acids (acetic and propionic) Carey C, Gregory T, Rupp W, Tatevossian A, Vogel GL (1986). The driving began to increase (by a total of about 18 mmol/L at 30 min, Table 2) forces in human dental plaque fluid for demineralisation and remineralisation of enamel mineral. In: Leach SA, editor. Factors in the CP samples; following this, these acid concentrations derelating to demineralisation and remineralisation of the teeth. Oxford creased. This observation apparently reflects a change from a homofermentive pathway to a heterofermentive metabolic pathway (UK): IRL Press Ltd., 163-173. as carbohydrate became limiting (Carlsson, 1984). Results from Carlsson J (1984). Regulation of sugar metabolism in relation to the feastand-famine existence in plaque. In: Guggenheim B, editor. Cariology several studies (Geddes, 1975, 1984; Edgaretal., 1981; Higham and Today. International Congress, Zurich, 1983. Basel (Switzerland): Karger, Edgar, 1989; Oliveby et al., 1990) have suggested that, initially, 205-211. following sucrose exposure, acetic acid concentrations in plaque decrease; this was also observed in some samples in this work Cole MF, Bowden GH, Korts DC, Bowen WH (1978). The effect ofpyridoxine, phytate, and invert sugar on production of plaque acids in situ in the (Tables 2 and 3). monkey (M. fascicularis). Caries Res 12:190-201. During the experimental time period (60 min), pH values remained quite low relative to the baseline values. This can, in part, Dawes C and Jenkins GN (1962). Some inorganic constituents of dental plaque and their relationship to early calculus formation and caries. be related to the relatively high total acid concentrations in plaque Arch Oral Biol 7:161-172. fluid which were 15 to 35 mmolfL greater than those found prior to sucrose exposure. During this 60-minute time period, however, no Edgar WM, Bowen WH, Cole MF (1981). Development of rampant dental caries, and composition of plaque fluid and saliva in irradiated primates. substantial production of ammonium ion was observed; in fact, its concentration initially decreased, following sucrose exposure. SimiJ Oral Pathol 10:284-295. larly, another study (Edgar et al., 1986) showed little change in Edgar WM, Dodds MWJ, Higham SM (1986). Changes in carboxylic acid and ammonia concentrations in plaque fluid within the first 23 min free amino acid profiles in human dental plaque after a carbohydrate challenge in situ. Biochem Soc Trans 14:977. following sucrose exposure. The importance of base (ammonia) production, however, may become evident at times greater than 60 Geddes DAM (1972). The production ofL(+) and D(-) lactic acid and volatile min following exposure to fermentable carbohydrate, as plaque acids by human dental plaque and the effect of plaque buffering and fluid pH and other constituents return to baseline values. In fact, we acidic strength on pH. Arch Oral Biol 17:537-545. previously found that resting plaque fluid samples from CF sub- Geddes DAM (1975). Acids produced by human dental plaque metabolism in jects, collected one to several hours after they had eaten, had situ. Caries Res 9:98-109. significantly higher ammonium concentrations than did similar Geddes DAM (1984). Current view ofplaque acidogenicity. In: Guggenheim samples from CP subjects (Margolis et al., 1988a). Further studies B, editor. Cariology Today. International Congress, Zurich, 1983. Basel in this area are required. (Switzerland): Karger, 199-204. Although only a limited number of site-specific samples have Gilmour MN, Poole AE (1967). The fermentative capabilities of dental plaque. Caries Res 1:247-260. been studied, it is clear from the present work that differences can be detected in the cariogenic potential of plaque fluid from white- Higham SM, Edgar WM (1989). Human dental plaque pH, and the organic acid and free amino acid profiles in plaque fluid, after sucrose rinsing. spots and sound surfaces in CP subjects. In fact, the differences in Arch Oral Biol 34:329-334. starved-plaque-fluid pH, calcium content of plaque residues, acid production, and DS(En) (before and following sucrose exposure) Lee CY, Margolis HC, Moreno EC (1990). Factors influencing the rate of enamel demineralization in vitro (abstract). JDentRes 69 (Spec Iss):302. between white-spot and sound surfaces pooled from CP individuals are similar to those between whole-mouth plaque fluids from CF Luoma H (1964). Lability ofinorganic phosphate in dental plaque and saliva. Its appearance in association with some other factors related to dental and CP groups. These differences may reflect different bacterial caries. Acta Odontol Scand 22 (Suppl 41):1-127. populations in the two sites considered. Thus, in similar samples, the in vitro pH-lowering potential and proportions of mutans strep- Margolis HC, Duckworth JH, Moreno EC (1988a). Composition of pooled resting plaque fluid from caries-free and caries-susceptible individuals. tococci were found to be greater in plaque samples from white spots JDent Res 67:1468-1475. than in samples from sound surfaces of CP individuals (Van Houte Margolis HC, Duckworth JH, Moreno EC (1988b). Composition and buffer etal., 1991). capacity of pooled starved plaque fluid from caries-free and cariesDespite the fact that the pooling of plaque samples may obscure some local differences, this investigation has shown that measursusceptible individuals. J Dent Res 67:1476-1482. able and significant differences exist between the plaque and plaque Margolis HC, Moreno EC (1992). Kinetics of hydroxyapatite dissolution in acetic, lactic, and phosphoric acid solutions. Calcif Tissue Int 50:137fluid compositions in samples from caries-free and caries-positive 143. human subjects. The identification and the measurement of such differences provide a unique insight into the etiology of caries Margolis HC, Murphy BJ, Moreno EC (1985). Development of carious-like lesions in partially saturated lactate buffers. Caries Res 19:36-45. formation.
Acknowledgment. WethankDr. P.M. Soparkar, Department ofClinical Trials, Forsyth Dental Center, for conducting the dental examinations. REFERENCES Abelson DC and Mandel ID (1981). The effect ofsaliva on plaque pH in vivo. JDent Res 60:1634-1638. Ashley FP (1975). Calcium and phosphorus concentrations ofdental plaque related to dental caries in 11- to 14-year-old male subjects. Caries Res 9:351-362. Birkeland JM (1970). Direct potentiometric determination offluoride in soft tooth deposits. Caries Res 4:243-255.
Moreno EC, Margolis HC (1988). Composition ofhuman plaque fluid. JDent Res 67:1181-1189. Moreno EC, ZahradnikRT (1974). Chemistryofenamel subsurface demineralization. J Dent Res 53:226-235. Muntz JA (1943). Production of acids from glucose by dental plaque material. JBiol Chem 148:225-236. Oliveby A, Weetman DA, Geddes DAM, Lagerlof F (1990). The effect of salivary clearance of sucrose and fluoride on human dental plaque acidogenicity. Arch Oral Biol 35:907-911. Pearce EIF (1982). Effect of plaque mineralization on experimental dental caries. Caries Res 16:460-471. Rankine CAN, Moreno EC, Vogel GL, Margolis HC (1985). Micro- analytical determination of pH, calcium, and phosphate in plaque fluid. JDentRes 64:1275-1280. Shaw L, Murray JJ, Burchell CK, Best JS (1983). Calcium and phosphorus content of plaque and saliva in relation to dental caries. Carries Res 17:543-548.
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Stephan RM (1944). Intra-oral hydrogen-ion concentration associated with dental caries activity. JDent Res 23:257-266. Tatevossian A (1987). Calcium and phosphate in human dental plaque and their concentrations after overnight fasting and after ingestion of a boiled sweet. Arch Oral Biol 32:201-205. Tatevossian A, Gould CT (1976). Methods for sampling and analysis of the aqueous phase of human dental plaque. Arch Oral Biol 21:313-317.
J Dent Res November 1992 Theuns HM, Van Dijk JWE, Driessens FCM, Groeneveld A (1983). Effect of time and degree of saturation of buffer solutions on artificial carious lesion formation in human tooth enamel. Caries Res 17:503-512. Van Houte J, Sansone C, Joshipura K, Kent R (1991). In vitro acidogenic potential and mutans streptococci of human smooth surface plaque associated with initial caries lesions and sound enamel. J Dent Res 70:1497-1502.
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