Composition of pooled resting plaque fluid from caries-free and caries-susceptible individuals.

The composition of pooled resting plaque fluid from 10 population samples, grouped according to age (8-11, 14-17, 18-25 years) and caries status (caries-free, CF, DMFS = 0; caries-susceptible, CS, DMFS greater than 10), was determined by means of ion chromatography. Subjects received a dental prophylaxis one week prior to plaque collection, abstained from oral hygiene for 48 hours, and did not eat or drink for at least one hour prior to plaque collection. Plaque samples from each group were pooled under mineral oil and centrifuged (15,000 g) for 45 minutes at ambient temperature. Supernatants were analyzed for organic acids, inorganic anions, mono- and divalent cations, and pH value. The ions Na+, NH4+, K+, Ca2+, Mg2+, Cl-, and phosphate were present in all samples. Acetic and propionic acids were predominant, comprising over 75% of the acid (anions) present under resting conditions. Succinic, lactic, formic, and butyric acids were present in lower concentrations. Within each age group, the mean values for pH and NH4+ concentration were higher in the caries-free group; the differences between mean values (CF: pH, 6.35; NH4+, 52 mmol/L; CS: pH, 5.85; NH4+, 37 mmol/L) were found to be statistically significant (p less than 0.05) when all data for the CF and CS groups were combined. Significantly higher concentrations of Mg2+ and butyric acid were also found for the combined CF data. Concentrations of all other constituents were similar. Calculations indicate that plaque fluid is supersaturated with respect to enamel mineral and dicalcium phosphate dihydrate, with a significantly higher degree of saturation with respect to enamel mineral in the CF group.(ABSTRACT TRUNCATED AT 250 WORDS)

Composition and buffer capacity of pooled starved plaque fluid from caries-free and caries-susceptible individuals.

The composition of pooled starved plaque fluid from six population samples, grouped according to age (8-11, 12-16, 8-17, 18-22 years) and caries status (caries-free, CF, DMFS = 0; caries-susceptible, CS, DMFS greater than 10), was determined by means of ion chromatography. Prior to plaque collection, all subjects abstained from oral hygiene for 48 hours and fasted overnight. In addition, selected subjects also received a dental prophylaxis one week prior to plaque collection. Plaque samples from each group were pooled under mineral oil and centrifuged at 15,000 g (ambient temperature) for 45 minutes. Supernatants were then analyzed for organic acids, inorganic anions, and mono- and divalent cations. Plaque fluid samples from CF and from the CS subgroups were also titrated with standard HCl for exploration of the inherent buffer capacity. The ions Na+ NH4+, K+, Ca2+, Mg2+, Cl-, phosphate, and carbonate were present in all samples analyzed. Formic, acetic, propionic, butyric, and trace amounts of isobutyric and isovaleric acids were detected, with acetic and propionic acids comprising between 85 and 94% of the total acids. Little (less than 1 mmol/L) to no succinic or lactic acid was detected in starved plaque fluid. No apparent difference was detected in the composition and saturation status of pooled starved plaque fluid from CF and CS individuals. These results suggest that caries susceptibility is not necessarily correlated with the plaque fluid composition of pooled starved plaque. In addition, no apparent difference in the inherent buffer capacities of these samples could be detected, consistent with the noted similarities in composition.(ABSTRACT TRUNCATED AT 250 WORDS)

The interpretation of CO2 equilibration data to obtain plaque fluid buffer capacities, and comparison with results obtained by titration.

Titration measurements of pooled plaque fluid buffering capacity (Shellis and Dibdin, 1988), which showed a broadly defined minimum at pH 7, were compared with recent curves published by Carey et al. (1988a), which they obtained by an ultra-micro CO2-equilibration technique and which suggested a quite different profile, peaking sharply at pH 7.1. When analyzed in a different, more conventional way, the raw measurements in the latter study become more consistent with our own results and with earlier findings of Tatevossian (1977). In particular, we conclude that the peak at pH 7.1 is an artifact, and that Carey et al. underestimated buffer capacities below pH 6.4 and above pH 7.4. Rationales for the two modes of analysis are compared, and possible reasons for the remaining differences between the re-analyzed CO2-equilibration results and the titration results are discussed. Suggestions for the improvement of the accuracy of the CO2-equilibration technique are put forward.

Composition of human plaque fluid.

The composition of pooled resting plaque fluid was determined in four groups of college-age students (18-22 years), each composed of 50 individuals, who abstained from oral hygiene for 36 hours and did not eat or drink for at least one hour prior to plaque collection. Plaque samples from each group were pooled under mineral oil in small centrifuge tubes and centrifuged at 37,000 g for one hour at 4 degrees C. Supernatants were then combined under mineral oil and centrifuged at 5000 g (4 degrees C) for 15 minutes. In general, the inorganic composition of plaque fluid in the four groups was quite similar and in agreement with values reported by other investigators, but quite different from those of saliva or serum. The mean composition was: Ca, 7.07 +/- 0.51 mmol/L; P, 23.2 +/- 5.3 mmol/L; Na, 18.6 +/- 2 mmol/L; K, 85.1 +/- 5.3 mmol/L; Mg, 3.9 mmol/L; Cl, 42.8 +/- 9 mmol/L; F, approximately 0.004 mmol/L; pH, 5.69 (5.63-6.01). Acetate, propionate, succinate, butyrate, lactate, and formate were determined in two samples analyzed, with acetate and propionate being the predominant acids found. It was also demonstrated, through the titration of one of the plaque fluid samples, that the observed buffer capacity in plaque fluid was mostly related to its organic acid composition. It was noted, however, that when the initial pH in plaque fluid exceeded 6.5, phosphate contributed significantly to the buffer capacity. The contribution of other soluble species (proteins, peptides, amino acids) to the observed buffering in plaque fluid appeared to be small.(ABSTRACT TRUNCATED AT 250 WORDS)

Plaque fluid as a bacterial milieu.

Studies of the extracellular, free concentrations of substrates, growth factors, inhibitors, and end-products of metabolism to which the intact plaque microflora is exposed in situ can assist in the understanding of factors controlling plaque pathogenicity. Information is becoming increasingly available from analysis of fluid separated by centrifugation of plaques collected at various intervals after an intra-oral pulse of dietary or experimental substrate, or different procedures or treatments having cariostatic potential. Such analytical results give more information than those obtained by analysis of aqueous or other extracts, because they yield values of substrate concentration representing those occurring at the bacterial cell surface. The largest body of information concerns extracellular levels of acid end-products of sugar catabolism in relation to food quality or sequence, and of amino acids and other products of nitrogen metabolism, in relation to studies of the detailed metabolic events of the Stephan curve, and of the demineralizing effect of the plaque environment. Areas where little information is available and which merit further study include plaque clearance of salivary and other components with anti-caries activity (e.g., antibodies, enzymes, fluorides, cations, other antimicrobials, etc.), and substrate concentrations to determine gradients for diffusion into and out of plaque.

Fluoride analysis in nanoliter- and microliter-size fluid samples.

A variety of techniques is described for measuring fluoride in volumes of from 0.005 to 5 microL, including: (1) micropipette procedures for transference and dilution of samples, (2) construction of miniature and micro fluoride-selective electrodes, and (3) methods for adapting standard electrodes for micro- and semi-micro volumes. These described techniques have a number of advantages, including speed of analysis, high accuracy, and adaptability to many types of fluid samples. Recent studies involving use of these procedures include the analysis of fluoride in: (1) plaque fluid samples from single sites before and after topical fluoride administration, (2) tooth mineral samples recovered by acid-etch or microdrill biopsy of enamel, and (3) fluid recovered from the interior of the tooth during simulation of the caries process.

The effects of experimental variations in dietary sugar intake and oral hygiene on the biochemical composition and pH of free smooth-surface and approximal plaque.

The biochemical compositions of free smooth-surface and approximal plaque were compared after 48 hours in the presence and absence of twice-daily toothbrushing and during ingestion of either high- or low-sugar diets in ten young adults. In addition, the effect of a single sugar intake on the pH of both types of plaque was investigated. The results confirmed previously reported differences in biochemical composition between free smooth-surface and approximal plaque, the concentrations of inorganic ions and carbohydrate fractions being generally lower in the approximal sample after all experimental regimens. The concentrations of calcium and phosphorus were doubled on the low-sugar diet compared with the high-sugar diet in both types of plaque. A significant decrease in carbohydrate concentrations was observed in free smooth-surface plaque in subjects on the low-sugar diet, but not in approximal plaque. Little variation was observed in the biochemical composition which could be attributed to the presence or absence of toothbrushing. The pH in free smooth-surface plaque was lower than that in approximal plaque five minutes after a sugar intake, but the situation was reversed two hours later. The results indicate that differences in composition and pH between free smooth-surface and approximal plaque are associated with reduced access to salivary and dietary influences due to diffusion limitation in the deeper layers of plaque.

The determination of extracellular fluid volume in whole dental plaque using potassium- or chloride-selective micro-electrodes.

This paper describes a procedure for the determination of the volume of water available for diffusion in whole plaque samples collected from single sites. In this procedure, known micro volumes of H2O were added to single-site plaque samples with calibrated nanoliter pipettes and the potassium or chloride concentration at each dilution measured with ion-selective micro-electrodes. By plotting the inverse of the concentration versus the volume of H2O added, one can determine the original volume of plaque fluid available for diffusion. Volumes determined in simultaneous measurements performed with chloride and potassium micro-electrodes were statistically indistinguishable. From the observed constancy in the initial potassium concentration in rat and human plaque fluid, a simplified macro method is suggested which allows for estimation of the extracellular fluid volume within 20% error in plaque samples.

Xylitol binding in human dental plaque.

Human dental plaque and whole saliva sediment were tested for their ability to bind 14C-labeled sucrose, fructose, glucose, sorbitol, and xylitol. Sucrose, glucose, sorbitol, and fructose were bound to all materials tested, in this decreasing order. The binding was strongest with plaque given a five-second ultrasonic shock, and lowest with salivary sediment. Xylitol was only insignificantly bound, indicating that plaque microorganisms possess specific recognition sites for xylitol to a very restricted extent.

Effects of single exposure to sugar on calcium and phosphorus concentrations of dental plaque.

The results from three studies on the plaque of children and adults indicated that the response of the plaque to a single five-minute exposure to sugar cubes or boiled sweets (candies) depended on the initial condition of the plaque. Thus, significant decreases in the levels of calcium and phosphorus in plaque were observed in previously starved plaques but not in plaque from participants without previous dietary sugar restriction.

Polyamines of dental plaque in caries-resistant vs. caries-susceptible adults.

Utilizing a sensitive liquid chromatographic system, polyamines were quantitated in three-day plaque from 13 caries-resistant (CR) and 35 caries-susceptible (CS) adults after they had fasted for 12 hours. The values for putrescine and cadaverine were significantly higher in the CR group. This may be attributed to a greater availability of salivary substrate precursors of polyamines and/or higher levels of biosynthetic decarboxylase activities in the CR subjects, or both.

Micro-analytical determination of pH, calcium, and phosphate in plaque fluid.

Micro-analytical techniques for the determination of calcium, phosphate, and pH in a small volume (less than 0.25 microliter) of plaque fluid are described and evaluated. The accuracy and the precision of the techniques were compared with those for standard macrotechniques applied to a large pooled plaque fluid sample. The results obtained for the micro-analysis of pooled plaque fluid were in excellent agreement with those obtained by macromethods. The described techniques were also used to analyze plaque fluid obtained from single quadrants of the oral cavities of five individuals. In this fashion, it was determined that, although a significant variation in plaque fluid composition exists between the quadrants, a greater variation exists between subjects. Analyses of plaque fluid obtained from six individuals, following sucrose exposure, were also conducted. The pH value of the fluid changed with time, following a typical Stephan curve, with a minimum value occurring between 15 and 30 minutes; following this, the pH increased to a value near that for resting plaque. An inverse relationship between pH and calcium and phosphorus concentrations was observed. It is noted that the described techniques are sensitive enough to carry out the above analyses both accurately and precisely using plaque obtained from a single quadrant.

A comparison of the potassium content and osmolality of plaque fluid and saliva, and the effects of plaque storage.

Previous determinations of osmolality and potassium concentrations in plaque fluid, much higher than those in saliva, suggest a restricted exchange between the two, which must be reconciled with recent findings of quite rapid diffusion in plaque. Possible reasons for the high values were considered, and of these the effect of solute leakage from bacteria to the plaque fluid during typical periods of storage was investigated. It was also shown that the osmotic pressure of plaque fluid could be measured quite accurately by vapor pressure osmometry on whole plaque samples without the need for centrifugation. Samples of plaque, or plaque fluid prepared by centrifugation at 12,000 g, were compared for osmolality or potassium content with matched samples prepared from plaque stored chilled or in liquid nitrogen. Saliva samples obtained just prior to plaque collection were also analyzed. Freshly collected plaque from overnight-fasted subjects had a plaque fluid osmolality of 156 +/- 35 as compared with 98 +/- 23 mOs/kg for saliva. Potassium in plaque fluid from freshly collected "mature" plaque was 40.6 +/- 5.1 as compared with 20.3 +/- 5.3 mmol/L for saliva, but for 1-2-day-old plaque from fasted subjects it was significantly lower (30.4 +/- 5.6 mmol/L). These values for plaque fluid are all much less than those previously found, and storage was found to cause a marked increase (range, 35-100%). Centrifugation at 12,000 g caused little change in plaque fluid osmolality but seemed to accelerate the rate of increase during subsequent storage.

Facts and artefacts in research on human dental plaque fluid.

In 1966, Jenkins suggested that the plaque fluid environment was likely to have higher concentrations of extracellular solutes than was apparent from analyses of total plaque concentrations. Early work on plaque fluid confirmed this contention, but some artefact was also generated by the prolonged centrifugation used for separation. The solute concentrations in plaque fluid mostly exceed those in saliva or crevicular fluid. Thus, the environmental conditions are distinctly different from those based on the assumption that saliva readily permeates films of dental plaque. In contrast, the presence of serum proteins suggests a crevicular input to plaque fluid. These data suggest that exchange between dental plaque and its environment is apparently restricted. Diffusion rates measured in dental plaque by different methods do not agree on how restricted it is. However, measuring diffusion in plaque introduces artefacts in packing density, a major determinant of the diffusion rate. The conditions used for collection and analysis have been reported to produce artefactual changes in plaque fluid potassium, a predominantly intracellular ion. Measurements of predominantly extracellular ions, such as calcium, are no less prone to artefact, whether based on ion-selective electrodes or on total calcium. We have much to learn about the fluid environment of the teeth and about dynamic changes in plaque fluid composition and properties during perturbations. Such information can give insights into pathological processes such as tooth demineralization and dental caries, calculus formation, and gingival inflammation.

An assessment of recent advances in the study of the chemistry and biochemistry of dental plaque fluid.

This paper discusses key points made during the symposium in the light of work carried out in other laboratories. It is emphasized that the unique importance of plaque fluid is that the net result of chemical changes induced by microbial activity is reflected in this medium, which is in intimate contact with the enamel surface, and that this medium is accessible to chemical and biochemical analyses. However, in order to assess the cariogenic potential of plaque, we must consider the properties of both whole plaque and plaque fluid together. Although it is apparent that results of plaque fluid composition are sensitive to both isolation and the storage procedures utilized, plaque fluid appears to be a distinct entity within the oral cavity. Technical advances have been made which allow for the determination of the activity of selected ions (hydrogen, calcium, phosphate, potassium, fluoride) in plaque fluid obtained from a single site within the mouth. It appears, however, that such data alone may be insufficient to define the cariogenic potential of plaque appropriately. Evidence is presented from which it can be concluded that, with use of pooled samples of plaque obtained from individuals with clear differences in caries experience, results on plaque and plaque fluid composition can be obtained which are consistent with noted differences in caries susceptibility. The importance of base production is also discussed, and it is noted that few studies have been carried out to elucidate the role of proteins found in plaque fluid. In conclusion, recent advances in the study of plaque fluid have provided new insights into the mechanism of caries formation which are also germane to the formation of dental calculi.

Fluoride in dental plaque and its effects.

Total plaque fluoride is in the range 5-10 mg/kg (ppm) on a wet-weight basis. The variability of literature data on plaque fluoride is partly ascribed to analytical problems, many assays being close to or below the concentration detection limit of the fluoride electrode. A change in classification of plaque fluoride compartments is necessary, since recent work indicates that there are two pools of plaque F: less than 5% of the total F is in plaque fluid as the free ion, and the large remaining portion of total plaque F is designated as bound F, with the total F being greater than 95% extractable by cold 0.5 mol/L perchloric acid. Sources of plaque fluoride include the diet, saliva, and crevicular fluid; enamel is unlikely to be a regular source for plaque F unless it is either coated daily with labile fluoride compounds, such as calcium fluoride, or released by demineralization. The location and nature of plaque bound F are not established, but the present evidence is consistent with an intracellular location. Bound F may be released by acids produced in plaque during sugar fermentation, but it is unlikely to reach ion concentrations high enough for sufficient time periods to exert significant inhibition of plaque acidogenesis. Epidemiological evidence showing correlations between pooled plaque F concentrations and caries prevalence in the plaque donors does not exclude the possibility of coincidental effects of water F on both caries and plaque F concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluoride in saliva and plaque following use of fluoride-containing mouthwashes.

The sensitivity of methodology for measuring the concentration of fluorine species in saliva and in plaque has been tested. Human subjects mouth-rinsed daily with aqueous solutions of NaF and Na2FPO3. Samples of unstimulated whole saliva and of plaque were collected twice weekly at least 18 hr after treatment application. Oral fluoride concentrations rose from placebo values for approximately two weeks before attaining equilibrium and returned to baseline when daily mouthrinsing was stopped. Mean elevated oral fluoride concentrations increased significantly with increasing applied NaF concentration in the range 0-1000 ppm F (0-0.053 mol/L). There appeared to be a linear relationship between saliva and plaque fluoride. The ability of fluoride treatments to sustain elevated oral fluoride levels between daily applications may be of major importance in caries control.

Physical and biochemical studies of Streptococcus mutans sediments suggest new factors linking the cariogenicity of plaque with its extracellular polysaccharide content.

Cultures of Streptococcus mutans MFe28 (serotype h) were grown with differing extracellular polysaccharide (EPS) content. Biochemical and physicochemical characteristics considered relevant to caries were measured. Acid production parameters measured in a pH-stat were: Vm = 0.76 +/- 0.14 mumol/g/sec (wet weight); apparent Km (acid production) = 100 mumol/L; molar yield = 1.97 +/- 0.25 mol acid/mol glucose. Acid anion inhibition of acid production was also noted. Buffering by the pure washed bacterial residue required approx. 112 mumol of base/g (wet weight) of residue to change the pH from 4 to 6.5, and this dropped almost to zero as the EPS content increased to 100%. Diffusion coefficients (D) in the residues were independent of EPS content over a wide range. When the effusion method was used, De (glucose) and De (acetate) were (3.26 +/- 0.6) and (5.05 +/- 0.8) x 10(-6) cm2/sec, respectively. The extracellular fluid fraction, measured by inulin exclusion, increased from 0.33 for the pure bacteria to 0.78 for the pure EPS. It is shown how, by these factors alone, and without any need for diffusion restriction, plaque EPS may lead to a lower pH at the tooth surface, thus increasing the cariogenic challenge.

Formation of calcium phosphates in saliva and dental plaque.

This is an X-ray diffraction study of the mineral phases in saliva and early dental plaque. The salivas studied came from patients with cystic fibrosis (CF), those with asthma, and heavy and light calculus formers. One-week old plaque was studied from individuals who are heavy, moderate, and light calculus formers.

Calcium, phosphorus, fluoride, and pH levels of human dental plaque from areas of varying fluoride levels.

Plaque calcium, phosphorus, fluoride, and pH in samples obtained from 149 life-long resident children, aged from six to seven and 12 to 13 yr, from one of three naturally fluoridated communities were determined. In general, smaller amounts of phosphorus and fluoride, and larger amounts of calcium and hydrogen ions (lower pH values) are associated with smaller amounts of fluoride in the drinking water supply. Significant differences among the mean pH, fluoride, and Ca/P concentrations both in the six-to-seven- and 12- to 13-year age group were found when comparing the results of the three areas with different levels of fluoride in their drinking water supply.