Analysis of the Buffering Systems in Dental Plaque

A semi-micro method was used for investigation of the buffering properties of whole plaque, plaque fluid, and washed plaque bacteria. Artifacts associated with titration of samples containing live bacteria were noted and their effects estimated. All three sample types showed minimal buffering in the region of neutrality, with much stronger buffering in the regions pH 4-5.5 and pH 8-9. For the range pH 4-7, almost 90% of the total buffer capacity of plaque appeared to be accounted for by macromolecules of bacterial cell walls and plaque matrix. Extracellular buffers in plaque fluid removable by centrifugation contributed up to 11%. These buffers (probably soluble proteins, peptides, organic acids, and phosphate) are, potentially at least, capable of exchange with saliva. In vitro, bicarbonate (dissolved in the extracellular fluid) contributed only 2-5% of total buffering; there was no evidence of formation of carbamino compounds. However, in vivo, salivary bicarbonate may be important as a continually replenished source of additional buffering.</jats:p

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 μmol/g/sec (wet weight); apparent Km (acid production) = 100 μmol/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 μmol of baselg (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 cm2lsec, 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. </jats:p

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 CO 2-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. </jats:p

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. </jats:p

A Quantitative Study of Calcium Binding by Isolated Streptococcal Cell Walls and Lipoteichoic Acid: Comparison with Whole Cells

Calcium-binding by surface components of oral bacteria may have important effects on remineralization/demineralization phenomena and plaque cohesion. Additionally, some species export large quantities of lipoteichoic acid, possibly as a protective measure. Measurement of calcium-binding can facilitate prediction of how this will effectively buffer plaque fluid calcium concentration and affect these processes. Using equilibrium dialysis, we measured calcium-binding capacities and affinities at pH 7.0 in isolated cell walls of Streptococcus downei, S. sanguis, and purified lipoteichoic acid (LTA) of S. sanguis. Mean binding capacities were: 56.5 μmol Ca/g wet weight for S. downei cell walls and 47.2 μmol Ca/g wet weight for S. sanguis cell walls, and 1.11 mol Ca/mol LTA phosphate were found. Mean dissociation constants (mmol/L) for cell wall calcium binding were 2.16 mmol/L ( S. downei) and 2.69 mmol/L ( S. sanguis). These constants were not significantly different from those for whole cells of the same species (Rose et al., 1993), but the dissociation constant for LTA (7.82 mmol/L) was significantly higher and suggested a different mode of binding. At neutral pH, at the known calcium concentration of plaque fluid, whole cells and cell walls are likely to be completely saturated with calcium, whereas free LTA is only 30% saturated. The large amounts of LTA exported by some sucrose-grown streptococci may therefore act as a calcium buffer and so protect the organisms against high local concentrations of calcium produced during demineralization. </jats:p