G.L. Vogel

A Microanalytical Procedure for the Determination of Calcium, Phosphate and Fluoride in Enamel Biopsy Samples

This paper describes analytical methods for the determination of calcium, phosphate and fluoride in successive layers of enamel removed from rat and human teeth by acid etching. These methods include: (i) simple, sensitive spectrophotometric methods for the determination of calcium and phosphate; (ii) an easily constructed miniature fluoride electrode, and (iii) an improved electrode adapter that permits one fluoride electrode to measure numerous microsamples very rapidly. The high sensitivity and simplicity of the analytical techniques described allows many types of microsamples of interest in dental research to be analyzed with an accuracy and speed heretofore attainable only with much larger samples.

Pharmacokinetics of Fluoride in Man and its Clinical Relevance

A large number of parameters will influence and mediate the activity and the pharmacological response of dental fluoride products after systemic or topical treatment. This report reviews some aspects of the pharmacokinetics of fluoride in man, fluoride bio-availability, plasma kinetics, and kinetics of fluoride in saliva and plaque fluid. Pharmacokinetic studies in growing dogs show that 90% of a single injected fluoride dose is retained shortly after birth, but at maturity it decreases to about 50%. The degree of fluoride accumulating in cal-cifying tissues seems to be strongly related to age. The bio-availability of fluoride from swallowed fluoride toothpaste is shown to be decreased if the toothpaste is ingested close to a meal. Several studies show that fluoride exerts its cariostatic effects through the liquid phase surrounding the enamel. The importance of fluoride in the fluid environment of the teeth and the kinetics of fluoride in saliva are discussed. Clinical studies using different slow-release fluoride systems indicate that they are promising cariostatic agents—in particular in-tra-dural slow-release devices and lozenges. A new micro-analytical method to study the kinetics of fluoride in plaque fluid collected from single tooth sites has been developed. Preliminary studies show that the clearance of fluoride from plaque fluid is slowest in the upper incisor region, followed by the molar region, and faster in the lower incisor region. A site-by-site study of the concentration of fluoride in plaque fluid after topical fluoride administration could be extremely beneficial in optimization of the methods and recommended safety regimens for fluoride therapy.

Ca, Pi, and F in the Fluid of Biofilm Formed under Sucrose

Calcium (Ca), inorganic phosphorus (Pi), and fluoride (F) concentrations are low in the whole plaque biofilm formed under exposure to sucrose. It was hypothesized that this would be reflected in the biofilm fluid, where these low values should greatly influence the de/remineralization process. Dental biofilms were formed in situ over enamel blocks mounted in palatal appliances and exposed 8 times/day to distilled water, glucose+fructose, or sucrose solutions for 14 days. While Ca, Pi, and F concentrations in the whole biofilms were significantly lower in the glucose+fructose and sucrose groups, no effect on biofilm fluid was observed, even after a cariogenic challenge. An increase in whole biofilm mineral ions was observed 24 hrs after the carbohydrate treatments were suspended, but this effect was also not observed in the fluid. These results suggest that there is a homeostatic mechanism that maintains biofilm fluid mineral ion concentration, regardless of its total concentration in the whole biofilm.

Fluoride in plaque fluid, plaque, and saliva measured for 2 hours after a sodium fluoride monofluorophosphate rinse.

Sodium monofluorophosphate (NaMFP) and sodium fluoride (NaF) are the two most common sources of fluoride used in currently marketed fluoride dentifrices. The purpose of this study was to investigate the effect of mouth rinses containing NaF or NaMFP on the concentrations of fluoride, or the MFP ion, in saliva, whole plaque, and plaque fluid. Twelve subjects abstained from tooth brushing for 48 h, fasted overnight, and then rinsed 1 min with 12 mmol/l (228 ppm [μg/g] F) NaF or NaMFP in the morning. Before the rinse and at 30, 60 and 120 min afterwards, upper and lower molar and premolar plaque samples and whole saliva samples were collected. Aliquots of plaque fluid and centrifuged saliva were obtained from these samples, and the whole plaque residue acid extracted. The F and MFP concentrations were then measured in these samples using ultramicro methods. For both rinses, a higher concentration of plaque fluid fluoride was found at lower molar sites while the reverse was true for the whole plaque fluoride. Furthermore, for both rinses, plaque fluid, whole plaque, but not salivary, fluoride concentrations were above baseline at 120 min. Following the NaMFP rinse, a substantial amount of unhydrolyzed MFP was found in plaque fluid and saliva. Although there was a very large range in these measurements, fluoride in plaque fluid (excluding fluoride in unhydrolyzed MFP) and whole plaque were significantly (p<0.05) greater after the NaF rinse at all time periods. In saliva, the NaF rinse produced a statistically significant greater salivary fluoride (excluding fluoride in unhydrolyzed MFP) only at 60 min. The lack of a clear correlation between these measurements and clinical studies suggest a novel mechanism may enhance the effectiveness of NaMFP dentifrices.

In vivo Fluoride Concentrations Measured for Two Hours After a NaF or a Novel Two-solution Rinse

The concentrations of fluoride in various samples from the oral environment were measured at timed intervals after a novel rinse or a NaF rinse, both containing a total of 12 mmol/L (228 ppm) fluoride. The novel rinse consisted of two solutions mixed just before application: Part A contained calcium chloride and sodium acetate; part B contained a hydrolyzable source of fluoride (sodium hexafluorosilicate) and sodium phosphate. Samples were obtained as follows: Single-site plaque-fluid samples were obtained by centrifugation of first-molar plaque; pooled whole-plaque samples were collected from second molars; centrifuged, pooled whole-saliva was collected by vacuum. All samples were analyzed by micro-analytical methods. Results showed that, compared with NaF, the two-solution rinse produced significantly higher salivary fluoride concentrations, plaque-fluid fluoride concentrations, and acid-extractable fluoride in the whole plaque by factors of about 4, 2, and 6, respectively, at 120 min. The results of this study suggest that the new rinse may provide a greater cariostatic effect at the same fluoride dosage than does a NaF rinse.

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 (<0.25 μl) 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.

Ca Pre-rinse Greatly Increases Plaque and Plaque Fluid F

Previous studies demonstrated that a Ca pre-treatment greatly increases salivary F from a subsequent NaF rinse. This study examines if these increases are found in plaque and plaque fluid F. Thirteen individuals accumulated plaque before rinsing with: (1) 12 mmol/L NaF (228 μg/g F), (2) 150 mmol/L Ca rinse, or (3) the Ca rinse followed by the F rinse. One hr later, plaque samples were collected, the plaque fluid was recovered, and the plaque residues were extracted 5 times with pH 6.8 or pH 4.8 buffers, and then by acid. The F in each extract after the Ca rinse/F rinse greatly exceeded the corresponding F from the NaF rinse. Consequently, the Ca rinse/F rinse increased the total plaque F and the plaque fluid F by 12x and 5x, compared with the NaF rinse alone. These and the previous salivary results suggest that a Ca pre-treatment may increase the cariostatic effects of topical F agents.

Increased Overnight Fluoride Concentrations in Saliva, Plaque, and Plaque Fluid after a Novel Two-solution Rinse

Recent studies showed that salivary, plaque-fluid, and whole-plaque fluoride were significantly higher 120 min after subjects rinsed with a novel two-solution rinse than after they rinsed with a NaF rinse of the same fluoride concentration. In this study, the persistence of these increases was investigated overnight, a period of time that is more clinically relevant. Improved analytical techniques for the ultramicro determination of whole-plaque and plaque-fluid fluoride from the same sample are also described. Thirteen subjects abstained from toothbrushing for 48 hrs and rinsed for 1 min with a 12 mmol/L (228 ppm) NaF or the two-solution rinse before bedtime. Samples were then collected the following morning before breakfast: (1) Saliva samples were either clarified by centrifugation or acid-extracted with 1 mol/L HClO4; and (2) single-site molar plaque samples were centrifuged to obtain plaque fluid and/or extracted with 1 mol/L HC1O4. Results showed that, compared with NaF, the two-solution rinse produced significantly higher fluoride concentrations in all samples: The concentration of fluoride in whole plaque and whole saliva following the new rinse exceeded concentrations found after the NaF rinse by factors of three and four, respectively, while in plaque fluid, the two-solution rinse produced about a two-fold increase over NaF values, which were near baseline levels. This increase, however, was only about 20% in centrifuged saliva. The increases in saliva and especially in plaque-fluid fluoride after the two-solution rinse indicate a greater remineralization potential, while the enhanced fluoride reservoirs found in plaque overnight after this rinse constitute a reserve that may release fluoride into the plaque fluid over an extended period of time.

Distribution of Fluoride in Saliva and Plaque Fluid after a 0.048 mol/L NaF Rinse

An ultramicro method has recently been described for measurement of plaque-fluid fluoride concentration (Vogel et al., 1990a). This method was used: (1) for exploration of the variation in fluoride concentration of plaque fluid collected from the same buccal tooth sites following a 0.048 mol/L NaF (0.2%) rinse, and (2) for examination of the distribution of fluoride in plaque fluid and saliva within one hour after this rinse. Results indicated an average coefficient of variation (CV) of 31% for plaque-fluid fluoride in triplicate samples recovered simultaneously from the buccal-proximal region of two teeth after the rinse. This was similar to the CV found for plaque-fluid fluoride from the same sites after separate administrations of the rinse. A strong linear correlation was found between salivary and plaque-fluid fluoride at 30 and 60 min after rinse administration, showing that plaque-fluid fluoride is influenced by the concentration of salivary fluoride after administration of this rinse. Plaque-fluid fluoride concentrations were higher than that in saliva at baseline, 30, and 60 min. Very large inter-site and inter-subject variations in plaque-fluid distribution were observed, with the central incisors showingthe slowest clearance. These variations suggest that an examination of plaque-fluid fluoride from specific tooth regions may be essential for understanding the effects of fluoride on the site-specificity of caries.

Composition of Plaque and Saliva Following a Sucrose Challenge and Use of an a-tricalcium-phosphate-containing Chewing Gum

Calcium phosphate concentrations in plaque, plaque fluid, and saliva play an important role in caries prevention. In this study, we used a microanalytical technique to examine the anticaries potential of a 2.5% (mass fraction) a-tricalcium-phosphate-fortified experimental gum by measuring the pH, free and total calcium, and total phosphate in plaque fluid, whole plaque, and saliva, and centrifuged saliva from 14 subjects who (1) accumulated plaque for 48 hours, (2) fasted overnight, (3) rinsed for 1 min with sucrose, and (4) chewed a control or experimental gum for 15 min. From these data, the hydroxyapatite (HAp) ion activity products (IAPHAp) of saliva and plaque fluid were calculated as a measure of tooth mineral saturation. Results, compared with those of the control gum, show significant increases in pH and in free calcium and phosphate concentrations in plaque fluid and saliva when the experimental gum was chewed following sucrose ingestion. These increases result in a rise in fluid saturation with respect to tooth mineral that, for plaque fluid, nearly cancels the decrease seen with the control gum after the sucrose rinse. This suggests that the experimental gum may be more effective than a conventional gum in ameliorating the cariogenic effects of sucrose. Similar statistically significant increases were also seen in the total calcium content of the plaque fluid, centrifuged saliva, whole saliva, and whole plaque, and in the total phosphate of whole plaque and whole saliva. These results suggest that the deposition of a mineral reservoir in plaque and saliva by the experimental gum may help resist future cariogenic challenges.