**2.1.2 Materials and methods**

52 Macro to Nano Spectroscopy

Saliva secreted into the mouth flows slowly as a thin film, over the tooth surfaces and mucosa and is cleared from the mouth by swallowing (Fig. 2). However, saliva does not flow equally throughout the mouth, and there are differences in the different areas. Measurement of the volume of saliva and velocity of the salivary film at different locations in the mouth are important for understanding the site-specificity of dental caries and

Using agar as an artificial-plaque, we have conducted studies on the five following items by measuring the clearance of potassium chloride from the agar using an atomic absorption

1) Salivary clearance from different regions of the mouth. 2) Salivary clearance in children with complete primary dentitions. 3) Influence of the location of the parotid duct orifice on oral clearance. 4) Effect of salivary flow rate on fluoride retention in the mouth. 5)

Very little research has been carried out on the rates of diffusion of substances into or from dental plaque in vivo. Primosch et al. (1986) studied topical fluoride distribution in the oral cavity and rates of clearance following different methods of dissolution of fluoride tablets. They found that after the chewing, sucking, or passive dissolution of the tablets, fluoride was not evenly distributed in the mouth, and that retention of fluoride was reduced by increased salivary flow rate. Thus, it would seem likely that the rate of renewal of the film of

The aim of this study was to determine the velocity of the salivary film by determining the rate of diffusion of potassium chloride from an artificial plaque at different sites in the

Estimation of the velocity of the salivary film at different locations in the mouth.

periodontal disease.

spectrophotometer.

Fig. 2. Salivary film and plaque

**2.1 Salivary clearance from different regions of the mouth** 

saliva over plaque must influence diffusion rates into and from plaque.

**2. Study** 

**2.1.1 Aim** 

mouth.


A 1-mol/L solution of potassium chloride was mixed with sufficient agarose (Electrophoresis Purity Reagent; BioRad Laboratories, Richmond, CA) to give a 1.0% solution which was heated until the agarose dissolved. The acrylic chambers(Fig. 3) to hold the gel were rectangular (16 mm in length, 8 mm wide, and 1.5 mm thick) with a cylindrical central depression (6 mm diameter and 1.5 mm depth).

Fig. 3. Diagram of design of the diffusion chambers

The weight of the agarose held in the center well of each chamber was measured six times using an electronic balance (FX-3200; A&D, Tokyo, Japan), and chambers in which the mean weight of agarose was more than 2 SD from the mean were excluded.

Two chambers were initially covered with a layer of Parafilm (American Can, Greenwich, Conn., USA), were attached bilaterally by floss to the teeth, with the gel surface away from the teeth. The chambers were attached to the upper first molars for measurement of the posterior sites (UPB) and to both upper incisors for measurement of the anterior site (UAB) (Fig. 4).

After temperature and salivary flow equilibration, the Parafilm was removed at time 0. The first diffusion chamber was removed from the mouth after being exposed to saliva for a selected period of time and the gel was transferred to flasks containing 400 ml of (100 ppm) sodium chloride. Subsequently the second chamber was removed and the potassium chloride extracted by the same procedure. The fluid was agitated intermittently for 90 min, and the potassium concentration was assayed by atomic absorption spectrophotometry (Shimadzu AA-6105, Kyoto, Japan). The times were chosen so that between about 30 and 60% of the potassium chloride would have diffused from the agarose discs. The initial KCI concentration in the agarose discs, which had not been placed into the mouth, was also measured.

Estimation of the Velocity of the Salivary Film at the Different Regions in the Mouth

**2.1.4 Result** 

p<0.001.

(min)

40

0

**2.1.5 Discussion** 

Fig. 5. Half-time when salivary flow rate is unstimulated

concentration in the saliva affected that in the gel.

10

20

30

– Measurement of Potassium Chloride in the Agar Using Atomic Absorption Spectrophotometry 55

The half-times in the mouth varied with locations and with salivary flow rate, as shown in Fig. 5 When the flow rate was unstimulated, the shortest halftimes occurred in the LALi site and the longest in the UAB site. In both groups, the difference was significant at

Locations

LALi LPLi UPLi LA B UPB LPB UAB

In this study, we measured the concentration of residual potassium in agarose gel to determine the velocity of salivary flow for the 7 different sites. The reason why potassium chloride was used as the target substance (with an agarose gel used as artificial plaque) is that it is readily soluble in water, harmless, has a low molecular weight enabling it to diffuse easily, is present at a low concentration (around 20 mM) in saliva and can be measured relatively easily. Because the potassium concentration in the agarose gel used in this study was much higher than that in the saliva of the subjects, it was unlikely that the potassium

The relationship between time and the quantity of potassium diffused from the gel into the saliva was pre-determined in a pilot study. Clearance was evaluated by determining the half-time, the time at which the concentration at time 0 is reduced to half, from the relationship between 3 time points, including time 0, and potassium concentration, as well as by comparing the mean half-time between different sites. Since the correlation between time and the quantity of potassium eluted from the agarose gel was found to decrease in the early and late phases of the test (Lecomte P, Dawes C, 1987), the time to hold the holder in the mouth was determined so that the half-time would be almost at the mid-point of the test. For the measurement of potassium concentration, sodium chloride solution was used as

the solvent to avoid errors in measurements due to ionization of potassium.

Fig. 4. Acrylic chamber attached to the upper central incised


The rate of potassium chloride clearance from the gels into a large, stirred volume was determined. One involved suspending the filled chambers in one liter of 100 ppm NaCl, stirred by a magnetic stirrer, either at room temperature or at 37℃. The diffusion chambers were taken from the fluid at selected time intervals and the gels transferred quantitatively with a sewing needle to flasks containing 500 ml of 100 ppm sodium chloride. The fluid was agitated intermittently for 90 min, since preliminary studies showed that the remaining potassium chloride was extracted from the gel in this time interval. The potassium concentration was also measured in identically prepared agarose discs which had not been put into the 100 ppm NaCl, to give the initial concentration.

A least-squares straight line was fitted, by computer, to the potassium concentration plotted against the square root of time. This gives a very good approximation of the theoretical clearance curve until about 65% of the diffusant has been lost from the gel (see 2-1-5). From the results, the half-time was calculated.
