Archs oral Bid. Vol. 36, No. I, pp. 525-521, Printed in Great Britain. All rights reserved
0003-9969/91$3.00+ 0.00 Copyright 0 1991Pergamon Press plc
1991
BETWEEN TOTAL AND IONIZED RELATIONSHIP CALCIUM CONCENTRATIONS IN HUMAN WHOLE SALIVA AND DENTAL PLAQUE FLUID S. MATSUO’ and
F. LAGERL#
‘Department of Dental and Public Health, Kanagawa Dental College, 82 Inaoka-cho Yookosuka, Kanagawa, Japan and ‘Department of Cariology and Clinical Research Center, School of Dentistry, Karolinska Institute, Box 4064, S-141 04 Huddinge, Sweden (Accepted 9 January 1991) Summary-Ionized and total calcium were determined with an ion-selective electrode in unstimulated whole saliva and in simultaneously collected plaque fluid. The mean ionic and total concentrations in saliva from 20 subjects were 0.53 and 1.03 mmol/l respectively, and in plaque fluid 0.64 and 1.20 mmol/l. The total calcium concentration in saliva was closely related to the total calcium concentration in plaque fluid (r = 0.95, p < 0.001) as was the ionized calcium concentration in saliva to that of plaque fluid (r = 0.84, p < 0.001). Key words: saliva, plaque fluid, dental plaque, calcium.
trations in resting whole saliva and in plaque fluid sampled on the same occasion.
INTRODUCTION
Calcium in the oral fluids is present in several fractions. Large fractions are calcium bound to inorganic ion species such as orthophosphate or carbonate, or to macromolecules such as proteins (Lagerliif and Lindqvist, 1982). Approximately half the total calcium in saliva is in ionic form, as shown by several workers using differing methods (e.g. Lightfoot and Coolidge, 1961; Vogel, Naujoks and Brudevold, 1965; Grsn, 1973; Maier er al., 1979; Lagerliif, 1980; Lagerlof and Ekstrand, 1982). Less is known about ionized and total calcium in dental plaque fluid (Tatevossian and Gould, 1976; Edgar, Bowen and Cole, 1981; Carey et al., 1986; Tatevossian, 1987; Moreno and Margolis, 1988), especially its relationship to that of total and ionized calcium in saliva. In most of these studies the calcium concentrations in plaque fluid were found to be considerably higher than those in saliva. The ionic calcium concentration of the oral fluids contributes to the stability of the dental hard tissues. Changes in calcium activity in the liquid surrounding the enamel crystals, composed mainly of hydroxyapatite, will influence the solubility of the tooth mineral by changing the degree of saturation with respect to hydroxyapatite. Calcium activity in a local area at or near the tooth surface is a function of the calcium concentration in the surrounding liquid. It seems likely that the salivary calcium level may influence the calcium level in the dental plaque by diffusion. The resulting changes could then have an effect on the calcium activity at the site of de- and remineralization of the tooth mineral. No data seem to be available on the relationship between ionized calcium in saliva and plaque fluid. Therefore, our aim now was to determine, in a human population, the total and ionized calcium concen-
MATERIALS
AND METHODS
Subjects
Twenty healthy volunteers, 15 females and 5 males aged 26-62 yr, participated in the study; all gave informed consent. They refrained from oral hygiene for 24 h before plaque collection. They were instructed not to eat or drink for 2 h before the appointment, which was between 9:00 and 11:OOa.m. Saliva collection
The subject was seated in a relaxed position with the head slightly inclined, allowing the saliva to accumulate in the floor of the mouth. The accumulated saliva was spat into a beaker and immediately analysed for ionized calcium as described below. Plaque fruid collection
To minimize salivary contamination, the subjects were instructed to swallow as much saliva as possible before plaque was collected. The teeth to be sampled were then isolated with cotton rolls in the vestibulum and under the tongue, and with a salivary ejector. Approximately lO-20mg of plaque material was removed from all the teeth with a spatula. Areas close to the gingiva and areas difficult to isolate from saliva were avoided. The collected material was immediately placed in the centre of a bacteria1 filter (PC Membrane 25 mm, 0.4 pm, Nucleopore, U.S.A.), which was folded around the plaque to form a cone. The filter was inserted into a plastic pipette tip (C20, Gilson, France), which was cut to fit into a soft polyethylene tube equipped with a lid (Microfuge Tubes 400 ~1 (7 x 50 mm), Beckman, Fullerton, CA, U.S.A.). The tube was centrifuged for 3 min at 874Og (Microfuge B, Beckman, Fullerton, CA, U.S.A.). The
525
526
S.
MATSLJO and F. LAGERL~F
filtrate, approx. 3-6 ~1, was immediately withdrawn into a glass capillary tube and analysed for ionized calcium as described below.
2.0 -
Calcium analysis
Ionized and total calcium were determined with a recently developed mini-electrode (Lagerlaf and Matsuo, 1991). In short, the electrode is based on the ionophore ETH 1001 (Fluka AG, Switzerland) dissolved in a polyvinyl chloride membrane placed over the orifice of a 200 ~1 plastic pipette tip inserted, together with a reference electrode, into a Plexiglas block maintained at 37°C. The sample, which may be recovered, is injected into a narrow channel in the Plexiglas block in order to achieve contact between the electrodes. Steady readings of the voltage between the electrodes were obtained in less than 10 s. The apparatus was standardized with solutions containing calcium in the appropriate range and with ion strength and pH similar to that of the sample: 50 mmol/l sodium chloride, 20 mmol/l potassium chloride and 3 mmol/l tris(hydroxymethyl)aminomethane buffer, pH 7.2. The sample was then analysed for ionized calcium. The sample was withdrawn and one part of an inorganic solution (50 mmol/l NaCl, 20 mmol/l KC1 and 3 mmol/l tris) and one part 10 mmol/l HCl were added, which resulted in a pH of the sample ranging from 3.54. This mixture was analysed for ionized calcium using standards of pH 4. As nearly all bound calcium is liberated at this pH, the reading of this analysis approximates to the total calcium concentration of the sample. Details of the method are given in Lagerliif and Matsuo (1991). RESULTS
The mean ionized calcium concentration in whole saliva collected from 20 subjects was 0.53 & 0.07 (SD) mmol/l and the total calcium concentration 1.03 5 0.30 mmol/l. There was also a significant correlation between the ionized and total saliva calcium concentrations (r = 0.78, p < 0.001). The ionized calcium concentration in the plaque fluid was 0.64 k 0.13 mmol/l, which was approx. 53% of the total calcium concentration (1.20 k 0.38 mmol/l). The correlation between the ionized calcium concentration in the plaque fluid and the corresponding total calcium concentration was significant (I = 0.80, p < 0.001). The total calcium concentration in saliva was closely related to the total calcium concentration in plaque fluid (r = 0.95, p < O.OOl), as was the ionized calcium concentration in saliva versus that of plaque fluid (r = 0.84, p < O.OOl),as shown in Fig. 1. Both the total calcium and the ionized calcium concentration were significantly higher in plaque fluid than saliva (p < 0.001, paired t-test). DISCUSSION
We found a strong correlation between the calcium concentrations of saliva and plaque fluid. This was expected, because the water phase of plaque and saliva form a continuum. Depending on the diffusion gradient, calcium will diffuse into or out of the
g
1.5-
i 5 Q,
1.0 -
Z ii:
0.5 -
, 0
I 0.5
Salivary
I
I
1.0
1.5
Ca (mm01 11)
Fig. 1. The relationships between ionized calcium concentrations in saliva and plaque fluid (O), and between total
calcium concentrations in saliva and plaque fluid (a). plaque. Therefore a dynamic equilibrium could quickly be established between the liquid phase of a thin integument and its surrounding liquid. A similar relationship between total calcium in saliva and whole plaque was found by Ashley and Wilson (1978) in young adults. We found also that both the ionized and total calcium concentrations in plaque fluid were higher than in saliva. There were no individual cases in which calcium levels in saliva were higher than in plaque fluid. Tatevossian and Gould (1976) and Tatevossian (1987) found high total calcium levels (mean 6.5 and 2.9 mmol/l, respectively). In the 1987 study the ionized calcium concentration was 1.53 mmol/l, which is considerably higher than we now found. Still higher values with large variations between individuals and sites have been found for total calcium in plaque fluid (Rankine et al., 1985; Moreno and Margolis, 1988). The lowest mean values for ionized and total calcium in plaque fluid, 0.9 and 3.1 mmol/l respectively, were found by Carey et al. (1986). The differences between our results and those of these other workers are not easily explained. Plaque fluid is usually defined as the extracellular free-water compartment. In practice, the plaque fluid may be defined by the separation procedure used. In the previous studies the sampling procedure and the analytical methods have differed (for review see Tatevossian, 1990). In our study the time between the sampling of the plaque and the completion of the analysis for ionized calcium was short, less than 4 min compared to at least 15 min in the previous studies. However, it seems very unlikely that the ionized calcium concentration in resting plaque fluid should be so much higher in plaque fluid than in saliva. Due to the diffusion gradient the difference between saliva and plaque fluid in ionized calcium should be close to equilibrium.
Ionized calcium in saliva and plaque No data seem to be available for the relationship between ionized calcium in saliva and plaque. The finding that the ionized calcium concentration in plaque fluid was significantly related to that of saliva and that the concentration level was only slightly higher in plaque fluid implies that data from studies on saliva may be applied to calculate saturation values for calcium phosphate (e.g. Lagerliif, 1983), or ion products for calcium fluoride (Lagerliif, Ekstrand and Riilla, 1988), in investigations of the stability of the dental hard tissues. The ionized calcium level in saliva or plaque fluid is approx. 50% of the total calcium (Lightfoot and Coolidge, 1961; Vogel et al., 1965; Maijer and Klassen, 1972; Gram, 1973). However, the ratio between the mean values of ionized and total calcium in plaque fluid varies between 2948% (Tatevossian and Gould, 1976; Carey et al., 1986; Tatevossian, 1987; Moreno and Margolis, 1988). This proportion is most probably dependent on the pH of the fluid, as in parotid saliva (Lagerllif, 1980). We did not monitor the pH of the saliva or the plaque fluid. However, it may be assumed that the pH of plaque fluid often differs considerably from the salivary pH. During a sugar challenge the pH of the plaque will decrease to low values. Bound calcium will be liberated, causing the ionized calcium concentration to increase to close to the total calcium values. The plaque we collected was in a resting stage and the pH may be assumed to be close to neutrality, which may be the reason for the fraction found, 53%. We have used a new method of calcium determination with satisfactory accuracy and precision (Lagerlof et al., 1988; Lagerliif and Matsuo, 1991). The precision in salivary samples was better than 7.1%. Influences from hydrogen or other ions known to interfere with ion-selective calcium electrodes were negligible, and a difference of 50mmol/l in ionic strength between the sample and the internal standard resulted in an analytical error of only 0.04 mmol/l (Lagerliif and Matsuo, 1991). Thus, the most likely error in the method may be related to the plaque sampling technique. Plaque was collected by the usual method, i.e. with a simple dental hand instrument. It is difficult to avoid contamination with small amounts of saliva. By carefully isolating the plaque from the saliva we assumed that this contamination was negligible. To our knowledge there is no method available to differentiate between saliva and plaque fluid in this context. The effect of filtration on the plaque fluid is difficult to evaluate. Preliminary studies showed that the filter used did not change the calcium concentration in small volumes of standard solution when compared to non-filtered solutions. However, the filtering procedure may have separated unfilterable calcium sources such as solid calcium phosphate or calcium bound to micro-organisms. The main advantage of using a bacterial filter is the short time taken to manipulate the sample compared to using microcentrifugation or microhaematocrit tubes, for which centrifugation times ranging from 15 to 60 min have been reported (see Tatevossian, 1990). We pooled the plaque material and the saliva from an individual; the calcium levels are therefore mean values for the whole dentition. There are probably AOB 36,7--D
527
local variations in plaque calcium, at least temporarily (Rankine et al., 1985). However, our aim was to investigate the relationship between salivary and plaque-fluid calcium. Our findings strongly suggest such a relationship. Acknowledgement-This
investigation was supported by grants from the Swedish Medical Research Council (Project No. 7203). REFERENCES
Ashley F. P. and Wilson R. F. (1978) The relationship between calcium and phosphorus concentrations of human saliva and dental plaque. Archs oral Biol. 23,69-73. Carey C. M., Gregory T. M., Rupp T., Tatevossian A. and Vogel G. L. (1986) The driving forces in human dental plaque fluid for demineralisation and remineralisation of enamel mineral. In Factors Relating to Demineralisation and Remineralisation of the Teeth- (Ed. Leach S. A.), DD. 163-173. IRL Press. Oxford. Edgar W. M., Bowen W. H. and Cole M. F. (1981) Development of rampant dental caries, and composition of plaque fluid and saliva in irradiated primates. J. oral Path. 10, 284-295. Gron P. (1973) The state of calcium and inorganic orthophosphate in human saliva. Archs oral Biol. l&1365-1378. Lagerliif F. (1980) Determination of ionized calcium in parotid saliva. Clinica chim. Acta 102, 127-135. Lagerlijf F. (1983) Effects of flow rate and pH on calcium phosphate saturation in human parotid saliva. Caries Res. 17, 403411.
Lagerlijf F. and Ekstrand J. (1982) The effect of flow rate on the ionized calcium concentration of human parotid saliva. Caries Rex 16, 324-328. Lagerlijf F. and Lindqvist L. (1982) A method for determination of concentrations of calcium complexes in human parotid saliva by gel filtration. Archs oral Biol. 27, 735-738.
Lagerliif F. and Matsuo S. (199 1) Ionselective minielectrode determination of ionic and total calcium concentrations in mixed saliva. Clinica. chim. Acza. In press. Lagerliif F., Ekstrand J. and Riilla G. (1988) Effect of fluoride addition on ionized calcium in salivary sediment and in saliva. Stand. J. dent. Res. %, 39944. Lightfoot L. and Coolidge T. B. (1961) Ionized calcium in saliva. J. dent. Res. 40, 282-286. Maier H., Coroneo M. T., Antonczyk G. and Heidland A. (1979) The flow-rate-dependent excretion of ionized calcium in human parotid saliva. Archs oral Biol. 24, 225-227.
Maijer R. and Klassen G. A. (1972) Ionized calcium concentration in saliva and its relationship to dental disease. J. Can. dent. Ass. 9, 66-69.
Moreno E. C. and Margolis H. C. (1988) Composition of plaque fluid. J. dent. Res. 67, 1181-l 189. Rankme C. A. N., Moreno E. C., Vogel G. L. and Margolis H. C. (1985) Micro-analytical determination of DH. calcium,.and phosphate in-plaque fluid. J. dent. Res.-64; 1275-1280. Tatevossian A. (1987) Calcium and phosphate in human dental plaque and their concentrations after overnight fasting and after ingestion of a boiled sweet. Archs oral Biol. 32, 201-205.
Tatevossian A. (1990) Facts and artefacts in research on human dental plaque fluid. J. dent. Res. 69, 1309-1315. Tatevossian A. and Gould C. T. (1976) The composition of the aqueous phase in human dental plaque. Archs oral Biol. 21, 319-323. Vogel J. J., Naujoks R. and Brudevold F. (1965) The effective concentrations of calcium and inorganic orthophosphate in salivary secretions. Archs oral Biol. 10, 523-534.
0003-9969/91$3.00+ 0.00 Copyright 0 1991Pergamon Press plc
1991
BETWEEN TOTAL AND IONIZED RELATIONSHIP CALCIUM CONCENTRATIONS IN HUMAN WHOLE SALIVA AND DENTAL PLAQUE FLUID S. MATSUO’ and
F. LAGERL#
‘Department of Dental and Public Health, Kanagawa Dental College, 82 Inaoka-cho Yookosuka, Kanagawa, Japan and ‘Department of Cariology and Clinical Research Center, School of Dentistry, Karolinska Institute, Box 4064, S-141 04 Huddinge, Sweden (Accepted 9 January 1991) Summary-Ionized and total calcium were determined with an ion-selective electrode in unstimulated whole saliva and in simultaneously collected plaque fluid. The mean ionic and total concentrations in saliva from 20 subjects were 0.53 and 1.03 mmol/l respectively, and in plaque fluid 0.64 and 1.20 mmol/l. The total calcium concentration in saliva was closely related to the total calcium concentration in plaque fluid (r = 0.95, p < 0.001) as was the ionized calcium concentration in saliva to that of plaque fluid (r = 0.84, p < 0.001). Key words: saliva, plaque fluid, dental plaque, calcium.
trations in resting whole saliva and in plaque fluid sampled on the same occasion.
INTRODUCTION
Calcium in the oral fluids is present in several fractions. Large fractions are calcium bound to inorganic ion species such as orthophosphate or carbonate, or to macromolecules such as proteins (Lagerliif and Lindqvist, 1982). Approximately half the total calcium in saliva is in ionic form, as shown by several workers using differing methods (e.g. Lightfoot and Coolidge, 1961; Vogel, Naujoks and Brudevold, 1965; Grsn, 1973; Maier er al., 1979; Lagerliif, 1980; Lagerlof and Ekstrand, 1982). Less is known about ionized and total calcium in dental plaque fluid (Tatevossian and Gould, 1976; Edgar, Bowen and Cole, 1981; Carey et al., 1986; Tatevossian, 1987; Moreno and Margolis, 1988), especially its relationship to that of total and ionized calcium in saliva. In most of these studies the calcium concentrations in plaque fluid were found to be considerably higher than those in saliva. The ionic calcium concentration of the oral fluids contributes to the stability of the dental hard tissues. Changes in calcium activity in the liquid surrounding the enamel crystals, composed mainly of hydroxyapatite, will influence the solubility of the tooth mineral by changing the degree of saturation with respect to hydroxyapatite. Calcium activity in a local area at or near the tooth surface is a function of the calcium concentration in the surrounding liquid. It seems likely that the salivary calcium level may influence the calcium level in the dental plaque by diffusion. The resulting changes could then have an effect on the calcium activity at the site of de- and remineralization of the tooth mineral. No data seem to be available on the relationship between ionized calcium in saliva and plaque fluid. Therefore, our aim now was to determine, in a human population, the total and ionized calcium concen-
MATERIALS
AND METHODS
Subjects
Twenty healthy volunteers, 15 females and 5 males aged 26-62 yr, participated in the study; all gave informed consent. They refrained from oral hygiene for 24 h before plaque collection. They were instructed not to eat or drink for 2 h before the appointment, which was between 9:00 and 11:OOa.m. Saliva collection
The subject was seated in a relaxed position with the head slightly inclined, allowing the saliva to accumulate in the floor of the mouth. The accumulated saliva was spat into a beaker and immediately analysed for ionized calcium as described below. Plaque fruid collection
To minimize salivary contamination, the subjects were instructed to swallow as much saliva as possible before plaque was collected. The teeth to be sampled were then isolated with cotton rolls in the vestibulum and under the tongue, and with a salivary ejector. Approximately lO-20mg of plaque material was removed from all the teeth with a spatula. Areas close to the gingiva and areas difficult to isolate from saliva were avoided. The collected material was immediately placed in the centre of a bacteria1 filter (PC Membrane 25 mm, 0.4 pm, Nucleopore, U.S.A.), which was folded around the plaque to form a cone. The filter was inserted into a plastic pipette tip (C20, Gilson, France), which was cut to fit into a soft polyethylene tube equipped with a lid (Microfuge Tubes 400 ~1 (7 x 50 mm), Beckman, Fullerton, CA, U.S.A.). The tube was centrifuged for 3 min at 874Og (Microfuge B, Beckman, Fullerton, CA, U.S.A.). The
525
526
S.
MATSLJO and F. LAGERL~F
filtrate, approx. 3-6 ~1, was immediately withdrawn into a glass capillary tube and analysed for ionized calcium as described below.
2.0 -
Calcium analysis
Ionized and total calcium were determined with a recently developed mini-electrode (Lagerlaf and Matsuo, 1991). In short, the electrode is based on the ionophore ETH 1001 (Fluka AG, Switzerland) dissolved in a polyvinyl chloride membrane placed over the orifice of a 200 ~1 plastic pipette tip inserted, together with a reference electrode, into a Plexiglas block maintained at 37°C. The sample, which may be recovered, is injected into a narrow channel in the Plexiglas block in order to achieve contact between the electrodes. Steady readings of the voltage between the electrodes were obtained in less than 10 s. The apparatus was standardized with solutions containing calcium in the appropriate range and with ion strength and pH similar to that of the sample: 50 mmol/l sodium chloride, 20 mmol/l potassium chloride and 3 mmol/l tris(hydroxymethyl)aminomethane buffer, pH 7.2. The sample was then analysed for ionized calcium. The sample was withdrawn and one part of an inorganic solution (50 mmol/l NaCl, 20 mmol/l KC1 and 3 mmol/l tris) and one part 10 mmol/l HCl were added, which resulted in a pH of the sample ranging from 3.54. This mixture was analysed for ionized calcium using standards of pH 4. As nearly all bound calcium is liberated at this pH, the reading of this analysis approximates to the total calcium concentration of the sample. Details of the method are given in Lagerliif and Matsuo (1991). RESULTS
The mean ionized calcium concentration in whole saliva collected from 20 subjects was 0.53 & 0.07 (SD) mmol/l and the total calcium concentration 1.03 5 0.30 mmol/l. There was also a significant correlation between the ionized and total saliva calcium concentrations (r = 0.78, p < 0.001). The ionized calcium concentration in the plaque fluid was 0.64 k 0.13 mmol/l, which was approx. 53% of the total calcium concentration (1.20 k 0.38 mmol/l). The correlation between the ionized calcium concentration in the plaque fluid and the corresponding total calcium concentration was significant (I = 0.80, p < 0.001). The total calcium concentration in saliva was closely related to the total calcium concentration in plaque fluid (r = 0.95, p < O.OOl), as was the ionized calcium concentration in saliva versus that of plaque fluid (r = 0.84, p < O.OOl),as shown in Fig. 1. Both the total calcium and the ionized calcium concentration were significantly higher in plaque fluid than saliva (p < 0.001, paired t-test). DISCUSSION
We found a strong correlation between the calcium concentrations of saliva and plaque fluid. This was expected, because the water phase of plaque and saliva form a continuum. Depending on the diffusion gradient, calcium will diffuse into or out of the
g
1.5-
i 5 Q,
1.0 -
Z ii:
0.5 -
, 0
I 0.5
Salivary
I
I
1.0
1.5
Ca (mm01 11)
Fig. 1. The relationships between ionized calcium concentrations in saliva and plaque fluid (O), and between total
calcium concentrations in saliva and plaque fluid (a). plaque. Therefore a dynamic equilibrium could quickly be established between the liquid phase of a thin integument and its surrounding liquid. A similar relationship between total calcium in saliva and whole plaque was found by Ashley and Wilson (1978) in young adults. We found also that both the ionized and total calcium concentrations in plaque fluid were higher than in saliva. There were no individual cases in which calcium levels in saliva were higher than in plaque fluid. Tatevossian and Gould (1976) and Tatevossian (1987) found high total calcium levels (mean 6.5 and 2.9 mmol/l, respectively). In the 1987 study the ionized calcium concentration was 1.53 mmol/l, which is considerably higher than we now found. Still higher values with large variations between individuals and sites have been found for total calcium in plaque fluid (Rankine et al., 1985; Moreno and Margolis, 1988). The lowest mean values for ionized and total calcium in plaque fluid, 0.9 and 3.1 mmol/l respectively, were found by Carey et al. (1986). The differences between our results and those of these other workers are not easily explained. Plaque fluid is usually defined as the extracellular free-water compartment. In practice, the plaque fluid may be defined by the separation procedure used. In the previous studies the sampling procedure and the analytical methods have differed (for review see Tatevossian, 1990). In our study the time between the sampling of the plaque and the completion of the analysis for ionized calcium was short, less than 4 min compared to at least 15 min in the previous studies. However, it seems very unlikely that the ionized calcium concentration in resting plaque fluid should be so much higher in plaque fluid than in saliva. Due to the diffusion gradient the difference between saliva and plaque fluid in ionized calcium should be close to equilibrium.
Ionized calcium in saliva and plaque No data seem to be available for the relationship between ionized calcium in saliva and plaque. The finding that the ionized calcium concentration in plaque fluid was significantly related to that of saliva and that the concentration level was only slightly higher in plaque fluid implies that data from studies on saliva may be applied to calculate saturation values for calcium phosphate (e.g. Lagerliif, 1983), or ion products for calcium fluoride (Lagerliif, Ekstrand and Riilla, 1988), in investigations of the stability of the dental hard tissues. The ionized calcium level in saliva or plaque fluid is approx. 50% of the total calcium (Lightfoot and Coolidge, 1961; Vogel et al., 1965; Maijer and Klassen, 1972; Gram, 1973). However, the ratio between the mean values of ionized and total calcium in plaque fluid varies between 2948% (Tatevossian and Gould, 1976; Carey et al., 1986; Tatevossian, 1987; Moreno and Margolis, 1988). This proportion is most probably dependent on the pH of the fluid, as in parotid saliva (Lagerllif, 1980). We did not monitor the pH of the saliva or the plaque fluid. However, it may be assumed that the pH of plaque fluid often differs considerably from the salivary pH. During a sugar challenge the pH of the plaque will decrease to low values. Bound calcium will be liberated, causing the ionized calcium concentration to increase to close to the total calcium values. The plaque we collected was in a resting stage and the pH may be assumed to be close to neutrality, which may be the reason for the fraction found, 53%. We have used a new method of calcium determination with satisfactory accuracy and precision (Lagerlof et al., 1988; Lagerliif and Matsuo, 1991). The precision in salivary samples was better than 7.1%. Influences from hydrogen or other ions known to interfere with ion-selective calcium electrodes were negligible, and a difference of 50mmol/l in ionic strength between the sample and the internal standard resulted in an analytical error of only 0.04 mmol/l (Lagerliif and Matsuo, 1991). Thus, the most likely error in the method may be related to the plaque sampling technique. Plaque was collected by the usual method, i.e. with a simple dental hand instrument. It is difficult to avoid contamination with small amounts of saliva. By carefully isolating the plaque from the saliva we assumed that this contamination was negligible. To our knowledge there is no method available to differentiate between saliva and plaque fluid in this context. The effect of filtration on the plaque fluid is difficult to evaluate. Preliminary studies showed that the filter used did not change the calcium concentration in small volumes of standard solution when compared to non-filtered solutions. However, the filtering procedure may have separated unfilterable calcium sources such as solid calcium phosphate or calcium bound to micro-organisms. The main advantage of using a bacterial filter is the short time taken to manipulate the sample compared to using microcentrifugation or microhaematocrit tubes, for which centrifugation times ranging from 15 to 60 min have been reported (see Tatevossian, 1990). We pooled the plaque material and the saliva from an individual; the calcium levels are therefore mean values for the whole dentition. There are probably AOB 36,7--D
527
local variations in plaque calcium, at least temporarily (Rankine et al., 1985). However, our aim was to investigate the relationship between salivary and plaque-fluid calcium. Our findings strongly suggest such a relationship. Acknowledgement-This
investigation was supported by grants from the Swedish Medical Research Council (Project No. 7203). REFERENCES
Ashley F. P. and Wilson R. F. (1978) The relationship between calcium and phosphorus concentrations of human saliva and dental plaque. Archs oral Biol. 23,69-73. Carey C. M., Gregory T. M., Rupp T., Tatevossian A. and Vogel G. L. (1986) The driving forces in human dental plaque fluid for demineralisation and remineralisation of enamel mineral. In Factors Relating to Demineralisation and Remineralisation of the Teeth- (Ed. Leach S. A.), DD. 163-173. IRL Press. Oxford. Edgar W. M., Bowen W. H. and Cole M. F. (1981) Development of rampant dental caries, and composition of plaque fluid and saliva in irradiated primates. J. oral Path. 10, 284-295. Gron P. (1973) The state of calcium and inorganic orthophosphate in human saliva. Archs oral Biol. l&1365-1378. Lagerliif F. (1980) Determination of ionized calcium in parotid saliva. Clinica chim. Acta 102, 127-135. Lagerlijf F. (1983) Effects of flow rate and pH on calcium phosphate saturation in human parotid saliva. Caries Res. 17, 403411.
Lagerlijf F. and Ekstrand J. (1982) The effect of flow rate on the ionized calcium concentration of human parotid saliva. Caries Rex 16, 324-328. Lagerlijf F. and Lindqvist L. (1982) A method for determination of concentrations of calcium complexes in human parotid saliva by gel filtration. Archs oral Biol. 27, 735-738.
Lagerliif F. and Matsuo S. (199 1) Ionselective minielectrode determination of ionic and total calcium concentrations in mixed saliva. Clinica. chim. Acza. In press. Lagerliif F., Ekstrand J. and Riilla G. (1988) Effect of fluoride addition on ionized calcium in salivary sediment and in saliva. Stand. J. dent. Res. %, 39944. Lightfoot L. and Coolidge T. B. (1961) Ionized calcium in saliva. J. dent. Res. 40, 282-286. Maier H., Coroneo M. T., Antonczyk G. and Heidland A. (1979) The flow-rate-dependent excretion of ionized calcium in human parotid saliva. Archs oral Biol. 24, 225-227.
Maijer R. and Klassen G. A. (1972) Ionized calcium concentration in saliva and its relationship to dental disease. J. Can. dent. Ass. 9, 66-69.
Moreno E. C. and Margolis H. C. (1988) Composition of plaque fluid. J. dent. Res. 67, 1181-l 189. Rankme C. A. N., Moreno E. C., Vogel G. L. and Margolis H. C. (1985) Micro-analytical determination of DH. calcium,.and phosphate in-plaque fluid. J. dent. Res.-64; 1275-1280. Tatevossian A. (1987) Calcium and phosphate in human dental plaque and their concentrations after overnight fasting and after ingestion of a boiled sweet. Archs oral Biol. 32, 201-205.
Tatevossian A. (1990) Facts and artefacts in research on human dental plaque fluid. J. dent. Res. 69, 1309-1315. Tatevossian A. and Gould C. T. (1976) The composition of the aqueous phase in human dental plaque. Archs oral Biol. 21, 319-323. Vogel J. J., Naujoks R. and Brudevold F. (1965) The effective concentrations of calcium and inorganic orthophosphate in salivary secretions. Archs oral Biol. 10, 523-534.
