Archs oral Bid. Vol. 34, No. 10, pp. 821-824, 1989 0003-9969/89 $3.00 + 0.

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Printed in Great Britain. All rights reserved Copyright 0 1989 Pergamon Press plc

PLAQUE FLUID pH, CALCIUM AND PHOSPHORUS

RESPONSES TO CALCIUM FOOD ADDITIVES IN A
CHEWABLE CANDY
C. A. N. RANKINE,’ T. J. F’RIHODA,’ K. R. ETZEL) and D. LABADIE~
‘L.S.U. School of Dentistry, 1100 Florida Avenue, New Orleans, LA 70119, ‘University of Texas Health
Science Center at San Antonio, San Antonio, Texas, ‘University of Pittsburgh School of Dental Medicine,
Pittsburgh, Pennsylvania and ‘Vinta Ouray Indian Health Center, Fort Duchesne, Utah, U.S.A.

(Accepted 22 March 1989)

Summary-Fourteen subjects between 7 and 17 years of age with an equal distribution of low and high
cartes activity were given: (1) a 10% sucrose rinse, (2) a reference candy, (3) a reference candy with 3%
dicalcium phosphate dihydrate, and (4) a reference candy with 0.75% calcium lactate on four different
occasions. Plaque samples were collected before and at 15-min intervals after the sucrose rinse or food
challenge for a period of 1h on each occasion. Plaque samples were centrifuged and the extracellular
plaque fluid analysed by a microtechnique for pH, total calcium and inorganic phosphorus concentration.
There was no significant increase in calcium and phosphorus in plaque fluid for the group using candy
with added calcium compared to the reference candy or sucrose rinse. There was no significant difference
between the measurements in subjects grouped as caries active or inactive. The results suggest no benefit
can be expected from adding dicalcium phosphate dihydrate and calcium lactate to candy to decrease
demineralization during a cariogenic challenge.

INTRODUCTION an individual rather than from pooled samples. Our

There is an immense body of data concerning the
effects of calcium and phosphate dietary supplements
in preventing animal and human dental caries. Sup-
plementation has been successful in reducing caries in
animals, but there is no conclusive information to
substantiate this effect in humans. Dicalcium phos-
phate reduced caries by 90% in animals (Stralfors,
1961); these findings have not been validated in
human trials (Averill and Bibby, 1964; Ship and
Mickelsen, 1964; Finn and Jamison, 1967). Calcium
lactate has also proved effective in reducing caries in
rats (McClure, 1960). An in vitro study (Shrestha,
Mundorff and Bibby, 1982) indicates that calcium
lactate reduces enamel solubility.
The effect of calcium and phosphate supplementa-
tion is to increase the buffering potential and decrease
the demineralization capacity of plaque (Nizel and
Harris, 1964) at the site of cariogenic challenge.
Jenkins (1966) and Edgar and Tatevossian (1971)
have estimated that between 25 and 35% of plaque
is extracellular fluid. The calcium and phosphorus
concentration in pooled samples of plaque fluid has
been quantified (Tatevossian and Gould, 1976); and
analysed after ingestion of a boiled sweet (Tatevos-
Sian, 1987) when no relative changes in total or
ionized calcium and inorganic phosphorus were de-
tected. Ashley (1975), in a study on ash samples of
plaque, reported a fall in whole plaque calcium and
phosphate after an exposure to sugar.
Recently, a microtechnique has been developed to
measure the pH, calcium and phosphorus concen-
tration of plaque fluid in a sample taken from a few
teeth within a single mouth (Rankine et al., 1985).
The advantage of this technique is that it allows
concurrent measurement of changes in plaque from
aim was now to examine the relative changes in
plaque fluid pH, total calcium and inorganic phos-
phorus induced by dicalcium phosphate dihydrate
and calcium lactate additives in a reference chewable
fruit-flavoured candy.
MATERIALS AND METHODS
Nine boys and five girls between the ages of 7 and
17 years with a mean age of 12.6 (SD 3.3) years were
studied. Subjects were screened and obtained from
the 6-month recall list of patients at the University of
Texas School of Dentistry at San Antonio. Each
subject and parent reviewed and signed a consent
form approved by the Institutional Review Board
for human subjects. The high caries activity group
(7 subjects) had four or more new carious lesions
during the preceding 6 months and the low caries
activity group (7 subjects) had no carious lesions for
the same period. The mean ages of the caries active
group and caries inactive group were 13.2 (SD 3.5)
and 11.9 (SD 3.2) respectively. All carious lesions
were restored before the study.
The test foods in this double-blind study were (1)
10 ml solution of 10% sucrose rinse as a control, (2)
a reference chewable fruit-flavoured candy, (3) the
reference candy with 3% dicalcium phosphate dihy-
drate and, (4) the reference candy with 0.75% calcium
lactate.
The individuals were asked to refrain from oral
hygiene for 48 h before plaque collection, and not to
consume any food or beverage 1 h before collection.
Five plaque collections were made at 15-min intervals
from time zero to 60min. After the first collection,
the subject was asked to rinse with the sucrose

821
822 C. A. N.
solution or chew 15 g of one of the three test candies
for 5 min and then swallow. Each subject was
also asked to swallow vigorously before plaque col-
lection to remove residual saliva. Small amounts of
plaque, equally distributed throughout the mouth,
were removed from the buccal, lingual and interprox-
imal supragingival surfaces of all teeth (with excep-
tion of the lower incisors) with a T 23 scaler
(American Dental Manufacturing Co., Missoula,
Mont., U.S.A.). After the initial collection process
with the 10% sucrose rinse, the individual was given
a dental prophylaxis, and asked to return at a later
date for a repeat procedure with a different foodstuff.
The sequence of testing the foods was the same for
each subject. The order of tests was: (1) the sucrose
rinse; (2) the candy with calcium lactate; (3) the candy
with dicalcium phosphate dihydrate, and (4) the
candy. The subjects were instructed to brush thor-
oughly after each test procedure rather than receiving
a dental prophylaxis. There was a minimum of 2-4
days between tests.
The plaque samples were placed immediately under
heavy mineral oil in a specially designed glass micro-
centrifuge tube (Rankine et al., 1985). The tube was
centrifuged at 40,OOOgat 4°C for 1 h. After centrifu-
gation, three layers were discernible: plaque residue
composed of cells and extracellular debris was at the
bottom; the mineral oil formed the top layer, and the
extracellular plaque fluid was the middle layer.
Samples of between 100 and 200 nl of plaque fluid
were retrieved from the microcentrifuge tube under a
dissection microscope with specially designed mi-
cropipettes (Rankine et al., 1985). Thus the microcen-
trifuge tube was broken at an inscribed line over the
heavy oil layer, approx. 2 mm above the plaque fluid.
A micropipette was gently inserted into the tube
through the oil into the plaque fluid and the sample
drawn up by slight suction. The first sample (approx.
150 nl) of plaque fluid was diluted into 300 ~1 of
distilled deionized water, and analysed for total cal-
cium and inorganic phosphorus concentration.
The second micropipette sample was placed on a
MI-408 needle pH electrode (Microelectrodes, Inc.,
Londonderry, N.H., U.S.A.) in a specially designed
Plexiglas cell filled with heavy mineral oil to prevent
dehydration, condensation and CO, loss. A microma-
nipulator was used to bring a modified reference
electrode in contact with the plaque fluid sample for
pH measurement.
Seventy-five microlitres of the first diluted sample
were mixed with an equal volume of malachite green-
ammonium molybdate reagent and analysed spec-
trophotometrically for inorganic phosphorus in a
microquartz cell. The total calcium content of the
remainder was analysed on a Perkin-Elmer Model
3030 atomic absorption spectrophotometer equipped
Table 1. Atomic absorption parameters
Step 1
(drying)
Temperature (“C) 110
Ramp time (s) 10
Hold time (s) 25
Internal aas flow mlimin 50
RANKME et al.
with a Perkin-Elmer Model HGA400 graphite fur-
nace. Pyrolytically coated graphite tubes (Perkin-
Elmer Corporation, Norwalk, Conn., U.S.A.) were
used in the furnace. Background emission correction
with a deuterium lamp was used during all analyses.
The wavelength (422.5), slit width (0.7 alternate) and
lamp current (15 mA) were all in accordance with
published analytical recommendations (Powell and
Tease, 1982). Calcium standards and plaque fluid
samples were introduced into the furnace using a
Perkin-Elmer AS-40 auto-sampler to provide opti-
mal analytical precision. Ten microlitre samples from
the remaining 225 ~1 diluted sample were siphoned
and placed in the graphite tube. Peak area during a
5-s atomization (integrated absorbance) was dis-
played and recorded on a Perkin-Elmer PR-100
printer. The operating conditions for the atomic
absorption spectrophotometer and the graphite fur-
nace unit are shown in Table 1.
This procedure for calcium analysis enabled mea-
surements between 10 and 250 parts per billion
calcium. System error from use of a standard was less
than 1%.
The statistical model for this experiment was
an analysis of variance for repeated measures. The
between-groups factor was a comparison of caries
active and caries inactive groups of patients. The
variables for the repeated measures design were pH,
total calcium or inorganic phosphorus and time (0,
15, 30, 45 and 60 min). An analysis of variance was
performed using time zero as the covariate in the
repeated measures model. The calcium means were
adjusted to correct for initial fluctuations in calcium
levels using standard computer software (Dixon,
1981) with time values being the covariate.
RESULTS
The mean pH changes for the 14 subjects with
sucrose and the three types of chewable fruit-
flavoured candy were similar (Table 2). Stephan pH
curves were derived; the analysis of variance for the
means showed no significant difference between the
tested foods.
The total calcium concentration in all tests showed
an initial increase, then a decline (Table 3). The
results were inverse in relation to the pH curves. The
plaque fluid changes in total calcium after all three
candies were similar. Note that at the 15-min interval
the plaque assay after the standard candy did not
peak, whereas the others peaked and were higher at
15-min intervals.
The significantly higher baseline of total calcium
for the candy with calcium lactate was probably
attributal to the dental prophylaxis being done after
the sucrose rinse and before the calcium lactate test
Step 2 Step 3 Step 4
(ashing) (atomizing) (conditioning)
1000 2600 2700
10 1 1
15 5 1
50 50 50
 Plaque responses to Ca added to candy 823

Table 2. pH changes in plaque fluid after carious challenge (mean f SD; n = 14)
PI-I
Test food 0 min 1Smin 30 min 45 min 60 min
Sucrose 6.14f0.66 5.01 kO.44 5.38+0.55 5.54kO.53 6.02kO.55
Candy 6.32 f 0.72 5.16 f 0.34 5.31 + 0.48 5.62 f 0.52 5.86 + 0.62
Candy with dicalcium phosphate dihydrate 6.34kO.68 5.17kO.36 5.32~040 5.60+0.40 5.81 +0.45
Candy with Ca lactate 6.45kO.54 5.11 rtO.41 5.36kO.34 5.62f0.41 5.62kO.41

Table 3. Calcium changes in plaque fluid after carious challenge (mean f SD; n = 14)
Total calcium (mM)
Test food 0 min 15 min 30 mm 45 min 60 min
Sucrose 2.50 + 0.79 4.48 + 1.75 3.90 &-2.08 3.59 + 1.53 3.27 + 1.12
Candy 2.50 f 1.73 3.93 f 3.19 4.01 f 1.85 4.07 + 2.19 2.70 + 1.91
Candy with dicalcium phosphate dihydrate 2.38 + 0.90 5.33 f 2.63 4.34 If 2.61 3.54 f 1.68 2.69 + 1.04
Candy with Ca lactate 3.63 + 2.15 6.08 + 3.25 4.99 k 2.12 5.01 k 1.77 4.29 k 3.20

in all 14 subjects. Prophylaxis should ideally have
been done at the start of the study. Therefore, use of
analysis for covariance to adjust for baseline differ-
ences permitted a more accurate comparison of the
three tested foods and sucrose (Fig. 1). The candy
with calcium lactate produced a borderline significant
increase (F = 5.1, d’ = 1, dfd = 38, p = 0.06) in total
calcium compared to the reference candy. The candy
with dicalcium phosphate dihydrate followed the
changes produced by calcium lactate, but was not
significantly different in effect from the sucrose con-
trol. The changes in concentration for phosphorus in
plaque fluid had no consistent pattern (Table 4).
There were no significant differences between the
caries active and inactive groups for all the variables
and foods tested. Overall, the caries inactive group
had higher (p = 0.09) calcium at the beginning of the
test than the caries active group.
DISCUSSION
The initial processes of cariogenesis take place at
the tooth-plaque interphase. The complexity of dem-
ineralization and remineralization in the lesion on the
enamel surface suggests that a better understanding
of plaque fluid is needed. It is the activity of various
biochemical constituents of this fluid that determines
the capacity of the tooth to resist demineralization.
s 5.0-
_fg 4.5-
E 4.0-
.2
+ 3.5-
0
5 3.0-
'i;
c 2.5,
0 Crmd” 0 candy w/caLects40
2.0-
. Cs"d"wlDCm .sucmw
These various constituents, such as pH, calcium
concentration, phosphorus concentration, protein
components and other major and minor trace ele-
ments, determine the activity in solution of basic
calcium phosphate salts which arises out of the degree
of saturation of the plaque fluid by enamel mineral.
Whether the constituents create an undersaturated or
supersaturated environment at the interphase will
determine the stability of that mineral. With under-
saturation, caries is initiated, whereas with supersatu-
ration the integrity of the tooth is maintained.
The concentrations of total calcium and inorganic
phosphorus in plaque fluid before our tests were
similar to those of Tatevossian and Gould (1976).
The pH changes formed a Stephan curve for the
sucrose rinse and the foods, but the subsequent
changes in total calcium and phosphorus were incon-
sistent with those in previous studies. Rankine et al.
(1985) demonstrated an increase in the total calcium
and phosphate after a sucrose rinse in humans. In the
macaque monkey, Edgar, Bowen and Cole (1981)
also observed a significant increase in total calcium
and no change in phosphorus in pooled samples of
plaque fluid after a sucrose challenge.
Our results and those of similar studies that have
demonstrated an increase in calcium in plaque after
a sucrose challenge raise the question of the origin of
the calcium increase with decreasing pH. It could
come from: (1) demineralization of the tooth, (2)
bacteria in plaque, (3) diffusion from saliva facilitated
by sucrose and coupled to binding of calcium by
plaque constituents, or (4) from extracellular mineral-
ized components within the plaque. It is conceivable
that a transient cariogenic environment is developed
soon after the sucrose challenge as indicated by the
lowest pH attained. Under these conditions enamel
could be demineralized, producing the observed in-
crease in calcium.
The pattern of change in inorganic phosphorus in
plaque fluid had large standard deviations, particu-
larly for the sucrose rinse. Dental prophylaxis at the

1.5 ! I I I I 1 1 1
start of the study probably could have eliminated
-10 0 10 20 30 40 50 60 70 this. Sources of inorganic phosphorus in plaque fluid
Minutes could be the tooth, saliva, intracellular fluid or
Fig. 1. Calcium changes in plaque fluid during a carious mineralized components within the plaque.
challenge after a baseline adjustment (n = 14). DCPD = With test foods containing a calcium additive, one
dicalcium phosphate dihydrate. might expect an increase of calcium in the plaque
824 C. A. N. RANKINE er al.

Table 4. Phosphorus changes in plaque fluid after carious challenge (mean f SD; n = 14)
Phosphorus (mM)
Test food 0 min 15 min 30 min 45 min 60 min
Sucrose 12.59 + 7.00 11.84 k 7.46 12.14 + 5.36 11.97 + 4.95 10.43 + 5.23
Candy 11.05~3.00 11.76k2.96 11.47k3.73 10.71 f4.10 9.59+2.60
Candy with dicalcium phosphate dihydrate 11.38 k 1.91 11.78 i 2.16 10.96 _+3.04 10.07 k 2.47 9.37 k 2.03
Candy with Ca lactate 12.79 k 3.10 13.14 + 4.34 12.35 + 4.03 12.36 k 4.33 10.77 + 3.24

beyond that found with a sucrose rinse and a refer-
ence food. This could produce conditions favourable
to the reversal of the physico-thermodynamic process
of deminerahzation; our results did not suggest this.
This does not preclude the possibility that our system
could create a change in binding potential of calcium,
or increase the percent of ionized calcium in plaque.
Further studies similar to ours and that of Tatevos-
sian (1987) need to be conducted on the concentra-
tion levels of ionized calcium and inorganic
phosphorus during a cariogenic challenge.
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