Changes in the concentrations of phosphates in human plaque after the ingestion of sugar with and without added phosphates

A,& ora1 B,nl Vol. Xl. pp. f817to 625. Pergamon Press 1975. Prmtsd in Great Brcun.

CHANGES IN THE CONCENTRATIONS OF PHOSPHATES IN HUMAN PLAQUE AFTER THE INGESTION OF SUGAR WITH AND WITHOUT ADDED PHOSPHATES A. Department

TATEVO~SAN*,

W. M. EDGAR

and G. N.

JENKINS

of Oral Physiology, University of Newcastle upon Tyne, Markham Laboratories, Upper Claremont Street. Newcastle upon Tyne NE2 4AJ. England

effect of ingesting sugar, with or without various phosphate supplements, on the phosphate concentration of plaque was investigated. Sugar containing 0.37 per cent of inorganic phosphate (I?i) did not consistently raise the Pi of plaque unless the initial plaque Pi was unusually low. This finding could be explained because the saliva stimulated by the sugar diluted the Pi so that its concentration was usually lower than that already present in the plaque. Sugar containing 1 per cent of certain organic phosphates which have been suggested as caries inhibitors did not invariably raise the total P of preformed plaque, but small increases in total P occurred when the sugar containing the phosphate was taken hourly during the development of the plaque. These variable and inconsistent results may explain why Pi supplements have usually been unsuccessful in reducing caries in man. Summary-The

INTRODUCTION

Many investigators have examined the caries-reducing potential in experimental animals of various inorganic or organic phosphates added as dietary supplements. Several inorganic phosphates (Pi) have been tested (reviewed by Nizel and Harris, 1964) but the soluble disodium salt has reduced caries most consistently. Various calcium phosphates have generally been less effective, and sometimes completely ineffective, and it seems illogical that several of the trials on human subjects have employed 2 per cent of the less active dicalcium phosphate added to the sugar and flour. Two of these trials in man gave completely negative results (Averill and Bibby, 1964; Ship and Mickelsen, 1964) and a third was negative except for teeth erupting during the trial (Averill, Freire and Bibby, 1966). Stralfors (1964) found a reduction in caries with 2 per cent of dicalcium phosphate added to the sugar and flour in the school lunches of 2262 children but the result was confused by the presence of 250 ppm of fluoride in the additive during the first half of the experiment. In addition, caries was measured only in the four upper incisors, which are the teeth probably most accessible to protective substances, and was thus not strictly comparable with the scores in the other experiments. In the experiments on human subjects who used sodium phosphate, 1 per cent of the acid salt (or a mixture of the acid and basic salts) was added to sugarcoated breakfast cereals. Although this was the only constituent of the diet containing the additive, subjects were permitted to eat as much of it as they wished and consumption was not limited to breakfast, except in one experiment on subjects in an institution. However, no attempt was made to modify the normal cereal-eating habits of the children. The results over 2 yr showed reductions of 24 and 43 per cent in the decayed, missing and filled surfaces (DMFS) increments in children examined by two investigators (Stookey, Carroll and * Present address College. Cardiff.

Physiology

Department,

University

Muhler. 1967). In adults. a 42 per cent reduction was found after 1 yr while, among institutionalized subjects of mixed age, a reduction of over 50 per cent was reported after 2 yr ingestion of the supplement (Brewer, Stookey and Muhler, 1970). Peterson (1969) was unable to confirm this result and found no caries reduction in children eating breakfast cereals supplemented with 1 per cent of a mixture of sodium dihydrogen and disodium hydrogen phosphate, Finn and Jamison (1967) compared the development of caries among three groups of children who, during 30 months, were instructed to use chewing gum containing sugar, sugar and dicalcium phosphate or neither, for several periods totalling lOOmin per day. The phosphate neutralized the caries-producing effect of the sugar because the latter two groups developed 19 per cent less caries than did the group receiving gum containing sugar, but this procedure is not strictly comparable with the addition of phosphate to a normal dietary constituent. Beneficial effects in reducing caries with a gum containing dicalcium phosphate were reported by Richardson et al. (1972). However, Ashley, Naylor and Emslie (1974a, b) found no reduction in caries incidence among a group of children aged 1 l-15 yr, consuming sweets containing 3 per cent dicalcium phosphate when compared with a control group consuming sweets without additive. The mechanism of action of dietary supplements of Pi has generally been assumed to be a local effect. Systemic phosphates fed by intubation exert little or no caries reduction in experimental animals (McClure and Muller, 1959; Stralfors, 1961; Ostrom and van Reen, 1963; McClure, 1965) although McDonald, Stookey and Muhler (1968) reported contrary results. The concentration of plaque phosphate was not measured in any of these dietary experiments so that it was not established whether it increased above the normal levels, which are known to be high (Dawes and Jenkins, 1962). Luoma, Hurskainen and Isokangas (1964) carried out one of the few attempts to examine changes in plaque chemistry in relation to dietary phosphate. They

618

A. Tatevossian, W. M. Edgar and G. N. Jenkins

found that a combination of 1.0 M sodium carbonate and 1.2M sodium dihydrogen phosphate added to sugar at the high level of 5 per cent (apparently = 0.63 per cent P in the sugar) raised the plaque Pi concentration by about 71 per cent when ingested by seventeen subjects with profuse plaque formation. However, in nine subjects who formed less plaque, no difference was observed in the mean concentration of Pi in plaque after the ingestion of sugar tablets with or without carbonate-phosphate additive. This limited result on the effect of ingested phosphate on plaque phosphate levels in man has been repeated and extended in the present work using sugar with various phosphate supplements at concentrations lower, and more likely to be acceptable, than those used by Luoma et a/. (1964). Reductions in caries have been reported following the addition of 1 per cent calcium sucrose phosphate (CSP) to the sugar and flour in children’s diets (Harris et al., 1967, 1968, 1969) and of l-2 per cent of various phytates and 1 per cent calcium glycerophosphate (CGP) in rats (Federov, 1961; McClure, 1963) and monkeys (Bowen, 1972). The effect of these supplements on the composition of the plaque has also been studied in the present work. a brief report of which has already been published (Tatevossian and Jenkins, 1969). MATERIALS

AND

METHODS

The first series of experiments was carried out on ‘&preformed” plaque, accumulated by refraining from tooth-brushing, usually for 24 hr. Plaque was collected initially for baseline measurement 1.5-2 hr after breakfast, from all surfaces of the teeth in opposite quadrants (e.g. left upper, LU, and right lower, RL). Plaque from the remaining quadrants was collected 5 min later, 2 min after the ingestion of the test materials. In the second series, the subjects cleaned their teeth as usual in the morning and the plaque was allowed to form during the experiment. In all experiments, plaque was collected by the method of Luoma (1964) with slight modification. usually with a plastic spatula made by attaching some Portex tubing to a dental chisel and shaping a spoon-shaped end in the tubing; metal spatulas were sometimes used. Each plaque sample was weighed quickly on a torsion balance (usually the whole procedure of collection and weighing was completed in 60 set), suspended in deionized water (500~1) using a vortex mixer, centrifuged (2000rev/min) and the clear supernatant containing the soluble components removed with a syringe. The plaque was washed by resuspending in a further 2004 of deionized water and the original extract and washings were pooled for the estimation of soluble and total phosphate by a micromodification of the method of Kuttner and Cohen (1927). The residue was digested by wet ashing and total P and N (by Nesslerization) estimated. The results are expressed relative to the N content or the wet weight of plaque. Calcium analyses were also carried out by atomic absorption spectrometry (Willis, 1960) but are reported only in those experiments in which calcium salts were used as the results did not affect the conclusions of the other experiments. (cc) Baseline experiments

on the lubility

of‘ phosphate

the plaque

Four experiments

were carried out in this series:

in

(1) The quadrants used for the subsequent experiments (i.e. RL + LU and LL + RU) were sampled in five subjects and the plaque P, and total P compared after aqueous extraction. (2) The effect of water rinses on plaque P, was tested in four subjects by holding three consecutive volumes of 7.5 ml of distilled water in the mouth for 1 min each, after the initial control sample of plaque had been removed. The remaining plaque was then collected immediately after the last rinse and assayed as described above. (3) The effect of rinsing for three consecutive I-min periods with 7.5 ml of a neutral buffered solution of sodium phosphate containing 0.37 per cent P (= 2 per cent CaHP0,.2H20, the concentration used in most of the dietary experiments) on plaque Pi concentration was tested, on the plaque collected 2 min after the last rinse. (4)The effect of sucrose was tested by measuring the Pi in plaque collected 2 min after the ingestion of three sugar lumps or immediately after a period of 15 min, during which time sugar lumps were sucked continuously. (h) The effect qf’suckirg sugar lumps contair~i~~ twious phosphates on the P content qf plaque The experiments consisted of sucking three sugar lumps, at 1 min intervals, care being taken to distribute the sugar and saliva throughout the mouth and to ensure that each lump was completely dissolved during 1 min. The second plaque sample was collected 2min after the last sugar lump had been swallowed. The supplements of Pi and sodium phytate (Sigma) were added to the sugar lumps (average wt 2.2g) as 200 ~1 of a solution or suspension containing 8.1 mg P (final concentration 0.37 per cent P, 5 2 per cent CaHP04. 2Hz0 but in one series, twice this concentration). Calcium sucrose phosphate (Colonial Sugar Corporation, Sydney, Australia) and calcium glycerophosphate (Sigma. grade XII approx 50 per cent p isomer and 50 per cent DLZ isomer) were added at a level of 1 per cent, as in the experiments of Harris et al. (1968) and Bowen (1972). The sugar lumps. moistened with the 200$ of additive solution but retaining their shape, were dried overnight in an oven at 37°C.

The effect of sucking sugar lumps, with or without incorporated Pi, on the Pi concentration of saliva was also tested. Ideally, the salivary Pi should have been measured periodically throughout the 3 min in which the sugar lumps were in the mouth but problems of sampling and ofavoiding particles of undissolved sugar containing Pi made this difficult. Two methods were eventually adopted: (a) the sugar lump and the saliva it stimulated during 1 min were allowed to accumulate in the mouth by inhibiting swallowing, then expectorated, the volume recorded and the Pi concentration estimated, (b) the sugar lump was dissolved in the mouth and the sugar-saliva solution was swallowed in the normal way. At the end of each minute, the small residue of the mixture in the mouth was expectorated and its Pi estimated. The relative value of these two methods is commented on later.

619

Plaque phosphate after phosphate ingestion Table 1. Comparison of various P fractions (pg/mgN) in plaque collected from diagonal quadrants Soluble fraction

Insoluble fraction Total P

Total P

pi

RU

LU

RU

LU

RU

Subject

L+L

I&

Lz

R+L

Lz

LU + RL

ME AM DR ww AT

45.6 55.4 30.5 36-7 28-6

46.8 25.7 35.1 36.7 27.2

53.2 76.6 50.1 43.2 36.3

53.8 45.7 46.9 45.8 38.6

118.5 116.0 109.3 129.6 96%

1@8 116.1 136.3 96.1 112.7

Mean of duplicate readings on pooled samples from paired diagonal quadrants. (d) Effects of sugar with added phosphates on the composition of plaque formed during the experiment

It seemed possible that phosphates might become incorporated in plaque if they entered the mouth while the plaque was forming. This was tested on subjects who had carried out their normal toothbrushing and therefore did not have a large accumulation of plaque when the experiment began. The subjects sucked seven sugar lumps, with or without phosphates contained within them, hourl:y throughout the working day after which samples of plaque were taken at about 4p.m. (i.e. 30min after taking the last sugar lump) from the two pairs of quadrants and treated as duplicates. The composition of the midday meal or snack, taken 3-4 hr before the plaque samples, was not rigorously controlled but was similar within subjects on the days of each experiment. An unavoidable difficulty in this experimental plan is that plaque for control and phosphate-containing sugar has to be collected on different days and is presumably likely to b: more variable than in the previous series of experiments in which plaque from opposing quadrants was collected on the same day before and after taking the sugar. RESULTS

(a) Baseline experiments

(1) A comparison of plaque from crossed quadrants in five subjects (Table 1) demonstrates that differences may exist in the concentration of Pi between the two pooled samples. In addition to the variations between quadrants and between subjects, these parameters Table 2. The effec:t of rinsing with water on plaque inorganic phosphate Subject NJ AT ME KJ Mean 0/0difference

Plaque Pi @gP/mgN) Control After rinse 34.1 38.5 64.4 60.3 38.5

Paired t-test p -: O%)l. Mean of duplicate readings from paired diagonal quadrants.

18.5 26.7 52.2 43.0 26.7 -31% on pooled samples

varied in the same subjects at different times, although each subject tended to have his own range of variation. (2) Three rinses with distilled water were followed consistently by a fall in Pi of the plaque which averaged 31 per cent (Table 2) and was of a similar order to the fall observed by Dawes and Jenkins (1962) after a glucose rinse. (3) Rinsing with a neutral solution of sodium phosphate buffer (0.37 per cent P) gave a 50-60 per cent rise (Table 3). (4) The effect of sucking three sugar lumps (without any additive) on the Pi concentration of plaque collected from 19 subjects 2 min after swallowing the last sugar lump is shown in Table 4. Great variability both in the magnitude and direction of the changes in concentration was found but the average difference was a drop of 11 per cent (from 53 to 47 pgP/mgN). In 7 samples the level fell, in 4 it rose and in 7 there was virtually no change. It may be significant that of the 7 samples showing a fall the average Pi before the sugar was significantly above the general average (76 falling to 49 pg/mgN) whereas in the four plaques in which the Pi rose, the initial Pi was below average (19 rising to 40 pg/mgN). Those with no change were intermediate (51 g/mgN). Phosphate in the insoluble fraction was not estimated in this series because it did not seem likely that this could be affected significantly by this procedure (Luoma, 1963). The change in Pi bore no relation to the weight of plaque collected. The ingestion of sugar lumps over a 15min period did not result

Table 3. Effect of rinsing with solutions of neutral sodium phosphate buffer (=@37 per cent P) on the inorganic phosphate of plaque collected 2 min later Subject DG AT BW NJ DR Mean % difference

Plaque Pi (pgP/mgN) Before rinse After rinse 78.5 48.9 76.8 96.5 47.0 69.5

134.5 83.3 128.9 137.1 70.3 110.8 + 59%

Paired t-test p < O@l. Mean of duplicate readings on pooled samples from paired diagonal quadrants.

620

A. Tatevossian, W. M. Edgar and G. N. Jenkins

Table 4. The effect of sucking 3 sugar lumps on plaque inorganic Plaques in which Pi cone fell Before After sugar sugar 125 24 48 118 60 53 &I 76

phosphate

45 22 62 33

20 10 24 24 19

2 min after ingestion

Plaque in which no significant change occurred After Before sugar sugar

Plaques in which Pi cone rose Before After sugar sugar

79 11 26 78 34 28

(ngP/mgN)

11 138 59 35 48 25 41

(+ ZO%,

73

12 146 56 39 50 23 36 45 51 (no change)

47

49 (-34%)***

51

Mean result of all subjects, 53 falling to 47 (- 11%). Paired t-test: *** p < 0001; * p < 0.05. in a more consistent or more pronounced fall than occurred with ingestion for 3 min (Table 5) although the five subjects happened to have low initial Pi concentrations in their plaque. (h) Effects of sugar with added phosphates on prejbrmed plaque The ingestion of 3 sugar lumps containing a neutral mixture of 2 per cent NaH,PO, and Na,HPO, (0.37 per cent P) failed to alter the average Pi concentrations in the aqueous extracts of 19 samples of plaque from 13 subjects (Table 6a). The responses of the individual plaques were, in most samples, related to their initial Pi. In 8 plaques with a high initial level, the Pi fell (from an average of 54 to 33 pg/mgN), in 2 it did not change, and in 9 with low values it rose (from an average of 26 to 42 pg/mgN). When sugar lumps containing double this concentration (i.e. 0.74 per cent P) were taken, an average rise in the plaque Pi in the soluble fraction of 21 per cent was found but this was not statistically significant at the 5 per cent level (Table 6b). Even with this concentration, 4 out of 9 samples showed a fall in Pi, and in 2 a substantial fall, which did not seem to be related to the initial Pi. Tricalcium phosphate was also ineffective in raising plaque phosphate (Table 6c), as was dicalcium phosphate. With sugar lumps containing sodium phytate, a small average rise (12 per cent) was detected in the solTable 5. The phosphate

uble total P fraction of plaque (Table 6d) but in only one was the rise considerable (27 per cent) and in 2 out of 8 samples a small fall in total P occurred. No significant change occurred in the P of the insoluble fraction of plaque. Calcium glycerophosphate was the only organic phosphate which caused a rise in total P of plaque in these experiments. The total P of the aqueous extract rose by 30 per cent and the total insoluble P by 20 per cent (Table 6e). No significant rise in either P fraction of plaque was detected after sucking sugar lumps containing calcium sucrose phosphate (Table 6f). (c) EfSects on salivary Pi The ingestion of sugar lumps without Pi led to a fall in the salivary Pi concentration (Table 7 column a) partly accounted for by the increased rate of flow which is accompanied by a reduced Pi concentration. The Pi concentration remained low for a considerable but variable time after the third sugar lump (Table 7 column a), presumably until saliva secreted by the gland during the sucking of the sugar was replaced by the slowly secreted, resting saliva with its higher concentration of Pi. A proportion of the observed fall in Pi concentration arose from the dilution of the salivary constituents by the sugar itself. For example, when I lump of sugar (2.2 g) was dissolved in 4 ml of saliva (a volume typical of that secreted in I min during the sucking of a sugar lump) the volume rose to 5.5 ml and the

cone of plaque before and 2min

after sucking sugar lumps for 15 min Insoluble

Subject AT WW AHT MH AM Mean % difference

Soluble fraction Inorganic P Before After sugar sugar 32.4 45.2 28.6 28.5 24.4 31.8

26.1 425 19.9 33.6 31.6 30.7 -3%

(pg/mgN) Total P Before After sugar sugar 37.5 53.8 32.8 32.7 28.7 37.1

28.8 52.5 288 44.1 31.6 37.2 0%

fraction

Before sugar

After sugar

128.0 121.7 96.0 88.6 115.8 110.0

123.5 100.8 101.1 1042 85.5 103.0 -6%

Plaque

Table

6. The effec;: of ingesting

phosphate

after phosphate

ingestion

three sugar lumps containing phosphate

various

621

phosphates

on plaque

calcium

and

Insoluble fraction No. of paired observations

Supplement (a) Neutral buffered sodium phosphate (0.37% P) (b) Neutral buffered sodium phosphate (0.74% P) (c) Tricalcium phosphate (0.37% P) (d) Sodium phytate (0.37% P) (e) Calcium glycerophosphate (1%) (f) Calcium sucrosz phosphate (1%) C = control;

Soluble fraction (pg/mgN) Inorganic P Total P Total Ca. C T C T C T

19

40.9

-

40.0

9

74.8 90.3 (+21%)

9

34.2 31.0 (-9%)

466

42.9

51.1

8

8

-

180.3 225.3 (+25%)

28.1

24.2 (- 14%)

178.1 196.7 (+ 3%)

57.6

29.8 45.0 (+ 51%)

(+ 12%) 87.5 113.8 (+30%)

28.3 34.4 (+ 227) 44.9 i3.4 (-3%)

86.0 83.8 (-3%) 144.8 173.7

34.1 26.1 (-23%)

42.3 48.4 (+ 14%)

28.1 31.8 (+ 13%)

173.3 179.3 (+ 3%)

41.0 (-4%)

6

102.6 116.3 (-12%) 45.7 (-2%)

( + WlJ

2min after ingestion.

Pi concentration fell from 0.24 to 016mg/ml. The magnitude of the fall in Pi concentration of saliva arising from dilution will clearly depend on its rate of flow. When the sugar containing P, was held in the mouth for 1 min, the maximum Pi concentration in saliva averaged only 1.7Omg per ml in 12 observations in 6 subjects (Table 7, column b). The 0.37 per cent P of the sugar was greatly diluted in the saliva to an average of one-third of its original concentration (8.1 mg Pi per lump dissolved in 5.5 vol of saliva = 0.15 per cent). When samples were taken at the end of each minute without the inhibition of swallowing, the peak Pi values were even lower (Table 7c) and below the average plaque fluid concentration as discussed later. (d) Effiectsqfsugur with added phosphates on the composition of plaquefbrming during the experiment The results of 2 1 experiments on 7 subjects showed that, even when the Pi was present throughout the plaque formation, the presence of Pi in the sugar lumps

did not cause a significant rise in the soluble P, concentration of the plaque (Table 8) and that an average fall of 18 per cent (p < 0.05) occurred in the total phosphate of plaque of the soluble fraction in 17 out of 21 experiments. In this series of experiments, the determination of the nitrogen of the plaque was omitted and the results are expressed on a wet weight basis. The control P levels may be compared using the factor of = 175 pg N per mg wet weight of plaque, derived from previous results, and are found to fall within normal limits. With phytate, 37 paired experiments on 14 subjects showed average increases of 5 and 18 per cent in the concentrations of Pi and total P, respectively, but neither increase was statistically significant and in some subjects a fall in total P was found. For the latter subjects, control values tended to be higher (mean = 1.33 pg/mg wet wt) than the general average. If those results showing a control total P level below 1.00 pg/ mg wet wt are selected, the effect of the phytate-con-

Table 7. Effect of sucking sugar lumps with and without (a) Sugar without

Before sugar After 1st sugar lump After 2nd sugar lump After 3rd sugar lump 1 min After sugar 2 min After sugar 7 min After sugar 12 min After sugar

T

(-2%)

T = test sample collected

Saliva collected

(+?$$) C

P,

Pi on the P, cone (mg/ml) of saliva

(b) Sugar containing in saliva secreted in 1 min

Pi

Mean from 12 expts and 6 subjects

Mean from 12 expts and 6 subjects

(c) P, sample at end of I min after natural swallowing Mean of 4 expts in 4 subjects

0.14 0.05 0.05 O-04 0.07 0.07 011 0.12

0.16 1.43 1.43 1.76 0.17 0.13 0.13 0.15

0.18 0.42 0.50 0.49 023 0.18 0.16 0.15

Pi cone of plaque fluid varies between

0.7 and 3.1 mgjml (Edgar and Tatevossian,

1971).

622

A. Tatevossian, W. M. Edgar and G. N. Jenkins taining sugar appears as follows: control, 0,62pg/mg wet wt, phytate 0,95pg/mg wet wt (53 per cent rise; 0.025 > P > 0.01). Calcium glycerophosphate and calcium sucrose phosphate, when incorporated at the 1 per cent level into sugar lumps, both led to small average increases in Pi of the soluble fraction of plaque (7 and 12 per cent respectively) neither of which was statistically significant However, the total P of the soluble fraction rose by 41 and 38 per cent respectively, both increases being highly significant. The total P of the insoluble residues of the plaque showed small rises averaging 20 and 11 per cent (not significant) after sucking the sugar lumps containing glycerophosphate and sucrose phosphate respectively. DISCUSSION

The baseline studies show large differences between the concentration of Pi in plaques from different individuals and that pooled plaque for one-half of the mouth may not give the same average values as that from the remainder. Consequently, it is impossible to detect an average change in the Pi of plaque from a group of subjects unless the group is fairly large and the findings consistent. Although the Pi of plaques from one individual tended to be similar to each other, occasional values showed wide variation. The 30 per cent fall in plaque P, following a rinse with distilled water shows that the plaque Pi can be removed, although it is surprising that contact with water for 3 min did not wash out the Pi more effectively. A possible reason for this may be that the structure of plaque, which presents many microchannels in which free water and dissolved substances would be held by large surface tension forces, may present a diffusion barrier which limits the changes in the deeper plaque layers. It is also clear that the fall in plaque Pi observed by Dawes and Jenkins (1962) after a glucose rinse could have been due mainly to a washing-out effect rather than the effect of bacterial metabolism and Pi uptake by the bacteria. The experiments in which the mouth was rinsed with sodium phosphate solution showed only a moderate rise in plaque Pi and this could also be explained by a limitation in diffusion for plaque and phosphate in addition to the already high level of Pi present in plaque fluid (Edgar and Tatevossian, 1971). The results of the experiments in which sugar lumps without Pi were sucked suggest that the effect on plaque Pi depends on the concentration of Pi already present. If this is high, the level tends to fall, because the increased rate of flow of saliva. in response to the sugar lumps, tends to decrease the salivary Pi concentration and could create a gradient favouring outward diffusion from the plaque, as reported by Luoma (1964). If the concentrations in the plaque were already low, this might not occur. There is no proved explanation for the rise in P, observed in the four plaques with initial low values but, in any case, more work would be required to establish this effect. It might be speculated that the absence ofa concentration gradient prevented outward diffusion of Pi and that the acid formed in the plaque, after the sugar, dissolved some Pi possibly from the enamel surface, as suggested by Ferguson and Thomas (1975) for their own similar findings. Although presumably similar changes

Plaque phosphate after phosphate ingestion occurred in the other groups of plaques, they may have been eclipsed by larger changes resulting from the con: centration gradients. The similar absence of a consistent rise in plaque Pi after ingesting 0.3:’ per cent P in sugar may be explained from the data collected on the concentrations of salivary P, during these experiments. In addition to the fall in salivary Pi concentration following the increased rate of flow (Table 7a), the Pi in the sugar is greatly diluted by the saliva and by the sugar itself, as mentioned above. Even when the whole of the Pi in each lump comaining the additive was allowed to accumulate in the mouth without swallowing, thus building up to higher concentrations than would occur during normal consumption, considerable dilution occurred by the rapidly flowing saliva. The resulting range of concentration of Pi (1.2-2.3 mg/ml) did not exceed that reported for plaque fluid itself: @7-3.1 mg/ ml (Edgar and Tatevossian, 1971). In other words, the Pi concentration of plaque is already so high that it is difficult to raise the saliva concentration so that it exceeds that of the plaque Auid. When the Pi in the sugar was not allowed to accumulate, the salivary Pi concentrations wert: even lower (Table 7c) and during normal eating the,y would presumably lie between these two values. The finding that in those plaques with low initial P,, the concentration did rise after ingesting sugar containing this supplement is consistent with this conclusion, as is the larger effect on plaque Pi (average rise of 21 per cent) of the higher concentration (0.74 per cent) of Pi in sugar reported in Table 6b and the 0.63 per cent used by Luoma et uI. (1964). The absence of a consistent effect on the Pi of plaque during the ingestion of sugar lumps containing D37 per cent Pi as tri- or dicalcium phosphate shows that insoluble particles in rugar are not readily incorporated into preformed plaque. As the Pi of saliva rises when sugar containing Pi is dissolved in the mouth, contrasting with the fall in salivary Pi with sugar alone (Table 7), it might be expected that fewer plaques would fall in Pi with the supplemented sugar than with the sugar alone. A slight tendency in this direction was observed; when sugar alone was sucked, the plaque Pi showed a non-significant fall which was absent when the sugar contained 0.37 per cent Pi and was reversed (an average rise of 21 per cent) when the sugar contained 0.74 per cent Pi. The result would be greatly influenced by the initial Pi of the plaques, however, and comparisons of the effect of sugar with and without Pi on plaques with similar initial Pi values would be necessary to establish this point. Relation between t,hese results and those of clinical ex-

prrirmwts on phosphate The finding that calcium phosphate does not readily enter plaque suggests a possible explanation for the negative results of.the insoluble CaHPO, used in some of the trials on caries in man. The reductions in caries reported by Stookey et al. (1967), Carroll et al. (1968) and Brewer et al. (1970) with sodium phosphate, although not confirmed in a similar experiment by Petersen (1969).areat first sight difficult to reconcile with the present work, parlicularly as these workers included the Pi in only one food. normally eaten once per day. However, the present results suggest that plaques initially low in Pi, which Ashley (1972) has stated (at least

623

in plaques from the upper posterior teeth) are associated with high caries rates, might increase in Pi after ingesting it with sugar. If this occurred, then Pi ingestion might benefit those most prone to caries but have little effect on those with a low caries increment, assuming that findings of Ashley are confirmed and apply to all the teeth. It is possible that in those experiments in which Pi supplements have reduced the average caries increment, the effect was exerted on those with plaques low in Pi. The initial Pi of plaque is unlikely to influence the entry of insoluble phosphates, which agrees with the finding reported in Table 6, and may explain why CdHPO, as an additive to sugar has not influenced caries. This work does not explain why Pi reduces caries in rodents. Possibly differences in the chemistry, permeability or thickness of plaque, which appear not to have been investigated, might account for these contradictory results. The differences in composition between human and pilocarpine-stimulated rat saliva reported by Ericsson (1962), and which he suggested might explain the contrasting effect of Pi on caries, have not been confirmed on unstimulated rat saliva (Tatevossian and Wright, 1974). The relation to organic phosphates in caries control The results in which sugar lumps containing sodium phytate were taken by subjects with a plaque already formed show that this substance does not readily permeate plaque in amounts which are detectable in the pre’sence of the great variations in organic phosphates in plaque. When the phytate was taken continuously throughout the formation of the plaque, a situation resembling the conditions in the mouths of subjects who clean their teeth regularly, a non-significant increase in the average organic phosphate concentration was found. As mentioned above, the experimental design, involving comparisons of plaques collected on different days, tends to lower the likelihood of observing significant changes in plaque chemistry. The failure to produce a significant rise in total P with phytate (Table 8) might be explained by the unusually high control values shown by some subjects in the phytate group; the absolute average increase in total P with phytate (0.18 pg/mg) was not greatly different from that with the other organic phosphates (0.25 and 0.23 pg/mg). It is also possible that some phytate may have diffused through the plaque and become bound to the enamel surface. With calcium sucrose phosphate, the increase in total P in plaque forming during ingestion of experimental sugar lumps was statistically significant. A complicating factor with this substance is that when water is added to the dry powder, comparable to what occurs when dissolving it in the mouth, it forms a viscous mass which is not readily dispersed (Colonial Sugar Refining Co. Ltd., 1970) and this may have led to its failure to enter a preformed plaque in detectable quantities. Calcium sucrose phosphate, however, is very soluble if added to a large volume of water with stirring, conditions unlike those in the mouth. It is possible that it became deposited on to the outer surface of preformed plaque but as a thin layer too small in quantity to detect. When the plaque was built up during the experiment, however, a deposit may have occurred on each successive layer of plaque (i.e. the plaque would contain seven layers as opposed to one).

624

A. Tatevossian, W. M. Edgar and G. N. Jenkins

Calcium glycerophosphate is the only substance which, in these experiments, entered both preformed and forming plaque, although calcium sucrose phosphate in rinsing solutions which do not become diluted by stimulated saliva as much as sugar lumps may be more likely to enter the plaque (Clarke and Fanning, 1971). These results show that, under the conditions of these experiments, the three organic phosphates tested, especially calcium sucrose phosphate and calcium glycerophosphate, enter human plaque when they are present during its development. This finding encourages the belief that all three substances might reduce caries in man, as they do in experimental animals. Results reported by others since these experiments were carried out have been contradictory. Bowen (1972) reported, in plaque from monkeys receiving sugar containing 1per cent glycerophosphate for 22 months, a rise in *he calcium but not in the phosphate concentration, whereas Brook, Gawthorpe and Winter (1973) found a significant rise in total P (p < @Ol) and a smaller rise in Ca (0.05 > p ( 0.1) in plaque from human subjects receiving 1 per cent calcium glycerophosphate in milk-cereal tablets. Stephen rt ul. (1973) on the other hand, found no change in the composition of plaque in children receiving tablets containing 20 per cent calcium glycerophosphate after meals for 3 weeks. The expIanation of these discrepancies is not clear but several possibilities exist. The experimental procedures have differed, including the time between taking the additive and collecting the plaque and whether the sample excluded plaque from the lingual surface of the lower incisor which is higher in Ca and P than plaque in general (Dawes and Jenkins, 1962; Ashley, 1974). There may be inherent difficulties in changing the composition of plaque arising from a selective permeability, alternatively, the differences may arise from technical difficulties of measuring changes in a material as variable in composition as plaque. Acknowled~~ymlents-We wish to thank the Medical Research Council for a grant to A.T.. to Mr. W. G. Wright and Mrs. D. Coutts for skilled technical assistance, to the Colonial Sugar Corp. for a gift of calcium sucrose phosphate and to the subjects for their cooperation,

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