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Buffering systems in the mouth
BUFFERING: W.
T~TJJFWIWAI,,
SYSTEMS
K.Sc.,
B.I>.S.,
IN THE
I).Prr~r,.,*
MOUTI3
SYIINEY,
L2~~~~~~~~~
Introduction IIE saliva possessesconsiderable ability to maintain its pH unchanged because of the presence of buffer substances whose eftlciency depends upon t,he relative amounts of acid (or alkali) and of buffer present. There are two aspects of the buffering action of the saliva in relation to the commonly accepted chemicoparasitic theory of dental caries (Miller). The first a.spect is the potential buffer capacity of saliva and the second is the effective buft’ering mechanism beneath and/or in the bacterial plaque on the tooth surface. The inability of saliva to reach certain sites in relation to the teeth (for example, interproximally and in pits and fissures on oeclusal surfaces) is an important limiting factor in the initiation. of dental caries. Some experimental observations on the permeability of capillary spaces by ionic solutions have been made by Kevin.’ His model experiments show how an obstruction, such as food, at the entrance to a capillary space hinders interchange of ions between the enclosed space and the exterior. A similar phenomenon would occur with bu-ffer solutions. Further, it has been claimed by Stralfor+ and Fosdick3 that ‘ ’ plaque ’ ’ ma,terial has an important buffering action. In the literature on the buffering properties of saliva, two terms are frequently used synonymously. These are buffer capacity and bufering efect. Bufer cupacity refers to the ability of a solution to resist change in pH on addition of acid or of alkali. It expresses the resistance to pH change at a given pH value, and can be calculated from a, knowledge of the concentration of the buffering systems in the solution. The slope of the titration curve at a particula(r pH value enables the buffer capacity at that value to be determined experimentally. Differences between the calculated and observed value for buffer capacity at several pH levels led Ericsson4 to suggest that proteins may play a,n important role below pH 6.0. Sellman and Wah Leungs also suggested tha,t protein might play a.n important role as a buffer at pH values below 6.0. The other term, buj‘“ering efSect, refers to the amount of acid (or alkali) required to change the pH of the solution from one value to another. The .__I
T
*Research
Officer.
Tnstitute
of
Dental
Research,
825
United
Denta.
Hospital.
BUFFERING
SYSTEMS
JN
MOUTH
829
range is chosen arbitrarily and for saliva the ranges pH: 7.0 to 6.0 and pI1 7.0 to 5.0 or 4.0 are used. So-called buffer capacity tests of saliva are, in fact, usually determinations of the buffering effect. Nevertheless, each of these terms, buficer capucity and bufel-ing e#jcect, merely relates to a different manner of describing the same property. The Buffer Systems in Saliva.-The substances in saliva which have been regarded as buffers are bicarbonate, calcium phosphate, protein, mucoid, and a,mmonia. Considerable attention has been given by various authors to the concentra.tions of calcium a,nd phosphate ions in saliva and to their buffering ability. The role of calcium and phosphate ions is related to the maintenance of the solid phase of the tooth insofar as an excess of either will prevent the solution of the tooth surface; in accordance with the law of mass action. Buffering action in saliva, as stated previously, is concerned with the maintenance of a relatively constant pI1 value in the mouth. When excess acid is present, it is only after the buffering systems have played their part that the pH can fall to values which affect the solubility of the mineral phase of the enamel. Of the compounds listed, ammonia has no buffering action in the strict sense, although as a base it does play a part in neutralization of acid. From the quantitative point of view, its role is not significant. Bicarbonate has been shown by several workers to be an important buffer in saliva. Ericsson” calculated the buffer capacity of bicarbonate and related the values to the observed values in several samples of resting saliva. Wah Leung” approached the problem in another manner. Using saliva from eleven subject 3, he studied the carbon dioxide-combining power of saliva under different pressures of carbon dioxide and, from the data, calculated the buffer capacity of the bicarbonate systern between pI1 6.8 and 7.2; this work showed bicarbonate to be the ma,in salivary buffer. Each of these methods has given information by what may be called an “indirect approach,” because the va,lues for the buffer capacity of bicarbonate and phosphate were calculated from their concentrations in the samples and compared with the values obtained for whole saliva frorn titration data. In another study, separation of the salivary buffers by stepwise elimination has enabled the role of each buffer system to be individually and directly observed.7x s Quantitatively, bicarbonate was again found to be the main buffer in saliva, and phosphate was found to play a less important role. It has been clearly demonstrated that bicarbonate and phosphate together account for nearly all the buffering properties of whole saliva, resting or activated. Furthermore, the difference in. buffering ability between resting and activated saliva is due to an increase in the bicarbonate content of the latter. The buffering mechanism of bica,rbonate and phosphate is well known. For the preservation of enamel surfaces, however, solution of bicarbonate may be as important as t,hat of calcium and phosphate ions. In bone the apatite Ions found in tissue fluid are crystals accurately reflect their environment.”
lt can be seen from the above t,h;tt any change: in pI1 will affect the ioniza.t,ion of both bie~rbonxte and lphosl)hate b(:(;irlls(; the respective viilues for K are constant. The precise values Car these constants under these conditions arc not, known. I’rotoin (rrnlcoid) >lntl the prcscnec of ot,her ions will have an important bearing on these values. I Iowever, on formation of enough acid to exhaust, these buft’e~, the only way in which buffering can continue is hy the irttrotluotion 01 a.cltfitiolial buffor. 4t is generally ;~ssumotl that, beneath the plaque, stltur:Lti(m conditions exist for the mineral s;rlhstance of enamel. When acid is formotl al, this site and the bnfllers have been exhausted, solution of the miltera phase occws in order t,o satisfy t,ht: solubility product. relationship. ‘I’he affinity for calcium ions exhibited hy protein, including mucoid, has an The solubility product o 1’ the importnnt, effect on t.he solubility relationship. tooth mineral (for instiznoe, hydroxyapatite) is not krtown beaausc of variation produced hy the environment, thus making it impossible to calc:uI~~te the effect o F given changes in the concentrations of ions under given conditions. Nevertheless, from basic princil)lcs, it WOuItl seem thMt ilCtCr tT~llOV~.l Off adsorbed ions the ortle~ ol’ solution of the ions in the crystal structure WOI~C~ depend largely upotl the size oE the ionic radii, the intcrionic forces, unrl the ionic composition of the surrounding mctliurn. The suggestion that proteirt and rrrucoid exert an important buffering This would bc expected in action I~low pl-I. 6.0 has not l~!n substantiated. view of the small concentrations of proteins in saliva and, in cornpnrison with bicarbonate and phosphate, these proteins exert il negligible effect. The same order o-f impodi~~lce was found for tho buffer systems in both resting and autivatcd diva obtained from two groups of thirty-two and thirty-six subjects, respectively. .Hicarhonate was the mqjor buffer, whereas I)hosl)hiite was tha minor one, and rnucoid had no detectable buffering effect. In resting saliva, however, the bic;trbonate values are more variilbl(: than in activated saliva. As a. buffer, phosphate is thwefora rrlorc important in resting saliva
BUFFERING
SYSTEMS
XN
in activated saliva. E‘urthermore, resting effect than activated saliva, because of its lower tion of salivary flow by chewin g paraffin wax saliva which contains greater concentrations of than
The Buffer
Systems
in Salivary
Sediment
MOUTH
831
saliva has a smaller buflering bicarbonate content. Stimulaproduces a la.rger volume of bicarbonate and sodium ions.lO
and Plaque.-Stralfors”
ex-
amined the buffering properties of salivary sediment and dental plaques alld found that these gave similarly-shaped titration curves for a given mass of each ma.terial. Plaque and sediment consist largely of bacteria, and their buffering properties might be attributed to their protein nature. However, a variable proportion of the buffering eEfect produced by salivary sediment was found to be due to adsorbed bicarbonate, whereas no phosphate was adsorbed.” On examination of plaque materia,l, no adsorption of bicarbonate was detected. Thus, the nature of the buffering systems in salivary sediment and in plaque would appea.wto be difYerent. Although sediment exerts a buffering effect, it is only a very small portion of the total buffering effect of the whole saliva. The buffering properties of plaque material must be considered also in relation to the ability of the bacteria within it to metabolize organic acids. The evidence indicates that the buffering power in plaque is attributable to the bacteria it contains. If mucoid is present it would not add to this buffering effect beea,use mucoid ha,s not been round to exert a buffering effect over a wide range of PH. The permeability of the plaque to ions would bc reduced by the presence therein of mucoid, and it is by this action on the diffusion rates of ions that mueoid could play a significant role.
Experimental Buffering Effect and Volume
Relation Between of Saliva.-A group of sixty-seven children a,t Hopewood H-ouse, Bowral, New South Wales, was used in. this study. The children’s home is a.pproximately seventy miles from the laboratory and it was necessary, therefore, to devise a method of collection and preservation of samples of saliva. Details of the procedure adopted and its value are given in an appendix to this article. It was found that a direct relation existed between the volume of five-minute samples of activated saliva and buffering effect (Fig. 1). From the statistical analysis, it can be seen that the correlation coefficient is positive, indicating a. direct relationship between the two variables. The value of the coe.fficient shows that this set of’ results could be expected to occur by chance only once in 1.,000 times. Therefore, the relation between volume and buffering cfFect is highly significant. This observn.tion for five-minute samples of saliva is in agreement with the results obtained by Dreizen a.nd his a.ssociatesLOwith thirty-minute samples of a.ctivated saliva.
Variation in the Buffering Properties of Saliva.--The effect of the following conditions on the buffering effect of saliva was observed : (I) prolonged chewing as may occur during a meal and (2) chewing paraffin wax for a similar period. Zk’urthermore, when changes in volume and/or buffering quality
nccmrred, observations The rela,tion between concentra?tion
were made of any changes in a particular buffer system. bicarbonate c~oncrntration and changes in the sodium inn
was also noted
.
activated
This
Fig.
is
l.-Correlation saliva.
approximately
Correlation
0.1
between volume and bufferingeffect of five-minute coefficient I‘ = +0.364 rvn-2 t E , where n is number of \iL i-2 0.364 v 65 = v 1 - 0.13241 0.364 x 8.06 p3,731. = 0.931 per cent level of significance, which is highly significant.
samples
of
observations
The fifteen children chosen as subjects were again from IIopewood House. subjects had a very low dental caries experience.*l 1. Effect of eating a meal: Saliva was collected immediately before and after the children had eaten a meal conta,ining grated raw beetroot and carrot, sliced raw cabbage, cauliflower, a,nd soya bean cooked with onions and tomatoes. This was followed by cubed fresh pineapple with junket and wheat germ. The meal was completed in approximately twenty min.utes. Paraffinactivated saliva (five-minute sa,mple) ‘was collected from each subject in a tube containing paraffin oil; it, was frozen at, -18 degrees and kept at t,his temperature until required for titration. These
BUIWERING
SYSTEMS
IN
833
MOUTH
In five of the fifteen subjects, the buffering effect ($1 7.0 to 6.0) was greater after the meal than before it. The other ten subjects did not show any appreciable change. After the meal, six subjects showed an increa,sed buffering power for the range of pl-I 7.0 to 4.0. It should be noted that the increased buffering power was not accompanied in each case by a larger volume 0.f saliva collected in the five-minute period (Table I). The same type of food produced different salivary responses in these subjects. TABLE
-
T . BUFFERING BUFFERING
SU:CT
1
0.56 (h;=
7.0,To
ERFECT
“.& 0.65
OF SALIVA (ML.,
/
0.02
($T
0.92
BEFORE N
ANII
Hd)
7.0,To
0.59
0.63
1.0
3t 4" 5
0.4 0.42 0.56
I!:: 0.56
0.67 0.69 0.89
&et 8 9
0.46 0.5 0.43 0.53
0.46 0.61 0.42 0.5 0.4 0.47 0.44 0.35 0.61 0.53
0.82 0.73 0.74 0.88 0.67 0.75 0.78 0.64 0.83 0.85
12
(A) 7.0 to
Refore meal; 6.0% and pH
(B) after 7.0 to 4.O.i
meal;
increased
A MEAL
-___---
VOLUME
4.yB) 1.05
0.37
0.46 0.47 0.38 0.48 0.50
AFTER
--
2*t
0.39
pl-I
EFFECT
?;r,
OF
(ML& 8
7
0.75 0.69 0.89
11 16 15
:i 15
0.73 0.97 0.76 0.85
189 13 18 12
12 SO 14 16
15
18 0.72 0.65 1.0 0.82 buffering
20 16 10 5 28
11 11 3 18 effect
after
eating
for
range
2. Effect of chewing para& wax: In the preceding experiment, the aubjects chewed for approximately twenty minutes without a consistent change in bnffering effect. If, instead of food, the subjects had chewed a. bland material such as paraffin wax, what changes might be expected in buffering effect during the sarne period and in the first 30 ml. of saliva produced? Activated saliva, was collected in four consecutive five-minute periods and on the following day from the same subjects in three consecutive 10 ml. fra,ctions. In each case the saliva was collected under paraffin oil and the samples were frozen (-18 degrees). The results of titration for eight subjects ase shown in Pig. 2. For each subject, the amounts of acid required to change the pF1 from 7.0 to 6.0, 5.0, or 4.0 are shown. With one exception, changes in buffering effect among the 10 ml. samples and the five-minute samples were similar for the three ranges of pH shown. Bach subject gave values for the buEering effect in the 10 ml. samples which were very different from those of the five-minute samples. In general the values for the 10 ml. fractions were less than the values for the five-minute samples. The buffering effect (pII 7.0 to 6.0) of the fourth five-minute sample was similar to that of the first fiverninute sample, although. differences occurred between these two and the In addition, the ehangcs in buffering second and. third five-minute samples. effect in the two series varied from one subject to another and showed no general trend.
removal of bicarbonate by carbon dioxide-free air below pI1 5.0.8 From the close relation bctwec~r the changes in ln&~ring efkct a.rld bicarbonate, it I’ollows that the remaining buf?er, phosphate, must remain virtually unchanged. Having found that bicarbonate was the only variable among tho anionic constituents of the salivary buffer systems, it was of interest, in view ol’ the observations by Dreizen and his associates’” on K’, Sa-! and IICIO’,,, to observe the concentrations of Na-. and K+. lkcause the curves I’OT the lbicarbonatc content and buffering effect were parallel or nearly so in each east, the values for bufkrirrg ci’fect were omitted to avoid undue complexity in the graphs.
Fig. a.---Variation samples of a&ivated 5.0. IPC, Buffering
saliva. effect;
in
buffering Values HCO’a,
effect and in rniMli&rs, bicarbonate.
bicarbonate 0.02 N
ALI- b
HCl
per
milliliter
COnCf?ntl”atiOn.
of’~a~
3 ‘for
and BH’
are conserutive (EL) 7.0 t0 6.0
an
fiveminute 7.0 (h)
b
to
b
a
l?ig. 4. Variation in buffering r:fCect and bicarbonate concentration. I, 2, anti 3 :LI’C Wig. 6: Iklation botwncn volume and bicarbonate soclium, and potassium concentrations of effect ; HCO’II bicxrbonatc. of saliva for pK (a) 7.0 to 6.0 and (b) 7.0 to 6.0. EC. liuffwing
The results
for the four five-minute samples (E‘ig. 6) showed that the X’a+ was parallel to that. of Ff CO’, in Xos. 1 and 3, whereas in the inde&her four subjects this was not the case. 111 general, l<+ levels VilI+fXl pendently of IICO’~, but in a Pew instances K’ values changed in a manner similar to l,bose for LSaA. When the volume was constant, as in the three consecutive 10 ml. wmples, In the bicarbonate (and therefore the bufferin g effoal ) was not constant. ;Icltlitiorr, no relation was lroted hetwem t,he volume of consecutive five-minute s:l mples oI’ sdivn. wn(l either its bicarbonate, Sa+, or* R+ cont,ent. ronrentralion
NPfo
Fig. fractions N I-TCI.
5.-Relation of activated
between saliva.
bicarbonate, NC&+ and
N+
sodiunl, values
in
anal potassium mE/l. TTCO’J
concentrations values as
milliliters
in
IO
ml. 0.02
hh
Fig.
F.--Kelation five-minute
between samples
of
volume and activated
bicitrbonate, saliva. Nm+.
sodium, K+. and
anti potassium HCO’:I values
as
concentrations in Fig.
5.
of
Salivary Buffer Tests and Dental Caries.--lkwxr’” has descCbed a, classifi~~aiion based on the FI~nonnt of acid rtquirccl to cha’nge the pH of a definite quantity of saliva from 1,l-T 7.0 I.0 6.0. These grxcles indiea.lc the soslv~pt~ibititp of t,he snhjcct to tlcnl al caldies, the correlation being rrradc i’rom D31P valacs for children ant1 young adults in whom the D&E value is related to caries activity. The results of a correlation of the buffering effect with the caries inritlencc of sixty-five children rrorn Hopewood FIouw is shown in Table IT. TT. I~TJFFE:KTNC:
TABLE
RFFRCT
AND
DENTAL
CARIES
TECIIHW~E
--__ NUMBERI" SUSCRPTlRIldTY
INDICATED
BUFFER
TESTY 2
_______I~_~rt + ++ +++
I mmulle Slightly susceptible Moderately suseeptilk I-Tiehlv suseeotible
R&p< ear~cs
A. Number slightly K. Number (++,
Total Per cent
++++
of immune (2) or suscepti hle ( +) in other grades +++,
i+++) --
susceptible
(B
--
x 100)
tOtR.1 :sObtained from of Dental Research,
results Sydney)
NEW
BY 0
I
13 13 10 1
1
Id3SIONS"
JANUARY,
Jhh’l:ART-,
1954 3 0 2 1 2
-1
4 3 4 1
-- 2 2 0 5 0
I
1
1%.%,
TO
4 1 1 0 0
/
5 2 0
0
0
0
0
0
0
ic,
7
2
2
2
2
11
5
5
3
0
0
37
12
7
-___ 5
2
2
30
42
72
GO
of clinical examinations anrl R. Harris (IJnitrd
carried Dental
out by Hospital,
H. R. Sullivan Sydney).
-
(Tnstituto
The results show tha.t in these children, in whom dental caries is a slow process and not very prevalent, I1 t,he buffer test of the saliva, places 30 per cent, o-f those who had no new lesions in the suseeptiblc cla.ss. Likewise, of those who had one, two, or three new carious lesions, 42, 72, and 60 per cent, respectively, were classified on the buffer test as susceptible. Two conclusions may be drawn from these results: first, that some of these children are cariesfree, although the buffer test shows them to be susceptible; second, others have new lesions, although they ase classified by the buffer test as caries-immune. Huch. observations show the limitations to which this test, like any other available test for caries susceptibility, is liable. Susceptibility to dental caries may be regasded as being due to a, disturba.nce of the dynamic equilibrium between acid production and neutralization on the tooth surface, the determining factor being the rate of acid formation rather than the capa.city for neutraliantion (that is, buffering effect). In the instances just mentioned, in those children who were classified wassusceptible to denta, caries on buffer tests but who did not have any new lesions, it is probable that lack of a powerful acidproducing system was the determining factor. Omission of the substrate for acid formation and not the lack of the enzyme systems of the bacteria, most probably accounts for the observations among these children. In those susceptible children who were classed as immune on the buEer test, the acidforming system probably was t,oo powerful for the buffer mechanism to counter-
BUiWhXlNG
SYSTEMS
IN
MOUTH
839
B’inally, considering the entire group, the results show that as the caries incidence increased, the buffer test became a. more reliable method for the assessment of susceptibility to dental caries.
act.
Summary I. The buffering systems in saliva, salivary sediment, and plaque are disBicarbonate is the major buffer in activated saliva, phosphate accounting fol* the remainder of the buffering effect. Salivary mucoid and bacteria have an insignificant buffer action in whole saliva. In resting saliva, the buffering effect is decreased because of the smaller concentration of bicarbonate in resting sa,liva than in activated saliva; therefore, phospha.te becomes relatively more important. Salivary sediment and plaque have different buffering systems. The former has a, variable amount of adsorbed bkrbonate which plaque does not possess. 2. The effect of eating a meal on the buffering power of the saliva of h constant increase or decrease in buffering fifteen subjects was examined. after the meal was not observed. 3. Paraffin-activated saliva, collected during a period simi1a.r to that taken for the meal, did not show any appreciable changes in bufFering properties, as determined by comparison of the first and la.st five-minute samples of saliva. During the test period, however, changes were observed which were paralleled hy changes in bicarbonate concentration. No consistent relation wa.s found between HCO’,, Na+, and K+ concentrations and volume of sample. 4. A significant relation was observed between buffering eft’ect for the sample, pH 7.0 to 6.0, and the volume of five-minute sarnples of activated sa.liva for a group of sixty-seven subjects. 5. The limitations of the buftering test in attempting to assess caries susceptibility were investigated in a group of sixty-five subjects. cussecl.
Appendix Effect of Freezing and Storage at -18 Degrees on the Buffering Properties and Content of Saliva.-If saliva is not rapidly chilled after collection, its buEering increases on standing. The problem of collecting saliva for buffer tests about miles away from the laboratory is therefore obvious. A method was required would enable collection of saliva from the group of children, transport of these samples to the laboratory, and storage without appreciable alteration in the buffering pro&rties of the samples. In a preliminary study, samples of activated saliva were collected under a layer of parafin oil and 2 ml. quantities were immediately placed in a cleep freex at -18 degrees. On titration after one, three, seven, fourteen, and twenty-out: days, it was found that the budering power had not appreciably altered (Table III). It freeze it, and transport the therefore became possible to collect saliva from the children, deep freeze containing the frozen samples to the laboratory and that, too, without risk of thawing. The
Mucoid power seventy which
Further
difficulties rapidity with by collecting the
tests were rude of the advantages of freezing saliva. For example, one of associated with the determination of the mucoid content of saliva is the which mucoid is depolymerieed by salivary mucinase. Knox~~ overcame this It is not always convenient to estimate the saliva under ice-cold conditions.
12
0.5 0.G 0.65
i3 14 15 16 17 18
mucoid degrees mtlcoid
0 . 5” i
0.75 0.45 0.4 of the sample way in which
6 _..
I
8
4 R 6 7 8 9 10 II
4 8 4 8 4 16 8 14 8 8 4
18 JO
0.6 0.6 0.48 i93 0:47
II::
Sl;U.JEC’l’
14 15 16 17
0.75
O:;i 0.6 0.5
--_ 0.5
content innnediat~ely after collection has been found to be a satisfactory content (‘l‘ablo TV).
12 13
0.51 0 52
of saliva, IO provent
\
(-
-..
and storage at -18 gross changes in
.-~
_....-_
--__8
4 2
4-8 2-4
8
4 & 4-8 4-8 2 2 2
: 7-2 4 4 8
4 4 4-x
References 1~ Kevin, 2. 3. 4. 5.
R. l3.: The Diet and Mastication : Their F:fl’ects 011 Diffusion and on the inception of Dental Caries, Dunedin, N.Z., 1951, Progress Printing Company. Stralfors, A. : The l3uffos Capacity of ~I)ontal Plaques, J. I). Ros. 27: 587, 1948. Some Xew Concepts Concerning the Role of Sugar in Dental Caries, Fostiick, J,. 8.: ORAT d ‘%X0., k (h”d, %bKk, AiTl) ORAl, ~‘h’~Tf. 5: 615, 1958. ISname-Apatii;tt Solubility, Acta odont. soandinav. 8: Supp. 3, 1949. b:ricssort, Y.: The Buffer Value of Saliva and Tts Relation to l)ontal Caries, Ao1.a odont. Sellman, S.: scandinav. 8: 244, 3949.
BUFFERING
SYSTXMS
IN
841
MOUTH
6. Wah
Leung, 8.: A Demonstration of the Importance o-F Bicarbonate as a Salivary Buffer, J. I). Res. 30: 403, 1951. 7. Lilienthal. B.: The Buffer Systems in Human Saliva. Dent. .J. Australia 25: 216, 1953. 8. Lilienthali B.: An Analysis of the Buffer Systems in ‘Saliva, J. 1). Res. (in press); 1%X. 9. Neuman, W. F., and Neuman, M. W.: The Nature of the Mineral Phase of Rone, Chem. Rev.
53:
1, 1953.
10. Dreizen, S:, Reed, A. I., Neidormeicr, W., and Spies, T. I).: Sodium Constituents of Human Salivary BuWers, J. D. Res. 32: 497, 1953. 11. Lilienthal, B., Goldsworthy, N. E., Sullivan, H. R., and Cameron, I). of the Children of Hopewood House, Bowrd, New South Wales: Dental Caries Extending Over Five Years (1947.1952), M. J.
and
Potassium
as
A.: The Fologj I. Observations On Australia 1: 878:
1953.
12. TIewar,
Margaret Caries. Part Standarclised 13. T
R.: Laboratory Methods for Assessing Susceptibility to II. Correlation of Results Obtairled by Clinical Examination Laboratory Methods, Dent. J. Australik 22: 24, 1950. Salivary Mucoids and Mucolytie Enzymes, Master of Science Sydney, 1951.
Dental and
by
Thesis,
T~TJJFWIWAI,,
SYSTEMS
K.Sc.,
B.I>.S.,
IN THE
I).Prr~r,.,*
MOUTI3
SYIINEY,
L2~~~~~~~~~
Introduction IIE saliva possessesconsiderable ability to maintain its pH unchanged because of the presence of buffer substances whose eftlciency depends upon t,he relative amounts of acid (or alkali) and of buffer present. There are two aspects of the buffering action of the saliva in relation to the commonly accepted chemicoparasitic theory of dental caries (Miller). The first a.spect is the potential buffer capacity of saliva and the second is the effective buft’ering mechanism beneath and/or in the bacterial plaque on the tooth surface. The inability of saliva to reach certain sites in relation to the teeth (for example, interproximally and in pits and fissures on oeclusal surfaces) is an important limiting factor in the initiation. of dental caries. Some experimental observations on the permeability of capillary spaces by ionic solutions have been made by Kevin.’ His model experiments show how an obstruction, such as food, at the entrance to a capillary space hinders interchange of ions between the enclosed space and the exterior. A similar phenomenon would occur with bu-ffer solutions. Further, it has been claimed by Stralfor+ and Fosdick3 that ‘ ’ plaque ’ ’ ma,terial has an important buffering action. In the literature on the buffering properties of saliva, two terms are frequently used synonymously. These are buffer capacity and bufering efect. Bufer cupacity refers to the ability of a solution to resist change in pH on addition of acid or of alkali. It expresses the resistance to pH change at a given pH value, and can be calculated from a, knowledge of the concentration of the buffering systems in the solution. The slope of the titration curve at a particula(r pH value enables the buffer capacity at that value to be determined experimentally. Differences between the calculated and observed value for buffer capacity at several pH levels led Ericsson4 to suggest that proteins may play a,n important role below pH 6.0. Sellman and Wah Leungs also suggested tha,t protein might play a.n important role as a buffer at pH values below 6.0. The other term, buj‘“ering efSect, refers to the amount of acid (or alkali) required to change the pH of the solution from one value to another. The .__I
T
*Research
Officer.
Tnstitute
of
Dental
Research,
825
United
Denta.
Hospital.
BUFFERING
SYSTEMS
JN
MOUTH
829
range is chosen arbitrarily and for saliva the ranges pH: 7.0 to 6.0 and pI1 7.0 to 5.0 or 4.0 are used. So-called buffer capacity tests of saliva are, in fact, usually determinations of the buffering effect. Nevertheless, each of these terms, buficer capucity and bufel-ing e#jcect, merely relates to a different manner of describing the same property. The Buffer Systems in Saliva.-The substances in saliva which have been regarded as buffers are bicarbonate, calcium phosphate, protein, mucoid, and a,mmonia. Considerable attention has been given by various authors to the concentra.tions of calcium a,nd phosphate ions in saliva and to their buffering ability. The role of calcium and phosphate ions is related to the maintenance of the solid phase of the tooth insofar as an excess of either will prevent the solution of the tooth surface; in accordance with the law of mass action. Buffering action in saliva, as stated previously, is concerned with the maintenance of a relatively constant pI1 value in the mouth. When excess acid is present, it is only after the buffering systems have played their part that the pH can fall to values which affect the solubility of the mineral phase of the enamel. Of the compounds listed, ammonia has no buffering action in the strict sense, although as a base it does play a part in neutralization of acid. From the quantitative point of view, its role is not significant. Bicarbonate has been shown by several workers to be an important buffer in saliva. Ericsson” calculated the buffer capacity of bicarbonate and related the values to the observed values in several samples of resting saliva. Wah Leung” approached the problem in another manner. Using saliva from eleven subject 3, he studied the carbon dioxide-combining power of saliva under different pressures of carbon dioxide and, from the data, calculated the buffer capacity of the bicarbonate systern between pI1 6.8 and 7.2; this work showed bicarbonate to be the ma,in salivary buffer. Each of these methods has given information by what may be called an “indirect approach,” because the va,lues for the buffer capacity of bicarbonate and phosphate were calculated from their concentrations in the samples and compared with the values obtained for whole saliva frorn titration data. In another study, separation of the salivary buffers by stepwise elimination has enabled the role of each buffer system to be individually and directly observed.7x s Quantitatively, bicarbonate was again found to be the main buffer in saliva, and phosphate was found to play a less important role. It has been clearly demonstrated that bicarbonate and phosphate together account for nearly all the buffering properties of whole saliva, resting or activated. Furthermore, the difference in. buffering ability between resting and activated saliva is due to an increase in the bicarbonate content of the latter. The buffering mechanism of bica,rbonate and phosphate is well known. For the preservation of enamel surfaces, however, solution of bicarbonate may be as important as t,hat of calcium and phosphate ions. In bone the apatite Ions found in tissue fluid are crystals accurately reflect their environment.”
lt can be seen from the above t,h;tt any change: in pI1 will affect the ioniza.t,ion of both bie~rbonxte and lphosl)hate b(:(;irlls(; the respective viilues for K are constant. The precise values Car these constants under these conditions arc not, known. I’rotoin (rrnlcoid) >lntl the prcscnec of ot,her ions will have an important bearing on these values. I Iowever, on formation of enough acid to exhaust, these buft’e~, the only way in which buffering can continue is hy the irttrotluotion 01 a.cltfitiolial buffor. 4t is generally ;~ssumotl that, beneath the plaque, stltur:Lti(m conditions exist for the mineral s;rlhstance of enamel. When acid is formotl al, this site and the bnfllers have been exhausted, solution of the miltera phase occws in order t,o satisfy t,ht: solubility product. relationship. ‘I’he affinity for calcium ions exhibited hy protein, including mucoid, has an The solubility product o 1’ the importnnt, effect on t.he solubility relationship. tooth mineral (for instiznoe, hydroxyapatite) is not krtown beaausc of variation produced hy the environment, thus making it impossible to calc:uI~~te the effect o F given changes in the concentrations of ions under given conditions. Nevertheless, from basic princil)lcs, it WOuItl seem thMt ilCtCr tT~llOV~.l Off adsorbed ions the ortle~ ol’ solution of the ions in the crystal structure WOI~C~ depend largely upotl the size oE the ionic radii, the intcrionic forces, unrl the ionic composition of the surrounding mctliurn. The suggestion that proteirt and rrrucoid exert an important buffering This would bc expected in action I~low pl-I. 6.0 has not l~!n substantiated. view of the small concentrations of proteins in saliva and, in cornpnrison with bicarbonate and phosphate, these proteins exert il negligible effect. The same order o-f impodi~~lce was found for tho buffer systems in both resting and autivatcd diva obtained from two groups of thirty-two and thirty-six subjects, respectively. .Hicarhonate was the mqjor buffer, whereas I)hosl)hiite was tha minor one, and rnucoid had no detectable buffering effect. In resting saliva, however, the bic;trbonate values are more variilbl(: than in activated saliva. As a. buffer, phosphate is thwefora rrlorc important in resting saliva
BUFFERING
SYSTEMS
XN
in activated saliva. E‘urthermore, resting effect than activated saliva, because of its lower tion of salivary flow by chewin g paraffin wax saliva which contains greater concentrations of than
The Buffer
Systems
in Salivary
Sediment
MOUTH
831
saliva has a smaller buflering bicarbonate content. Stimulaproduces a la.rger volume of bicarbonate and sodium ions.lO
and Plaque.-Stralfors”
ex-
amined the buffering properties of salivary sediment and dental plaques alld found that these gave similarly-shaped titration curves for a given mass of each ma.terial. Plaque and sediment consist largely of bacteria, and their buffering properties might be attributed to their protein nature. However, a variable proportion of the buffering eEfect produced by salivary sediment was found to be due to adsorbed bicarbonate, whereas no phosphate was adsorbed.” On examination of plaque materia,l, no adsorption of bicarbonate was detected. Thus, the nature of the buffering systems in salivary sediment and in plaque would appea.wto be difYerent. Although sediment exerts a buffering effect, it is only a very small portion of the total buffering effect of the whole saliva. The buffering properties of plaque material must be considered also in relation to the ability of the bacteria within it to metabolize organic acids. The evidence indicates that the buffering power in plaque is attributable to the bacteria it contains. If mucoid is present it would not add to this buffering effect beea,use mucoid ha,s not been round to exert a buffering effect over a wide range of PH. The permeability of the plaque to ions would bc reduced by the presence therein of mucoid, and it is by this action on the diffusion rates of ions that mueoid could play a significant role.
Experimental Buffering Effect and Volume
Relation Between of Saliva.-A group of sixty-seven children a,t Hopewood H-ouse, Bowral, New South Wales, was used in. this study. The children’s home is a.pproximately seventy miles from the laboratory and it was necessary, therefore, to devise a method of collection and preservation of samples of saliva. Details of the procedure adopted and its value are given in an appendix to this article. It was found that a direct relation existed between the volume of five-minute samples of activated saliva and buffering effect (Fig. 1). From the statistical analysis, it can be seen that the correlation coefficient is positive, indicating a. direct relationship between the two variables. The value of the coe.fficient shows that this set of’ results could be expected to occur by chance only once in 1.,000 times. Therefore, the relation between volume and buffering cfFect is highly significant. This observn.tion for five-minute samples of saliva is in agreement with the results obtained by Dreizen a.nd his a.ssociatesLOwith thirty-minute samples of a.ctivated saliva.
Variation in the Buffering Properties of Saliva.--The effect of the following conditions on the buffering effect of saliva was observed : (I) prolonged chewing as may occur during a meal and (2) chewing paraffin wax for a similar period. Zk’urthermore, when changes in volume and/or buffering quality
nccmrred, observations The rela,tion between concentra?tion
were made of any changes in a particular buffer system. bicarbonate c~oncrntration and changes in the sodium inn
was also noted
.
activated
This
Fig.
is
l.-Correlation saliva.
approximately
Correlation
0.1
between volume and bufferingeffect of five-minute coefficient I‘ = +0.364 rvn-2 t E , where n is number of \iL i-2 0.364 v 65 = v 1 - 0.13241 0.364 x 8.06 p3,731. = 0.931 per cent level of significance, which is highly significant.
samples
of
observations
The fifteen children chosen as subjects were again from IIopewood House. subjects had a very low dental caries experience.*l 1. Effect of eating a meal: Saliva was collected immediately before and after the children had eaten a meal conta,ining grated raw beetroot and carrot, sliced raw cabbage, cauliflower, a,nd soya bean cooked with onions and tomatoes. This was followed by cubed fresh pineapple with junket and wheat germ. The meal was completed in approximately twenty min.utes. Paraffinactivated saliva (five-minute sa,mple) ‘was collected from each subject in a tube containing paraffin oil; it, was frozen at, -18 degrees and kept at t,his temperature until required for titration. These
BUIWERING
SYSTEMS
IN
833
MOUTH
In five of the fifteen subjects, the buffering effect ($1 7.0 to 6.0) was greater after the meal than before it. The other ten subjects did not show any appreciable change. After the meal, six subjects showed an increa,sed buffering power for the range of pl-I 7.0 to 4.0. It should be noted that the increased buffering power was not accompanied in each case by a larger volume 0.f saliva collected in the five-minute period (Table I). The same type of food produced different salivary responses in these subjects. TABLE
-
T . BUFFERING BUFFERING
SU:CT
1
0.56 (h;=
7.0,To
ERFECT
“.& 0.65
OF SALIVA (ML.,
/
0.02
($T
0.92
BEFORE N
ANII
Hd)
7.0,To
0.59
0.63
1.0
3t 4" 5
0.4 0.42 0.56
I!:: 0.56
0.67 0.69 0.89
&et 8 9
0.46 0.5 0.43 0.53
0.46 0.61 0.42 0.5 0.4 0.47 0.44 0.35 0.61 0.53
0.82 0.73 0.74 0.88 0.67 0.75 0.78 0.64 0.83 0.85
12
(A) 7.0 to
Refore meal; 6.0% and pH
(B) after 7.0 to 4.O.i
meal;
increased
A MEAL
-___---
VOLUME
4.yB) 1.05
0.37
0.46 0.47 0.38 0.48 0.50
AFTER
--
2*t
0.39
pl-I
EFFECT
?;r,
OF
(ML& 8
7
0.75 0.69 0.89
11 16 15
:i 15
0.73 0.97 0.76 0.85
189 13 18 12
12 SO 14 16
15
18 0.72 0.65 1.0 0.82 buffering
20 16 10 5 28
11 11 3 18 effect
after
eating
for
range
2. Effect of chewing para& wax: In the preceding experiment, the aubjects chewed for approximately twenty minutes without a consistent change in bnffering effect. If, instead of food, the subjects had chewed a. bland material such as paraffin wax, what changes might be expected in buffering effect during the sarne period and in the first 30 ml. of saliva produced? Activated saliva, was collected in four consecutive five-minute periods and on the following day from the same subjects in three consecutive 10 ml. fra,ctions. In each case the saliva was collected under paraffin oil and the samples were frozen (-18 degrees). The results of titration for eight subjects ase shown in Pig. 2. For each subject, the amounts of acid required to change the pF1 from 7.0 to 6.0, 5.0, or 4.0 are shown. With one exception, changes in buffering effect among the 10 ml. samples and the five-minute samples were similar for the three ranges of pH shown. Bach subject gave values for the buEering effect in the 10 ml. samples which were very different from those of the five-minute samples. In general the values for the 10 ml. fractions were less than the values for the five-minute samples. The buffering effect (pII 7.0 to 6.0) of the fourth five-minute sample was similar to that of the first fiverninute sample, although. differences occurred between these two and the In addition, the ehangcs in buffering second and. third five-minute samples. effect in the two series varied from one subject to another and showed no general trend.
removal of bicarbonate by carbon dioxide-free air below pI1 5.0.8 From the close relation bctwec~r the changes in ln&~ring efkct a.rld bicarbonate, it I’ollows that the remaining buf?er, phosphate, must remain virtually unchanged. Having found that bicarbonate was the only variable among tho anionic constituents of the salivary buffer systems, it was of interest, in view ol’ the observations by Dreizen and his associates’” on K’, Sa-! and IICIO’,,, to observe the concentrations of Na-. and K+. lkcause the curves I’OT the lbicarbonatc content and buffering effect were parallel or nearly so in each east, the values for bufkrirrg ci’fect were omitted to avoid undue complexity in the graphs.
Fig. a.---Variation samples of a&ivated 5.0. IPC, Buffering
saliva. effect;
in
buffering Values HCO’a,
effect and in rniMli&rs, bicarbonate.
bicarbonate 0.02 N
ALI- b
HCl
per
milliliter
COnCf?ntl”atiOn.
of’~a~
3 ‘for
and BH’
are conserutive (EL) 7.0 t0 6.0
an
fiveminute 7.0 (h)
b
to
b
a
l?ig. 4. Variation in buffering r:fCect and bicarbonate concentration. I, 2, anti 3 :LI’C Wig. 6: Iklation botwncn volume and bicarbonate soclium, and potassium concentrations of effect ; HCO’II bicxrbonatc. of saliva for pK (a) 7.0 to 6.0 and (b) 7.0 to 6.0. EC. liuffwing
The results
for the four five-minute samples (E‘ig. 6) showed that the X’a+ was parallel to that. of Ff CO’, in Xos. 1 and 3, whereas in the inde&her four subjects this was not the case. 111 general, l<+ levels VilI+fXl pendently of IICO’~, but in a Pew instances K’ values changed in a manner similar to l,bose for LSaA. When the volume was constant, as in the three consecutive 10 ml. wmples, In the bicarbonate (and therefore the bufferin g effoal ) was not constant. ;Icltlitiorr, no relation was lroted hetwem t,he volume of consecutive five-minute s:l mples oI’ sdivn. wn(l either its bicarbonate, Sa+, or* R+ cont,ent. ronrentralion
NPfo
Fig. fractions N I-TCI.
5.-Relation of activated
between saliva.
bicarbonate, NC&+ and
N+
sodiunl, values
in
anal potassium mE/l. TTCO’J
concentrations values as
milliliters
in
IO
ml. 0.02
hh
Fig.
F.--Kelation five-minute
between samples
of
volume and activated
bicitrbonate, saliva. Nm+.
sodium, K+. and
anti potassium HCO’:I values
as
concentrations in Fig.
5.
of
Salivary Buffer Tests and Dental Caries.--lkwxr’” has descCbed a, classifi~~aiion based on the FI~nonnt of acid rtquirccl to cha’nge the pH of a definite quantity of saliva from 1,l-T 7.0 I.0 6.0. These grxcles indiea.lc the soslv~pt~ibititp of t,he snhjcct to tlcnl al caldies, the correlation being rrradc i’rom D31P valacs for children ant1 young adults in whom the D&E value is related to caries activity. The results of a correlation of the buffering effect with the caries inritlencc of sixty-five children rrorn Hopewood FIouw is shown in Table IT. TT. I~TJFFE:KTNC:
TABLE
RFFRCT
AND
DENTAL
CARIES
TECIIHW~E
--__ NUMBERI" SUSCRPTlRIldTY
INDICATED
BUFFER
TESTY 2
_______I~_~rt + ++ +++
I mmulle Slightly susceptible Moderately suseeptilk I-Tiehlv suseeotible
R&p< ear~cs
A. Number slightly K. Number (++,
Total Per cent
++++
of immune (2) or suscepti hle ( +) in other grades +++,
i+++) --
susceptible
(B
--
x 100)
tOtR.1 :sObtained from of Dental Research,
results Sydney)
NEW
BY 0
I
13 13 10 1
1
Id3SIONS"
JANUARY,
Jhh’l:ART-,
1954 3 0 2 1 2
-1
4 3 4 1
-- 2 2 0 5 0
I
1
1%.%,
TO
4 1 1 0 0
/
5 2 0
0
0
0
0
0
0
ic,
7
2
2
2
2
11
5
5
3
0
0
37
12
7
-___ 5
2
2
30
42
72
GO
of clinical examinations anrl R. Harris (IJnitrd
carried Dental
out by Hospital,
H. R. Sullivan Sydney).
-
(Tnstituto
The results show tha.t in these children, in whom dental caries is a slow process and not very prevalent, I1 t,he buffer test of the saliva, places 30 per cent, o-f those who had no new lesions in the suseeptiblc cla.ss. Likewise, of those who had one, two, or three new carious lesions, 42, 72, and 60 per cent, respectively, were classified on the buffer test as susceptible. Two conclusions may be drawn from these results: first, that some of these children are cariesfree, although the buffer test shows them to be susceptible; second, others have new lesions, although they ase classified by the buffer test as caries-immune. Huch. observations show the limitations to which this test, like any other available test for caries susceptibility, is liable. Susceptibility to dental caries may be regasded as being due to a, disturba.nce of the dynamic equilibrium between acid production and neutralization on the tooth surface, the determining factor being the rate of acid formation rather than the capa.city for neutraliantion (that is, buffering effect). In the instances just mentioned, in those children who were classified wassusceptible to denta, caries on buffer tests but who did not have any new lesions, it is probable that lack of a powerful acidproducing system was the determining factor. Omission of the substrate for acid formation and not the lack of the enzyme systems of the bacteria, most probably accounts for the observations among these children. In those susceptible children who were classed as immune on the buEer test, the acidforming system probably was t,oo powerful for the buffer mechanism to counter-
BUiWhXlNG
SYSTEMS
IN
MOUTH
839
B’inally, considering the entire group, the results show that as the caries incidence increased, the buffer test became a. more reliable method for the assessment of susceptibility to dental caries.
act.
Summary I. The buffering systems in saliva, salivary sediment, and plaque are disBicarbonate is the major buffer in activated saliva, phosphate accounting fol* the remainder of the buffering effect. Salivary mucoid and bacteria have an insignificant buffer action in whole saliva. In resting saliva, the buffering effect is decreased because of the smaller concentration of bicarbonate in resting sa,liva than in activated saliva; therefore, phospha.te becomes relatively more important. Salivary sediment and plaque have different buffering systems. The former has a, variable amount of adsorbed bkrbonate which plaque does not possess. 2. The effect of eating a meal on the buffering power of the saliva of h constant increase or decrease in buffering fifteen subjects was examined. after the meal was not observed. 3. Paraffin-activated saliva, collected during a period simi1a.r to that taken for the meal, did not show any appreciable changes in bufFering properties, as determined by comparison of the first and la.st five-minute samples of saliva. During the test period, however, changes were observed which were paralleled hy changes in bicarbonate concentration. No consistent relation wa.s found between HCO’,, Na+, and K+ concentrations and volume of sample. 4. A significant relation was observed between buffering eft’ect for the sample, pH 7.0 to 6.0, and the volume of five-minute sarnples of activated sa.liva for a group of sixty-seven subjects. 5. The limitations of the buftering test in attempting to assess caries susceptibility were investigated in a group of sixty-five subjects. cussecl.
Appendix Effect of Freezing and Storage at -18 Degrees on the Buffering Properties and Content of Saliva.-If saliva is not rapidly chilled after collection, its buEering increases on standing. The problem of collecting saliva for buffer tests about miles away from the laboratory is therefore obvious. A method was required would enable collection of saliva from the group of children, transport of these samples to the laboratory, and storage without appreciable alteration in the buffering pro&rties of the samples. In a preliminary study, samples of activated saliva were collected under a layer of parafin oil and 2 ml. quantities were immediately placed in a cleep freex at -18 degrees. On titration after one, three, seven, fourteen, and twenty-out: days, it was found that the budering power had not appreciably altered (Table III). It freeze it, and transport the therefore became possible to collect saliva from the children, deep freeze containing the frozen samples to the laboratory and that, too, without risk of thawing. The
Mucoid power seventy which
Further
difficulties rapidity with by collecting the
tests were rude of the advantages of freezing saliva. For example, one of associated with the determination of the mucoid content of saliva is the which mucoid is depolymerieed by salivary mucinase. Knox~~ overcame this It is not always convenient to estimate the saliva under ice-cold conditions.
12
0.5 0.G 0.65
i3 14 15 16 17 18
mucoid degrees mtlcoid
0 . 5” i
0.75 0.45 0.4 of the sample way in which
6 _..
I
8
4 R 6 7 8 9 10 II
4 8 4 8 4 16 8 14 8 8 4
18 JO
0.6 0.6 0.48 i93 0:47
II::
Sl;U.JEC’l’
14 15 16 17
0.75
O:;i 0.6 0.5
--_ 0.5
content innnediat~ely after collection has been found to be a satisfactory content (‘l‘ablo TV).
12 13
0.51 0 52
of saliva, IO provent
\
(-
-..
and storage at -18 gross changes in
.-~
_....-_
--__8
4 2
4-8 2-4
8
4 & 4-8 4-8 2 2 2
: 7-2 4 4 8
4 4 4-x
References 1~ Kevin, 2. 3. 4. 5.
R. l3.: The Diet and Mastication : Their F:fl’ects 011 Diffusion and on the inception of Dental Caries, Dunedin, N.Z., 1951, Progress Printing Company. Stralfors, A. : The l3uffos Capacity of ~I)ontal Plaques, J. I). Ros. 27: 587, 1948. Some Xew Concepts Concerning the Role of Sugar in Dental Caries, Fostiick, J,. 8.: ORAT d ‘%X0., k (h”d, %bKk, AiTl) ORAl, ~‘h’~Tf. 5: 615, 1958. ISname-Apatii;tt Solubility, Acta odont. soandinav. 8: Supp. 3, 1949. b:ricssort, Y.: The Buffer Value of Saliva and Tts Relation to l)ontal Caries, Ao1.a odont. Sellman, S.: scandinav. 8: 244, 3949.
BUFFERING
SYSTXMS
IN
841
MOUTH
6. Wah
Leung, 8.: A Demonstration of the Importance o-F Bicarbonate as a Salivary Buffer, J. I). Res. 30: 403, 1951. 7. Lilienthal. B.: The Buffer Systems in Human Saliva. Dent. .J. Australia 25: 216, 1953. 8. Lilienthali B.: An Analysis of the Buffer Systems in ‘Saliva, J. 1). Res. (in press); 1%X. 9. Neuman, W. F., and Neuman, M. W.: The Nature of the Mineral Phase of Rone, Chem. Rev.
53:
1, 1953.
10. Dreizen, S:, Reed, A. I., Neidormeicr, W., and Spies, T. I).: Sodium Constituents of Human Salivary BuWers, J. D. Res. 32: 497, 1953. 11. Lilienthal, B., Goldsworthy, N. E., Sullivan, H. R., and Cameron, I). of the Children of Hopewood House, Bowrd, New South Wales: Dental Caries Extending Over Five Years (1947.1952), M. J.
and
Potassium
as
A.: The Fologj I. Observations On Australia 1: 878:
1953.
12. TIewar,
Margaret Caries. Part Standarclised 13. T
R.: Laboratory Methods for Assessing Susceptibility to II. Correlation of Results Obtairled by Clinical Examination Laboratory Methods, Dent. J. Australik 22: 24, 1950. Salivary Mucoids and Mucolytie Enzymes, Master of Science Sydney, 1951.
Dental and
by
Thesis,