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Factors in Human Saliva Correlated With the Occurrence of Salivary Calculus
F A C T O R S I N H U M A N S A L IV A C O R R E L A T E D W I T H T H E O C C U R R E N C E O F S A L IV A R Y C A L C U L U S B y B E N J A M I N T E N E N B A U M , B .S ., D .D .S ., and M A X W E L L K A R S H A N , P h .D ., N e w Y ork City
IT T L E is known of the exact chem. ical and physical processes concerned in the formation of salivary calculus. Hypotheses involving carbon dioxide loss, colloidal changes, bacterial action and the operation of the enzyme phosphatase have been based almost entirely on experiments in which calculus-like masses were pro duced in vitro. A detailed review of these hypotheses was recently published by Rosebury and Karshan.1 From the work o f Hall and Westbay2 and of Bibby,3 it appears that the pu of the medium is unimportant in the forma tion of artificial deposits from stagnating saliva. The studies o f Naeslund,4 on the other hand, suggest that local increases in alkalinity of saliva, brought about by bac terial action, may be of importance in the formation of calculus. Presumably, pre cipitation would then result from an in crease in P 0 4 ions, derived from H 2P 0 4 and HPO4 ions. It has been suggested that the enzyme phosphatase, which is capable of liberat ing inorganic phosphate from organic phosphate compounds in saliva, may play a role in the formation of calculus. The
L
(Read before the N ew York Section of the International Association for Dental Research, June 4, 1936.) (This study was conducted with the aid of a grant for dental research from the Carnegie Corporation.) (From the Department of Biological Chem istry, College of Physicians and Surgeons, and School of Dental and Oral Surgery, Columbia University.)
Jour. A .D .A . & D. Cos., Vol. 24, August 1937
enzyme has been found by Adamson5, in human gum tissue and by Smith6 in des quamated epithelial cells in saliva. That the protein content of the me dium may be related to the formation of these deposits is suggested by the work of Karlstrom and M iller,7 who found that precipitation of calcium salts was mark edly reduced by the addition of soluble protein. Since the main inorganic unit in sali vary calculus appears to be tertiary cal cium phosphate, it is reasonable to suppose that the concentration of calcium and phosphate in saliva is related to the tend ency to form these deposits. Precipita tion of tertiary calcium phosphate would depend on the type of phosphate ion pres ent, which in turn is dependent on the hydrogen-ion concentration of the me dium. Hence, a relationship may exist between the pu of saliva and calculus formation. In view of these considerations, it is surprising that no satisfactory effort seems to have been made to correlate individual differences in salivary pu, calcium, inor ganic phosphate and protein with differ ences in tendency to form calculus. The present study is an attempt to ascertain whether such a correlation exists. Fortynine cases, persons ranging in age from twenty to fifty-two years, have been stud ied thus far. Twenty were calculus-free and had not had dental prophylactic treat ment for at least one year. In the remain ing cases, there were varying degrees of
!255
The Journal of the American Dental Association and T h e Dental Cosmos
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Inorganic Phos phorus
Cc.
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7 .1s
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7 .3 7 7 .4 3 7 .1 3 7 .4 8
7 .3 7
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Age
Cal cium
Inorganic Phos phorus
M
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7 .18 6.50
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First Sample
S3
Num ber of Analy ses
T able
1 .— D ata
on
S a l iv a r y
A n alyse s
of
C a l c u l u s - F ree
and
C alcu lu s
G rou ps
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First Sample
Second Sample
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Tenenbaum and Karshan— Salivary Calculus
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METHODS
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Saliva, stimulated to flow by the chew ing of paraffin, was collected in three successive portions, from one to three hours after breakfast. The first portion (1 to 2 cc.), collected under paraffin oil, was used to determine the initial p a ; the second portion (10 to 12 cc.) was used for determination of calcium, inorganic phosphate and protein; the third portion (1 to 2 cc.), again collected under oil, was used for a second fin determination. The analytical procedures were as follow s: Total Calcium.— Four cubic Genii meters of saliva was added to 1 cc. of 40 per cent trichloracetic acid. After standing for ten minutes, the mixture was centrifuged and 3 cc. of supernatant fluid was analyzed as described by Krasnow, Karshan and Krejci .8 Phosphate.— The phosphate content was determined, on a separate portion (0.5 cc.) of the supernatant fluid used for the determination o f total calcium, t t by Tisdall’s9 method for determining inorganic phosphate in blood, Protein.— The procedure described by Karshan10 was used. The pa was determined colorimetrically on saliva diluted 1 : 10, with bromthymol blue as the indicator; 0.25 cc. of a 0.04 per cent solution of the sodium salt of the indicator was mixed with 4.5 cc. of water under oil, and 0.5 cc. of saliva then added. The statistical method employed10’ 11 for the interpretation of results is based on the assumption of homogeneity of the two groups studied; that is, on the “ null” assumption that there is no difference between the two groups. A ll the values in each class (total calcium, inorganic phos-
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Standard d ev iatio n of m ean
04 O
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deposits. Three sets of analyses were performed in 75 per cent of the cases; two sets were performed in 14 per cent.
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20
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Calculus
Calculusfree
Calculus
first sample
pH
second sample
pH
79
0 .2 1 0 .18
7 .3
0 .19 7
0.22
6 9.9
7.21
7 .0 9
79
52
6 .9 9
52
294
79
29
Calculus
7 5.5
1 .74
13.6
314
1 .8
1.12
1.0
W
Standard Deviation
11.8
6 .7
5.1
Group Mean (M )
52
20
Calculusfree
Protein mg. %
79
29
79
52
49
29
20
Number of Analyses
19
Calculus
Calculusfree
Group
No. of Cases (N)
CalculusInorganic free phosphate Calculus mg. %
mg. %
Calcium
Class of Value
0 .0 3 4
0 .0 4 9
0 .03 7
0 .0 5
13.2
17.3
0 .3 3
0 .4 5
0 .2 1
0 .2 3
Standard Deviation of Mean (o -M )
7 .2 6
7 .0 4
302
12.9
6.1
Class Mean (Mo)
0 .0 3
0 .0 3
10.52
0 .2 9
0 .1 9
Difference Deviation of Class Mean (crMo)
+
-
+
-
0 .0 4
0 .05
0 .0 5
0 .0 5
0 .04 5
0 .0 5 7
0 .04 7
0 .05 8
16.9
-
8
0 .4 4
0 .5 4
0 .2 8
0 .3 0
20.2
0 .7
1.1
0.6
1 .0
Standard Deviation of Difference (o-D )
+ 12
+
-
+
-
Difference of Means (D)
T a b l e 2 .— C o m p a r i s o n o f S a l i v a r y A n a l y s e s o f C a l c u l u s - F r e e a n d C a l c u l u s G r o u p s
<
0 .9
1.7 to 1
1.7 to 1
2.7 to 1
1.1
0 .9
1.4 to 1
1 to 1
0.8
0 :5
1 to 1
8 to 1
1.6
0.6
21 to 1
27 to 1
1,033 to 1
Odds Against Random Occurrence of Difference
2 .0
2 .1
3 .3
° "D
Signifi cance Value D
1258 The Journal of the American Dental Association and The Dental Cosmos
Tenenbaum and Karshan— Salivary Calculus phate, protein and pu) in the two groups (calculus-free and calculus) were there fore pooled and each group mean ( M ) was compared with the pooled class mean ( M 0) to test this assumption. By divid ing the difference between these means by the standard deviation of the difference, a significance value was calculated, from which the odds against the random oc currence of the difference were obtained from tables12 devised in accordance with the theory of probability.
1259
calculus. T h e mean p n value was lower and the mean protein content was higher in the calculus-free than in the calculus group, but, on account of the variability within each group, the differences be tween the means in these respects are not statistically significant. Studies along these lines on unstimu lated saliva are in progress. B IB LIO G R A P H Y 1. R o s e b u r y , T h e o d o r , a n d K a r s h a n , M a x : A n n D ent., 3 :37, M a r c h 1936. 2. H a l l , I v a n C ., a n d W e s t b a y , C l a y t o n : D . Cosmos, 6 7 :1 1 5 , F e b r u a r y 1925. 3. B i b b y , B a s i l G . : D . Cosmos, 7 7 :6 6 % , July 1935. 4. N a e s l u n d , C a r l : D . Cosmos, 6 8 :1 1 3 7 , December 1926. w ell
RESULTS AND CONCLUSIONS
T h e data for individual cases are given in Table 1. A summary of the analytical 'data and the statistical analyses are given in Table 2. The most striking difference between the calculus-free and calculus groups appeared in the calcium content of the saliva. The mean value for cal cium in the calculus-free group was lower than that in the calculus group, the for mer differing significantly from the class as a whole. This indicates that there is a significant difference in calcium between the two groups. The mean value of in organic phosphate was likewise lower in the calculus-free than in -the calculus group, and the difference between the mean of the calculus-free group and the class as a whole is probably significant. These data indicate that the calcium and, probably, the phosphate content of saliva are correlated with the occurrence of
5. A d a m s o n , K . T . : A u stra l. J . E xp e r. B io l. & M . Sc., 6 :2 1 5 , June 1929.
6. S m i t h , G . H .: A ustral. J . E xp e r. B io l. & M . Sc., 7 :4 5 , June 1930. 7. K a r l s t r o m , S a m , a n d M i l l e r , E d g a r G ., J r . : J . D . Res., 8:453, J u n e 1928. 8. K r a s n o w , w ell,
Fran ces;
K arshan ,
M ax
a n d K r e j c i , L a u r a E .: J . L ab . & C lin .
M e d ., 17:1148, August 1932. 9. T i s d a l l , F r e d e r i c k F . : J . B io l. Chem ., 5 0 :3 2 9 , February 1922.
10 . K a r s h a n , M December 1936. 11. R
osebury,
axw ell
: J . D . Res., 1 5 :3 8 3 ,
T heodor,
and
K arshan ,
M a x w e l l : A rc h . Path. & L a b . M e d ., 2 0 :6 9 7 ,
November 1935. 12. P e a r l , R a y m o n d : “ Introduction to M ed ical Biometry and Statistics.” Philadelphia: W . B. Saunders Co., 1930. 630 W est One Hundred Sixty-Eighth Street.
IT T L E is known of the exact chem. ical and physical processes concerned in the formation of salivary calculus. Hypotheses involving carbon dioxide loss, colloidal changes, bacterial action and the operation of the enzyme phosphatase have been based almost entirely on experiments in which calculus-like masses were pro duced in vitro. A detailed review of these hypotheses was recently published by Rosebury and Karshan.1 From the work o f Hall and Westbay2 and of Bibby,3 it appears that the pu of the medium is unimportant in the forma tion of artificial deposits from stagnating saliva. The studies o f Naeslund,4 on the other hand, suggest that local increases in alkalinity of saliva, brought about by bac terial action, may be of importance in the formation of calculus. Presumably, pre cipitation would then result from an in crease in P 0 4 ions, derived from H 2P 0 4 and HPO4 ions. It has been suggested that the enzyme phosphatase, which is capable of liberat ing inorganic phosphate from organic phosphate compounds in saliva, may play a role in the formation of calculus. The
L
(Read before the N ew York Section of the International Association for Dental Research, June 4, 1936.) (This study was conducted with the aid of a grant for dental research from the Carnegie Corporation.) (From the Department of Biological Chem istry, College of Physicians and Surgeons, and School of Dental and Oral Surgery, Columbia University.)
Jour. A .D .A . & D. Cos., Vol. 24, August 1937
enzyme has been found by Adamson5, in human gum tissue and by Smith6 in des quamated epithelial cells in saliva. That the protein content of the me dium may be related to the formation of these deposits is suggested by the work of Karlstrom and M iller,7 who found that precipitation of calcium salts was mark edly reduced by the addition of soluble protein. Since the main inorganic unit in sali vary calculus appears to be tertiary cal cium phosphate, it is reasonable to suppose that the concentration of calcium and phosphate in saliva is related to the tend ency to form these deposits. Precipita tion of tertiary calcium phosphate would depend on the type of phosphate ion pres ent, which in turn is dependent on the hydrogen-ion concentration of the me dium. Hence, a relationship may exist between the pu of saliva and calculus formation. In view of these considerations, it is surprising that no satisfactory effort seems to have been made to correlate individual differences in salivary pu, calcium, inor ganic phosphate and protein with differ ences in tendency to form calculus. The present study is an attempt to ascertain whether such a correlation exists. Fortynine cases, persons ranging in age from twenty to fifty-two years, have been stud ied thus far. Twenty were calculus-free and had not had dental prophylactic treat ment for at least one year. In the remain ing cases, there were varying degrees of
!255
The Journal of the American Dental Association and T h e Dental Cosmos
Q s - * ! f r < 'O C O vO C S C O C O O O C O C O C O e S C O ,-0
'—
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Cc.
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Case
Num ber of Analy ses
'O O 'f o o o L o - 'C O 'O r i M O i N N n c o O N '^ r o O M w r 'i O N M r i o ^
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6 .8 0
7 .1s
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7 .2 0 7 .1 7
6.66
7 .3 2
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Second Sample Case
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Cc. Mg./100
Age
Cal cium
Inorganic Phos phorus
M
7 .18 7 .18
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Protein
First Sample
S3
Num ber of Analy ses
T able
1 .— D ata
on
S a l iv a r y
A n alyse s
of
C a l c u l u s - F ree
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C alcu lu s
G rou ps
Protein
First Sample
Second Sample
1256
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Tenenbaum and Karshan— Salivary Calculus
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METHODS
q
Saliva, stimulated to flow by the chew ing of paraffin, was collected in three successive portions, from one to three hours after breakfast. The first portion (1 to 2 cc.), collected under paraffin oil, was used to determine the initial p a ; the second portion (10 to 12 cc.) was used for determination of calcium, inorganic phosphate and protein; the third portion (1 to 2 cc.), again collected under oil, was used for a second fin determination. The analytical procedures were as follow s: Total Calcium.— Four cubic Genii meters of saliva was added to 1 cc. of 40 per cent trichloracetic acid. After standing for ten minutes, the mixture was centrifuged and 3 cc. of supernatant fluid was analyzed as described by Krasnow, Karshan and Krejci .8 Phosphate.— The phosphate content was determined, on a separate portion (0.5 cc.) of the supernatant fluid used for the determination o f total calcium, t t by Tisdall’s9 method for determining inorganic phosphate in blood, Protein.— The procedure described by Karshan10 was used. The pa was determined colorimetrically on saliva diluted 1 : 10, with bromthymol blue as the indicator; 0.25 cc. of a 0.04 per cent solution of the sodium salt of the indicator was mixed with 4.5 cc. of water under oil, and 0.5 cc. of saliva then added. The statistical method employed10’ 11 for the interpretation of results is based on the assumption of homogeneity of the two groups studied; that is, on the “ null” assumption that there is no difference between the two groups. A ll the values in each class (total calcium, inorganic phos-
O
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cent 0 cases
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Os O O
Standard d ev iatio n of m ean
04 O
£
0 e 's S E « | g _c
59 04
deposits. Three sets of analyses were performed in 75 per cent of the cases; two sets were performed in 14 per cent.
g. S t -a -|j « u " g
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20
29
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Calculus
Calculusfree
Calculus
first sample
pH
second sample
pH
79
0 .2 1 0 .18
7 .3
0 .19 7
0.22
6 9.9
7.21
7 .0 9
79
52
6 .9 9
52
294
79
29
Calculus
7 5.5
1 .74
13.6
314
1 .8
1.12
1.0
W
Standard Deviation
11.8
6 .7
5.1
Group Mean (M )
52
20
Calculusfree
Protein mg. %
79
29
79
52
49
29
20
Number of Analyses
19
Calculus
Calculusfree
Group
No. of Cases (N)
CalculusInorganic free phosphate Calculus mg. %
mg. %
Calcium
Class of Value
0 .0 3 4
0 .0 4 9
0 .03 7
0 .0 5
13.2
17.3
0 .3 3
0 .4 5
0 .2 1
0 .2 3
Standard Deviation of Mean (o -M )
7 .2 6
7 .0 4
302
12.9
6.1
Class Mean (Mo)
0 .0 3
0 .0 3
10.52
0 .2 9
0 .1 9
Difference Deviation of Class Mean (crMo)
+
-
+
-
0 .0 4
0 .05
0 .0 5
0 .0 5
0 .04 5
0 .0 5 7
0 .04 7
0 .05 8
16.9
-
8
0 .4 4
0 .5 4
0 .2 8
0 .3 0
20.2
0 .7
1.1
0.6
1 .0
Standard Deviation of Difference (o-D )
+ 12
+
-
+
-
Difference of Means (D)
T a b l e 2 .— C o m p a r i s o n o f S a l i v a r y A n a l y s e s o f C a l c u l u s - F r e e a n d C a l c u l u s G r o u p s
<
0 .9
1.7 to 1
1.7 to 1
2.7 to 1
1.1
0 .9
1.4 to 1
1 to 1
0.8
0 :5
1 to 1
8 to 1
1.6
0.6
21 to 1
27 to 1
1,033 to 1
Odds Against Random Occurrence of Difference
2 .0
2 .1
3 .3
° "D
Signifi cance Value D
1258 The Journal of the American Dental Association and The Dental Cosmos
Tenenbaum and Karshan— Salivary Calculus phate, protein and pu) in the two groups (calculus-free and calculus) were there fore pooled and each group mean ( M ) was compared with the pooled class mean ( M 0) to test this assumption. By divid ing the difference between these means by the standard deviation of the difference, a significance value was calculated, from which the odds against the random oc currence of the difference were obtained from tables12 devised in accordance with the theory of probability.
1259
calculus. T h e mean p n value was lower and the mean protein content was higher in the calculus-free than in the calculus group, but, on account of the variability within each group, the differences be tween the means in these respects are not statistically significant. Studies along these lines on unstimu lated saliva are in progress. B IB LIO G R A P H Y 1. R o s e b u r y , T h e o d o r , a n d K a r s h a n , M a x : A n n D ent., 3 :37, M a r c h 1936. 2. H a l l , I v a n C ., a n d W e s t b a y , C l a y t o n : D . Cosmos, 6 7 :1 1 5 , F e b r u a r y 1925. 3. B i b b y , B a s i l G . : D . Cosmos, 7 7 :6 6 % , July 1935. 4. N a e s l u n d , C a r l : D . Cosmos, 6 8 :1 1 3 7 , December 1926. w ell
RESULTS AND CONCLUSIONS
T h e data for individual cases are given in Table 1. A summary of the analytical 'data and the statistical analyses are given in Table 2. The most striking difference between the calculus-free and calculus groups appeared in the calcium content of the saliva. The mean value for cal cium in the calculus-free group was lower than that in the calculus group, the for mer differing significantly from the class as a whole. This indicates that there is a significant difference in calcium between the two groups. The mean value of in organic phosphate was likewise lower in the calculus-free than in -the calculus group, and the difference between the mean of the calculus-free group and the class as a whole is probably significant. These data indicate that the calcium and, probably, the phosphate content of saliva are correlated with the occurrence of
5. A d a m s o n , K . T . : A u stra l. J . E xp e r. B io l. & M . Sc., 6 :2 1 5 , June 1929.
6. S m i t h , G . H .: A ustral. J . E xp e r. B io l. & M . Sc., 7 :4 5 , June 1930. 7. K a r l s t r o m , S a m , a n d M i l l e r , E d g a r G ., J r . : J . D . Res., 8:453, J u n e 1928. 8. K r a s n o w , w ell,
Fran ces;
K arshan ,
M ax
a n d K r e j c i , L a u r a E .: J . L ab . & C lin .
M e d ., 17:1148, August 1932. 9. T i s d a l l , F r e d e r i c k F . : J . B io l. Chem ., 5 0 :3 2 9 , February 1922.
10 . K a r s h a n , M December 1936. 11. R
osebury,
axw ell
: J . D . Res., 1 5 :3 8 3 ,
T heodor,
and
K arshan ,
M a x w e l l : A rc h . Path. & L a b . M e d ., 2 0 :6 9 7 ,
November 1935. 12. P e a r l , R a y m o n d : “ Introduction to M ed ical Biometry and Statistics.” Philadelphia: W . B. Saunders Co., 1930. 630 W est One Hundred Sixty-Eighth Street.