FACTORS INVOLVED IN THE REACTION CHANGES OF HUMAN SALIVA.*

BY GUY W. CLARK AND KENNETH L. CARTER.t

(From the Division of Biochemistry and Pharmacology, Medical School, University of California, Berkeley.)

(Received for publication, February 28, 1927.)

The fact that deposits of calculus on the teeth frequently lead to irritation at the gingival margin has caused many to believe that these deposits and the subsequent irritation constitute the initial phase in the development of one form of pyorrhea. Al- though the relationship of the reaction of the saliva to deposition of calculus (tartar) on the teeth has been the object of many investigations, the exact chemical and physical processes involved in the formation and deposition of calculus are as yet unexplained. Marshall’s (1) work on the “salivary index” was a study of the buffer value and did not give any idea of the true hydrogen ion concentration of the saliva. Using a hydrogen electrode, Foa (2) and Kirk (3) made the earliest attempts to determine the true reaction of saliva. It is obvious however, that the presence of carbon dioxide, ammonium salts, and proteins would make it difficult to determine accurately the initial reaction. The use of the open hydrogen electrode in such studies is therefore a question- able procedure. More recent work on the reaction of saliva is that of Starr (4) with observations on 610 specimens from 228 healthy normal subjects; that of Bloomfield and Huck (5) with 102 samples from 52 healthy subjects, and that of Bunzell (6) dealing with 274

* This work, carried out under the auspices of the original California Stomatological Research Group, was supported in part by grants from the Carnegie Corporation, the American Dental Association, and the Associ- ated Radiographic Laboratories of San Francisco.

f The material submitted in this paper forms part of a thesis submitted by Kenneth L. Carter in partial fulfilment of the requirements for the de- gree of Master of Science in the Graduate School of the University of California.

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392 Reaction Changes of Human Saliva

subjects of varying ages. All of these investigators employed calorimetric methods. While Starr found variations from pH 5.75 to 7.05, 86 per cent were between pH 6.35 and 6.80. The median, mean, and mode’ were at practically the same point, 6.60, showing that a sufficient number of subjects had been taken to approximate, very closely, a normal distribution. Bunzell separated his subjects according to sex and age. For 50 school boys he found an average pH of 6.64 and for 50 school girls an average of 6.62. For 50 female medical students the average was 6.49; for nine aged men (averaging 69 years) the average was 6.24, and for a group of aged women (averaging 74 years) the pH averaged 6.00. Bunzell’s work indicates that the saliva becomes more acid with advancing age and that sex seems to have no, bearing on the reaction.

Those who believe that caries is primarily an external process stress the solvent action of acids in the saliva. There is, however, little or no experimental proof involving pH determinations to support this idea. On the contrary, Lothrop and Gies (7), Pohle and Strcbinger (8), and Gans (9) find no correlation whatever between existing dental conditions and the reaction of an indi- vidual’s saliva.

The work reported in this paper includes first, a comparative study of the reaction of saliva in the glands with that freshly expectorated; and second, a study of the changes in reaction resulting from the incubation of saliva, attempting thereby to simulate as far as is possible natural conditions for the action of bacteria and enzymes.

Methods.

A small hydrogen electrode was first considered but the idea that it was necessary to keep the saliva in a closed system in order to prevent loss of carbon dioxide, together with presence of am- monium salts and proteins, led us to adopt a calorimetric method. To accomplish the first part of the problem it was necessary to devise a single piece of apparatus which would serve both as a cannula and as the tube in which the reaction could be determined directly. The apparatus developed for this purpose is shown in

1 Median, mean, and mode are used in a statistical sense.

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G. W. Clark and K. L. Carter 393

Fig. 1, Parts I, II, and III. The cannula part is calibrated in tenths of a cc. from the tip of the cannula to 0.4 cc. The comparator, or barrel part, is calibrated in 0.1 cc. from the upper stop-cock to 1.4 cc. These figures were used because it was found that 0.4 cc. of indicator to 1.0 cc. of saliva or buffer gave the maximum color difference with these small diamet,er tubes. The test-tubes were cut from the same piece of Pyrex tubing as was the barrel of the cannula, so that the diameter, thickness of wall, and tint of glass

“0rl”“‘gJlT~ RrNovRRLr

FIG. 1.

were exactly the same. The comparison was made in the block shown in Part III; the removable section A permitted the cannula to be placed directly in the block.

Buffer Xtandards.-The series of buffer standards were made from the phosphate mixtures of Sorensen (10) as described by Clark (11). Baker’s C.P. salts were recrystallized. The values given by Clark were plotted as two curves and interpolation made to secure the proportions of the mono- and dibasic salts for a

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394 Reaction Changes of Human Saliva

series OI buffers, ranging from 5.2 to 7.8, varying by 0.1 pH unit. These standards were kept in Pyrex flasks which had been coated inside with a mixture of equal parts of beeswax and para.ffin. Prior to use they were checked against a hydrogen electrode.

Indicators.-Brom-thymol blue was the indicator most used since it covers the pH range between 6.0 and 7.8. For ranges from 5.2 to 6.8 brom-cresol purple was used. The 0.04 per cent solution of each indicator was prepared according to Clark’s (11) directions.

Procedure for Determining the pH of Xaliva.-The cannula- comparator was completely filled with mercury by raising the reservoir. After drawing in 0.4 cc. of indicator, the lower stop- cock was closed and the cannula inserted into the duct (or into the flask of incubated saliva). The mercury reservoir was then lowered a few cc. and the stop-cock partially opened, and saliva was drawn in until the mixture reached the 1.4 cc. mark on the barrel. From 2 to 5 minutes of most careful manipulation were necessary to obtain 1 cc. of saliva by cannulation of one of the salivary glands. Both stop-cocks were then closed, the com- parator gently shaken, and comparison made with the proper buffer standards. These standards were made up by adding 0.8 cc. of indicator to 2.0 cc. of buffer solution. A tube of saliva was placed behind each buffer tube and a tube of water behind the barrel of the cannula containing the saliva. All comparisons were made with a Palo Daylight lamp as background. The above apparatus was used in making all pH determinations because of its convenience and, more important, the small amount of saliva needed. It also obviated any changes in the carbon dioxide content which ordinary pipetting might introduce.

Although Michaelis and Pechstein (12) and Starr (4) showed that dilution of saliva has but little effect on pH, we preferred to eliminate this factor entirely and used only undiluted saliva.

Carbon Dioxide.-The method of Van Slyke (13) was used.

EXPERIMENTAL.

In Table I data are presented showing the differences in reaction between the saliva as it is obtained directly from the glands and that freshly expectorated. If one considers the extensive changes

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G. W. Clark and K. L. Carter 395

in the reaction of saliva produced by various types of stimulation of the salivary glands (14-16), the differences in pH (0.1) shown in Table I seem negligible. A comparison of the pH of quiescent and paraffin-activated saliva is shown in Table II. In these determinations a sample of quiescent saliva was taken and the pH determined. Meanwhile a piece of paraffin was placed in the mouth and after a very brief period of chewing, never exceeding 2 minutes, the reaction of this activated saliva was determined. As can be seen the rise in pH of the activated sample may be as

TABLE I.

Comparison of the pH of Cannulated and Expectorated Saliva.

K. C ......... M. E ......... K. C .........

Cannulated. Expectorated.

Parotia gland. Sublmguel gland.

PH pff PH

6.40 6.50 6.40 6.52

6.25 6.35

Difference.

PH

0.10 0.12 0.10

TABLF: II.

Comparison of pH of Resting and Parafin-Activated Saliva.

Subject. I Resting. Paraffin-activated. Difference.

G. C ................... K. C., ................ H. U .................. K. C ................... L. L ...................

Pff PH pff

7.15 7.30 0.15 5.95 6.80 0.85 6.95 7.35 0.40 6.10 7.10 1.00 6.40 7.30 0.90

much as a whole unit. The slight difference between cannulated2 and freshly expectorated samples, together with the difficulties encountered in cannulating the glands, made it undesirable to pursue further this part of the investigation.

Since it required about 40 minutes to collect adequate amounts of saliva it seemed necessary to determine the changes taking

2 It should be pointed out that the cannulated samples were obtained under the influence of several types of stimuli (pressure, pain, psychic, etc.) and it is possible that the secretions of the different salivary glands are actually more acid than is indicated by our results.

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396 Reaction Changes of Human Saliva

place during that interval. A definite decrease in carbon dioxide was anticipated but the actual findings showed only a very slight loss, even when the saliva stood in an open container. Data, typical of the results obtained, are presented in Table III. While Findlay (17) found that carbon dioxide was more soluble in colloidal solutions, it does not seem that such an explanation could account for retention of the gas in saliva, considering that this fluid contains around 20 volume per cent, while 0.06 per cent (usually 0.04) is the highest value we can assign to this gas in laboratory air. Henderson and Stehle (18) found the carbon dioxide tension in the tissues of the mouth to be 54 mm. (58 mm. in saliva itself) therefore, when saliva is exposed to the air where the carbon dioxide is but 4 X 1O-4 per cent of the total tension, one would expect appreciable loss of the gas. The matter became

TABLE III.

Changes in Carbon Dioxide Content when Saliva is Kept in Open Container. Subject I<. C., Nov. 17, 1925.

Time incubated.

hrs

At once. 3 1 1Q

Carbon dioxide.

vol. per cent

27.4 26.5 26.0 26.9

PH

7.10 7.15 7.30 7.70

more clear when determinations were made on saliva which was kept closely stoppered; here, instead of a lower or a constant value, there was a definite increase. It was evident that carbon dioxide was being formed in the saliva and in the open vessels the gas formed was enough to replace that which was being lost, hence a nearly constant value was always found. It was also noticed that the formation of carbon dioxide was more vigorous in the thicker, heavier salivas than in the thin type. We were fortunate in securing a sample of very thick, sticky saliva from subject R. L. during an intense paroxysm of calculus deposition. The data presented in Table IV show the rapidity and extent of the carbon dioxide formation.

It was thought that the formation of carbon dioxide was too, rapid to be attributed to bacterial action. In order to ascertain

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G. W. Clark and K. L. Carter

more definitely the mechanism of this carbon dioxide formation it was necessary to select a substance which would be strongly germicidal for the ordinary microorganisms in the buccal cavity without inhibiting the action of any enzymes present. Accord- ingly, an excess of chloroform was added to half of a fresh sample

TABLE IV.

Changes in Carbon Dioxide Content When Saliva Is Kept in Closed Container.

Subject R. L., Nov. l&1925.

Time incubated. Carbon dioxide.

min. vol. POP cent At once. 22.3 30 23:l 60 26.0

PH

7.00 7.25 7.25

TABLE V.

Chloroform Does Not Inhibit Carbon Dioxide Production in Saliva (Open Container).

Time incubated.

At once. 30 min. 1 hr. 2 hrs.

Carbon dioxide.

Chloroform-treated.

vol. per cent

39.9 39.5 39.9 36. I*

.I Control.

vol. per cent

39.6 39.3 39.9 36.4*

* As can be seen from the data presented in Tables I to V and Figs. 2 to 4, the production of carbon dioxide is quite rapdd from the time of collection up to approximately 2 hours; subsequently, from the 2nd to the 51 h hours, there is usually a drop in the carbon dioxide content which in turn is fol- lowed by a steady increase in the content of the gas. These changes can be seen most readily by glancing at Figs. 3 and 4. Ilad Hall and Westbay followed the changes in the carbon dioxide content in conjunction with their observations on the pH, they would no doubt have noted the changes men- tioned above.

of saliva and, with the untreated portion as a control, the carbon dioxide changes followed through 2 hours of incubation. Since the control and the chloroform-treated samples show similar and concurrent variations, it is evident that chloroform did not in- hibit the gas formation (see Table V). Had preservation been

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398 Reaction Changes of Human Saliva

effected there should have been a noticeable decrease in the carbon dioxide content of the samples containing chloroform. The rapidity of the increase in carbon dioxide and the failure of chloro- form to check the gas formation suggest very strongly that bac- terial action is not responsible for the immediate production of carbon dioxide in t’he saliva. Since enzymes are “poisoned”3 by most of the heavy metals, such as mercury, experiments were made to determine the effects of mercuric chloride on the gas formation. Mercuric chloride (1 cc. of a 1 per cent solution per 100 cc. of saliva) was added to one-half of a fresh sample of saliva and with the other portion as a control: changes in the carbon dioxide content were followed as was done with chloroform. The

TABLE VI.

Mercuric Chloride Inhibits Production of Carbon Dioxide in Saliva (Open Container).

Subject. Time incubated.

L. L.. . . . . . “ . . . . . . . . . . . “ . . . . . . .

K. C.. . . ‘I . . . . . . . . . ‘I . .

hrs. vol. percent nol. per cent At once. 14.5 14.5 1 10.7 13.5 2 9.8 15.5 At once. 27.8 28.3 1 25.4 28.4 2 26.2 28.5

-

Carbon dioxide.

Morcuric chloride- treated. Control

results, given in Table VI, show that gas formation was inhibited by the mercuric chloride. The results presented in Tables III, IV, and VI suggest very strongly that the production of carbon dioxide is due principally to enzymatic action.

3 Since the composition of enzymes is unknown it is impossible to ex- plain the so called poisoning by mercury. As could be expected, we found considerable material precipitated whenever mercuric chloride was added to saliva. After removal of this precipitate by centrifuging, the super- natant fluid gave a black precipitate with hydrogen sulfide, indicating that we had added an excess of the reagent. These heavy metal-protein corn- plexes are appreciably soluble in physiological salt solution (0.9 per cent NaCl) and in this condition are relatively unstable. We are, therefore, unable to exclude decomposition of the Hg-protein complex by the hydrogen sulfide.

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G. W. Clark and K. L. Carter 399

As shown in Tables III and IV a rise in pH generally occurred simultaneously with increases in the carbon dioxide content of incubated saliva. Hall and Westbay (19) observed similar changes in about half of their samples and suggest loss of carbon dioxide as the possible cause. However, as we have pointed out in the preceding pages, it is not possible to correlate the changes in pH with changes in the carbon dioxide content. This anoma- lous condition shows that other agents are increasing the pH more effectively than carbon dioxide can decrease it. Although con- siderable ammonia is present in saliva (20-23), the extent to which it might form during incubation was not known and it occurred to us that production of this compound might partially or wholly

I ’ I I

3 6 HOUfJ

22

FIQ. 2.

account for the peculiar changes in pH. If the ammonia content of the saliva were due to bacterial action upon the urea present, as stated by Schmitz (20), the formation should be inhibited more or less completely by chloroform. The diverse action of chloro- form and mercuric chloride upon carbon dioxide production in saliva led us to repeat the experiments with these preservatives to determine their action upon the formation of ammonia. For this purpose a composite sample of paraffin-activated saliva was divided into three portions; to one was added an excess of chloro- form, to the second the customary amount of mercuric chloride, while the third portion was retained, untreated, for control. The results, given in Fig. 2, show that there was a very marked and rapid increase in the ammonia content of the control sample, over

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400 Reaction Changes of Human Saliva

300 per cent in 6 hours. Chloroform partially inhibited the formu-

tion of ammonia, an increase of only 44 ‘per cent in 6 hours in contrast to the complete failure in inhibiting the formation of

Vd f&r 637 f 20 I5

IO

5

z4

12

ZO

6.8 66

FIG. 3.

carbon dioxide. On the other hand, mercuric chloride was as effective in inhibiting ammonia production,4 as was observed in

4 A number of experiments was conducted to check the ammonia values obtained by permutit absorption with those obtained by aeration. The results showed that the more rapid permutit method is dependable and that it may be used safely with complex fluids such as saliva. Experiments with solutions of ammonium sulfate and saliva also showed that the amount of mercuric chloride used had no effect either upon the absorption by the permutit or subsequently on Nesslerization. Under existing conditions

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G. W. Clark and K. L. Carter

the experiments in carbon dioxide formation. The results with chloroform are contrary to those of TJpdegraff and Lewis (23) and place some doubt on the view expressed-by Schmitz (20) that the total ammonia in the saliva is the result of bacterial action

FIG. 4.

upon the urea. Having found definite increases in the ammonia content of incubated saliva, the next experiments included simul- taneous determinations of ammonia, carbon dioxide, and pH.

it is also unlikely that complex ammonium-mercury compounds were formed, certainly not in amounts greater than the inherent analytical errors.

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402 Reaction Changes of Human Saliva

Saliva was collected from several subject,s (either resting or paraffin-activated) and, after mixing thoroughly, the composite dividided into two equal portions; mercuric chloride was added to one-half and the other portion kept as a control. The results, given in Figs. 3 and 4, show the extent of the concurrent varia- tions in the pH, carbon dioxide, and ammonia. Considering first the samples treated with mercuric chloride, it can be seen that, with an almost constant, value for ammonia, there has been a gradual rise in pH which is apparently associated with a progres- sive loss of carbon dioxide. However, in the corresponding un- treated samples both the ammonia and carbon dioxide increased several per cent without any significant changes in pH. (Con- sidering the changes at the end of 9 and 10 hours, COZ increased 1.8 volume per cent; pH increased 0.05 unit in resting saliva. In paraffin-activat,ed saliva, CO, increased 6.3 per cent; ammonia nitrogen increased 7.8 mg. ; pH increased 0.30.)

The results of a large number of analyses5 show that resting saliva contains from 20 to 30 per cent as much carbon dioxide (as bicarbonate) and from 3 to 6 times as much inorganic phosphate as does blood plasma. Accepting an average pH of 6.6 (4-6) (9), it is evident that saliva is a well buffered mixture and would be able to neutralize large amounts of hydrogen or hydroxyl ions without showing appreciable changes in the pH. In this con- nection it should be noted that Bloomfield and Huck (5) gave human subjects as much as 20 gm. of sodium bicarbonate per day without effecting any changes in the pH of the saliva.

If deposits of calculus are associated with changes in reaction, it would seem that definite increases in the pH should cause measurable decreases in the amount of calcium held in solution. A number of calcium determinations made in conjunction with the data presented in Figs. 3 and 4 exhibited no such relationship. Thus in one instance the determinations made 12 hours apart showed an increase in the carbon dioxide of 9.2 volumes per cent, a decrease in the pH of 0.3 unit, with a 30 per cent decrease in the calcium content. When the pH reaches 8.0 or more, it is quite evident that much of the calcium would be precipitated (Ca mucinate, Ca3(P0&). Although the pH may subsequently de-

6 These figures were obtained from a large number of analyses of saliva (24, 25).

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G. W. Clark and K. L. Carter 403

crease by a whole unit or more, the concentration of hydrogen ions is never sufficient to redissolve the precipitate. This ex- planation would also a.pply to the results reported by Hall and Westbay (19).

SUMMARY.

The results of the experiments reported in this paper indicate that:

1. The saliva obtained by direct cannulation of the human parotid and sublingual glands is but slightly more acid, 0.1 pH, than freshly expectorated resting saliva.

2. Although the volume per cent of carbon dioxide is much higher in paraffin-activated saliva, it varies in much the same manner as in resting saliva.

3. Samples of either resting or paraffin-activated saliva may stand for several hours without showing appreciable changes in the carbon dioxide content. This is explained by an equilibrium bet,ween the carbon dioxide escaping and that being formed.

4. The formation of carbon dioxide is probably the result of enzymatic action.

5. There is apparently some ammonia formed by bacterial action. It is thought, however, that most of it is the result of enzymatic action.

6. There is no demonstrable relationship between the pH, the volume per cent of carbon dioxide, and the ammonia cont.ent. The pH changes in saliva apparently involve other constituents than those studied. To complete this aspect of the work properly it will be necessary to make complete analyses of a large number of samples of saliva.

BIBLIOGRAPHY.

1. Marshall, J. A., Am. J. Physiol., 1914, xxxvi, 260. 2. Foa, C., Arch. jisiol., 1905-06, iii, 369. 3. Kirk, E. C., J. Allied Dent. Sot., 1914, ix, 186. 4. Starr, H. E., J. Biol. Chem., 1922, liv, 43, 55. 5. Bloomfield, A. L., and Huck, J. G., Bull. Johns Hopkins Hosp., 1920,

xxxi, 118. 6. Bunzell, H. H., Colgate and Co. Bulletin No. 1, New York, 1923. 7. Lothrop, A. P., and Gies, W. J., J. Allied Dent. Xoc., 1910, v, 262; 1911,

vi, 65.

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404 Reaction Changes of Human Saliva

8. Pohle, E., and Strebinger, E., Deutsch. Monatschr. Zahnheilk., 1922, xl, 306.

9. Gans, L. F., J. Am. Dent. Assn., 1926, xiii, 222. 10. Siirensen, S. P. L., Ergebn. Physiol., 1912, xii, 393. 11. Clark, W. M., The determination of hydrogen ions, Baltimore, 2nd

edition, 1922. 12. Michaelis, L., and Pechstein, H., Biochem. Z., 1914, lix, 77. 13. Van Slyke, D. D., J. Biol. Chem., 1917, xxx, 347. 14. Chittenden, R. H., and Richards, A. N., Am. J. Physiol., 1898, i, 461. 15. McClelland, J. R., Am. J. Physiol., 1922-23, lxiii, 127. 16. Marshall, J. A., Am. J. Physiol., 1917, xliii, 212. 17. Findlay, A., and Creighton, T., J. Chem. Sot., 1910, xcvii, 536.

Findlay, A., and King, G., J. Chem. Sot., 1913, ciii, 1170. 18. Henderson, Y., and Stehle, R. L., J. Biol. Chem., 1919, xxxviii, 67. 19. Hall, I. C., and Westbay C., Dent. Cosmos, 1925, lxvii, 115. 20. Schmitz, H. W., J. Lab. and Clin. Med., 1922-23, viii, 78. 21. Hench, P. S., and Aldrich, M. J., J. Am. Med. Assn., 1922, lxxix, 1409. 22. Morris, J. L., and Jersey, V., J. Biol. Chem., 1923, lvi, 31. 23. Updegraff, H., and Lewis, H. B., J. Biol. Chem., 1924, lxi, 633. 24. Clark, G. W., and Shell, J. S., Dent. Cosmos, 1927, in press. 25. Clark, G. W., and Levine, L., Am. J. Physiol., 1927, in press.

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Guy W. Clark and Kenneth L. CarterSALIVA

REACTION CHANGES OF HUMAN FACTORS INVOLVED IN THE

1927, 73:391-404.J. Biol. Chem. 

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