STUDIES OF HUMAN MIXED SALIVA.

I. THE DETERMINATION OF THE HYDROGEN ION CONCEN- TRATION OF HUMAN MIXED SALIVA.*

BY HENRY E. STARR.

(From the Robert Hare Laboratory of Chemistry, School of Medicine, University of Pennsylvania, Philadelphia.)

(Received for publication, June 24, 1922.)

INTRODUCTION.

Variations in the composition of human mixed saliva, i.e. saliva as it normally is found in the mouth, consisting of a mixture of the secretions of the submaxillary, sublingual, parotid, and buccal glands, have been noted in various conditions of health and disease by countless investigators. The salivary reaction, particularly, has been the subject of many reports for almost a century.

In 1835, Donne (1) reported that although he found the saliva normally alkaline to litmus, he had found it acid in many diseases, including encepha- litis. In 1844, Wright (2) attempted to elaborate a diagnostic system of salivary analysis. About the same time, Simon (3) wrote at length on “morbid saliva” as distinct from “normal.” Among other studies of a similar character were those of Binet (4) in 1884, Sticker (5) in 1889, Die- minger (6) in 1898, Michaels (7) in 1902, and Fleckseder (8) in 1916. Since 1901, Kirk has been the chief exponent of the concept of “the saliva as an

* This investigation was undertaken at the request and with the coopera- tion of the Directors of the Psychological Laboratory and Clinic of this university, to determine if the salivary reaction might be of value as an addition to the usual clinical tests and measurements em$oyed in making a psychological diagnosis.

The writer desires to express his sincere thanks for their kindly criti- cism, advice, and cooperation to Dr. Glenn E. Cullen, Dr. Edward C. Kirk, Dr. John Marshall, Dr. E. B. Twitmyer, and Dr. Lightner Witmer. The advice of Dr. Cullen, particularly with reference to the technique employed for pH and COn determinations and in the preparation of the manuscript, has been especially helpful and is greatly appreciated.

43

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Determination of Salivary pH

index of faulty metabolism” (primarily in relation to the etiology of dental caries) and has conducted and fostered many investigations (9-14j. In 1916, Marshall (15-19) studied the salivary reaction in various physiological and pathological conditions, including dementia praecox and epilepsy, by means of a titration technique (“salivary factor” and “total neutralizing power”) which he had developed for the study of dental caries. In 1918, Ludlum (20) reported two sets of litmus paper reactions of successive specimens of saliva derived from two subjects whom he regarded as repre- senting different types of insanity. The chemistry of the saliva, espe- cially as a factor in dental hygiene, and the quantitatively determined salivary reaction in relation to the nature of substances taken into the oral cavity, has been intensively studied by Gies (21-28), Pickerill (29), and Prinz (30-32). Since 1919 Sullivan and his colleagues (33, 34) have been investigating the saliva of pellagra patients at various stages of the disease, employing refined quantitative methods, with especial reference to sul- focyanate content.

The peculiarly contradictory findings of many investigators, the innervation of the salivary glands by both cranial and sym- pathetic nerves, and the variation in quality and quantity of secretion depending upon the nerve stimulated (see Heidenhain (35) and Langley (36)); the adaptation of secretion to the ingested substance and the reflex secretion arising from purely psychic stimuli (Pawlow, 37); together with the dry mouth of fear (con- sidered by Cannon, 38) and the drooling of the idiot:-these factors render the problem of correlation between salivary composition and metabolic disorder a promising field for research.

Almost all of the investigators mentioned were interested in the salivary reaction as the principal variant. The early investi- gators employed the crude litmus paper test, reporting specimens simply as “acid” or “alkaline.” Probably the first reliable quantitative determination of the degree of acidity or alkalinity of human mixed saliva was that of Chittenden and Ely (39) who, in 1883, found the average alkalinity for fifty-one specimens as determined by titration with cochineal as indicator, to be 0.08 per cent “expressed as sodium carbonate.”

A subsequent series by Chittenden and Smith (40) published in 1885 gave a mean of 0.097 per cent similarly expressed. Schlesinger (41) ob- tained somewhat lower findings, reporting, in 1891, an average of 0.032 per cent, also expressed as sodium carbonate. Other investigators have re- ported on the alkalinity of the saliva expressed in terms of sodium hydrox- ide, among which may be mentioned Szabo (42) who, in 1900, found the

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Henry E. Starr

alkalinity of the saliva to vary between 0.058 and 0.064 per cent. Cohn (43) also in 1900, reported the alkalinity as varying from 0.002 to 0.048 per cent

expressed as NaOH. Probably the most extensive series of determinations of both “acidity” and “alkalinity” of human mixed saliva was that con- ducted by Gies (26) in 1916. In his report, Gies makes the statement that his results “have shown that salivary secretiorr varies, both qualitatively

and quantitatively, within limits that are independent of any mechanical stimulus or any dental condition “-a highly suggestive observation from the point of view of the present investigation.

In each of the above mentioned researches, all quantitative determina-

tions were made by titration, the indicators most frequently employed being litmus, cochineal, lacmoid, methyl orange, .p-nitrophenol, and phenolphthalein. The work was, consequently, on the “quantity factor” rather than the “intensity factor” of the acidity.

Very few investigations of the actual hydrogen ion concentration of saliva have been reported. Foa (44) is usually cited in this connection, and his findings, by the electrometric method, were reported in 1906. He found the mixed saliva before and after eating to have a hydrion concen-

tration corresponding respectively to pH 8.2 and 8.3. Kirk (12) published a series of determinations made by means of the

Hildebrand hydrogen electrode in 1914. His findings were pH 7.9, 5.6, 6.4, 7.7, 8.1, 7.5, 6.4, 6.2, and 8.6.

Michaelis and Pechstein (45) in the Course of an investigation of con-

ditions affecting the activity of salivary diastase, in 1914, reported electro- metric determinations of the hydrion concentration of three samples of saliva, undiluted; two samples diluted with “gewbhnlichem” distilled

water (presumably containing some CO*); and one specimen diluted with recently boiled freshly distilled water (i.e., approximately carbon dioxide- free). In each instance of dilution 1 volume of saliva was diluted with 9 volumes of water. Their results were: undiluted saliva, pH 6.79, 6.91, and 6.92; saliva diluted with “gewijhnlichem” distilled water, pH 6.65 and

6.34; saliva diluted with freshly boiled recently distilled water, pH 7.01. Their conclusion (Michaelis and Pechstein (45), p. 92) is “Da die neutrale Reaktion bei 7.07 (18”) liegt, so ist also der Speichel leicht sauer.”

A careful study of the literature has revealed only the above cited researches of Foa, Kirk, and Michaelis and Pechstein as stating definitely the hydrion concentration of human saliva, electrometrically determined. As to calorimetric determinations, Graham (46) made some pH determinations in an investigation of litmvs as an indicator of the salivary reaction within certain limits. In 1920, Bloomfield and Huck (47) in the course of an investigation of the bacteria of the oral cavity, calorimetrically determined the pH of a number of specimens of saliva. Unfor- tunately in the former investigation the saliva was dialyzed and

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46 Determination of Salivary pH

in the latter it was centrifuged, both procedures no doubt resulting in loss of COZ and resultant increase in pH, as indicated in a subsequent section. It is of interest to note in this connection that in 1916 Gies pointed out that centrifugation of mixed saliva results in increase of- the supernatant liquid’s alkalinity to phenolphthalein (25).

The suggestive fluctuations of the “quantity factor” of acidity of mixed saliva as reported by many investigators, the meagerness

. of data upon the “intensity factor” or hydrion concentration, especially as regards calorimetric determinations thereof, together with a realization of the ease with which such determinations might be made by adapting the technique instituted by SZrensen (48)) Henderson and Palmer (49)) Clark (50)) and Cullen (51, 52) led directly to the experiments reported in this article.

EXPERIMENTAL.

Methods.

The method which has been found most satisfactory and which has been employed throughout the present investigation (except when otherwise stated in the text) is briefly as follows.

The saliva is collected under oil’ in a clean receiver. 1 cc. is then re- moved by means of a pipette and transferred to a test-tube containing 9 cc. of freshly boiled distilled water (pH 6.6 to 6.7) and 1 cc. of 0.01 per cent aqueous solution of dibromothymolsulfonephthalein also under oil. The saliva and diluent are then mixed thoroughly by stirring with the flattened end of a glass rod beneath the level of the supernatant oil layer.’ After the diluted saliva is of uniform virage throughout,* the pH is determined by comparison with suitable standards against a milk-glass background.

Notes.

1. Preliminary to using any article coming into contact with the saliva -test-tube, pipette, etc.-it is washed and rinsed thoroughly with dis- tilled water and then tested with a few cc. of dilute indicator solution (e.g., 0.001 per cent brom-thymol blue).

1 As in Cullen’s method for determination of plasma pH (see Cullen, G. E., J. Biol. Chem., 1922, lii, 501).

2 The term “virage” was suggested by S$rensen and adopted by Clark (Clark, W. M., The determination of hydrogen ions, Baltimore, 1920, 38).

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Henry E. Starr

2. When distilled water was boiled vigorously for a few minutes and then allowed to cool to about 18°C. in a tightly closed glass-stoppered flask, the pH was approximately pH 6.6 to 6.7. Further boiling may result in a higher pH. However, pH 6.6 to 6.7 has been chosen as standard for the water employed in this investigation, as it is more readily attained and maintained than is a higher pfi value.

3. Brom-thymol blue as employed serves very well for the determination of hydrion concentrations between pH o ‘.% and 7.2, thus including the nor- mal range of salivary pH. For values above pH 7.00 phenol red (0.005 per cent) has been employed to advantage in the same manner. The actual quantity of indicator added is such as to give quite distinguishable virages over the range above indicated. The percentage of the solution employed is such that 1 cc. may be added instead of t,he usual drop or drops. 1 cc. may be measured more accurately by means of a pipette than can a drop by means of a rod or dropper. Moreover, the solution is thereby rendered so dilute that a slight error in the measurement thereof will have but a negligible effect upon the total amount of indicator added, thus tending t,o keep more nearly standard the conditions of intensity and brilliance of virage.

In lieu of preparing for each test a mixture of I cc. of indicator and 0 cc. of distilled water (pH 6.6 to 6.7), 10 cc. of a stock indicator solution of one-tenth the strength of that cited may be employed, in which case the pH of the diluent is obvious without, further testing.

4. SGrensen’s standards of .~/16 primary potassium phosphate and secondary sodium phosphate solutions, prepared from Merck’s chemicals were employed. They covered the range from pH 5.0 to 8.0 at int,ervals of 0.1 pH. Readings were made to 0.05 pH. Each standard is prepared in a manner similar to that employed for the salivary pH determinations and is kept sealed in hard glass ampules for more or less “permanent” use. It is necessary to check up the series from time to time by comparison with freshly prepared standards-a precaution employed weekly during the present investigation.

The use of the Walpole comparator, or the method of superposition, is seldom necessary as the saliva is but rarely colored. It is, however, fre- quently quite turbid and it is because of this interfering turbidit,y that dilution and the milk-glass backing are employed.

Dilution.-The saliva is diluted in the ratio of 1 : 9 with dis- tilled water, which is practically the same dilution as employed by Michaclis and Pechstcin (45) and also by Bloomfield and Huck (47). To determine the effect of such dilution upon the salivary pH, a quantity of saliva was collected under oil and portions of it were transferred by means of a pipcttc to a scrics of test-tubr ti containing (also untl(>r oil) such anlounts of distilled water as to render the proportion of saliva to water succcssivcly: 1 part of

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48 Determination of Salivary pH

saliva to 0, 1, 3, 5, 7, 9, 12, 15, and 20 parts of water. In each tube there had been previously placed and mixed with the water such a quantity of 0.01 per cent brom-thymol blue solution that the indicator was always present in the proportion of 1: 10 with respect to the total quantity of diluted saliva. Fifty specimens of saliva were thus investigated. The results are indicated in Fig. 1. The initial pH of the salivas examined ranged from 5.90 to 6.85. Temperature was at all times between 18 and 20°C.

k I

Irl

2ODL w UO.OL

13 5 7 9 12 15 20 PARTS I-40 ADDED TO 1 PAR-f SALIVA

FIG. 1. Dilution curve of human mixed saliva. Increases in pH given in 0.01 pH terms, are average values. The mean deviation from the aver- age increase at plateau level was less than 0.05 pH.

It is evident that the saliva undergoes a slight increase in pH on dilution up to a proportion of 1:3 or 1:5, when a plateau is reached and maintained at least until the dilution is 1: 12. The slight increase noted (in 94 per cent being 0.05 pH or less ) is of relatively little importance in this investigation, inasmuch as readings were made only to 0.05 pH.

It should be noted, however, that dilution of the saliva in the ratio of 1:5 was accompanied by an increase in 30 per cent of the specimens of 0.05 pH and 6 per cent increased 0.10 pH. The values of pH found by this method of dilution should, therefore, probably be reduced by 0.05 pH to obtain a value more closely approximating that of the undiluted saliva. As to the actual hydrion concentration corresponding to the values found

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Henry E. Starr 49

by the calorimetric method, no definite formula can be given, inasmuch as electrometric determinations to correlate the methods (involving deter- mination of protein error) have not yet been made. In the light of Cullen’s work on the calorimetric determination of the pH of blood plasma (52), it is probable that the method herein described yields somewhat too high pH values. In the present investigation, however, this is of relatively slight import inasmuch as the determinations were made for comparison of pH value found for different specimens of saliva when the same method was employed.

Collection of Specimen. Injluence of Parafin as Activator.

The work of Gies, Marshall, and others indicates that “acti- vated” saliva is quantitatively more alkaline than “normal resting saliva.” Although it appeared most probable that the same conditions would be found with regard to the salivary pH, the assistance which paraffin might give in facilitating the collection of specimens in quantity rendered it advisable to make a de& nite trial of its usefulness.

Each subject was seated comfortably before a rack containing three test-tubes, each containing about 1 cc. of oil and a thistle tube leading below the surface of the oil. The saliva was allowed to accumulate in the mouth during 5 minute periods, during which time the mouth was kept closed. The first sample was taken 5 minutes after the subject had seated himself and allowed the saliva to collect normally prior to ejecting it through the funnel tube into the test-tube below the surface of the oil. After ejection of the first sample, the subject was at once given a paraffin cube, previously tested for neutrality, which he chewed fairly vigorously for a second 5 minute period. He then ejected the accumulated saliva, removed the paraffin and “rested” 10 minutes, when he again allowed the saliva to accumulate normally, ejecting it at the expiration of the third 5 minute period. The salivary pH was always determined immediately after ejection. The results thus obtained in a series of ten experiments on five subjects appear in Table I.

The data in Table I show clearly that the use of even so Inert an activator as paraffin is precluded in obtaining specimens of saliva, inasmuch as in every instance its use resulted in an increase in pH of from 0.10 to 0.60 pH. In each instance, it may also be noted, the pH decreased to approximately its initial value after removal of the paraffin and 10 minute rest.

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50 Determination of Salivary pH

TABLE I.

The Effect of Chewing upon the Snlit~ary pli.

Subject.

IFT

IiII

IS

IG

BMT

Reaction of humnn mixed snliw collected in 5 min. periods.

Ikforc chewing.

pII

6.40 G.l.35

While ct1rwin.c. 10 min. afterchewinn.

p II

6.55 6.60

6.25 O.S5 6.30 6.35 G.iO 6.30

G.40

6.40 G.S3

G.i5 G.% (i 00

G.i5 G.55

G.iO i.05

6.35 (3.75

G.95

i.00 i.00

6.80 G.SO G.SS

-

Treatvlent of Specimen njtcr Ejection.

I3:spcrimcnts were made to cleterminc how long nftcr cjrction from the mouth human mised saliva may bc exposed to air at ordinary temperatures without undergoing change in hydrion concentration. It appeared probnblc that the salivary pH would increase more or less rapidly from loss of CO?, and that this loss would be prcventccl by covering the saliva with oil, a precaution found necessary by Bloomfield and Huck (47).

Each subject was instructed to retain the saliva as it collected naturally in his mouth until he had a “mouthful” and then to eject it into a small thistle tube leading below the surface of a few cc. of oil in a clean test- tube, in the usual manner. The quantity varied n ith different individuals, some apparently regarding 1 cc. as a “mouthful, ” \vhilc others furnished as much as 10 cc. at a single ejection. With care, abolit 5 cc. could usually

be collected. The pII of the saliva was determined immediately after ejection. Approsimately half of the saliva was then drawn by means of a pipette from below the surface of the oil and placed in a similar test-tube

exposed to the air. Lit intervals the p11 of the saliva in cnch tuhc was

determined until the supply of the particular spccimcn, exclusive of dregs, n-as cshausted.

Tnclvc spccimcns wcrc thus csnminctl. Only two of those which wrc untlcr oil sl~owcl any incrcnsc nftcr standing for 20

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Henry E. Starr 51

minutes, and they increased by but 0.05 pH. Of the portion exposed to the air, all had increased after 20 minutes exposure from 0.05 to 0.40 pH, and three-fourths of them increased 0.05 to 0.10 pH after but 2 minutes exposure.

It is obvious, therefore, that mixed saliva speedily increases in pH when allowed to stand exposed to air at ordinary room tem- peratures, and that oil employed as indicated in the preceding description of the general method is a necessary and adequate protection.

Determinations without Oil. Method B.-In certain investigations in the earlier stages of the present research oil was not employed and the saliva was mixed with diluent and indicator by pouring rapidly from one test-tube into another for a period of less than 1 minute. This resulted uniformly in loss of CO2 and increase in pH, as shown by the results of a series of 100 parallel determinations made in this manner and by the method employing oil as described in the text. The average increase was 0.15 PI-E, and the average deviation therefrom was less than 0.05 PH. Since the dilution effect is approximately 0.05, the findings of the non-oil method, as employed, may be regarded as uniformly 0.20 pH higher than the pH of the original undiluted saliva. Findings obtained by this method will be reported in certain instances in this series, but will always be accom- panied by the statement that they were obtained by the non-oil method.

It should be noted that the non-oil method is not recommended, for lack of care in mixing, or undue prolongation of the pouring can lead to an error of 0.45 pH, or more. 0.15 pH is presented as the increase arising from the method as employed in this investigation.

Centrifugation.-Since simple exposure to air results in loss of CO2 and consequent increase in pH, it would seem evident that centrifugation, in the usual manner,3 results in even greater loss of COZ. In 1916, Gies (26) reported that centrifugation of saliva results in decreased acidit,y of the supernatant layers to phenol- phthalein and increased acidity at the lower levels. The following determinations were carried out to determine the effect of centrifu- gation on mixed saliva, in terms of pH.

20 cc. of mixed saliva were collected from each subject, who was allowed to eject several “mouthfuls” to obtain that quantity. The saliva was col- lected below a 2 cc. layer of oil in a graduated 25 cc. cylinder in which had

For adequate measures to be employed in the centrifugation of carbon dioxide-containing solutions to avoid loss of carbon dioxide, see Cullen, G. E., J. Biol. Chem., 1922, lii, 508.

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52 Determination of Salivary pH

been placed 2 cc. of the brom-thymol blue solution, the ejections being

made into a funnel tube leading below the level of the oil. When the desired amount of saliva had been collected it was thoroughly mixed with the indicator by stirring in the usual manner. 11 cc. were then trans- ferred by means of a pipette to a 15 cc. centrifuge tube containing 1 cc. of oil, below which the saliva and indicator mixture were introduced. The

tube was then stoppered tightly with a rubber cork leaving an air-space of about 2 cc. A second 11 cc. portion was placed in a similar centrifuge tube withaut a protective oil layer and uncorked. The pH was determined by

comparison with standards in the same type of centrifuge tubes. Both tubes containing saliva were then centrifuged for 10 minutes at the rate of 2,500 revolutions per minute, when they were removed, the pH of each was again determined and any difference in pH at different levels in the tubes carefully noted. The sediment was then stirred thoroughly with the

supernatant liquid in each tube and the pH again determined.

This procedure was repeated on ten specimens obtained from four subjects. The initial pH values of the specimens ranged from pH 6.55 to 6.85. After centrifugation, all the specimens showed an increase in pH of the supernatant liquid. In the open tubes, the increment was from 0.15 to 0.55 pH and averaged 0.30. In the stoppered tubes the increment was from 0.05 to 0.20, averaging less than 0.15 pH. No definite pH value can be given for the lowest stratum of semisolid material which displayed a virage considerably below the limits of accuracy of the indicator employed; i.e., below pH 5.2. After mixing this material, which was mark- edly present in all but two specimens, with the supernatant liquid, the resultant mixture, in the open tubes, invariably showed a lower pH than did the unmixed supernatant liquid, but not as low as the initial salivary pH. Thus, after mixing, in the open tubes the pH was still 0.10 to 0.30 pH higher than the initial value. In the corked tubes, the mixture was 0.05 to 0.10 higher in pH value than was the initial value.

It is evident, therefore, that centrifugation as employedresults in loss of COz and precipitation of a substance of greater hydrion concentration than the supernatant liquid, both of which effects result in increased pH of the supernatant liquid. This increase is greater in the case of saliva exposed to the air than in that partially protected by oil and stopper. In the latter the CO2 no doubt came into partial equilibrium with the CO2 in the oil and air-space above the liquid, with consequently less loss of COZ.

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Henry E. Starr

If centrifugation is to be employed at all, it is obvious that rigid precautions must be taken to avoid loss of carbon dioxide. More- over, precipitation of the acidic sediment (termed “acid mucinate” by Gies (24)) may lead to erroneous findings as to pH value.

CONCLUSION.

A method for the calorimetric determination of the hydrogen ion concentration of human mixed saliva is described, together with the precautions necessary. Activators, such as paraffin, result in increase of salivary pH.

BIBLIOGRAPHY.

1. DonnB, A., Arch. med., 1835, viii, series 2, 53, 147. gen.

2. Wright, Der Speichel in physiologischer, diagnostischer und thera- peutischer Beziehung, Vienna, 1844.

3. Simon, J. F., Physiologische und Pathologische Anthropochemie mit Beriicksichtigung der eigentlichen Zoochemie, Berlin, 1842.

4. Binet, P., Sur la sueur et la saliva dans leur rapport avec 1’6liminatioq Paris, 1884.

5. Sticker, G., Deutsch. med. Ztg., 1889, suppl., No. 18. 6. Dieminger, H., Beitriige zur Kenntniss des menschlichen Mundspeichels

in gesunden und pathologischen Varhaltnissen, Dissertation, Wtire- burg, 1898.

7. Michaels, J. P., Sialo semeiology, Philadelphia, 1902. 8. Fleckseder, R., 2. Heilk. Abt. inn. Med., 1916, xxvii, 231. 9. Kirk, E. C., Dent. Rev., 1903, xvii, 383.

10. Kirk, E. C., Dent. Cosmos, 1910, lii, 729. 11. Kirk, E. C., Dent. Summary, 1914, xx&, 35. 12. Kirk, E. C., Dent. Cosmos, 1914, lvi, 1. 13. Kirk, E. C., Dent. Cosmos, 1915, lvii, 33. 14. Kirk, E. C., Crowell, W. S., and Appleton, J. L., Jr., J. Allied Dent.

Sots., 1914, ix, 186. 15. Marshall, J. A., Am. J. Physiol., 1914-15, xxxvi, 260. 16. Marshall, J. A., Dent. Items of Interest, 1916, xxxviii, 116. 17. Marshall, J. A., Am. J. Physiol., 1916, xl, 1. 18. Marshall, J. A., Dent. Cosmos, 1916, lviii, 1225. 19. Marshall, J. A., Dent. Cosmos, 1917, lix, 33. 20. Ludlum, S. D., Med. Clin. of North America, 1918, ii, 3, 895. 21. Gies, W. J., J. Allied Dent. Sots., 1911, vi, 334. 22. Gies, W. J., and Lothrop, J. Allied Dent. Sots., 1911, vi, 65. 23. Gies, W. J., J. Allied Dent. Sots., 1914, ix, 346. 24. Gies, W. J., J. Allied Dent. Sots., 1916, xi, 273. 25. Gies, W. J., and Shepard, L. A., J. Allied Dent. Socs., 1916, xi, 275. 26. Gies. W. J., J. Allied Dent. Sots., 1916, xi, 488.

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27. Gies, W. J., J. Allied Dent. Sots., 1916, xi, 659. 25. Lonenstein, G. A., and Gies, W. J., Proc. Sot. Exp. Biol. and Afed.,

1918-19, xvi, 53. 29. Pickerill, H. I’., The prevention of dental caries and oral sepsis, Phila-

delphia, 1st edition, 1910; New York, 2nd edition, 1919. 30. Prinx, II., Dent. Cosmos, 1918, Ix, 140. 31. Prinz, H., Denf. Cosmos, 1918, lx, 197. 32. Prinz, II., Dent. Cosmos, 1918, Ix, 287. 33. Sullivan, M. X., and *Jones, Ii. Ii, Pub. Health Rep. 5&S, 1919, 3. 34. Sullivan, M. X., and Dawson, P. R., J. Biol. Chem., 19%21, xlv, 473. 35. Heidenhain, R., in Hermann, L., Handbuch der Physiologie, 1883,

v, pt. 1. 36. Langley, J. N., J. Physiol., 1889, x, 291, 433. 37. Pawlow, J. P., The work of the digestive glands, London, 1902. 38. Cannon, \V. B., Bodily changes in pain, hunger, fear, and rage, New

York and London, 1915. 39. Chittcnden, R. H., and Ely, J. S., Am. Chem. J., 1882-83, iv, 329. 40. Chittenden, II. H., and Smith, H. E., Tr. Conneclicul Acad. Arfs and

SC., 1882-85, vi, 343. 41. Schlesinger, A., Virchows Arch. path. Anal., 1891, cxxv, 146, 340. 42. Szabo, J., Ungar. med. Presse, 1900, v, 653. 43. Cohn, M., Deutsch. med. Worh., 1909, xxvi, 68. 44. Foa, C., Arch. fisiol., 1905-06, iii, 369. 45. Michaelis, L., and Pechstein, H., B&hem. Z., 1914, lix, 77. 46. Graham, L. W., Denf. Cosmos., 1919, lxi, 409. 47. Bloomfield, A. L., and Huck, J. G., Bull. Johns Hopkins Hosp., 1920,

xxxi, 118. 48. S$rensen, S. P. L., Ergebn. Physiol., 1912, xii, 398. 49. Henderson, L. J., and Palmer, W. W., J. Biol. Chem., 1912-13, xiii, 398. 50. Clark, W. hl., The determination of hydrogen ions, Baltimore, 1920. 51. Van Slyke, D. D., Stillman, E., and Cullen, G. E., J. Biol. Chem.,

1919, xxxviii, 167. 52. Cullen, G. E., J. Biol. Chem., 1922, Iii, 501.

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Henry E. StarrHUMAN MIXED SALIVA

HYDROGEN ION CONCENTRATION OFTHE DETERMINATION OF THE

STUDIES OF HUMAN MIXED SALIVA: I.

1922, 54:43-54.J. Biol. Chem. 

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