Boston University
OpenBU
http://open.bu.edu
Theses & Dissertations
Dissertations and Theses (pre-1964)
1955
The chemical composition of
human saliva.
Chauncey, Howard Haskell
Boston University
http://hdl.handle.net/2144/8548
Boston University
BOSTON UNIVERSITY
GRADUATE SCHOOL
Dissertation
THE CHEMICAL COMPOSITION OF HUMAN SALIVA
by
Howard Haskell Chauncey
(B.S., Tufts College, 1949; A.M., Boston University, 1952)
Submitted in partial fulfilment of the
requirements for the degree of
Doctor of Philosophy
1955
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ACKNOWLEDGEMENTS
The investigations presented herein have been carried out
in the Department of Biochemistry, Boston University School of
Medicine, Boston, and in the School of Dentistry, Tufts College, Boston,
Massachusetts.
To Professor Fabian Lionetti for his continued interest and
to Professor Vincent F. Lisanti for the arrangements he made in
bringing this work to completion, I am pleased to have this opportunity
of expressing my gratitude for their never-failing support.
I would like to thank Professor Z. Hadidian for his many
suggestions and for permission to use his laboratory facilities.
I am
further indebted to Drs. R. A. Winer and A. Mesrobian, Mr. P. Weiss,
Mr. G. Atkins, Mr. S. Green, Mrs . I. Mahler, Miss R. Hill, Miss P.
Lorina, Miss M. Murphy, and Miss V. M. Johnson for their technical
assistance.
Grateful acknowledgement is due to Drs. William Fishman,
Howard Lind, and Irwin Sizer for their aid and suggestions.
These investigations have been supported in part by contracts from the
Office of Naval Research - NR 183-005, N7onr-46303.
LIST OF ILLUSTRATIONS
Chapter I
Colorimetric Methods for the Determination of Whole Saliva
Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Distribution Frequency Histograms of Whole Saliva Enzymes
Acid and Alkaline Phosphatase . • • . • . • • . • . . • . . • • • • • . • . .
Total Esterase and Cholinesterase • • • . . • • • • • • • • • • . • • • .
Lipase and Sulfatase . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .
Beta-D-Galactosidase and Beta-Glucuronidase . • • • • • • • • .
Hyaluronidase and Lysozyme . • • • . • . • • • . . • . . • . • • • • • • • • .
Mean, Range of Activity, and Source of Whole Saliva Enzymes . .
3
6
7
8
9
10
12
Chapter II
Inorganic Components of Parotid Saliva . • • . • • • • • • • . • • . . • • . • • .
Inorganic Components of Whole Saliva . • • • • • • . • . . . . . . • • • • • • . •
Secretion Rate of Inorganic Components of Stimulated
Parotid Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Organic Components of Saliva • • . . • • • • • . • • • • • • • • • • • . • • • • • • • •
pH and the Cationic and Anionic Balance of Stimulated
Parotid and Whole Saliva . • • . • • • • • • • • • • • • . • • . • • • • • • • • •
Results of Salivary Protein Analyses by Former Investigators..
Nitrogenous Components of Saliva • • • • • • • • • • • • • • • • • • • • • • • • • • •
Salivary Enzyme Titers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Serum Enzyme Titers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Population Distribution o,f Whole Saliva Components . . • . • . . . . • •
Population Distribution of Parotid Saliva Components • • . • . • . • • .
Population Distribution of Serum Components . • • • • • • . • • • . • • • . •
Comparison of Whole Saliva Sodium Values to Results of
Former Investigations . . . • . . . . . . . . . . . . . . . .. . . . . . . . • . . . .
Comparison of Whoie Saliva Potassium Values to Results
of Former Investigations . . • . • • • . • • • • . • • • . • • • • • • . • • • • . .
Summary of 11 t 11 Test of the Differences Between Whole
and Parotid Saliva Components • • • . • • • . • . • • . • • • • • . • • • • • •
27
28
29
30
32
33
34
36
37
39
40
41
43
44
55
Chapter III
Calibration Curve of Beta-Naphthol Estimation
Effect of pH on the Reaction Velocity • • • • • • • . . • . • . • • . • • . • • • • • •
60
61
b. Lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
c. Cholinesterase . . . . . . . . . . . . . . . . . . . . . . . . . .
d. Sulf.a tas e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Oxidizing Enzymes ..•••.••......•..••..•...
4. Proteolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . .
5. Miscellaneous Enzymes
a. Urease . . . . . . . . . . . .
b. Carbonic Anhydrase ..••..••••..•.••.....
c. Mucinas·es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
x v ii
xviii
x viii
xix
. ................... .
XX
x xi
xxi
xxi
xxi
Presentation of Investigations
Chapter I.
The Determination, Distribution, and Origin of
Whole Saliva Enzymes ....•.•..
. ........................ .
.....•..• . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Methods and Materials
l. Esterases
.......................
a. Acid Phosphatase
b. Alkaline Phosphatas.e .•.•••.•••..•.••.•.....
c. Total Esterase ...•...
d. Pseudocholinesterase
e. Lipase . . . . . . . . . . . . .
f. Sulfatase .••
2.
. ..................... .
...............................
Carbohydrases . ............................. .
a. Beta- Gal acto sidas e . ...................... .
b. Beta-Glucuronidase
c. Hyaluronidase .•...
d. Lysozyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
......................
l. Range and Mean Activity ...................... .
Parotid Saliva E n zymes ..•
.................... .
1. Range and Mean Activity
..................
B. Whole Saliva Enzymes
C.
D. Oral Microorganisms as Sources of Enzymes in
Whole Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . .
E. Discussion . •
. .. ............................... .
1
2
5
6
6
7
7
8
8
9
9
9
10
10
11
11
12
12
13
13
C h apter II.
Comparison of the Composition of Whole
and Parotid Saliva .••..•.••..••••
A. Methods and Materials ..
18
1. Inorganic Components ..•••••..••.••••...•.•
a. Sodium ...•
b. Potassium
c. Calcium
d. Chloride
e . Bicarbonate
f. Phos-p horus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Organic Components . • • . . . • . • . . • . • . . . . . . . . . .
a. Phosphorus
..••.
b. Lactic Acid
3. Nitrogenous Components
a . Total Proteins ..•.•.
b. Mucoid Nitrogen ....•...••.•..•......•..
c. Non Protein Nitrogen
4. Enzyme Activity .•••.•.•
a. Acid Phosphatase .••••••••..••.••..•..•.
b. Total Esterase ..
c. Pseudocholinesterase
d. Li pa·s e .... • . . . . . . . .
e. Beta-Glucuronidase
20
21
21
21
. ................... .
. ..................... .
27
.........................
38
B. Range and Mean Values
Distribution Analysis
19
19
19
19
20
20
22
23
23
23
25
25
25
25
25
25
...... ..............
c.
17
D. Discussion of Results
Chapter III. Characterization of Parotid Saliva Acid
Phosphatase . . . . . . . . . . . . . . . . . . . . . . . .
42
57
A. Methods and Materials .
58
B. Enzyme Characteristics •••..••.••.••.••.•••••.
l . pH Optimum ...••.
2. Substrate Optimum
3. Reaction Order . . . . . . . . . . . . . . . . . . . . . . .
4. Maximum Velocity and Michaelis-Menten
Dissociation Constant .••.••..•...•...•.
5. Temperature Coefficient of Activation .•••.•..
60
60
61
63
70
c.
79
Discussion ..•.
69
Chapter IV.
Characterization of Parotid Saliva Total
Esterases .•••.•
A. Methods and Materials .••
B. Enzyme Characterization
82
83
. .................... .
1. pH Optimum . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . .
2. Effect of Substrate Concentration
3. Reaction Order . . . . . . . . . . . . . . . .
4. Temperature and Enzyme Activity
\
C. Discussion ••..
85
85
86
88
91
92
Epilogue
..................................
Abstract .• . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . .. .. .. . . . . . .
References
.......................................
General Discussion
Autobiography . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
xxii
xxiv
xxix
liii
CONTENTS
Prologue
General Introduction
Literature Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
11
A. Inorganic Components .•.••..••.••••...••.•.•.
1. Sodium .••
2. Potassium
3. Calcium ..
4. Phosphorus
5. Chloride .•.
6. Bicarbonate
7. Thiocyanate
8. Trace Elements . . . . . . . . . . . . . . . . . . . . . . . . . . .
a. Magnesium . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
b. Copper . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . .
c. Cobalt
d. Iodine
e. Bromine
vii
vii
vii
B. Organic Components ..•.•. . •..•..•.•.••••.•••.
vii
1. Lactic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Organic Phosphorus ..•..•...•.•.....•..•.. .
vii
viii
ii
iii
iii
iv
iv
vi
vii
vii
vii
Vll
C. Nitrogenous Components ..•.•.•..••.••.•..•.•.
viii
1. Total Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v iii
ix
2. Proteins ..•
3. Non Protein Nitrogen .•.•.•..•.••.....•••.•.
X
D. Enzymic Components ....•..••.••.•••.••••.•..
xii
1. Carbohydrases
a. Amylase .••.
b. Maltase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
c. Invertase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
d. Beta-Glucuronidase
e. Hyaluronidase
f. Lysozyme
2. Esterases . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . .
a. Phosphatases ..•••••...•...•.........•..
xii
x iii
){ iii
x iii
x iv
xiv
xiv
x iv
x iv
Effect of Varying Substrate Concentration on the
Reaction Velocity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kinetics of the Enzymatic Hydrolysis of Beta-Naphthyl
Phosphate by Salivary Phosphatase .•.••••..•.••.....•.
II
II
II
II
Reaction Velocity and Substrate Concentration ..........•.•..
Velocity Constant and Enzyme Concentration ..•........••...
Determination of the Optimum Substrate Concentration ...••••.
Effect of Enzyme Concentration on the Reaction Velocity
Effect of Substrate Concentration on the Energies of
Activation and Inactivation • . • • . . . • . • • • . . . . • . . • • . . . . . . .
Effect of Temperature on the Reaction Velocity . . • • • • . . . . • . . • .
Temperature Coefficient (Ql o) . • • • • • • • • • • • • • • • • • • • • • • • • • • . . •
Heat Denaturation of Parotid Saliva Acid Phosphatase . . . • • • . • .
Determination of the Energy of Inactivation . • • • . . . • . . . • . • . . • .
Plot of S against S/v for the Effect of Temperature on the
Maximum Velocity and Michaelis-Menten Constant . . • • • .
Effect of Temperature on the Michaelis- Menten Constant
and the Maximum Velocity . . • • . • • . • . • . . • . . • . . . . . • . . • • .
62
64
65
66
68
68
69
70
72
73
74
75
76
77
78
Chapter IV
pH Activity Curve of Parotid Saliva Total Esterases .••.•..•••
Effect of Substrate Concentration ..•.•.••.•.••..•.•..•......
Kinetics of Parotid Saliva Total Esterases .•..••..•..••..••••
Substrate Concentration and the Reaction Velocity
Effect of Enzyme Concentration on the Reaction
Velocity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effect of Temperature on the Reaction Velocity ..•••..•.•••..
86
87
88
89
90
91
i
GENERAL INTRODUCTION
Saliva as obtained from the oral cavity is a mixture containing
the contributions of the various salivary glands, the oral tissues, microorganisms, and ingested substances.
Evidence has been obtained in-
dicating that saliva is a requisite for the proper maturation of erupted
teeth; based on the finding that animals with extirpated major salivary
glands are more prone to the carious process. (142a, b).
If saliva does have a protective function, as expounded by
many investigators , this protective function is not evidenced to the same
degree in all individuals.
In order to determine the possible presen ce of
protective agents in saliva and to clarify those processes which may
account for the apparent natural resistance to caries present in certain
persons, further information concerning the basic physiology of saliva
and the salivary glands is necessary.
Certain of the salivary components appear to be i n dependent
of the saliva flow rate, while others, sodium, potassium, bicarbonate,
proteins, and pH, are found to be influenced by the rate of secretion.
In
turn, the salivary flow rate is influenced by numerous systemic factors
which include; dehydration, infectious diseases, sleep, emotional factors,
mental abnormalities, nervous disorders, and the presence of various
drugs.
Whether or not these systemic conditions, by their indirect
effect on the salivary constituents, can contribute to the initiation of
oral pathology has not
bee~
indicated.
However, recent observations
have noted certain changes in the salivary components which do occur
,..
during systemic disease and subsequent investigations have emphasized
the interrelationships which might bring about these changes.
The work herein presented has had as its goal: the enlargement of the spectrum of salivary constituents; the determination of the
ii
b a se levels of the various components measured; and the elaboration
of their origin, characteristics, and interdependence.
LITERATURE REVIEW
A divergence of opinion exists concerning the physiological
role of saliva.
Certain early observers ascribed immunity to caries to
salivary protection, while others believed that the occurrence of caries
was due either to the absence of a protective mechanism in saliva or even
to a deleterious effect by the saliva itself.
Attempts to understand the
manner in which the saliva influenced the oral structures imposed a need
for information concerned with its composition.
Although hampered by
the lack of precise methods for the analysis of salivary components,
which rendered inaccurate the results of many early investigations,
certain observations as well as their observers were of outstanding
merit.
Limits have thus been set, us i ng for the most part, newer or
more reliable procedures which presented the greatest accuracy in the
evaluation of the constituents measured.
Inorganic Components
Sodium and Potassium - Although the most comprehensive studies of
salivary sodium and potassium have been limited to the past three decades, their presence was noted in whole and parotid saliva at least as
early as 1913 (Howe 1913a, b).
Observations by Ferris, Smith, and
Graves (1923); Clark et al. (1927); Becks (1929); and Mathis (1934) were
made using whole saliva samples from a limited number of persons and
in general were subject to the inaccuracies inherent in the gravimetric
iii
methods employed.
The development of more accurate procedures, zinc
uranyl acetate for sodium and the perchlorate butyl alcohol method for
potassium, afforded Brown and Klotz (1937); McCance (1938}; and
Vladosco and Bellea ( 1938) a more precise means for measuring these
electrolytes in whole saliva.
Flame photometry, with its high degree of accuracy and
rapidity of testing, provided the means for mass quantitative analysis
of salivary sodium and potassium.
Lorinczy and Karoly ( 1948);
Tekenbroek and Woldrung (1952); deTraverse and Coquelgt (1952); and
Hoffmeister and Albrecht (1953) measured the normal sodium and potassium titers in resting whole saliva, while Dreizen et al. (1953)
observed as well the changes which were brought about by salivary
stimulation.
Conscious of the variations in the ionic components of blood
due to certain systemic disturbances, other investigators (73, 79, 97,
109 , 266, 270) have studied the fluctuations of whole saliva sodium and
potassium which occur in patients with ascites, cirrhosis of the liver,
congestive heart failure, and adrenal cortical diseases.
Calcium and Phosphorus - Realization of the relationship between mineral metabolism, the endocrine system, nutrition, and systemic manifestations was the impetus to the initiation of studies concerned with
the calcium and phosphorus content of saliva.
Many investigations were
carried out in the belief that oral pathology was a result of metabolic
malfunctions which could be diagnosed by observing the variations in
these salivary components.
Ferris et al. ( 1923); Pattison ( 1926);
Clark and Levine (1927); Raskin (1927-28); and numerous other authors
attempted to establish the normal range and mean values for these constituents.
Evaluation and comparison of the results of these early
iv
authors is difficult due to their use of dissimilar technical procedures
and often outright disregard for the general health of the individuals
tested.
In 1934 Becks and Wainwright established standard procedures for salivary collection, treatment, and analytical methods which
are still followed.
They measured the calcium and phosphorus content
of 650 normal healthy persons and recorded the titers of resting whole
saliva and the changes which occurred upon stimulation.
To date their
work still stands as the most complete study of these two components
in whole saliva and is considered as the foundation for all subsequent
observations.
More recent efforts (12, 13, 14, 180, 224, 267) have been
focused on the use of radioactive isotopes for the elaboration of the
existence of mechanisms which transport calcium and phosphorus to
the enamel and dentine via the saliva.
Chloride and Bicarbonate- Howe (1912) observed the presence of
chloride in whole saliva and the individual glandular secretions.
A
method for chloride determination by titration with standard silver
nitrate solution was described, but no values for salivary chloride
were given.
In 1923 Ferris et al., using the Meyers (1921) adaptation
of the Volhard-Harvey method, measured the chloride content of three
persons subsisting on controlled diets.
The Whitehorn principle
( 1920-21 ), where the chloride of protein free filtrates is precipitated
by silver nitrate in the presence of nitric acid and the excess of silver
titrated with standard thiocyanate solution, appeared to be the procedure
of preference for most of the succeeding investigators.
Employing this
method Clark et al. (1927) examined the chloride content of resting
whole saliva in five physically sound subjects who were under strict
v
dietary conditions.
Clark and Levine (1927) could detect no consistency
in salivary chloride under regulated dietary conditions nor during
fasting.
Greater than normal variations occurred in saliva activated
by chewing paraffin than in resting saliva, while ingested chloride did
not readily i n crease the salivary levels.
The results from Brown and
Klotzrs (1934) efforts to correlate the chemical composition of the oral
secretions with rate of flow indicated that when the eighteen persons
studied were divided into three groups based on flow rate, low, medium,
and high; the high group had a mean chloride content which was almost
twice that exhibited by the low group.
The investigations of Mathis
(1934) and Vladesco (1938), although contributing to the establishment
of the normal range, were limited in the number of individuals tested.
McCance ( 1938) in testing the effect of salt deficiency on the composition
of body fluids encountered no consistent change in the concentration of
chloride in resting saliva.
Donneddu (1943) after comparing the sodium
chloride content of normal subjects and gastric patients, both fasting
and with salt loading, found that the sodium chloride of saliva varied in
the same manner as the hydrochloric acid of the gastric juice.
Persons
with hyperchlorhydria of the gastric juice exhibited values above the
cont r ol group, while the saliva of patients with histamine resistant
a n achlorhydria contained less sodium chloride than the controls.
Intra-
venous injections of thirty percent sodium chloride did not significantly
change the salivary level in any case.
Similarly, Lorinczy and Karoly
( 1948) evidenced no significant difference in salivary chloride in resting
saliva taken before meals, after meals, after the administration of
quinine, and after the imbibition of sucrose.
A parallel trend between
pH and chloride was recorded in the study of Popova-Milashevskaya
(1950).
White et al. (1950) pointed out a lowering of salivary chloride
vi
which was associated with congestive heart failure.
Salivary bicarbonate, whose presence was implied by
Howe (1912) and proven by Ferris et al. (1923), was first measured by
Clark et al. (1927).
Their determination of carbon dioxide, bound as
bicarbonate, was performed by the method of VanSlyke (1922), but
unfortunately no precautions were taken to prevent the changes which
occur on exposure to air.
The importance of bicarbonate was not
realized until Wah Leung (1951) indicated its role as a salivary buffer.
Sand (1951-52) established that the bicarbonate in saliva was the result
of the metabolic activity of the salivary glands and could be used as an
index of glandular activity.
No indication of the normal bicarbonate
levels in whole saliva was available until the study of Dreizen et al.
(1953) on human salivary buffers, where they presented data showing
the content of resting whole saliva and the increases resulting from
stimulation.
Thiocyanate - Thiocyanate was the first salivary component to be
analysed quantitatively.
Lodholz ( 1904) in a discussion of its glandular
origin relegated to the parotid glands the role of chief secretor of this
compound.
Its chemical origin and clinical significance were the ob-
jects of numerous speculations.
Howe (1909) realizing the inadequacies
of previous investigations attempted to establish standards for the
normal salivary content.
Attention was focused upon the possibility that the thiocyanate of saliva might exert a retarding influence on the carious destruction of teeth, either directly, by an inhibitory effect on the microorganisms concerned in carious production, or by interfering with plaque
formation and thus prevent localization of the caries producing agencies.
By 1915 a decrease of interest in thiocyanate had occurred and Kirk
(1915) in a report on the chemistry and physiology of saliva stated that
vii
thiocyanate was considered to be a normal excretory product without
influence in dental disease.
The recent increase in the use of thio-
cyanates as a hypotensive drug and the subsequent renewal of interest
concerning normal salivary levels led Fischmann and Fischman ( 1948)
to present one of the most reliable and uniform set of data on this
component in normal and hypertensive persons.
Trace Elements - In addition to the forementioned inorganic constituents of saliva there are electrolytes which are present only in small
or trace quantities.
Magnesium has been detected in amounts less than
one mg. percent by Krasnow, Oblatt, and Hirschfield ( 1938) and
Lorinczy and Karoly ( 1948).
Dreizen et al. (1952) have found saliva to
contain microgram quantities of copper and cobalt, the former being
present regularly while the latter was present only occasionally.
The occurrence of iodine in whole saliva has been noted by
several investigators (49, 50, 210), who found inorganic iodine to be
selectively concentrated by the salivary and gastric glands.
Most
recent concern has been with the role of salivary iodine and its relation
to hyperthyroidism (276) and thyroxine production (85).
Other authors
have given evidence for the presence of bromine in human saliva (249).
Organic Components
Lactic Acid - Salivary lactic acid originates from two major sources;
the secretion with saliva by the glands and its formation in whole saliva
and dental plaques by salivary and bacterial enzymes.
While the latter
theory has been substantiated by Summerson and Newwirth (1941);
Volker and Pinkerton {1947); and Muntz (1943), comparatively little
attention has been given to the glandular formation of lactic acid.
Mollman (1935) was the first to record the production of lactic acid by
viii
the salivary glands.
in the glands.
However, he asserted that it originated exclusively
Vladesco ( 1936) noted that lactic acid was always present
in stimulated whole saliva, even after an aqueous mouth rinse.
In
opposition to Mollman 1 s assumption that the glands were the sole source
of lactic acid, were the results of Helstrom and Ericsson ( 1952) who
observed that although lactic acid was present in the mandibular saliva
of only one of the several individuals tested, the parotid secretions of
these same persons always contained this component.
Furthermore,
whole saliva values which were similar to those evidenced in the parotid
saliva, increased markedly after a sucrose mouth rinse.
Koch ( 1944)
found the whole saliva of forty persons to contain an average lactic acid
level which was higher than their mean blood laCtic acid content.
Vais
and Tarnopolfskaya (1951) claimed that children with multiple-carious
teeth showed in their saliva obtained before meals, a level of lactic acid
that was three times that found in children without caries.
Fujishiro
( 1952) indicated that variations in salivary lactic acid were proportional
to the rate of salivary secretion and blood concentration.
His observa-
tion that salivary levels were lower than the blood content was in
direct contrast to the findings of Koch.
Organic Phosphorus - Although a few of the authors cited in the review
of inorganic phosphorus have also measured the organically bound
phosphorus of saliva, comparatively little investigation on this constituent has been done.
The most recent studies, by Eggers-Lura (1947,
1948) and Davies and Rae (1948), have involved a dispute as to the
correctness of the applied methods of measurement.
Nitrogenous Components
Total Nitrogen - Pavlov (1888, .185) in determining quantitatively the
ix
total nitrogen of saliva, during rest and activity, noted that the appearance of nitrogenous substances in the saliva was concomitant with a
decrease of nitrogen in the secretory gland.
Loss of nitrogen by the
gland, however, was much less than the increase in salivary nitrogen.
The difference was explained as a glandular replacement which was
equivalent to at least one-half the original loss.
This assumption was
confirmed when Henderson ( 1900) in a precise investigation of the
total nitrogen of the secreting gland noted after the initial loss, the
establishment of a nitrogen equilibrium during the period of activation,
The first work to be concerned with a survey of whole saliva
nitrogenous fractions was performed by Ferris, Smith, and Graves
( 1923) in their measurement of total nitrogen, mucin nitrogen, demucinized nitrogen, urea nitrogen, ammonia nitrogen, and non protein
nitrogen.
Results presented from three cases include values for mucin,
ammonia, and non protein nitrogen which are in agreement with later
authors.
Total nitrogen and total proteins were low in comparison to
more recent work.
Clark et al. ( 1927) studied the whole saliva total
nitrogen of five prison subjects and concluded that diet had no influence
on the reaction of saliva.
Although observing a correlation between
certain inorganic components of saliva and rate of secretion, Brown and
Klotz ( 1934) found no relationship between flow rate and salivary total
nitrogen.
Goldberg and Lee (1953), in a determination of the total
nitrogen content of stimulated saliva fractions, found the supernatant of
centrifuged whole saliva contained twice as much total nitrogen as the
precipitate.
Proteins - In 1935 Karshan , measured the total proteins of stimulated
whole saliva, in order to observe the possibility of a correlation between
protein content and dental caries.
No correlation was noted, but the
X
normal range and mean for the group were presented.
Bram.kamp
(1936 , 1937) investigated the total proteins and urea of stimulated
parotid saliva and found that the total protein content of the parotid
secretion tended to rise when the flow rate was increased.
In an effort
to find some relationship between salivary nitrogenous components and
dental pathology, investigations on the protein content of whole saliva
were carried out by Krasnow et al. (1938): Gorlin (1948); and Deakins
et al. (1941).
The results of these studies evidenced no conclusive
correlation between oral condition and saliva proteins, although the
latter authors observed a rough linear relationship between flow rate
and protein level.
Dreizen et al. (1952) in a study of the constituents
of human saliva buffers measured whole saliva proteins under resting
and stimulated conditions.
Their results indicated that the protein con-
tent decreased on stimulation.
Non Protein Nitrogen - The first accurate determinations of salivary
ammonia are generally attributed to Wurster (1889), who presented the
results of two cases.
Observations on resting and active glands and
the distribution of nitrogen between mucin and nonmucinous substances
by Anrep (1920-21), led him to conclude that the mucin of saliva was
entirely derived from material prestored in the gland and that the
nonmucin nitrogen was obtained from the body fluids.
Hench and
Aldrich ( 1923), in measurements of ammonia and urea in whole saliva,
encountered the fact that the ammonia of saliva increased at the expense
of urea.
The combined urea and ammonia nitrogen values were found
to approximate that of the urea nitrogen of the blood.
In urea retention,
the combined nitrogen of saliva (urea and ammonia) always increased
with an increase of blood urea nitrogen.
Morris and Jersey (1923)
reported the presence of amino acids, creatinine, and uric acid in
xi
whole saliva.
Values for salivary ammonia and urea were also pre-
sented, but their main interest lay with uric acid, which they believed
represented the actual cellular activity and could serve as an index of
glandular metabolism.
Barnett and Bramkamp (1929) in an effort to
determine the influence of secretion rate on the urea concentration
(urea and ammonia) in whole and parotid saliva observed decreases in
urea upon stimulation.
Greater decreases occurred in the whole saliva
urea than in the parotid saliva urea concentration.
Mean values re-
ported for stimulated whole and stimulated parotid salivas were almost
identical.
The non protein nitrogen of paraffin stimulated whole saliva
as investigated by Updegraff and Lewis (1924) proved to be 37o/o of the
normal blood level and was found to be comprised, at least in part, of
ammonia nitrogen, urea nitrogen, and uric acid nitrogen.
Youngburg
( 1935- 36) examined saliva in order to note the variations in the ammonia
concentration of resting whole saliva in "normal" individuals (presumable with a low caries rate) and in persons having active dental caries.
A comparison of values showed no characteristic difference in ammonia
content for the two groups.
Determination of ammonia nitrogen in the
salivas of pernicious anemia subjects and normal subjects indicated that
there was no effect related to the "alkaline tide".
The work of Evans
(1951), who measured the ammonia in the resting saliva of 208 persons,
was in agreement with the results obtained by Youngburg.
Sullivan and
Storvick (1950), however, upon investigating paraffin stimulated saliva,
from 572 college freshmen, found ammonia to be absent in some cases.
Moreover, they obtained a mean level that was considerably lower than
that experienced by either Youngburg or Evans.
In order to explain the presence of ammonia in whole saliva,
various hypothesis have been offered:
selective secretion of ammonia
by the gland and transformation of urea into ammonia under the influence
X l1
of the gland.
The first would seem less tenable because the amount of
ammonia in t h e blood is small, while the second would liken the gland
to the kidney in preserving the alkaline reserve of the organism and
necessitate the presence of urease in the gland.
The most accepted
theory at present, that saliva as secreted contains little or no
ammonia, is based primarily on the observations of Prinz (1921) and
Bliss (1937).
The former found that fresh saliva collected from the
duct of one of the secretory glands showed little ammonia, while whole
(mixed) saliva as found in the mouth contained a much greater concentration.
The latter author observed that calculus from the teeth con-
tained urease and he believed that the formation of ammonia was due
to bacterial action.
Cary (1946) in order to determine whether ammonia
was present in saliva as it came into the mouth and not due to the conversion of urea and other nitrogenous material by the bacterial enzymes
of the mouth, tested submaxillary and parotid saliva.
He concluded
that the salivary glands had neither an excretory nor an secretory
function in the formation of ammonia, but that the source was mainly
urea and proteins.
Kesel et al. (1947) found the source of ammonia N
in whole saliva not to be due entirely to urea, but that the amino acids
present, through bacterial deamination, contributed to its formation.
Jacobsen and Kesel (1950) noted increases in the ammonia N content of
incubated whole saliva at the expense of the other salivary nitrogenous
fractions.
Enzymic Components
Carbohydrases - Of the group classified as carbohydrases; amylase,
maltase, invertase, beta-glucuronidase, hyaluronidase, and lysozyme
have been found in human saliva.
xiii
Amylase
The amylolytic ability of human saliva was first d,iscovered
by Leuchs ( 1831).
The designation "ptyalin" was applied by Berzelius
(1840) to the salivary enzyme which had the ability to hydrolyze starch.
A crude isolate of salivary amylase was first prepared by Mialhe and
Cohnhein (1865).
( 1948).
The enzyme was later crystallized by Meyer et al.
The nature of the starch splitting reaction was established by
Myrback and Svanborg (1953).
Jytte ( 1954) was able to determine the
amino-acid composition of salivary amylase.
The relationship of salivary amylase to normal and patholo gical conditions of the oral cavity has been the object of numerous investigations.
The presence of amylase in parotid saliva and the increase in out-
put due to salivary stimulation was observed by Pickerill ( 1924), who
advanced the theory of a protective action by the enzyme.
Robb et al.
(1921) were i n agreement with Pickerill and believed the function of
amylase to be the cleaning of the teeth by the removal of starch.
Human
salivas exhibit considerable variation in their amylase activity (Deakins
et al. 67; Hubbell 130; Turner and Crowell 257; and Pratt and Eisenbrandt
192).
The use of the amylolytic index of saliva as an indicator for dental
caries susceptibility is highly disputed.
Hubbell (1933) and Florestano
et al. (1941) noted greater amylolytic activity in the saliva from carious
individuals as opposed to the findings of Marshall-Day (1934); Turner and
Crane (1944); and Sullivan and Storvick (1950) of an inverse relationship
between amylase content and dental caries.
On the other hand, McClure
(1939); Bergeim and Barnfield (1945); Barany (1947); and Hess and Smith
( 1948) reported no significant difference in the production of reducing
sugars by saliva from carious and noncarious individuals.
Maltase and Invertase
The presence of maltase in saliva was established by Tauber
X1V
and Kleiner ( 1933}, who noted that maltose was hydrolyzed after incubation with saliva.
uals.
Considerable variation occurred in different individ-
The presence of invertase was indicated by Volker 1 s (1950}
measurement of an increase in reducing substance concentration after
the addition of sucrose to saliva.
Similarly, Helstrom and Ericsson
( 1952} found the whole saliva lactic acid content to increase after a
sucrose mouth rinse.
According to the results of Bourquelot (1910}
and Lisbonne (1910), salivary invertase appeared to be a metabolic
product of the microorganisms which inhabit the oral cavity.
(3-Glucuroni dase, Hyaluronidase, and Lysozyme
Beta- glucuronidase was first noted in saliva by Fishman et
al. ( 1948}, who believed it to be a secretory product of the glandular
epithelium.
Salivary hyaluronidase (Lisanti 1950} and
f3- glucuronidase
have been found to be produced by certain of the oral bacteria (Mahler
and Lisanti 1952; Lorina et al. 1954).
The lysozyme activity of saliva
was first demonstrated by Fleming ( 1922}.
Penrose ( 1930} found that
lysozyme was active against Micrococcus lysodeikticus but ineffective
against many pathogenic organisms.
Holzknecht and Poli (1950) re-
ported saliva as having eight times the lysozyme content of serum.
Purification of salivary lysozyme was done by Roberts et al. ( 1938).
Phosphatases
Esterases - The possible relationship between salivary phosphatase
activity and the formation of dental caries and oral calculus led numerous
investigators to study the origin of these enzymes.
Hexosediphosphatase,
the first enzyme of saliva observed to have the ability to hydrolyze
XV
esters of phosphoric acid, was discovered by Demuth (1925).
(1936) confirmed its presence in saliva.
Giri
A pH optimum of 7. 0- 7. 2
was noted by both authors.
Adamson ( 1929) noted the presence of a phosphomonoesterase in gingival tissue which had an optimum pH of 9. 0.
He found
that saliva had a phosphorus containing complex, probably in the nature
of an ester, which was capable of being hydrolyzed by the gingival
tissue enzyme with the liberation of phosphorus.
The presence of a
phosphomonoesterase in saliva, but with an optimum in the acid
range, was shown by Smith (1930); Giri (1936); Glock et al. (1938};
Rae (1941); Eggers-Lura (1947); Dentay and Rae (1949); Fitzgerald
(1952); and Helman and Mitchell (1954).
The origin of the salivary
phosphomonoesterase has been a highly controversial subject.
Smith
(222) and Adamson (2) were of the opinion that the phosphatase came
solely from desquamated epithelial cells.
Umeno (1931) stated that the
enzyme was absent in saliva, but was present in the salivary glands.
Glock, Murray, and Pincus (104), unable to demonstrate phosphatase
in seitz filtered saliva and finding only slight activity in submaxillary
saliva, believed its presence to be due to epithelial cells and common
mouth organisms.
On the other hand, Zander (1941) and Valloton
(1942) showed phosphomonoesterase activity in gingival tissue only in
the endothelial cells of capillaries and subepithelial gingival tissue.
None was present in the epithelium of the gingival tissue.
From these
results it was inferred that the enzyme in saliva could be derived only
from bacteria or from tissue during inflammation.
Cabrini and
Carranza ( 1951) later substantiated the findings of Zander and Valloton.
Eggers-Lura (76) in an effort to determine the source of the
salivary phosphomonoesterase tested gingival tissue, plaque, enamel,
xvi
dentine, bone, and whole, parotid, and submaxillary saliva.
All were
active at pH 9. 4 and 5. 5, except for enamel and dentine which exhibited no activity at the acid pH.
acid range.
Saliva itself was more active in the
Dentay and Rae (69), like Glock et al. (104), found no
activity in Seitz-filtered saliva.
Although the residue from centrifuged
whole saliva had almost as much activity as the original mixture, t h e
clear supernatant contained only slight activity.
This, in addition to
their failure to find phosphomonoesterase activity in oral Lactobacillus
acidolphilus, led them to state; "saliva as secreted by the glands contains no phosphatase.
The small amount of phosphatase activity in
saliva is probably derived from cellular debris and food residues,
with a lesser contribution by microorganisms".
Aroused by the pre-
vious discovery of actinomyces in the matrix of oral calculus,
Ennever and Warner (1952) examined eleven strains of oral actinomyces
for phosphatase activity using a culture plate method.
strains tested were positive.
All eleven
Fitzgerald, opposed to the conclusions of
Dentay and Rae (69) that microorganisms played only a minor role in
the elaboration of salivary phosphatase, examined numerous strains of
oral bacteria.
He observed that streptococci, micrococci, Aerobacter
aerogenes, and lactobacilli had pronounced activity, both in the acid
and alkaline range.
Since no activity was evidenced in either parotid
saliva or bacteriological filtered saliva he expressed the opinion that
the major portion of saliva phosphatase was due to oral microorganisms.
He felt this supposition was supported by his finding of a correlation
between the lactobacillus count and the phosphatase values.
However,
Helman and Mitchell (116) were unable to find a significant relationship
between phosphatase activity and lactobacilli.
Moreover, they found
no difference in the phosphatase levels of persons with and without
calculus.
xvii
That the oral cavity contains other enzymes capable of
hydrolyzing phosphoric acid esters was shown by Axrr.tacher (1932),
who measured the enzymatic breakdown of pyrophosphate by saliva.
Cabrini and Carranza (52) demonstrated histochemically an enzyme 1n
gingival epithelium which was able to liberate phosphorus from nucleic
acid and a lecithinase in the subepithelial tissue, which had a pH
optimum of 9. 2.
From the results of the forementioned investigations,
it appears that saliva contains at least three phosphatases: hexosediphosphatase, pyrophosphatase, and phosphomonoesterase; the latter
being derived chiefly from oral microorganisms with minor contributions attributed to glandular secretion and ingested substances.
Lipase
Scheer ( 1928) investigated human salivary lipase and
found that this enzyme was present in infants as early as the first
month.
Scheer believed the function of salivary lipase was to free the
oral cavity of fatty materials.
After demonstrating the lipolytic
activity of saliva, by its ability to change the surface tension of a
saturated solution of tributyrin, Peluffo ( 1929) studied the effect of
inhibitors and investigated the enzyme's characteristics.
Koldajew
and Pik ul ( 1929) studied the lipase of dog saliva, which they obtained
through fistulae placed in the sublingual, submaxillary, and parotid
ducts.
The greatest activity was exhibited by the secretion collected
from the parotid gland.
pendent of amylase.
Salivary lipase activity proved to be inde-
Although Katzenstein ( 1929) noted an inverse
relationship between the lipase content of saliva and flow rate; the rate
of secretion being influenced by disease, Koebner (1931) found no
deviations of the lipase titer from normal, in several kinds of liver
xviii
disease.
Koebner, however, did find increases in the lipolytic titer
during pregnancy and lactation and decreases in persons with diabetes
mellitus.
There was close agreement by the above authors as to the
properties of human salivary lipase; an optimum pH between 7. 0 and
7. 8 and heat lability, the enzyme was destroyed at 65 degrees C.
Cholinesterase
The absence of acetylcholine (Gibbs 1935; Winterstein and
Ozer 1948) and the inability of saliva to hydrolyze acetylcholine (Bloch
and Nechles 1938; Gal and Adler 1948) has led to the belief that human
saliva is devoid of cholinesterase activity.
On the other hand, both
acetyl and pseudo cholinesterase have been detected in the salivary
glands of rats (Ord and Thompson 1950) and in the peripheral . autonomic
ganglia of the parotid glands of cats (Koelle 1950).
Hines and McCance
(1953) pointed out that whole and parotid saliva from pigs contained a
pseudo cholinesterase.
It would thus appear, from the evidence at
hand, that human saliva, in contrast to other mammals, does not
contain enzymes capable of splitting choline esters.
Sulfatase
According to Fromageot (1950) the following sulfatases
exist:
myrosulfatase, chondrosulfatase, glucosulfatase, and phenol-
sulfatase.
The designation phenolsulfatase has been recently changed
to arylsulfatase in order to include those enzymes which hydrolyze
sulfuric esters possessing an aromatic nucleus.
Pincus (1950) in
demonstrating the possible contribution. of sulfatases to the formation
of dental caries established the presence of sulfatase in proteus bacteria obtained from carious teeth.
In enumerating the processes which
xix
occur in caries, he included depolymerization of sulfated polysaccharides, release of sulfate, hydrolysis of collagen, and an increase
of solubility of calcium salts in the dentine.
Candeli and Tronieri
( 1951) pointed out that the sulfatase of Proteus and Staphlococcus
aureus which attacked beef costal cartilage, with the liberation of
sulfate ions, was also able to attack the glycoproteins of undemineralized enamel and dentine.
The enzyme responsible for this hydrolysis
of the organic sulfates of teeth would thus be categorized as either a
glucosulfatase or a chondrosulfatase.
Oxidizing Enzymes - The presence of catalase (Deakins 1941) in saliva
has been established, but the exact nature of the other oxidizing enzymes which exist in human whole saliva has not been defined.
Mosimann and Sumner (1951) observed a strong reaction for the saliva
of man and other mammals using hydrogen peroxide with guaiacol,
p-cresol, and pyrogallol.
Activity was noted in both the solution and
sediment from saliva and they were able to obtain a tenfold concentration by ammonium sulfate and alcohol fractionation.
the catalyzing agent as being a peroxidase.
They reported
In contrast, MacDonald
and Smith ( 1909) found that human saliva had the ability to oxidize
guaiacum, pyrogallol, chloral hydrate, and hydroquinone without the
addition of hydrogen peroxide.
Herlitzka (1910) found hydrogen perox-
ide to be inhibitory towards the oxidation of guaiac resin by saliva.
Additional reports of oxidative enzymes in saliva have been tended by
Ville and Mestresat (1912) and Besancon-Justin and Monceaux (1923).
These findings lead one to conclude that human saliva probably contains
at least three different oxidizing enzymes i the two iron containing
enzymes catalase and peroxidase, which have hydrogen peroxide as a
common substrate and a phenol oxidase, which while utilizing similar
XX
hydrogen donors as the peroxidase, catalyzes the direct oxidation of
its substrate by atmospheric oxygen and is a copper containing enzyme.
Proteolytic Enzymes - Willstatter et al. (1929) stated that the salivary
glands differed from secreted saliva in their enzymic constituents.
The salivary glands of man and other mammals were found to contain
proteinases and peptidases.
The proteinases were tryptic and
catheptic in nature, while peptic activity was lacking.
In human saliva
the complexity of proteolytic action was declared to be due to the
presence of epithelial cells, lymphocytes, leucocytes, and bacteria in
suspension.
After centrifugation salivary tryptic activity diminished
85%, the difference being found in the sediment.
That microorganisms
were an actual source of salivary protease was shown by cultures of
salivary bacteria proteolytic activity at pH 8. 0, though practically none
at pH 4. 5.
Willstatter, Bamann, and Rohdewald (272) produced a
proteolytic enzyme from leucocytes which appeared to be identical with
the one found in the saliva.
Thus, they were of the opinion that the
proteases present reflecte d in a measure the proteolytic equipment of
the lymphoid-lymphatic tissue of the salivary glands, their occurren ce
in the saliva being more or less adven titious.
Voss (1931) found that
the human parotid gland secreted a proteolytic enzyme which could
hydrolyze casein.
The activity varied with different individuals and
was greatest in those secretions which were yellowish in color, viscous,
and turbid.
Contrary to the findings of Willstatter et al. (272) for
whole saliva, the proteolytic activity of the parotid secretion was not
found in the sediment afte r centrifugation but in the clear supernatant
liquid.
No proteolytic activity could be conclusively demonstrated in
fresh sublingual- submaxillary saliva.
That the pH optimum for the
parotid enzyme was in the neighborhood of 8. 0 was taken as evidence of
xxi
its tryptic nature.
In 1935 Fantl and Weinman ( 1935a, b) showed that
saliva contained an enzyme which was capable of digesting the proteins
found in normal saliva.
Subsequent investigation (268, 269) indicated
that the salivary proteins, as well as other soluble proteins, were
digested by this enzyme.
A study of the saliva of caries-susceptible
and caries-immune individuals showed that the saliva of the immunes
had more of the proteolytic enzyme factor than in the susceptibles in
about 80o/o of the cases (269).
When the saliva was filtered the filtrate
contained no proteolytic enzyme.
Bacteria cultivated from the mouth
did not seem to influence proteolysis.
Other Enzymes - In addition to the already enumerated enzymes, various other enzymes have been reported to be present in saliva.
Vladesco (1932); Singer (1951), Ballantyne et al. (1952), Stephan (1940),
and Hine and O'Donnell (1943) found urease in human whole saliva.
Peptidase (Warfield, 1911); mucinase (Alfonsini, 1946); and collagenase
(Sreebny and Engel, 1951) activity have also been noted in saliva.
Rapp (1946) observed the presence of carbonic anhydrase, while Van
Haeff (1915) found saliva to contain an enzyme which could separate
hydrogen sulfide from horse radish.
\
CHAPTER I
The Determination, Distribution, and Origin of
Whole Saliva Enzymes
The enzymatic hydrolysis of synthetic substrates, whose
end products are capable of forming dyes, has been used for quantitative histochemical and physiological fluid determinations.
These
have been employed to study the effects of local and systemic disturbances (39, 61, 88, 106, 110, 135, 234, 280) and may be utilized in
evaluating the role of enzymes in normal and disease processes of the
oral cavity.
Numerous enzymes have been previously demonstrated in
saliva.
They include:
amylase, invertase, maltase, carbonic an-
hydrase, urease, oxidases, catalase, proteolytic enzymes, lipase,
phosphatases , lysozyme, and hyaluronidase (66, 94, 104, 155, 163,
172, 187, 197 , 229, 232, 254, 256).
While phosphatase, amylase
and lysozyme have aroused the most interest of the enzymes reported
to be in saliva, no agreement has been reached concerning their role in
various pathological conditions of the oral cavity.
Similarly, no effort
has been made to establish the normal range of activity of the salivary
enzymes.
Enzymes in the oral cavity may origin ate from several
sources: the major and minor salivary glands, bacteria, oral tissues,
and ingested substances.
It is of primary importance therefore to
have some fundamental knowledge concerning the various sources of the
enzymes present.
The measurement of a spectrum of enzymes using various
esters and der ivatives of a single chromogenic substance, beta-naphthol,
2
affords a means of comparing the various enzymes of saliva; while the
simultaneous determination of the activities of the ten enzymes included
in this study, on the same saliva sample permits the evaluation of the
possible interrelationship between individual enzyme systems.
Thus
the determination of the presence, range of activity, and origin of
several enzymes affords a better understanding of the factors distinguishing the normal from the abnormal in oral conditions.
The presence of certain hydrolytic enzymes in whole saliva
was tested.
Once their presence was established, whole saliva samples
were taken from 56 persons and the range of activity and the mean
activity of these enzymes were determined.
Parotid saliva was tested
for the presence of these enzymes; mean and range of activity were
also determined.
Inoculums of whole saliva were incubated in beef
brain-heart infusion broth to establish qualitatively whether or not
the flora of the oral cavity were capable of elaborating these enzymes.
The enzymes measured were:
acid phosphatase, alkaline
phosphatase, total esterase, cholinesterase, lipase, aryl-sulfatase,
beta-D-galactosidase, beta-glucuronidase, hyaluronidase, and lysozyme.
Procedure
The subjects were given code numbers, and twenty-five
ml. of paraffin stimulated saliva were collected in sterile bottles
from each individual.
The salivas were obtained immediately on
arising before toothbrushing, eating, or mouth rinsing.
All enzyme
activities were assayed from fresh saliva specimens with the exception
of beta-D-galactosidase and beta-glucuronidase, which were determined
on stored frozen samples ( -l 0°C for 10 days) .
The final volume of
saliva used for each colorimetric test system is given in Table l.
(56a)
. ., 3
These variations were necessary in order to· bring the quantity of dye
released fr o m the substrates during incubation within the range of the
colorimeter.
Hyaluronidase activities of the saliva samples were determined by a modification of the viscoslln.etric method (164).
zyme activities were measured turbidimetric-ally (223).
Lyso-
The remaining
enzyme activities were determined by the colorimetric methods of
Seligman and his co-wo.r kers, (6Z, 198, 206, 2.14, 215) modified for use
with saliva.
The -chromogenic hydxolysis product was either beta-
naphthol or a substituted naphthol.
When combined with tetrazotized
diorthoanisidine these produced a water insoluble dye which was extracted with ethyl acetate o·r chloroform and measured photocolorimetrically (Klett-Summerson).
TABLE I
ENZYME
VOLUME
OF
SALIVA
(ML.)
ACid phosphatase
0.05
Alkaline phosphatase
Esterases (total )
Cholinesterase
1.00
Lipase
Sulfatase (aryl)
0.10
2.00
.B- D-galactosidase
1.00
,8-glucuronidase
3.00
0.05
3.00
SUB·
STRATE
CONCENTRATION
(FINAL)
(MG. PER
S UBSTRATE
Monosodmm ,8-naphthylphosphate
Monosodium ,8-naphthylphosphate
,8-Naphthyl-acetate
,8-Carbonaphthoxycholine
iodide
,8-Naphthyl-laurate
Potassium 6-benzoyl-2naphthylsulfate
6-Bromo-2-naphthyl -.BD-galacto-pyranoside
Sodium 8-benzoyl amino
2-naphthyl glucuronide
PERIOD
DIAZONIUM
OFIN·
SALT CON·
CUBATION CENTRATION
TRICHLORO·
AT 37° C.
(MG. PER
ACETIC ACID
CONCENTRATION
(HOURS)
c.c.)
EXTRACTION
AGENT
c. c.)
0.1
pH
BUFFER
4.8
0.2 M acetate
2.0
4.0
40% (1.0 c.c.)
Ethyl acetate
0.1
9.1
0.1 M verona!
1.0
4.0
40% (1.0 c.c.)
Ethyl acetate
O.Ql
0.108
7.4
7.4
0.1 M verona!
0.1 M verona!
1.0
1.0
4.0
2.0
40 % (1.0 c.c.)
80% (1.0 c.c.)
Ethyl acetate
Ethyl acetate
0.071
0.25
7.4
6.1
0.1 M verona!
0.1 M acetate
5.0
24.0
4.0
1.0
40 % ( 1.0 c.c.)
40% ( 1.0 c.c.)
Ethyl acetate
Ethyl acetate
0.15
5.0
0.1 M acetate
2.0
1.0
80% (2.0 c.c. )
Chloroform
0.20
5. 0
0.1 M acetate
4.0
1.0
80% ( 1.0 c. c.)
Ethyl acetate
The substrate solution (1. 0 ml. f or sulfatase,
s. 0
ml. for
the others.) was mixed with the required saliva dilutions in a 20 ml.
test tube and incubated at 370C for the time periods- indicated in. T'a ble
1.(~6a)
After incubation the pH was adjusted with alkaline salt solutions (6Z,
Zl4) to bring the pH to the optimum for coupling with the diazonium salt,
One milliliter of freshly prepared solution of tetrazotized diorthoanisidine
4
was added to each tube.
After three to fifteen minutes a trichloro-
acetic acid solution was added to facilitate extraction of the dye from
the protein complex and to prevent decomposition of the excess
diazonium salt (Table 1).
The dye was extracted by the addition of
10.0 ml. of either ethyl acetate or chloroform (Table 1).
were then thoroughly shaken to extract the dye.
The tubes
The resulting emul-
sion was broken by centrifugation at 1500 rpm for fifteen minutes.
The colored organic layer was transferred with .a pipette to a Klett
tube for measurement of its color density with a photoelectric
colorimeter at 540 m).l.
The method of Rutenburg, Cohen, and Seligman (26) was
adapted for the determination of .aryl sulfatase activity of saliva.
Twenty-five (25. 0) mg. of the sulfatase substrate (6-benzoyl-2~
naphthyl sulfate) was dissolved in 100 ml. of acetate buffer, pH 6. 1
(0. 1M).
Two cc. of whole saliva was added to each tube containing
lee. of the substrate solution.
at 370C for 24 hours.
The tubes were shaken and incubated
Upon removal from the incubator lee. of 0. 2M
sodium dihydrogen phosphate was added to each tube.
A freshly pre-
pared solution of diazo blue B (lee. of a 1 mg. /cc. solution) was
added and the tubes were shaken and allowed to stand at room temperature for 15 minutes to permit complete color development.
One
cc. of 40 per cent trichlord:tcetic acid solution was added and the
color (dye) was extracted with 10 ml. of ethyl acetate as described in
the previous procedures.
The procedure for the determination of beta- glucuronidase
activity consisted of first dissolving 20. 0 mg. of 8-amino benzoyl-2naphthyl glucuronide in 80 ml. of distilled water.
100 ml. with acetate buffer (0 . 1M), pH 5. 0.
This was diluted to
Five ml. of this substrate
solution was mixed with 3. 0 ml. of whole saliva and the mixture was
.!")
incubated at 370C for four hours.
After removal from the incubator
0. 5 ml. of 0. 2M trisodium phosphate solution was placed in each tube.
One cc. of a lmg .
Icc.
solution of d iazo blue B was added and 5
minutes was allowed for color development.
acetic acid was used to stop the reaction.
One cc. of 8 0o/o trichloro-
The dye was extracted with
10.0 ml. of ethyl acetate and the Klett readings taken.
Color density was converted to micrograms of beta-naphthol
or substituted beta-naphthol from standard curves.
The curves were
prepared by adding varying amounts of these compounds to pooled
saliva followed by immediate color development with d iazo b lue B.
One unit of activity is defined as the amount of enzyme
whic h liber a tes the color equivalent of ten mg. of beta-naphthol or
substituted beta-naphthol during the period of incubation at 37°C.
This
unitage was used to facilitate comparison of oral levels of the various
enzymes.
The hyaluronidase activity is expressed as the percent of
the half-time , that is , the time required to achieve a SO% decrease in
viscosity of the substrate in 45 minutes at 25°C. ( 164).
The lysozyme activity of saliva was determined by comparison to a standard solution containing 1 I 100, 000 of crystalline egg
white lysozyme (Delta) which had a reaction time of 1. 25 minutes in
0. 85% NaCl at pH 6. 8.
The units reported were calculated by dividing
the reaction time of the individual salivas by the r e action time of the
standard (1. 25) and multiplying the reciprocal of this value by fifty.
Since the quantity of saliva tested was 2. 0 ml. this expressed the
activity as the amount found in l 00 ml. of saliva.
Results
Enzyme Activity of Whole Saliva
()
Fig. 1.
....... ·::·
10
··- ""'""..,.,---------------~
9
·:·.::.··:
.: ·..
7
6
1---- ------t....
::: . ::.:
. . ·: -: .. ::1--- - - - - - - - - - - - - - - - - - f
.. ·
4
.
.· ·: . :::.:·.
.··:·
·. .....
.
:··~
[:: ::m~(.· ·.
3
2
-
., ··
t\ •.
1-
0
PHOSPHATASE---~
Mean 5.55
RCIIIIJI 0.5-13.0
'--ACID
8
L.:
<
.00
0 .9
1.0
1.9
· .. · ,:.:
:,.;::.:,.:
2.0
2.9
4.0
4.9
3.0
3.9
5.0
5.9
-~ . ~:.,
t. ;, ·T··· . :.::-::_;:
6.0
6.9
7.0
l9
8.0
8 .9
9.0
9.9
10.0
10.9
11.0
11.9
12.0 13.0
12.9 13.9
Acid PhosphafQse
UNITS PER 100 MI. SALIVA
10 ~----------------------------------------------------------~
9
ALKALINE PHOSPHATASE
Mean .478
RaiiQe .025- 1.11
8
51-------f
41------~~:
;:_:-:·:1
31------+
..·
=: . •,
..
21------>:: .: (; ·
o~~~~~~~~~L~
'·J~
.00
.08
.08
.159
.160
.279
.240 .320 .400 .480 .1160
.319 .399 .479 .!158 .839
.640
.719
ALKALINE PHOSPHATASE
UNITS · PER 100 MI. SALIVA
Fig-. 2.
.7'20
.799
.100 .880 .960 1.040
.879 .959 1.039 Lll9
Fig . 3.
•or-----------------------------------------------~
9 ~-----------------------------8
1---------,.,,...,..,..,,...,.,,..--v ··: '::::-.-.·.:
~--t: .. ,
1
TOTAL ESTERASE
Mean 3 .23
Range .0.10-7.50 -----~
.
1 - - - - - - - - r . t ::·.· ... . . .. 1---- .:
; ..., . <.
,.,.,.,.,------1, .
6 t------...
- . :·:P :.
·:::·. ·:::::· . ..
-~ -:·
. .
"3
2
.
. ..
· ·.:
·..
.,·
l;
.., .
..
··: -
..
:: ·
i: :
t---i~·r,. r-~ r ;, /; ,, .•. •. '=
r--- ... ~---r; - -:· .·. ~> ·<··J."?-- . ·. >.}L -":1 ~
· ·: r-- 1·· .
1----
c::
:= ::·. • .-_) :
·;·: • :'
Y· · . •. :•.- ·••::'·,·. : ·:•••.:.;.· ' .:· .·:t[,,...,.
. ··.j
,.,.,.fi:""J
-ml·---i
••
.:
•:
-,; ~-.., _ ,·; :i::::.'~ ·:·.:,_.) .::,··: :.,, ..:· ~ : ·, ;.· ;.:\: ,):
0~--~~~~~~~~~~~~~~~~~~~~~--~
~~ -~-:
0 .0
.!19
0.60
Ll9
1.2.0 L80 2.40 3.00 3.60 4.20
1.19 2.39 2.99 3.59 4 .19 4.79
TOTAL
4.80 !1.40 6.00 6 .60 7.20
!1.39 5.99 6.59 7.19 7.79
ESTERASE
UNITS PER 100 MI. SALIVA
18
17
16
~
___
r . · ·or--------------------------------------------~
: .:
-
:,.
I!I
14
•1---------~--
13
•• . · ;':;
12
1- ;·. ·;,
II
f-
10
9
r--
8
f-
CHOLINESTERASE
Mean .053
---------i
Range .000-.221
·:,: ~
!--- ,,.~.
.. . . .-~~·.<~----------~---------------------i
. ·. :~
7 -
~
i[][l:r!:-=.': -Jt~=:'" 'l;n·t-.
6-
43
2. -
.---}t il·--·
- J___F_T__: .-:----1 --t
If-
oL-~~~~~~~~~~~~~~~~~~4=~~
.000 .017 D34 .051 .011 D85 .102 .lit .llle .1~3 .170 .117 .204 .221
.016
.033
.050 .017 .o84 .101
Jll
·' "
~2
J69
.116
.203
220 .23T
CHOLINESTERASE
UNITS PER 100 MI. SALIVA ·
Fig . 4.
\
... 8
Fig. 5.
10
9
LIPASE
8
Mean 1.18
Ranoe 0.20-2.70 - - - - - - - . 1
7
6
II
4
:.···-·. ·':
3
·
.·· ··:
2
·. ·
.:
. ..
:
..
;·.·
.
·.'·
T'::l
f:. :..:J
. ·_.,_
0
.00
.19
.20
.39
.40
.59
.60
.79
.80
.99
1.00
1.19
120
1.39
1.40
1.59
1.60
1.79
1.80
1.99
2.00 2.20
2.19 2.39
2.40 2.60
2.59 2.79
LIPASE
UNITS PER 100 MI. SALIVA
15
14
~--------------------------------------------------~--,
13
SULFATASE
12
Mean .191
Ranoe .025-.465
10 1-----
- -- - - - - - - - - f
9
8
UNITS PER 100 MI. SALIVA
F ig. 6.
9
Fig . i .
I~
...
1-
13 f12 1II
~,
~-
-
GALACTOSIDASE
Mean 1.07
Ranqe.04-2 .76
1-
10 1 - - - - --
o-
.. . ..
--------f:·. ·::1-- - - - - - --
---------1
9 18 f-
7
1-
6 f~
~-
4 1-
-------------1
~
3 1-
.,
.=;p
2 1-
· :: r,; ·.
.· ·.
1-0
...f - - --
. ·.
r,·.·.
.00
J9
.20
.39
t40
.59
.60
.79
.80 1.00 1.20 1.40 1.60
.99
Ll9 1.39 1.!59 1.79
fJ, o-GALACTOSIDASE
UNITS PER 100 MI. SAUVA
1.80
1.99
2.00 2.20 2.40
2.19 2.39 2.!59
2.10
2.79
18
l.
17
16
..
15
1
. - - - - -- - -
14
f3 - GLUCURONIDASE - - - - - - 1.
Mean .117
Ranoe .ooo-.533
..
13
12
II
r :·.
10 f - ) :· ..
9
J•.
v..: ::. ·.
: ~rr:·. > ·.:·. ·.·:j,"'....,..,:r - - - - - - -- -- - - -6
1-- . ·. :>• .
r·
.. ..
4
1-- . :··
. .:.f-- - - --
L' .·. F=.·.:
'
o
1---~e
- - -- - - - - - - - -- ----1
~
~---rf.:-.r,-,...,.
. ·.•.
·. ·. .~
-----1
.. .:.
. ... ...
:; •
t=+
.· - -.,-..
'..j ---- ---1
L_~~·~:~d·~;·.~:·~r~·~~~~:·~·. . L~~~.: ~·: j~----~r;:~::~J_____~r7l
.:~·· ~
.000
.040
,041
.081
.082
.122
.123
.1 63
.164 .205
204 . 245
.246 .287 .328 .369
.286 .3 27 .368 .409
~ -
GLUCURONIDASE
UNITS PER 100 MI. SALIVA
F ig. 8.
.410
.450
.451
.491
.492 .533
.532 .573
10
F ig . 9.
lOr-------------------------------------------------------~
9 1 - - - - - - - - - - - - - HYALURONIDASE - - - - 1
Mean 293'4
0 - 53'4 _ _ _ _ _-t
8 . - - - - - - - - - - - - - --.,..,,.,.,.,--RanQe
. ....··.
i:.. ·. :~i
7 t--~,:-~·-·:~_-;.1----------t:··· .. :~t----r.:'!'._..:C:O:..
l-------1
"'l
··••
6
r- :, ... ·]-----------!'~:· .·.. :_,1-,.,.,,---1;.. :.
c
:···:·::
: :
3
:~ .· ·.: ]------f"·{.:;:· :··-<:.t-----f~:r,; _.·:; : -~
i'::
I
0
·.·::
.
-b~~
,, .,..,..,
_
.----t
: 't"; .. :::
·.{ .<
:!----.,.,.,_,.,. ,_:,""' ... :: . _:'!·- :,·. :-: :.
- I-' . :'J-----,.,..,..,.f"'
.:-:-::
1/... t - - - - 1
·.·. - ·,, .."::-
:l - - - - - 1
2-
(::
.....
'" '··
0 .0
3.9
.:
r-:.··~
4 .0
7.9
8.0
11. 9
12.0
15.9
16.0
19.9
~
.:::: .·. ·l. .
20.0 24.0 28.0 32.0 36.0 40.0 44.0 48.0
23.9 27.9 31.9 35.9 39.9 43.9 47.9 51.9
HYALURONIDASE
PERCENT OF HALF TIME
52 .0
55.9
50r-------------------------------------------------------~
LYSOZYME
Mean 10.89
Ran9e .37-62.50
UNITS PER 100 MI. SALIVA
Fig. 10.
1l
Phosphatases:
The salivas of 56 adults contained a mean acid phospha-
tase activity of 5. 55 units/100 rnl. of saliva, with a range of 0. 5 - 13.0
units I 100 rnl. (F ig. 1).
The saliva of these same persons contained a
mean of 0. 478 units of alkaline phosphatase activity per 100 rnl., with
a range of 0. 025- 1.11 unitsllOQ rnl. of saliva. (Fig. 2) (56a) .
Esterase, Serum cholinesterase, Lipase:
The mean total esterase
activity of the saliva samples studied was 3. 23 unitsllOO rnl., with a
range of 0.10- 7. 50 unitsllOO rnl. (Fig. 3).
Of the 56 salivas tested
16 had no demonstrable cholinesterase activity.
The range of cholin-
esterase activity was from 0. 000 to 0. 221 unitsllOO rnl., with a mean
activity of 0. 053 units (Fig. 4).
The lipase activity varied from 0. 20
to 2. 70 unitsllOO ml., the mean activity being 1.18 unitsllOO ml.
of saliva (Fig. 5).
Sulfatase, Beta-D-galactosidase, Beta-glucuronidase:
The range of
aryl-sulfatase activity was from 0. 025 to 0.465 unitsllOO ml. and had
a mean activity of 0. 191 units I 100 ml. (Fig. 6).
Beta-D- galactosi-
dase activity of frozen saliva was found to have a range of 0. 04 2.76 units/100 ml., with a mean of l. 07 unitsl100 ml. (Fig. 7).
In
t h e determination of the beta- glucuronidase activity, three persons
were found to have no detectable activity in their saliva.
The mean
activity was determined to be 0. 117 unitsllOO ml., with a range of
0. 000 to 0. 533 unitsl100 ml. of frozen saliva. (Fig. 8).
Mucopolysaccharidase Activity:
Of the saliva samples studied 7 persons
were found to have no hyaluronidase activity.
Two of these were among
the three in which no beta- glucuronidase could be demonstrated, while
six of the seven were in the group showing no cholinesterase activity.
The mean hyaluronidase activity was 29. 3%, with a range of 0. Oo/o to
53. Oo/o (Fig. 9).
variable.
The lysozyme activity was found to b e extremely
This activity ranged from 0. 37 to 62.50 unitsllOO ml., with
12
an average activity of 10.89 units/ 100 ml. of saliva.
However, almost
one-half the subjects tested fell in the range 0. 00 - 4. 90 units/100 ml.
(Fig. 10).
TABLE
II
SOURCE
ENZYME
RANGE
OF ACT I VITY
MEAN
ACTIVITY
WHOLE
SALIVA
PAROTID
SALIVA
5.55
0.5-13.0
+
A cid phosphatase
0.478
0.025-1.11
+
Alkaline p h osphatase
3.23
0.10-7.50
+
Total esterases
0.000-0.221
0.053
+
Cholinester ase
0.20-2.70
1.18
+
Lipase
0.191
0.025-0.465
+
Sulfatase
0.04-2.76
1.07
+
/3 -D -Galactosidase
0.000-0.533
0.117
,8-Glucur ouid ase
+
10.89
0.37-62.50
+
Lysozyme
0.0 % -53.0%
Hyaluronida se*
29.3 %
+
• Acti vities for a ll enzymes except hya luroni dase a re expr essed as
saliva .
ORAL
BACTERIA
+
+
+
+
+
+
+
+
+
+
+
~
+
+
+
uni ts per 100 m i. of
Parotid Saliva: Sterile parotid saliva, obtained by cannulation with a
parotid cup (Curby 63), was shown to contain acid phosphatase,
esterase, cholinesterase, lipase, beta- glucuronidase, and lysozyme.
Sterility was verified by plating an aliquot on horse blood agar plates.
A random sampling of ten persons from the original group
showed that the acid phosphatase activity ranged from 0 . 250 to 0 . 770
units/100 ml. of parotid saliva, with a mean activity of 0.423 units.
The mean total esterase activity was 0. 034 units/100 ml. with a range
of 0. 012- 0. 065 units/100 ml.
Cholinesterase activity ranged from
0 . 023- 0. 043 units/100 ml . , with a mean activity of 0. 033 units/100 ml.
Lipase activity was found to have a mean activity of 0. 142 units and
ran ged from 0 . 025 - 0. 258 units/100 ml ,
concentration in parotid saliva.
Lysozy me was found in high
It ranged from 25 - 136 units I 100 ml.,
while the mean activity was 67 units.
While we could find no evidence
of beta- glucuronidase activity in parotid saliva, samples tested for us
in Dr. William Fish man's laboratory exhibited the presence of an
enzyme capable of hydroly zing phenolphthalein glucuronide.
The ran-
dom sampling demonstrated that the parotid activity ranged from 17 to
13
175 units ( F ishman) per 100 ml. of parotid saliva.
In the studies on parotid saliva no demonstrable activity was
found for alkaline phosphatase, aryl sulfatase, beta-D- galactosidase,
and hyaluronidase either in individual samples or in pooled parotid
saliva which had been lyophylized and reconstituted at three times
normal concentration.
Oral Enzymes of Bacterial Origin: In order to determine whether the
difference in the enzyme activity of whole and parotid saliva could be
attributed to the oral flora, inocula (one loopfull), of whole saliva from
each of the 16 subjects, were placed in beef brain-heart infusion broth
(Difco) and incubated for 24 hours at 37°C.
Using sterile incubated
broth as a control, the broth cultures were tested for enzyme activity
in the same manner as whole saliva.
These results indicated that the
microorganisms found in saliva were capable of producing acid phosphatase, alkaline phosphatase, esterase, cholinesterase, lipase, beta-Dgalactosidase, beta- glucuronidase, and hyaluronidase.
Effect of Freezing on Salivary Enzymes:
Comparative measurements
using frozen ( -10°C) saliva demonstrated decreases in activity for
cholinesterase (1 Oo/o), lipase (1 0-25%), esterase (0-15%), aryl sulfatase
( 15- 35%), and beta- glucuronidase (50%).
No change was observed for
beta-D-galactosidase and lysozyme, but an increase of 40-50% was
found in acid phosphatase activity and 10-20% for hyaluronidase activity.
Alkaline phosphatase demonstrated unexplained variable activity.
Re-
peated determinations showed increases to 35o/o in certain salivas while
others showed consistent decreases of 20%.
Variations of less than 1 O%
were within the range of reproducibility of the techniques employed.
Discussion
The presence of both acid and alkaline phosphatase in whole
14
saliva has been demonstrated.
While repeated determinations on
parotid saliva showed no evidence of alkaline phosphatase activity, the
acid phosphatase activity of parotid saliva was approximately 100/o of that
found in whole saliva, indicating that most of this enzyme is either of
exogenous origin or due to the secretions of the other glands.
Gal and Adler (99) were unable to demonstrate the presence
of either cholinesterase or a cholinesterase inhibitor in saliva.
The
determinations were done on whole and filtered saliva, but no account
was made of bacterial contributions.
Ord and- Thompson (183) in
measuring the rate of hydrolysis of acetylcholine, acetyl-beta-methylcholine and benzoylcholine by different tis sues of the rat found that
salivary glands contained approximately equal proportions of true and
pseudocholinesterase.
Koelle (146) found both cholinesterase types in
the peripheral autonomic ganglia of the parotid gland.
Bacteria have
also been noted to possess cholinesterase activity (248).
Cholinesterase
activity was found in whole saliva, parotid saliva, and in broth cultures
of the mixed flora obtained from the oral cavity.
However, the parotid
cholinesterase titer represented 60% of the activity of whole saliva.
Oral esterase (total) wa s
found to be of both glandular and bacterial
origin, with the parotid gland activity contributing about one per cent.
The lipase activity of parotid saliva amounted to about 10% of the whole
saliva lipase titer.
This enzyme was also produced by the oral
bacteria.
Sulfatase (190) was found in whole saliva but not in the
parotid secretion or in the oral microorganisms by our method.
Since
mammalian tissues contain aryl sulfatase it is possible that its presence
is due either to the cellular debris in saliva or to the other glandular
secretions.
However, work by other authors has indicated that certain
bacteria can elaborate these enzymes (9).
15
Beta-D-galactosidase activity has been found in certain plants,
molds, and mammalian tissues as well as in various strains of bacteria,
(Lactobacillus delbrueckii, Escherichia coli) (246).
This enzyme,
although found in whole saliva, did not appear to be present in parotid
saliva.
The bacteria inhabiting the oral cavity were found capable of
producing this enzyme and because of their high activity may be responsible for most of its production.
Fishman et al. (89) have found beta-glucuronidase activity in
saliva and suggested that it was secreted by the glandular epithelium.
Becker and Friedenwald (15) in a histochemical study of beta-glucuronidase
found it to be present throughout the entire submaxillary gland, and
present only in the tubular epithelium of the sublingual glands.
The data
of the present study indicate that the parotid activity of this enzyme was
from 5 to 20% of the amount found in whole saliva.
Organisms have
been isolated from the oral cavity capable of producing this enzyme (158).
There appeared to be a difference in the type as well as the amount of
activity of enzymes from both sources.
The parotid enzyme did not
appear to be capable of hydrolyzing the substrate employed.
However,
it was hydrolyzed by whole saliva to a greater degree than was phenolphthalein glucuronide.
Some of the bacteria of whole saliva were found
to be capable of producing a beta-glucuronidase which hydrolyzed both
the phenolphthalein glucuronide and 8-benzoyl-2-naphthyl glucuronide.
These preliminary experiments show that there may be a difference in
the action of this enzyme, depending on its source, glandular or bacterial, and that this action may be selective.
Skrotskii et al. (221) have shown lysozyme to be present m
the saliva of 95%- 98% of 336 patients, without any relation to the
condition of the mouth.
In the present work on 56 persons, all the
saliva samples contained lysozyme although to a highly varying degree.
16
However, the activity of parotid saliva was considerably higher than
that found in whole saliva.
This may be due to adsorption of the enzyme
on mucin, whose content is relatively higher in whole saliva and may
also be caused in part by the inhibitory effect of certain inorganic ions
such as chloride (218).
Hyaluronidase, present in whole saliva, but absent from
parotid secretion, is evidently of bacterial origin.
Mahler and Lisanti
have isolated four types of hyaluronidase producing streptococci from
the oral cavity (164).
The measurement of a spectrum of enzymes under the
described conditions has afforded comparisons of the contributions of
the activities from glandular, and bacterial sources as well as the
total obtained in the admixed (native) state.
The facility of simultaneous
measurements of these activities in single salivary samples allows for
detection of significant simple or multiple variations.
CHAPTER II
Comparison of the Composition of Stimulated Whole and
Parotid Saliva
Chapter I indicated that the parotid gland secretion contributed a part of six of the ten enzymes present in whole saliva, with the
remaining share of these six enzymes apparently derived from the oral
flora, cellular debris, and the other salivary glands.
In order to
further determine the part whole relationship of the parotid secretion to
whole saliva, various inorganic, organic, nitrogenous, and enzymic
components of stimulated whole and parotid saliva were measured.
An
aqueous mouth rinse was employed to minimize the effect of oral
microorganisms and cellular debris.
One enzyme, beta- glucuronidase,
was determined in whole saliva both before and after the washing process
as an indicator for the effectiveness of the mouth rinse.
Blood obtained
from the test subjects was examined for enzyme titer so that serum and
saliva enzyme levels could be compared using the same substrate for
both, while the saliva levels for the inorganic, organic, and nitrogenous
components could be compared to the established normal serum values.
Becks and Wainwright (18-29, 259-264) by their own efforts,
and in conjunction with an appraisal of the results tended by previous
workers, have attempted to establish the normal range and means for
salivary calcium and phosphorus.
However, there is no systematization
of the data on almost all of the other known components, and in many
cases sufficient data is not available to attempt a correlation of the
findings.
On reviewing the literature and attempting to deterw..ine the
normal range for these components, one is aware of a constant lack of
18
regard for biometrics.
Statistical evaluations were determined, in
many instances, employing no precautions about the normality of the
population distribution or the number of cases involved.
In several
publications which proposed that the variations of certain components
measured in persons with pathological conditions were significant, only
five or six subjects had been examined.
With the above-mentioned shortcomings in mind, the data
accumulated in the investigation of the comparative chemical properties
of human whole and parotid saliva are presented in a manner which is
believed to alleviate these difficultie$.
METHODS AND MATERIALS
Collection of Saliva and Serum:
Twenty-five ml. of whole saliva were obtained on arising,
before eating or toothbrushing.
The participating persons rinsed their
mouths thoroughly with tap water and whole saliva specimens were
collected by stimulation with paraffin.
Care was exercised to discard
the first 5 ml. in order to avoid dilution due to the rinsing process.
Parotid saliva was then collected from each subject by
means of a parotid cap (63).
chicle.
The stimulation agent used was flavored
When the flavor of the bolus being chewed was lost, it was
discarded and a new bolus was introduced; a standard bolus size was
constantly maintained.
The collection tubes, graduated at 12.5 ml. and 25. 0 ml.
levels, were immersed in ice water and the parotid saliva flow rates
were measured by recording the length of time required for the secretion to reach the 12.5 and 25.0 ml. marks.
Ten ml. of blood were withdrawn from each person and
19
allowed to clot.
After centrifugation the supernatant serum was removed
for testing.
All serum and saliva samples were kept refri g erated until
used.
Serum was tested. for acid phosphatase, total esterase, and
pseudocholinesterase levels.
and nitrogenous
com~onents
Whole and parotid saliva enzyme titers
were measured.
The above process, with the exception of the blood withdrawal,
was repeated the subsequent day using the same group of individuals .
Samples obtained the second day were examined for inorganic components,
total phosphorus, lactic acid content and pH.
An addition al 5. 0 ml.
saliva sample, for the determination of bicarbonate and pH, was collected
under a layer of paraffin oil.
Determination of Inorganic Constituents:
Calcium - The Clark- Collip (57, 58) modification of the Kramer- Tisdall
(148, 235) principle was employed, where the calcium was precipitated
directly as the oxalate and the latter titrated with potassium permang anate.
I
On the basis of Sendroys (21 7) results showing that the direct precipitation as oxalate from diluted serum gave acceptable results, and that
preliminary removal of protein was not necessary, the saliva calcium
was determined directly.
Calcium was recorded as mg. per 100 ml.
of saliva.
Sodium and Potassium - Sodium and potassium were measured using
quantitative flame photometry.
The procedure employed was that
suggested by Natelson (182) modified for use with saliva.
A solution of
the material (0. 2 ml. saliva diluted to 25.0 ml. with distilled water and
containing 50 ppm lithium sulfate as the internal standard for sodium
determinations and 250 ppm Liz S04 for potassium determination) was
20
tested and the galvanometer readings compared to standard solutions of
sodium and potassium.
The results were recorded as mg. o/o.
Chloride - The chloride content of whole and parotid saliva was determined using Benotti's (32, 273) modification of the VanSlyke principle
(245).
Saliva proteins were oxidized, and the chloride precipitated, by
wet digestion with concentrated nitric acid in the presence of silver
nitrate.
The excess silver was then titrated with thiocyanate.
Salivary
chloride was expressed as mg.% for whole and parotid saliva.
Bicarbonate - The bicarbonate content of whole and parotid saliva was
determined by the titration method of VanSlyke (243, 244), where the
sample to be measured was treated with an excess of standard acid
(0. OlN HCl) which was titrated back with standard alkali (0. OlN NaOH)
to the original pH of the sample as obtained.
One ml. of saliva was
substituted for plasma and the determination done as described by
Hawk, Oser, and Summerson ( 114).
The salivary bicarbonate content
was expressed as mg. o/o of bicarbonate ion.
Phosphorus - The total phosphorus content of whole and parotid saliva
was determined using the following modification of the digestion methods
presented by Davies and Rae (65) and King (141).
1. 5 ml. of saliva were
diluted with 4. 5 ml. of distilled water and 1. 0 ml. aliquots were placed
in test tubes.
1. 2 ml. of 60% perchloric acid were added to each tube and
the mixture was digested for thirty minutes in a boiling water bath.
The tubes were then allowed to cool and 4. 0 ml. of water were added
with mixing.
Next 1. 0 ml. of a 5% solution of ammonium molybdate
was added and the tubes were centrifuged for five minutes at 2500 rpm.
5. 0 ml. of the resulting mixture were placed into Klett tubes and the
phosphorus content determined employing the method of Fisk and
2t
Subbarow (96), where inorganic phosphate which has reacted with
molybdate is reduced with 1-amino 2 naphthol 4 sulfonic acid (ANSA)
and the intense blue color which develops is measured.
One ml. of
ANSA reagent (113) was added to each tube and after allowing the tubes
to stand 20 minutes the color density was measured in a Klett Photoelectric colorimeter using a 660 m)l filter.
Values were compared to
blanks and a standard which were treated in the same manner.
The
blanks contained l. 0 ml. of water while l. 0 ml. of a standard solution
containing 0. 01 mg. P/ml. was used for comparison of color density.
Inorganic phosphorus was measured by adding 8. 0 ml. of
10"/o trichloroacetic acid to 2. 0 ml. of the previously diluted saliva.
The mixture was shaken well and centrifuged for five minutes at 2500
rpm.
F ive ml. of the supernatant were pipetted into a test tube and
l. 0 ml. of Molybdate II reagent (113) was added.
The tube was then
centrifuged for ten minutes at 3000 rpm and the supernatant fluid
removed, placed into a Klett tube and mixed with 0. 5 ml. of ANSA.
The color density was measured after allowing five minutes for complete color development.
Blanks and standard solutions were prepared
as described for total phosphate,
Total organic phosphorus was determined as the difference
between total and inorganic phosphorus.
Lactic Acid - The salivary lactic acid content was measured according
to the method of Barker and Summerson (10) where the glucose and
other interfering materials of the protein free filtrate are removed by
the Van Slyke- Salkowski method of treatment with copper sulfate and
calcium hydroxide.
An aliquot of the resulting solution is heated with
concentrated sulfuric acid to convert lactic acid to acetaldehyde which
is then determined calorimetrically by reaction with p-hydroxydiphenyl
22
in the presence of copper ions.
Although the recommended procedure indicates the removal
of proteins with either tungstic acid, trichloroacetic acid, or zinc
hydroxide, preliminary experiments with known amounts of lactic acid
added to parotid saliva indicated that the small amount of protein
present did not appear to influence the measurement.
Whole saliva
which contains debris, contributed from several sources, did not give
as accurate results.
In order to alleviate this difficulty whole saliva
mixed with 32% trichloroacetic acid in a 1:2 ratio (3. 0 ml. saliva, 6. 0
ml. TCA) was filtered, and the filtrate tested.
Results were recorded
in mg.%.
Measurement of pH - Specimens of stimulated whole and parotid saliva
were collected in tubes containing a layer of paraffin oil to prevent
loss of carbon dioxide and subsequent change of pH.
A graduated 1. 0
ml. pipette was inserted below the layer of oil and 1. 0 ml. of saliva
removed for measurement in a Beckman model G glass electrode pH
meter.
Measurement of Nitrogenous Components:
The Micro-Kjeldhal method (162) was employed for the
determination of the nitrogen content of the various protein and non
protein nitrogenous components of whole and parotid saliva.
Total nitrogen was measured by the above stated procedure
where duplicate samples of saliva were digested with catalyst plus hot
sulfuric acid and the nitrogen converted to ammonium sulfate.
The
ammonia liberated by the addition of alkali was distilled into a boric
acid solution and titrated directly with standard acid.
The non protein nitrogen content of saliva was determined
23
using the Somogyi ( 225) method of deproteinization.
One ml. of the
saliva to be tested was placed in a 15 ml. conical centrifuge tube and
diluted with 1. 0 ml. of distilled water and 1. 0 ml. of 1. 8% zinc sulfate.
One ml. of 0. 1 N sodium hydroxide was added and the tube was shaken
vigorously.
rpm.
The tube was then centrifuged for fifteen minutes at 2, 000
Duplicate 1. 0 ml. samples of the supernatant fluid were pipetted
into Micro-Kjeldhal digestion tubes and the nitrogen content measured.
This procedure was employed in lieu of tungstic acid or trichloroacetic
acid precipitation because of the low protein content of saliva.
When
only small amounts of protein are present these latter two methods
were found to be inadequate for complete precipitation of the protein
substances.
Salivary albumin was determined using the method of
Wolfson et al. (275) modified for use with saliva.
One ml. of saliva
was pipetted into a conical fifteen ml. centrifuge tube and 2. 0 ml. of
34. 5% sodium sulfate were added.
to stand for five minutes.
This mixture was shaken and allowed
If after centrifugation for fifteen minutes at
2, 000 rpm. the liquid was not cleared of the precipitate which formed
on the addition of the sulfate, 1. 0 ml. of span ether was added and the
tubes stoppered and reshaken and centrifuged.
One ml. samples of
the albumin containing solution were removed and the nitrogen content
measured.
The mucoid components of whole saliva were separated out
using the method of Glass (1 02).
2. 0 ml. of whole saliva were mixed
with 2. 0 ml. of 10% sodium hydroxide and 1. 0 ml. of distilled water.
Five ml. of 32% trichloroacetic acid were added and the tube shaken
and left at room temperature for ten minutes.
The solution was then
filtered with Whatman #2 filter paper and the filtrate collected.
Five
ml. of the trichloroacetic acid filtrate were placed in a conical fifteen ml.
24
centrifuge tube.
After the addition of 5. 0 ml. of acetone the tube was
stoppered, inverted twice, and placed in a water bath at 37 degrees for
ninety minutes.
mucoids.
rpm.
This procedure permitted the precipitation of the
The tube was then centrifuged for fifteen minutes at 2, 000
The acetone solution was decanted, the precipitate washed once
with acetone, and the tube finally inverted on a filter paper in order to
allow the acetone to drain.
Sodium hydroxide, 5. 0 ml. of a 0. 1 N
solution, was added to the precipitate and the mixture stirred to insure
complete solution of the mucoid.
Two ml. of this solution were placed
in a digestion tube and the nitrogen content determined.
Since repeated samples showed no mucoid to be present, the
total protein nitrogen of parotid saliva was calculated as the difference
between the total nitrogen and the non protein nitrogen.
For whole
saliva it was necessary to subtract the mucoid nitrogen and NPN in
order to obtain the total protein nitrogen.
Albumin nitrogen was de-
termined by analysis of the fluid remaining after precipitation of the
globulin and mucoid fractions with sodium sulfate, making the appropriate correction for non protein nitrogen.
Globulin in parotid saliva
was estimated by subtracting the albumin nitrogen from the total
protein nitrogen content, while whole saliva values were obtained by
subtraction of albumin nitrogen and mucoid nitrogen from the total
protein nitrogen.
Results were reported in mg. o/o for total protein, albumin,
· and globulin.
The nitrogen value for each fraction was multiplied by
the factor 6. 25 which allowed conversion to protein, based on the
assumption that the average protein contains 16o/o nitro g en.
nitrogen and mucoid nitrogen were recorded as m g. o/o N.
Non protein
25
Estimation of Enzyme Activity:
Acid Phosphatase - The method for the phosphatase determination of
serum was that described by Seligman et al. (214), while saliva was
measured using the modification described in Chapter I.
For the
determination of the acid phosphatase activity of whole saliva, 1. 0 ml.
of saliva was diluted with 19. 0 ml. of distilled water and 1. 0 ml. of this
0. 05 dilution was tested.
In the determination of parotid saliva phos-
phatase activity 0. 5 ml. of saliva was tested directly.
Total Esterases and Pseudocholinesterase - Total esterase activity of
serum was measured according to the method of Ravin, Tsou, and
Seli g man ( 198).
Whole and parotid saliva were determined using the
modification described in Chapter I, substituting the following saliva
volumes.
Whole saliva total .e sterase activity was measured using
1. 0 ml. of an 0. 05 dilution of saliva and distilled water, while a 3. 00
ml. sample of parotid saliva was found to produce the most accurate
estimation under the test conditions.
Cholinesterase activity of whole
saliva, parotid saliva, and blood serum was determined in the same
manner as the total esterases, substituting beta-carbonaphthoxycholine
iodide, as substrate, for the beta-naphthyl acetate.
The saliva volume
used was 3. 0 ml. for both whole and parotid saliva.
Lipase - The lipase activity of saliva was determin ed using the method
of Seligman and Nachlas (215) (Chapter I) which employs beta-naphthyl
laurate as substrate.
Activity was measured following the described
procedure but substituting 1. 0 ml. of an 0. 10 ( 1. 0 ml. of saliva diluted
with 9. 0 ml. of distilled water) dilution for whole saliva and 1. 0 ml. of
undiluted parotid saliva.
Beta-Glucuronidase - Since the substrate previously used (Chapter I),
2G
8-amino benzoyl naphthyl glucuronide was unavailable, beta- glucuronidase activity was measured by the method of Fishman et al. (89).
The
volume of whole and parotid saliva employed was the same as the
blood serum volume suggested by this technique.
Beta- glucuronidase
units are reported as the amount (mg.) of phenolphthalein liberated by
100 ml. of enzyme solution in one hour at 37 degrees.
Measurement of Enzyme Activity
One unit of enzyme activity, when using beta-naphthol compounds as substrate, is defined as that amount of enzyme which liberates
the color equivalent of 10 mg. of beta-naphthol during the stated incubation period at 37 degrees.
100 ml. of saliva and serum.
Final results are recorded as units per
Thus the determination of enzyme ac-
tivity necessitate the preparation of calibration curves prepared with
beta-naphthol and tetrazotized diorthoanisidine (Diazo Blue B) in the
presence of the saliva and serum proteins.
The amounts of beta-naphthol used for the determination of
the calibration curves were 0, 10, 20, 30, 40, 50, and 60 meg.
Six
determinations in all were done for each concentration and the mean
value was used in the plot of color density against concentration.
In
each case the proper amount of saliva or serum was added to correct
for the loss of color due to the adherence of dye to the saliva proteins.
In each series of enzyme determinations at least two control tubes were included.
In these tubes buffered substrate only was
added and the tubes were incubated for th
indicated time period.
Upon
removal the proper dilution of saliva or s rum was added, depending
on the enzyme activity measured, to the c ntrol tubes and these tubes
were treated as were the tubes for the en yme determination.
Since
the control or blank tubes indicate the am unt of spontaneous hydrolysis
27
during the incubation period they were us d to adjust the photoelectric
colorimeter (Klett-Summerson) to the n
point (zero).
Parotid Saliva:
Cations - The arithmetic mean (M) of th
be .2. 9 mg.
o/o
with a standard deviation o
range of O. 9 - 5. 0 mg. o/o.
mg.
o/o
The average
with a range extending from 8. 0 t
calcium content was found to
0. 8 mg. o/o and an obgerved
odium value was 48.2
112. 0 mg.
o/o.
+ 26.4
Potasgium.
values ranged from 54.0 - 187 . 0 mg.o/o, the mean value and standard
deviation were found at 83. 9 + 2.0. 6 mg.
TABLE I
INORGANIC COMPONENTS OF PAROTID SALIVA
NUMBER
IN
GROUP
OBSERVED
RANGES
mg . o/o
MEAN
mg.%
STANDARD
DEVIATION
mg . %
MEDIAN
mg.%
Calcium
51
0.9-5.0
2.9
0. 8
3.0
Sodium
51
8. 0 - 112. 0
48.2
26.4
48.0
Potassium
51
54.0 - 187.0
83.9
20.6
81. 0
Chloride
51
23. 1 - 166.2
81.9
34,0
85.0
Bicarbonate
50
27.4 - 205. 0
109.4
46.7
101. 0
Phosphorus
40
5.0-18.1
9.5
2.8
9.2
COMPONENT
Anions - Parotid saliva chloride had a mean value of 81. 9 mg. o/o and a
standard deviation of 34. 0 mg. o/o.
Recorded values ranged from
28
23.1 - 166. Z mg. o/o.
Bicarbonate had an average value of 109.4
mg. o/o, while the content varied from Z7. 4 to 2.05. 0 mg. o/o.
Inorganic
o/o, with a stand-
phosphorus was found t-o have a mean value of 9. 5 mg.
ard deviation of 2. 8 mg.%.
+ 46.7
The total obse.rved range extended from
5. 0 to 18. 1 mg. o/o.
Whole Saliva:
Cations - The mean c.aldum content was 14.5 mg.% with a standard
deviation of 3. 7 mg. o/o and .a range of 8. 8 - · 26.0 mg.%.
Whole s-aliva
sodium was found to vary from 10.0 - 98. 0 mg.%, with an average
.±. 17.4 mg. o/o. The p.o tassium titer
had a mean and standard deviation of 74. 3 .± 13. 2.. mg. o/o, with a range
valu.e and atandard deviation of 33.6
of 52. 0 - 114. 0 :mg. o/o.
TABLE II
INORGANIC COMPONENTS OF WHOLE SALIVA
COMPONENT
NUMBER
IN
GROUP
OBSERVED
RANGES
mg. o/o
MEAN
mg.%
STANDARD
DEVIATION
mg.%
MEDIAN
mg.%
Calcium
45
8.8-26.0
14.5
3. 7
14. 1
Sodium
45
10. 0 - 98. 0
33.6
17. 4
28.0
Potassium
45
52. 0 - 114.0
74.3
13.Z
73.0
Chloride
45
39.8-157.7
71.0
22.8
68.5
Bicarbonate
44
17.1-127.2
71.4
25.0
73.5
Phosphorus
38
6.2-13.5
9. 7
1.9
9.8
Anions - Whole saliva chloride had an average value of 71. 0
+ 2.2.. 8
mg.%.
The lowest and highest observed values we.re 39.8 and 157.7 mg.%.
Bicarbonate content had an arithmetic mean of 71.4 .mg. o/o, a standard
deviation of 25. 0 mg. o/o, and ranged from 17. 1 - 12.7. 2 mg. o/o.
Inorganic
29
phosphorus had a range extending from 6. 2 to 13.5 mg.%.
The mean
and S.D. were ·9 . 7 t 1. 9 mg. o/o.
Although mg.% values are the usual manner of expressing
measureme.n ts of mineral concentration for body .fluids:, certain
authors (20) fe·el that this pro·cedure is- inadequate when dealing with an
excretory and/or secretory product such as saliva.
It is their conten-
tion that a more valuable method of expressing salivary components is
by recording these components as. amount per time nnit, as a measure
of rate of se.c retion (mg. /hr. value).
While no re.cord was made of the rate. of flow of whole
saliva, parotid saliva. flow rates. were accurately determined.
Table III
contains the mean, median, and dispersion values. oi inorganic constituents of the pa.r otid s·ecretion calculated in mg . ./hr.
TABLE III
SECRETION RATE OF INORGANIC COMPONENTS OF STIMULATED PAROTID SALIVA
- Mean, Median, and Dispersion Values (mg. /hr.) -
NUMBER
IN
GROUP
OBSERVED
RANGES
mg . /hr.
Calcium
46
0.4-4.6
Sodium
46
Potassium
COMPONENT
MEAN
mg. /hr.
STANDARD
DEVIATION
mg . /hr .
1.5
0.9
1. 7 - 84.4
25. 1
20.0
46
16.2-131.
38.4
18. 7
Chloride
46
8.0-96.8
38.8
23 . 1
Bicarbonate
46
7.9-214.
57.0
44.9
Phosphorus
40
1.2-12.4
4.3
2. 1
30
Or
Parotid Saliva.:
The
s.p horus. content w as found to
0/o. The observed
be 6. 8 mg.% and had a standard deviatio
of 2. 4 mg.
range was 2. 3 to 16 . 1 mg.%.
iva lactic acid had a mean
value of 2. 1 mg.% with a standard deviat on oi 1. 7 mg.%.
and lowest observed values were 7 . 6 and 0 . 2 mg.%.
Results expr es.sed
as units/hr. are recorded in Table IV.
TABLE IV
ORGANIC COMPONENTS OF
ALIVA
Parotid Saliva
COMPONENT
NUMBER
IN
GROUP
OBSERVED
RANGES
mg . ')(.
Phosphorus
40
z. 3
Lactic Ac i d
51
O. Z- 7 , 6
- 16. 1
STANDARD
DEVIATION
ma. 'J(.
MEA
mg .
MEDIAN
mg.')(.
6.
Z.4
6. 6
z.
1.7
1.6
Whole Saliva
Phosphorus
38
0 . 7 - 13 . 9
4.
Z.4
3. 7
Lac tic A c id
45
0 . 1 - 11.9
1.
z. z
1. 1
SECR E TION R ATE OF ORGANIC COMPONENT
SALIVA
-Mean , Median , and Diaperaion V
OF STIMULATED PAROTID
uea (mg . /hr . )-
NUMBER
IN
GROUP
OBSERVED
RANGES
ma./hr .
Phoaphorua
40
0. 5 - 7 . 1
3. 1
1.0
Lactic A cid
46
0. I - 7 . 6
I,Z
I.7
COMPONENT
g . /hr.
The highest
STANDARD
DEVIATION
mg . /hr .
3f
Whole Saliva:
Total organic phosphorus of whole saliva. had a mean value
and standard deviation of 4. 0
+ 2. 4
variance was from 0. 7 - 13. 9 mg.
from 0. 1 to 11. 9 mg.
o/o,
mg.
o/o.
o/o.
The observed content
The lactic acid content ranged
with a mean value and S.D. of 1, 8
..±
2, 2 mg. o/o.
pH and the Cationic and Anionic Balance of Stimulated
Parotid and Whole Saliva
Various factors are capable of influencing the buffer
capacity of saliva.
Marshall (166) and other investigators (56, 94,
228) have noted that stimulated saliva has a greater neutralizing
power than resting saliva.
In the investigation of the bufier systems
of saliva Wah Leung (258) has demonstrated the importance of bicarbonate, which plays a primary role, with phosphates and protein adding
minor contributions.
Dreizen et al, (74) have stated that much of the
increase in buffer capacity of stimulated saliva is probably due to
increases in the quantity of sodium and bicarbonate produced as a
result of the stimulation process.
In the investigation herein presented, routine analyses of
the pH of whole and parotid saliva were made, using the previously
described precautions to prevent changes due to loss of carbon dioxide.
The mean parotid saliva pH was 7. 44 with a standard deviation of 0, 18.
The observed range of values was from 7. 00 to 7. 87.
Whole saliva
presented a greater degree of variation: values ranging from 7.128, 13.
The mean and S.D., 7. 64
+ 0, 20 were only slightly higher
than that recorded for parotid saliva.
32
Table V presents the pH values of parotid and whole saliva
and thoae components (mEq/ 1} whi.ch probably play a role in the salivary
buffer systems.
TABLE V
pH AND THE CATIONIC AND ANIONIC BALANCE OF STIMULATED
PAROTID AND WHOLE SALIVA
pH
NUMBER
IN
GROUP
OBSERVED
RANGES
PAROTID
50
7. 00 - 7 . 87
WHOLE
44
7 . 12-8 . 13
TYPE OF
SALIVA
STANDARD
DEVIATION
MEDIAN
7 . 44
0. 18
7 . 42
7. 64
0 . 20
7 . 61
MEAN
CATIONS
TYPE
OF
SALIVA
PAROTID
WHOLE
CALCIUM
mEq/1
M
S. D .
SODIUM
mEq/1
M
S. D.
1.5
20 . 9+11.5
21.5 + 5.3
43 . 9 + 17 . 1
14 . 4 +
19 . 0+3.4
40. 7 + 12. 8
+ 0.4
7 . 24.:!: 1.9
POTASSIUM
mEq/1
M
S. D .
7.6
TOTAL
mEq/1
M
S. D .
ANIONS
TYPE CHLORIDE BICARBONATE PHOSPHORUS LACTIC ACID
OF
mEq/1
mEq/1
mEq/1
mEq/1
SALIVA M
S.D.
M
S.D .
M
S. D .
M
S. D.
PROTEINS
mEq/1
M
S.D.
PAROTID
23 . 3.:!: 9 . 6
17 . 9.:!:7 . 5
5,5.:!:1.6
0, 2.:!: 0, 2
0 , 4 + 0, 3
WHOLE
19. 9.:!:6.4
11.7+4.1
5 . 6.:!:1.1
0.2.:!: 0.2
0 . 4+0.2
Total
mEq/ 1
M
S . D.
PAROTID
47 . 4.:!:19.2
WHOLE
37.7.:!:12.0
Nitrogenous Components
In earlier investigations (Table VI) of the protein content of
whole saliva various authors have reported con centrations ranging from
80 mg. o/o (160) to 564 mg.% (137) .
Most of these investigators have
utilized methods. which allow either precipitation and subsequent measurement (of the salivary proteins) by modified Micro-Kjeldhal techniques
for nitrogen determination or the direct meas.uremerit of tyrosine with
phenol reagent.
The latter procedure allows e'stimation of proteins on
the basis of th.e tyrosine - protein ratio of 1 to 21.
Permitting the
assumption that controls. have been used to· c.orrect for the presence of
free tyrosine in saliva, .little account has been made for the mucin which
is normally pre.sent.
Mucin or salivary mucoid, while having a protein
moiety is considered to· be a glycoprotein. due to its carbohydrate prosthetic group.
If the nitrog.e n or tyrosine present in mucin is thus in-
cluded in these measurements, erroneo.us results. will be o.btained in the
conversi on to total protein .
In order to eliminate this. difficulty in the present study,
mucin nitrogen as well as· the non protein nitrogen fraction of whole
saliva was. determined.
By employing this procedure a more accurate
measurement of the true (simple) protein content of whole saliva could
thus be obtained.
TABLE VI
NUMBER
IN
GROUP
RANGE
mg."/o
MEAN
mg."/o
Alcohol
precipitation
82
80 - 210
134
Krasnow
et al.
Tyrosine
ratio
95
Karshan
Tyrosine
ratio
100
Gorlin
Tyrosine
ratio
35
AUTHOR
Lorthrop and
Gies
METHOD
Deakins et
al.
Nitrogen
determination
Bramkamp
Nitrogen
det e rmination
20
362
S.D.
mg."/o
TYPE
OF
SALIVA
restin g
w hol e
35
restin g
whole
189 - 564 .
286
6. 3
sti mulated
whole
175- 442
280
65.9
stimulated
whole
87.5-378.0
68. 75- 362. 50
256
stimulated
w hole
stimulated
parotid
34
The mean whole saliva protein value. of the 40 individuals
tested was 146.4 mg.%, with a standard deviation ·of 65. 1 mg. o/o.
Observed values ranged from 2.5. 0 to 293.8 mg.%.
(Table VHA)
Pa.rotid saliva total p-rot.ein ranged !rom 2.6. 2 to 623.8 mg.%,
I
with a standard deviation and m.e.an value of 179.0
.± 117.0 mg.%.
Re-
corded values fo-r non protein nitrogen, mucin nitrogen, albumin, and
globulins are presented in Table VII B.
TABLE VII A
NITROGENOUS COMPONENTS OF WHOLE SALIVA
NUMBER
IN
GROUP
RANGE
mg . "/o
40
56 . Z- Z93 .8
37
0 . 0- 163.1
Globulins
37
0.0-Z33 . 8
Non Protein
Nitrogen
40
Mucin Nitrogen
40
COMPONENT
Total Proteins
. Albumin
S. D.
mg . ,.,
MEDIAN
mg . .,
146 , 4
65. I
135.9
68 .- Z
4Z . 7
6Z . 5
8Z. 7
41.9
66.9
MEAN
mg, %
z-
46.4
30.3
6. 8
30.0
3. 5 -
Z4. 5
14 . 1
4.9
13.8
19 .
TABLE VII B
NITROGENOUS COMPONENTS OF PAROTID SALIVA
COMPONENT
NUMBER
IN
GROUP
RANGE
mg.%
MEAN
mg .'/o
S. D.
mg .%
Total Proteins
46
Z6 . Z - 6Z3. 8
179 . 0
117 . 0
Albumin
46
0 . 0 - 336.
61. 9
84.3
30 .6
Globulins
46
0. 0 -
z
401. z
117 . 0
9Z . I
91. 9
Non Protein
Nitrogen
46
10 . 4 -
44 . 8
Z4.0
7.7
Z5 . 6
MEDIAN
mg . %
156.
z
TABLE VII C
SECRETION RATE OF NITROGENOUS COMPONENTS OF STIMULATED PAROTID SALIVA
COMPONENT
NUMBER
IN
GROUP
Total Protein•
46
Non Protein
Nitrogen
46
RANGE
mg ./ hr .
MEAN
mg. / hr .
S. D.
mg ./ hr .
6.8-359.1
86 . 7
75.8
3.9-ZZ . 5
II. Z
5.8
MEDIAN
mg . / hr .
If the mean mucoid nitrogen value of whole saliva is as·sumed
to be due to the simple proteins present, and hence multiplied by the
conversion factor 6. 25, a value of 88.4 mg.% is recorded.
When this
"incorrect product" is added to the "true" protein value of 146. 4
mg.% a figure (234. 8) which approaches those reported by the forementioned authors is obtained.
While the literature concerning the non protein nitrogen
(NPN) fraction of saliva is scant, a comparison of existing values (67)
and those determined in this study show similar results.
Updegraff and Lewis (239) in studying the NPN (ammonia
N, urea N, and Uric acid) of 67 persons reported a range of 5. 6 to
26.7 mg.%, with a mean value of 13.0 mg.%.
Malhado (165) has
observed that salivary urea is almost identical with blood urea (10 to
20 mg.% N), while Bergman and Barg (35) have recorded that salivary
urea had a range of 30-50 mg.%.
(15- 25 mg.% N).
Data concerning the exact quantitative measurement of
salivary mucin has not been reported except for Glass (102), the
author of the method employed in this study, who found the salivary
mucin content to be between 30 and 500 mg.%.
ENZYME ACTIVITY
The enzyme levels of parotid and whole saliva as presented
1n Table VIII show that the whole saliva enzyme titers are higher than
the levels demonstrated by these same enzymes in the parotid secretion,
even though mouth rinsing was instituted in an attempt to minimize
in£1 uencing factors, such as oral bacteria and cellular debris.
,,
TABLE VIII
PAROTID SALIVA ENZYME TITERS
NUMBER
IN
GROUP
E:-I Z YM E
41
A c i d P hos phataae
OBSERVED
RANGE
U/100 mi.
MEAN
U/100 mi.
S . D.
MEDIAN
0 . 100-1.130
0. 432
0. 190
0.430
0.044
o. 033
Tot a l Esteraaes
46
0.006 - 0.216
0.050
Ch o linesterase
49
0 . 013 - 0 . 860
0. 036
0 . 014
0 . 035
L i pas e
49
0 . 000-0 . 165
0 . 032
0.042
0.022
Be ta- Glu c uron i dase
41
0. 000 - 350 . 0
84 . 85
64 . 80
69 . 00
S.D .
MEDIAN
l. 98
3 . 50
WHOLE SALIVA ENZYME TITERS
NUMBER
IN
GROUP
E NZY ME
Acid P hosphatase
35
OBSERVED
RANGE
U/100 ml.
MEAN
U/IOOrnl.
0 , 50
- II. 00
3.94
- 13.40
Total Esterases
45
l. 20
3.69
2.35
3 . 10
Cholinesterase
39
0 . 046 - 0. 212
0. 105
0.037
0.095
L 1pase
43
0 , 60
l. 55
I. 03
I. 25
Be ta-Gl ucuronidaa e
(before rinsing)
40
!68 . 0- 2390 . 0
770.8
495 . 8
641.0
Beta-Gl ucuronidaae
(after rinsing)
43
74 . 0 - 1000.0
462 . 8
239 . 9
468 . 0
- 5. 00
Serum values for acid phO'sphatas.e and beta-glucuronidas-e,
contained in Table
IX, fall into the ranges reporte.d as normal by
previous investigators (89, 214).
Serum cholinesterase and total
esterase values while presenting a range and mean higher than those
previou:s.ly established (198), exhibit normal population distribution
patterns.
Thia appears to indicate possible variation in the measure-
ment techniques when used by different iny·estigators.
The results.,
however, show the absence of abnormal valu.e s. which might affect the
levels of similar or the same enzymes excreted and/ or secreted by the
salivary gland·s.
37
TABLE IX
BLOOD SERUM ENZYME TITERS
NUMBER
IN
GROUP
ENZYME
OBSERVED
RANGE
U/100 ml.
MEAN
S.D.
MEDIAN
Acid Phosphatase
35
0.20- 1.50
Total Esterases
43
37.6- 110.4
71. 3
16.6
76.0
Cholinesterase
41
48. 0 - 99. 2
71. 9
15.6
75.2
Beta-Glucuronidase
37
114.0 - 880. 0
263. 7
150.8
216.0
0.94
0.24
1. 00
Comparison of the mean whole saliva enzyme levels, Table
VIII, where the saliva collection was made after a water rinse, to
similar results (Chapter I) in which the stimulated whole saliva was
obtained before mouth rinsing, shows certain changes..
The greatest change is that manifested by acid phosphatase
which s.hows average decrea.se of 1. 61 units/100 ml. (5. 55 to 3, 94
units/100 ml. ).
Total esteras.es and lipase exhibit only minor varia-
tions, while there was an average increase in cholineste.rase titer of
0, 05Z units per 100 ml.
When considering the possible effect of mouth rinsing on
the salivary enzyme levels, it is normal to assume that a decrea.se
should result since much of the activity present is due to bacterial
.metabolism and cellular debris.
This is confirmed by the values
' recorded for beta-glucuronidase (Table VITI) before and after rinsing
(decrease of 40%).
Similarly acid phosphatase levels show a 29%
decrease under those levels reported in Chapter I.
The reasons for the remaining enzymes (total esterases,
38
cholinesterase, and lipase) not acting in the same manner can at this
time be left only to speculation.
Distribution Analysis
Tables X, XI, and XII summarize the chi square tests of
goodness of fit and the distribution plottings on each of the saliva
(whole and parotid) and blood serum components.
The two tests used for this analysis were interpreted
together in a combined judgment in order to gauge the relation of the
distribution to normality.
The chi square test of goodness of fit is
extremely sensitive to differences in interval frequency - far more
sensitive than visual inspection.
Since the Nfs are small in the
present study (under 55 in all cases) and chi square cannot be applied
to expected frequencies with magnitudes under five, the scores in the
"tails" of the distri'.bution were added into one common tail frequency
at each end.
The effects of highly extreme scores were essentially
"washed out" by this technique.
Visual inspection, conversely, while
only mildly sensitive to differences in interval frequency, yields high
awareness of extreme deviate scores.
The two techniques taken
together in a combined judgment results in a good estimation of the
distribution normality or abnormality and picture where difficulties,
if any, probably lie.
Knowledge of the distribution analysis is desirable for a
number of reasons.
First, for its own sake it is often illuminating
to know the general shape of the data.
Second, many statistical opera-
tions depend, at least in part, upon whether or not the data can be
considered normal.
Parametric statistical techniques are more
sensitive than their equivalent non-parametrical counterparts.
Using
parametric techniques when they are not in order leads to spuriously
WHOLE SALIVA
COMPONENTS
N
'1..2
d. f.
ACID PHOSPHATASE
35
7.73
3
p
LEVEL
>.05< .06
RESULT
U ORDERLI NE.
NORMAL
TOTAL ESTERASE
LIPASE
{l- GLUCURONIDASE •
45 '
43
5.08
16.46
3
>.10
2
<.01
NORMAL
A~NORMAL
VISUAL SCATTER ANALYSIS
SHAPE
~
.
a. ••
ONE VERY EXTREME POSITIVE SCORE
PLUS A KURTOTIC IM BALANCE IN
MODAL CATEGORY
UNUSUAL CROWDING INTO LOWER
CATEGORIES AS IF RELATIVELY NORMAL
6..
~
COMBINED JUDGMENT AND COMMENTS
PHQ I\ABLY
NOH~ tA L
C UR VES LOWER END WERE CUT OFF
PROI\ 1\ ULY AUNOHMAL
LOWEH END O F SCALE
NOT EXTENDED SUFFI C I ENTLY
EXTREME POSITI V E S KEW ; MAN Y
A JJ !'OI<t..LAL
EX T REME POSITIVE S C ORES
-wOULD BE NORMAL WITH RO OM AT LOW
END OF SCALE FOR EXTREME SCORES
40
3.95
2
> .10
NORMAL
43
6.53
4
>.10
NOR MAL
CHOLINESTERASE
39
8.51
2
< .02
ABNORMAL
HYALURONIDASE
46
3.63
4
>.30
NORMAL
40
1.54
3
> .50
NORMAL
TOTAL PROTEINS
40
8.48
3
<.05
ABNORMAL
ALBUMIN S
36
1.84
3
> .50
NORMAL
GLOBULINS
37
25.75
3
< .01
ABNORMAL
MUCOID
40
6.35
3
> .05
NORMAL
CALCIUM '
45
4.65
3
>.10
NORMAL
~
45
4!18
4
> .30
NORMAL
&...._
WOCJLD BE NORMAL WIT H MORE ROOM AT LOW
END OF SCA LE FOR EXTREME SCORES
POTASSIUM
45
2 .29
3
> .50
NOR MAL
.A..
CLDRVE NORMAL, BU T NO ROOM AT LOW END
OF SCA LE FOR EXTREME SCORES
NOR MAL
CHLORIDE
45
9.30
4
> .05<.10
B ORDERLINE
NORMAL
MOD ERATE IM 11 A L ANCE , PLUS SL IG H T POSITIVE
S KE.....V . • .•• ONE VER Y EX T HEME NE GAT I V E SCORE
PROAA!lLY NORMAL
BICARBONATE
44
6.90
3
>.05<.10
B ORDERLINE
NORMA L
INORG ANI C PHOSPHORUS
38
6.12
2
< .05
A U NORMAL
ORGANIC PHOSPHORUS
38
10.09
3
< .02
A U NORMAL
LAC TATE
45
-
-
-
pH
44
3
> .10
(BEFORE RIN SING)
f3 - GLUCURONIDASE
(AFTER RINSING) '
NON PROTEIN NITROGEN
1-- ---
SOOIUM
1 --
1-- -
---
f--4 . ~4
N OR!\1 A L
..•
...£.
SLIG HT IM BA L ANCE IN MODE,
BUT RE LATI VELY SY MMETRICA L
MCJDERATE POSITIVE SKEW, SOM E KURTOTIC
::IM BALANCE, PLUS TWO VERY EXTREME
NEGATIVE DEVIATE SCORES
•
•_.....
......
6.
PRO I3 A P. L Y A ll NOHMAL
SLIGHT NEGATIVE SKEW
NORMAL
NORM A L
NORMAL
UNUSUAL IMBALANCE
A B NORMAL
S LIGH T POSITIVE S KEW
NORMA L
&.i
6
NORMAL
-
~
~
NOR MAL
'
CROWDING IN LOW CATEGORIES
I : MBALANCE WITH LI TT LE VISIBLE SKEW
S LI G H T POS I TI V E S KEW
RE L ~T I VELY
SYMMETRICAL. SOME IMBALANCE
R~ L AT I VELV
S Y MME T RICAL , WITH SLIG I! T
POLYKURTOT JC IM UA LA NC E
ABNOR MAL
EXTREME POS ITI VE SK E W
RE LATI VELY NORMAL
RELA T I VELY NORM AL
NOR MAL
CURVE CUT OFF AT LOWER TA IL nY
LACK OF LOW SCO R E CATI<:GO HII::S
NO UMAL
PROUABLY NOH MAL
M. ODE RATE KUR T O T IC IM BALAN C E .. .. T WO
EX THEME N EG A T I VE S C OH E S
AUNOHM.AL
QUES T IONAULF: DISTRIBUTION
Z ERO C AT EGOR Y USURPS ALMOS T
I!ALF Tl IE S CO R l:S
AUNORMAL
EXTREME N EGATIVE IM BALA NC E
M •ODERA TE I M UALA!'I CE DUE T O S PA R C ITY
OF" C ASES IN LO 'N f:H C A TEG ORI ES
RELA T I VE L Y NORM AL
SLI G H T NF:GA fiVE S KEW
c..;......
~-
p
RESULT
GENERAL'
SHAPE !
> .30
NORMAL
~
3
>.50
NORMAL
14.07
3
< .10
ABNORMAL
49
-
-
-
j3 - GLUCURONIDASE
41
13.6
2
< .06
ABNORMAL
CHOLINESTERASE
49
1.21
4
> .50
NORMAL
NON PROTEIN NITROGE N
46
1.64
3
> .50
NORMAL
TOTAL PROTEINS
46
15.36
3
< .01
ABNORMAL
PAROTID SALIVA
COMPONENTS
N
x2
d.f .
FLOW RATE A
46
3.75
4
ACID PHOSPHATASE
41
2.10
ESTERASE I
46
L IPASE
LEVEL
~
a...
I.
~
•
...6...
..a._
VISUAL SCAT "'TfER ANALYSIS
NOR
COMBINED JUDGMENT AND COMMENTS
NORMAL
MAL
SLIGHT POSOITIVE SKEW
NORMAL
MARKED PC.SITIVE SKEW
ABNORMAL
CONSIDERABLE POSITIVE SKEW
ZERO
CATEGOR~
USURPS ALMOST
HALF TH...:.E SCORES
ABNORMAL
EXTREME POSITIVE IMBALANCE
MODERATE P · OSITIVE SKEW
POSSIBLE. NOR MAL
NORMAL E ; .XCEPT FOR
TWO EXTREME mtllEGATIVE SCORES
NORMAL
NORM A L
NOR MAL
POSITIVE SKEW PLUS KURTOTIC
IMBAL...ANCE
ABNORMAL
~
ALBUM INS
46
-
-
~
-
GLOBULINS
46
8.80
4
< .05>.01
BORDERLINE
FLOW RATE B
46
4.69
2
< .05>.01
BORDERLINE
NORMAL
CALCIUM
51
1.77
4
> .50
NORMAL
SODIUM
51
3.18
4
> .50
NORMAL
POTA SS IUM
51
19.92
4
< .01
ABNORMAL
CHLORIDE
51
1.73
3
> .50
NORMAL
BICARBONATE
50
1.78
4
> .50
NORMAL
INORGANIC PHOSPHORUS
40
.22
2
> . 50 1
NORMAL
ORGANIC PHOSPHORUS
40
6.53
3
< .05>.01
BORDERLINE
NORMAL
L ACTATE
51
4.07
3
> .20
NORMAL
pH
50
8.04
4
< .05>.01
BORDER LINE
NORMAL
I
NORMAL
ZERO CATEGORY: USURPS ALMOST
HALF TH:a: SCORES
ABNORMAL
EXTREME POSITIVE IMBALANCE
~
IMBA L ANCE PLUTS POSITIVE SKEW
PROBABLY ABNORMAL
.£.....
RELATIVEL Y NORM.I!aL EXCEPT F OR FOUR
EXTREME NE-c;ATIVE SCORES
PROBABLY NORMAL
QUESTIONABLE DISTRIBUTION
...
•-...•
•
6
.......-....
~
NO~MAL
N ORMAL
NO FIR MAL
NORMAL
IMBALANCE PLt:IJS POSITIV E SKEW
ABNORMAL
RELATIVE::LY NORMAL
NORMAL
RELATIV E:LY NORMAL
N OR MAL
RELATIVELY NOR.t.,..o...{AL, .. .. A FEW HIGH
POSITI VE SCORE ; S CAUSE A SLIGHT
POSITI'""VE SKEW
N O RMA L
ONE VERY EXTRE~E NEGATIVE SCO RE
N O RMA L
MOD ERATE POSI~IVE SKEW DUE TO
THREE EXTREME: POSITIVE SCORES
PR O BABL Y NO RMAL
SLIGHT NEGATIVE SKEW IS PROBABLE ALTHO UGH
I T APPEARS REL ATIVE L Y SYMMETRICAL
SLIGHT KURTO•TIC IMBALANCE
PR O BABLY NORMAL
-·
(j
~·
BLOOD SERUM'
COMPONENTS
N
'/.._2
d.f.
p
LEVEL
ACID PHOSPHATASE
35
5.44
2
>.05<.10
TOTAL ESTERASE
43
4.43
4
> .30
:\'OR MAL
CHOLINESTERASE
41
6.25
3
> .05
:-./OR MAL
37
6.16
2
<.05
AB:-JORMAL
RESULT
nORD ERLI NE
NORMAL
- -- - "-
fJ- GLUCURONIDASE
-
...
GENERAL
SHAPE
•
•
~
VISUAL SCATTER ANALYSIS
COMBINED JUDGMENT AND COMMENTS
SOME IMBALANCE IN THE NEGAT I VE RA~GE
PLUS A SLIG HT r-:'EGATIVE SKEW
PROJ\A t\ L Y :'ljQR MAL
NORMAL
!'1/0RM ,\L
NORMA.L
NOH. MAL
EXTREME POSITIVE SKEW
ABNORMAL
~
r-
42
high results.
Failure to use them when their assumptions can be
met is wasteful of data.
Furthermore, in explorative work it may be
of considerable advantage to know what the data look
like so that
information to be gathered in the future may be obtained in such a
manner as to be more normal and more "polishedrr.
For example,
if a distribution with an extreme positive skew is found (scores at the
low end of the measurement scale are too close together as compared
with scores higher on the s.cale) and it is des.ired to gather more data
on this variable in the future, the distribution analysis suggest that
the data be recorded in some new measurement index which would
push the high end of the present scale closer together and separate
further the lower end (square root, log transformation, etc.).
A
corollary to this is the necessity of "polishing" the procedure, especially at the low end of the scale, so as to insure that the small
differences at the low end of the scale which are given great weight,
are really accurate enough to be meaningful.
Discussion
Whole Saliva:
Sodium and Potassium- The authors included in Tables XIII and XIV
analyzed the salivas of 83 and 112 patients respectively, in their
studies on the sodium and potassium content of human saliva.
These
investigations were the most dependable of the sodium and potassium
analyses recorded, in that the methods employed allowed accurate
measurement and the data was presented in a comprehensive manner.
Results varied from 7. 6 to 205. 0 mg.
153. 0 mg.
o/o for sodium, and from 22. 0 to
o/o for potassium. Even though in the majority of cases the
mean and ran ge of the values were reported, no effort was made to
43
determine the standard deviation.
Fortunately, sufficient data was
available in so.me cases to allow the calculation o{ the means and
standard deviations, where lacking, and permit their inclusion in
th:eBe tables.
TABLE X Ill
WHOLE SALIVA SODIUM
AUTHOR
METHOD
OF
DETERMINATION
NUMBER
OF
GROUP
RANGE
mg. 'Yo
MEAN
mg. '!o
S.D.
TYPE
OF
SALIVA
4.28
Resting
McCance
Uranyl Zinc
Acetate pptn.
5
lZ . O- ZZ.O
15.60
Dreizen
et al.
Flame
Photometry
5
15.0- 195.0
43.9
Dreizen
et al.
Flame
Photometry
Z5
7.6-55.0
23.6
11. 0
Brown and
Klotz
Uranyl Mg
Acetate pptn.
18
16.5-131.1
58.8
35.2
Dreizen
et al.
Flame
Photometry
5
Z9. 0 - 205 . 0
87.0
Dreizen
et al.
Flame
Photometry
Z5
32. 7 -
Present
Study
Flame
Photometry
45
1 o. 0 - 98. 0
zoo.
1
Resting
Resting
Stimulated
Stimulated
80.9
33.5
Stimulated
33.6
17.4
Stimulated
44:
TABLE XIV
WHOLE SALlY A POTASSIUM
AUTHOR
METHOD
OF
DETERMINATION
NUMBER
OF
GROUP
RANGE
mg . "/o
MEAN
mg.%
S.D.
TYPE
OF
SALIVA
Wach
Sodium Cobalti
Nitrite pptn.
2.9
2.2. .0-12.7 .0
74.8
McCance
Sodium Cobalti
Nitrite pptn .
5
75 . 0-116.0
86.0
Flame
Photometry
5
48. 0 - 153. 0
80.4
Flame
Photometry
2.5
41. 1 - 96. 2.
71. 9
15.2
Resting
Perchlorate
Butyl Alcohol
18
57.2-115.9
83.0
13.2.
Stimulated
58 . 0 - 133 . 0
83.0
Dreize n
et al.
Dreizen
et al.
Brown and
Klot z
Resting
17. 3
R eating
Resting
Dreizen
et al.
Flame
Photometry
5
Dreizen
Flame
Photometry
2.5
54.0-98.1
73.0
11. 7
Stimulated
et al.
Present
Study
Flame
Photometry
45
52. 0 - 114.0
74 . 3
13.2
Stimulated
Stimulated
The whole saliva sodium and potassium values. p.res·ented in
Tables XIII and XIV indicate an increase in the sodium level of stimulated saliva over that contained in resting saliva.
Potassium values
appear to be relatively .stable a:nd do not show any noticeable change
upon stimulation.
Although the potassium titers obtained in the present
study agree quite well with the m .e asurements of previous authors, the
sodium levels of stimulated whole saliva are considerably lower than
those. presented by the forementioned investigators.
The small num-
ber of total pe.rsons observed, as well as the highly va.riable nature of
salivary .sodium, for both the resting and stimulated secretions,
indicates the need for further measurements in order to determine its
45
normal physiological range.
Calcium- In their critical review of the literature on the analysis of
salivary calcium, prior to their own study, Becks and Wainwright
(21) noted v alues for resting saliva ranging from 3. 7 (205) to 17. 8
(143) mg. per cent, while activated saliva presented a range of 2. 2
(131) to 14.0 mg. per cent (125).
Becks (20) in his report on the
normal calcium level of saliva indicated that the resting saliva calcium
content, of the 650 healthy individuals tested, ranged from 2. 4 to 1 1. 3
mg. per cent.
per cent.
The mean and standard deviation were 5. 8 _2: 1. 4 mg.
The population distribution approached a symmetrical
curve which h ad a comparatively low skewness value; typical of that
presented by a normal population distribution.
Investigation by
Becks, Wainwri ght, and Young (30, 31) revealed a mean calcium
content of 5. 5 7 mg. per cent (6. 8 8 m g . /hr.) for paraffin stimulated
whole saliva.
More recent observations (45, 213) on the calcium content
of resting and s t imulated saliva have tended to confirm the results of
Becks and Wainwright; range and means, and the finding that large
variations in calcium occur in the same person and in different individuals.
Becks and Wainwright (24, 28) believed that saliva was changed
quantitatively with respect to calcium by any type of stimulation; the
calcium (mg. per cent) having a slight tendency to decrease with
increased secretion rates.
The reverse, however, occurred for the
calcium content in mg. /hr. values.
Comparison of the whole saliva calcium data contained in
the present study to that obtained by other investigators shows a wide
divergence in values.
The values herein presented are of a much higher
magnitude than t hose found in similar studies.
This variation is
probably due to the lack of centrifugation or filtration of the saliva
samples before analysis.
That erroneous high values can result from
whole saliva which has had no preliminary centrifugation has been observed by Becks and Wainwright (21).
Phosphorus - Becks (18) in his discussion of former studies on salivary
phosphorus recorded the ranges for the total and inorganic phosphorus
of resting saliva as 8. 7 to 18. 1 mg. per cent and 6. 2 to 25. 6 mg. per
cent respectively .
Stimulated saliva total phosphorus had a range of
5. 2 to 22.1 mg. per cent, while the inorganic phosphorus ranged from
8. 8 to 15.4 mg. per cent.
Wainwrightts (262) study of the inorganic
phosphorus content of the resting saliva from 650 persons recorded an
average value of 16.8
+ 6. 4
mg. per cent, while the lowest and highest
observed values were 6. 1 and 71. 0 mg. per cent.
Eggers-Lura (76) in his measurement of the various phosphorus fractions of resting saliva reported the following means and
standard deviations:
Total phosphorus, 20.4
inorganic phosphorus, 14. 9
5. 5
+ 3. 4
+ 4. 2 mg. per cent;
mg. per cent; and organic phosphorus,
+ 3. 9 mg. per cent.
The results presented by Davies and Rae (65} did not
include the standard deviation or the mean, while the publication by
Dreizen et al. (73) failed to indicate the range, mean, or standard
deviation.
Where these values were absent, values were calculated
from the available data and are presented in the ensuing discussion.
Davies and Rae (65} determined the total phosphorus and organic phosphorus of 35 persons.
The difference between the total phosphorus
and organic phosphorus values was us.ed as a measure of the inorganic
phosphorus.
Total phosphorus was found to range between 9. 3 and
18. 7 mg. per cent, with a mean and standard deviation of 12. 9
+ 2. 5
mg.
per cent.
Organic phosphorus had a mean value of 0. 9 mg. per cent
and a standard deviation of+ 0. 3 mg. per cent; with the observed values
having a range of 0. 4 to 1. 8 mg. per cent.
The inorganic phosphorus
(by difference) showed a range of 7. 8 to 17.4 mg. per cent and an average and standard deviation of 12.0 + 2. 5 mg. per cent.
No indication
was given whether or not the sal iva samples were obtained by stimulation.
Dreizen et al. (73) measured, what may be assumed to be the
inorganic phosphorus fraction of both resting and stimulated saliva in
twenty-five individuals.
Resting saliva presented a range of 15.2 to
38. 1 mg. per cent and had a mean value and standard deviation of
22.7 _:!: 6. 2 mg. per cent.
+ 4. 1 mg. per cent.
Stimulated saliva had a . mean value of 16.4
The range was from 10.5 to 25. 1 mg. per cent.
In the present study the inorganic P of stimulated saliva
had a range of 6. 2 to 13.5 mg. per cent; mean and standard deviation
were 9. 7 + 1. 9 mg. per cent.
These values while falling into the
general range for inorganic Pare noticeably lower than those presented
by the forementioned authors.
That salivary stimulation and the re-
sulting increased rate of secretion can cause a lowering of the inorganic
P level, mg. per cent, has been noted by Becks, Wainwright, · and
Young (30, 31).
In measuring the inorganic Pin the stimulated saliva
from twenty-five persons, Becks and his co-workers found the mean and
standard deviation to be 11. 76 + 1. 98 mg. per cent; with a range of 6. 8
to 16. 2 mg. per cent.
Similarly, the results of Dreizen et al., although
excessively high for both resting and stimulated saliva, exhibits an
average decrease upon stimulation.
On the other hand, Sellman (216)
did not believe that salivary inorganic phosphorus was influenced to any
great degree by the stimulation process.
As seen in Table IV the average organic phosphorus content
of the group studied was 4. 0 + 2. 4 mg. per cent.
The lowest and highest
48
observed values were 0. 7 and 13. 9 mg. per cent.
These values are
similar to those evidenced by Eggers-Lura, but are much higher than
the levels obtained by Davies and Rae, whose low results may be due
to the hydrolysis of the organic phosphorus during their storage period.
The total phosphorus of stimulated saliva had a range of
9. 9 to 22.9 mg. per cent, an average value of 13.8 mg. per cent, and
a standard deviati on of 4. 3 mg. per cent.
Eggers-Lura (76) commented
on the Davies and Rae procedure for the determination of total phosphorus,
which was employed for the present study, and stated that lower than
actual values may result from the use of perchloric acid as a digestion
agent which may convert liberated phosphate into inorganic pyrophosphate, a compound which gives no color in the colorimetry.
That such
a conversion did not occur in the present study is suggested by the
comparatively high organic phosphorus values obtained.
Chloride - The previously existing data, pertaining to the salivary
chloride levels, has been limited to a total experimental population of
thirty-three persons.
Clark and Shell (60) tested five individuals and
noted a range of 27 to 147 mg. o/o and a mean of 55 mg. o/o, using resting
whole saliva.
Brown and Klotz (4 7, 48) employing eighteen persons
recorded values ranging from 24 to l 00 mg.
51. 22 mg.
o/o,
for paraffin stimulated saliva.
a mean of 72. 8 mg.
o/o
o/o,
with a mean value of
Vladesco (252) observed
and a range of 40. 0 to 119.5 mg.
o/o
for salivary
chloride using deproteinized resting whole saliva from ten persons.
These findings, as reported by the above authors, are similar to the
whole saliva chloride values presented in Table II; which show a range
of 39.8 to 157. 7 mg. o/o and a mean value of 70. 99 mg.
o/o,
for the 45
persons investigated.
In light of recent findings; that congestive heart failure is
associated with lowered sodium and chloride levels (270), and that the
saliva of patients with hyperchlorhydria and histamine resistant anachlorhydria ( 72) show abnormal levels of sodium and chloride, it would
seem of prime importance that more information be forthcoming concerning this anion.
Bicarbonate - Dreizen et al. (73) in measuring the ionic contents of the
buffer systems of saliva recorded the bicarbonate levels of resting and
paraffin stimulated whole saliva.
A range of 21. 2 - 65. 3 mg. o/o, with a
mean and standard deviation of 39. 3
saliva.
+ 18.2
.± 11. 4
mg. o/o was found for resting
Stimulated saliva had a mean and standard deviation of 96. 0
mg. o/o.
The range extended from 49. 5 to 118.8 mg. o/o.
Although
the mean bicarbonate value for stimulated saliva, as reported by these
authors, is 34o/o higher than the mean level found in the present study,
Table II, the observed ranges are close.
Lactic acid- Determination of the normal , physiological, lactic acid
content of whole and parotid saliva has been almost completely neglected
by workers in this country.
Foreign investigators have evidenced some
interest in salivary lactic acid, but there is still insufficient basis for
the calculation of normal titers.
Vladesco (251) reported finding salivary levels from 4 to
14 mg. o/o, while Koch (144) observed t h at the whole saliva from fasting
males contained an average content of 15.7 mg. o/o.
Fujishiro (98)
measured the concentration of lactic acid in human whole saliva and
noted that the salivary levels were lower than those of serum.
He
further stated that the salivary lactic acid showed variations proportional
to the rate of secretion and the blood concentration.
Hellstrom and
Ericsson (115) studied the production of salivary lactic acid and the
inhibition of its formation.
They noted values for fasting whole saliva
that ranged from 0. 81 to 4, 86 mg. o/o, with an average value of 2. 25 mg.
o/0 •
The range and mean values presented in Table IV are in
agreement with the findings of Vladesco and Hellstrom and Ericsson.
However, Koch's mean value of 15. 7 mg.
o/o appears to be disproportion-
ately high and may be the result of lactic acid formation by the oral
flora.
pH - Brawley (43, 44) in a review of the previous literature cited the
pH of normal resting saliva as ranging from 4. 9 to 8. 3,
His own
analysis, using a monocolor colorimetric method, of the pH levels of
3405 subjects showed an average value of 6. 75; with a range of 5. 6 to
7. 6 (median 6. 84).
No significant difference was noted between the
average morning pH and afternoon pH.
Experiments showing that the pH of resting saliva was
lower than the pH of saliva obtained under muscular movement, as in
mastication or during the actual chewing process, were carried out
by Starr (228), Carlson and McKistry (54), and McClelland (170).
Later work by Champion (56) and othe r authors confirmed these original
observations and indicated the independence of salivary pH from blood
buffer changes.
Soyenkoff and Hinck (226) measured the pH in nine persons
after an aqueous mouth rinse and found the resting saliva. to have a
range of 6. 37 to 6. 89; average pH was 6. 64.
For paraffin stimulated
saliva the pH range was 6. 97 to 7. 32, with a mean value of 7. 13.
Eisenbrandt (78) found the mean pH of resting saliva to be 6. 72
while Dewar (71) noted a mean pH of 6. 83
+ 0. 32. for
+ 0. 24,
the resting saliva
of 25 persons.
The results as reported in Table V indicate a pH range of
7. 12 to 8. 13 and a mean value of 7. 64 for stimulated saliva.
These
values indicate a lower hydrogen ion concentration than that normally
51
experienced with resting saliva.
Such results are indeed expected when
considering the processes which occur in the oral cavity.
The meta-
bolism of oral microorganisms and their production of pH lowering
acids possibly can account for the low pH levels in saliva obtained after
long periods of stagnation (overnight).
However, this was not the case
under the conditions employed by Soyenkoff and Huck, where mouth
rinsing was instituted.
Sufficient data has been issued by various
authors to assume that the pH of resting saliva, the admixture of all
the glandular secretions, is normally below pH 7. 0.
Once the stimula-
tion process is initiated new principles must be applied.
In view of
the known increases of sodium and bicarbonate in stimulated saliva, and
the influence t h at these ions have on the salivary pH levels, higher
values, close to those seen in Table V should be expected.
Although
Soyenkoff and Hinck did observe an increased pH after stimulation, their
results are slightly lower than those of the pres.ent study.
If their ob-
servations on nine persons had been extended to include a larger group
similar results might have been recorded.
Nitrogenous Components -
Comparison of the information garnered in
the present study with the findings available from previous investigations
on the nitrogen containing components of saliva has been included in the
pres entation of the results.
In the determination of the various protein
components of saliva, albumins and globulins, certain liberties have
been taken.
The procedure described was a method for the separation of
serum protein fractions which had been modified for use with saliva.
Although the fraction which precipitated on the addition of sodium sulfate
appeared to be globulins, further confirmation by electrophoretic
patterns should be a future requisite to their analysis.
No information
concerning the concentration of salivary albumin or globtdins has been
- ...
--
.....
··--- -
-
·-
previously presented, but Farroni (84) found that the saliva of normal
individuals contained slight amounts, in regard to serum, of serum
albumin and gl obulins, which were present together as well as separately.
Enzymes - Due to the different substrates employed by various authors
for the determination of the enzyme activities of saliva no valid comparison of the salivary levels can be made.
:Parotid Saliva
Initial investigations of the parotid secretion, by Howe (127),
indicated the presence of sodium, potassium, calcium, and magnesium.
Howe (128) also described methods for the measurement of salivary
phosphate, chloride, and calcium, but values for these components
were not presented.
Bramkamp (42) measured the chloride content of
saliva, using paraffin and lemon as stimulatory agents.
Stimulated
parotid saliva had a mean chloride value of 143. 7 mg.%; with a range
of 68 to 206 mg. %.
Faster flow rates and higher chloride levels were
obtained when lemon was used for stimulation.
When the results were
separated according to the agent used, the chloride content of the
lemon activated saliva had a range of 179 to 206 mg.
190 mg. %.
% and a mean of
The paraffin stimulated saliva had an average value of
97 mg.%, with a range extending from 68 to 134 mg.%.
The latter
values, obtained with paraffin activated saliva, are similar to those
found in the present study (Table I).
Bramkampts data which included
secretion rates, allowed calculation of the amount of chloride secreted
per unit time.
The average rate for paraffin stimulated saliva was
37. 83 mg./hr., while lemon stimulated saliva had a mean rate of
254. 26 mg. /hr.
The mean rate for paraffin stimulated chloride is
almost identical with the value recorded in Table III, but the amount of
chloride produced upon stimulation with lemon is amazingly high.
Although this change in secretion rate due to different agents appears
to be valid, caution must be exercised in accepting these results because
of the absence of information regarding the length of the collection
period.
Paraffin is known to produce a relatively steady rate of flow
over prolonged periods of stimulation.
However, this is not true with
lemon, which tends to cause extremely high initial flow rates which
decrease upon continued stimulation.
Howe (129) mentioned the presence of proteins, albumin
and globulins, in parotid saliva, but again salivary levels for these
substances were lacking.
Bramkamp (41) measured the saliva protein
content of one individual (Table VI), but unfortunately this did not
allow comparison with the results presented in Table VIII.
These two
citations are the only available information on the protein components
of parotid saliva.
Bramkamp (42) also measured the urea concentra-
tion of the parotid secretion and found values ranging from 8 to 16 mg.
per cent for stimulated normal saliva.
The hydrogen ion concentration has received the most
attention of all the parotid saliva components.
Nash and Morrison
(181) observed values varying from pH 6. 0 to 7. 9 for resting saliva,
while Schmidt-Nielson (212) found the pH of resting parotid saliva to
vary from 5. 45 to 6. 06 (average 5. 81) and also noted that various
stimuli caused the pH to increase greatly.
The influence of the rate of
secretion on the pH was confirmed by Zagami and DeStefano (279), who
recorded values from pH 5. 3 to 7. 7, depending mainly on the intensity
of the stimulus and the speed of secretion.
reported by Infantellini ( 133).
Similar results were also
The observed pH range of 7. 00 to 7. 87
(mean 7. 44) for stimulated saliva, as seen in Table V, is thus in
agreement with the findings of these other investigators.
54
"t" testst
The following table (12A) summarizes the ''t" test and other
statistics pertinent to an evaluation of the difference between the means
of each variable i n parotid saliva versus its connterpart in whole saliva.
The first column of the table cites the phi coefficients of
correlation computed between each of the two variables.
Where signifi-
cant, the phi is reported exactly; where non- significant, the phi is reported as zero.
The two columns following the phi 1 s summarize the F
tests of homogeneity of variance for i n dependent samples (appropriate
where phi equals zero).
Where these tests of homogeneity show that the
variances are homogeneous, the most sensitive form of the ''t" test may
be used; that is, the form with the pooled deviations error term.
Where
heterogeneous, however , the less powerful form of the "t" test with the
non-pooled error term must be us.ed plus the Cochran- Cox "table"
correction.
Use of these parametric ' 1t 11 tests depend on the s h apes of
the two. distributions being relatively similar in construction.
Where
they deviate too considerably from either one another or normality, a
non-parametric test for unpaired samples becomes appropriate; for
example, the Man n- Whitney "U" test or Festinger 1 s "d" test.
Wh ere phi 1 s are reported as significant the appropriate "t"
test is the matched groups technique.
However, this method can only
be used when difference scores are plotted and shown to be fairly normal
in distribution.
When this distribution deviates from normal to any
considerable extent it becomes necessary to use a non-parametric device;
for example, the Sign test or Wilcoxon 's test for paired replicas.
In one case, that of Esterase, it was not necessary to meet
the assumption s of the more sensitive parametric device, since the differences between the means was so large that the less powerful but simpler
non-parametric Sign test would be highly significant.
55
TABLE
lZA
SUMMARY OF "t" TEST ON THE SIGNIFICANCE OF THE DIFFERENCES
BETWEEN WHOLE AND PAROTID SALIVA COMPONENTS
Variable
()
Acid Phosphatase
+.00
Total Esterase
+.35
p
Result
Method*
.£. 01
Heterogeneous
A
+.00
.£. 001
Heterogeneous
c
~=7 ·.
Beta-Glucuronidase
+.00
~.
Heterogeneous
A
Cholinesterase
+.54
Lipase
B
01
B
14. 1
Significant
L.Ol
Significant
L....
01
Significant
141
L..
01
Significant
=1
I!-.
01
Significant
4.0
L.01
Significant
L. 01
/1.
)l.
=0
5
+.00
>. 10
Homogeneous
D
Total Proteins
+.00
' . 01
Heterogeneous
A
14.4
Albumins
+.00
>. 10
Homogeneous
c
b=2. 1 ). 10
Globulins
+.00
i,.. 01
Heterogeneous
A
19.4
Calcium
-.42
E
Sodium
+.00
"'· 0 l
Heterogeneous
A
B
Potassium
+.42
Chloride
+.00
Bicarbonate
+ . 37
Inorganic P
+.00
"'· 02
Organic P
+.00
Lactic Acid
+.00
+.00
>.10
c
Homogeneous
Final Result
L. 01
Non Protein Nitrogen
pH
•
p
Significant
Non Significant
01
Significant
6.2
.t... 01
Significant
15.3
L.01
Significant
01
Significant
A=9
L..
L.
~=1.8).06
Non Significant
B
ft=ll
L.Ol
Heterogeneous
A
2.4
). 10
Non Significant
>· 10
Homogeneous
D
l.Z
). 10
Non Significant
). 10
Homogeneous
D
0.7
>.50
Non Significant
>. 10
Homogeneous
D
5.4
"'. 01
Significant
Significant
Statistics Utilized
A = non-pooled error term "t" test
and Cochran - Cox correction .
D
B = non-parametric sign test
for paired replicas .
E = "t" tests applied to matched
groups technique.
C = non-parametric Mann- Whitney
"U" for unpaired samples.
regular pooled deviations
error term "t" test.
5H
Table IX presents the parotid saliva and whole saliva enzyme
titers.
Parotid saliva contains 11 per cent of the acid phosphatase
titer; 1 per cent of the total esterases; 34 per cent of the cholinesterase;
2 per cent of the lipase; and 18 per cent of the beta- glucuronidase level
as found in the whole saliva from the same group of pers-on s.
In
Chapter I similar results were observ e d for acid phosphatase, total
esterases, and beta-glucuronidase.
In considering the similarity and
variances of the enzyme titers two conditions should not be overlooked;
t h e mean parotid saliva enzyme levels as calculated in Chapter I were
from a much smaller group, and different conditions of collection
prevailed in the collection of the whole saliva.
CHAPTER III
Characterization of Parotid Saliva Acid Phosphatase
Numerous investigations have been made to determine the
source of the phosphomonoesterase in the human oral cavity.
Various
early workers, Smith (222); Adamson (2) ; Glock, Murray, and Pincus
(104); were of the opinion that the salivary phosphomonoesterase resulted
from the desquamation of epithelial cells.
On the other hand, Zander
(28); Valloton (241); and Cabrini and Carranza (52) were unable to
demonstrate phosphatase in the epithelium of gingival tissue and believed
the enzyme in saliva could be derived only from bacteria or subepithelial gingival tissue during inflammation.
The ability of oral microorganisms to produce phosphomonoesterase was shown by Glock, Murray, and Pincus (104}; Eggers-Lura
(76); Dentay and Rae (69); Fitzgerald (91); Ennever and Worner (80};
and Fosdick (95}.
Among t?-e organisms which hydrolyzed esters of
phosphoric acid were:
actinomyces, lactobacilli, streptococci, micro-
cocci, and Aerobacter aerogenes.
Pronounced activity was observed
at both acid and alkaline ranges.
Although the whole saliva phosphomonoesterase has a pH
optimum in the acid range (76, 101, 104, 116, 196, 222), activity has
also been noted at alkaline pH levels (76, 116}.
Previous investigation,
Chapter I, has shown that whole saliva and cultures of oral microorganisms were able to attack monosodium beta-naphthyl phosphate both
at pH 4. 8 and pH 9. 1.
Parotid saliva was active at the acid pH, but
repeated testing showed no demonstrable alkaline activity.
The acid phosphatase levels of the parotid secretion were
found to be similar i n titer to that found in human serum (Chapter II}.
r:o
,) 0
It was this latter finding which led to the present study of the properties
of the parotid saliva phosphomonoesterase.
Characterization of this
enzyme would thus permit comparison with the other phosphomonoesterases present in the various body tissues; serum, bone, muscle, and
prostate gland, and aid in its identification.
MATERIALS AND METHODS
Collection of Saliva:
Human male parotid saliva was obtained with the aid of the
parotid cap (Curby 66).
The orifice of Stetson's duct was wiped with
sterile gauze and 70o/o alcohol and the caps retained by the buccal
mucosa surrounding the duct on each side of the mouth.
was initiated by the chewing of flavored chicle.
Stimulation
The resulting parotid
secretion was collected in tubes immersed in iced water; all saliva was
pooled and kept refrigerated.
The pooled samples could be kept for
one week without loss of activity.
Measurement of Phosphatase Activity:
The method used was that of Seligman et al. (214), employing an aqueous solution of mono sodium beta-naphthyl phosphate as
substrate.
Determinations of phosphatase activity are normally carried
out at 37 degrees and pH 4. 80 using 0.1 M acetate buffer; however, it
was found necessary to use a 1. 2 M acetate buffer since the 0. 1 M
solution was unable to maintain the desired pH levels with saliva.
The
pH value recorded are those of the final mixtures, including substrate
and enzyme.
After incubation the solution was made alkaline by the
addition of 1. 0 M Na2
co 3
and the color development proceeded as
described below.
Assays were run in triplicate, and substrate blanks were
59
obtained by incubating the buffered substrate in the absence of saliva
which was added to t h e tubes immediately before the addition of the
tetrazotized diorthoanisidine.
Measurements were made in the
Klett-Summerson against the appropr i ate blanks.
Estimation of Beta Naphthol :
Beta naphthol (226) was determined by means of the water
insoluble complex which forms on the addition of tetrazotized diorthoanisidine (Diazo Blue B) at alkaline pH.
This insoluble purple azo dye
was extracted with ethyl acetate and the color density measured.
The
beta naphthol was dissolved in 5 mi. of ethyl alcohol and diluted with
distilled water to give a final concentration of 1. 4 x 10- 4 M.
The
calibration curve (Fig. 1) was prepared by adding 0 . 5 mi. of parotid
saliva and 2. 5 mi. of 0. 1 M acetate buffer (pH 4. 8) to 2. 5 mi. of
standard beta naphthol solutions, followed by the addition of sufficient
1. 0 M sodium carbonate to raise the pH to the range 7. 4 - 7. 8.
The color was developed and extracted with ethyl acetate
and five mi. of the colored organic liquid was removed by pipette.
The color density was measured in a Klett photoelectric colorimeter,
using a 540 mp green filter.
60
500
>-
1--
(j)
z
~ 300
_J
<l
2
1-Q_
200
0
100
0
10
20
3('
40
50
-()lg ) (3 - NAPHTHOL
Figure 1. - Calibration curve of beta-naphthol estimation.
RESULTS
pH Activity Curves of Parotid Saliva!
The pH activity curve was determined for parotid saliva over
the range 2. 70 - 5. 98 in 1. 2. M acetate buffers at a substrate concentration of 8. 9 x 10- 4 M .
F or the determination of the optimum pH, 0. 5 ml.
of saliva was added to 2. 5 ml. of substrate solution and 2. 5 ml. of
1. 2 M acetate buffer resulting in a final substrate concentration of
4.1 x 10- 4 M and an acetate concentration of 0. 54 M.
~
~
j
""'
0
=>..
""::::;
- ,0
15 N
0
w
0
t(
a:
""::::;
CD
~
8
12
""~
--'
,
0
I
16
I
I
9
c..
I
c..
.
r:
4
0
2
I
en..
6
--'
>I
1I
>
i=
<(
c..
(f)
0
<(
z
?i:
c..
<(
3 ~
a
0
0
30
• 0
4 .0
E.O
pH
F igure 2. - Effect of pH on reaction velocity.
Incubated for 2 hrs. at 37 degrees in 0 . 54 M
acetate buffer at varying pH values. Initial substrate concentration 8 . 9 x lo-4 M. Final volume
5. 5 ml. containing 0. 5 ml. of saliva. The two
determinations A (0) and B (e) were obtained
from the same pool of saliva but testing was done
on successive days.
The results shown in Fig . 2 indicate that the pH optimum
for parotid saliva acid phosphatase was 4. 57 with beta-naphthyl phosphate as substrate.
Optimum Substrate Concentratiom
In order to determine the optimum substrate concentration a
series of experiments were carried out, in each of which 2. 5 ml. of
acetate buffer pH 4 . 57 and 0. 5 ml. of saliva were incubated for 2 hours
at 37 degrees with 2. 5 ml. of sodium beta-naphthyl phosphate solution
and the beta-naphthol liberated was measured colorimetrically by the
method described.
The beta-naphthyl phosphate solutions used were
such that the initial concentrations of substrate in the system ranged
from 0.45 to 17.86 x 10-4M at the beginning of the experiment.
0
~~
--''
0.;.
0:: u
o E
>-:X:
w
><.
~ ?.
0
:X:
a.
)3
~ ~-
iE
iE
~
10
I
<Il.
>-
~
5
>=
(.)
<(
50
10.0
15.0
20.0
SUBSTRATE CONCENTRATION (M x i0-4 )
Figure 3. - Effect of varying substrate concentration on the
reaction velocity.
, plot of reaction velocity against substrate concentrationj
, the above , data plotted according to the equation derived by
Lineweaver and Burk (154),
s
v
=1 s
v
+ Km'
v
where S is the substrate concentration, v and V the observed and
maximum velocities respectively, and Km the .M ichaelis-Menten
constant.
The results obtained are shown in Fig. 3 from which it appears.
that the maximum velocity of the acid phosphatase activity occurred
between the initial substrate concentrations of 8. 92 and 13.38 x
lo- 4
M.
If the experiment is plotted according to the method of Lineweaver
and Burke (154) the Michaelis-Menten constant, Km and the maximum
velocity, Vmax, can be calculated from the data.
In the experiment
shown the value of Km is 3. 21 x lo- 4 , while the V max is 24.69
meg. /hr.
Effect of Time - Order of Reaction:
In the preceding determinations of the effect of pH and
substrate concentration the experiments were carried out using 0. 5 ml.
of saliva as enzyme source.
However, in order to bring the optical
density of the dye formed by the reaction of liberated beta-naphthol and
tetrazotized diorthoanisidine within range of the colorimeter after long
intervals of incubation, at the higher substrate concentrations, the
saliva volume was halved.
Pooled parotid saliva was diluted with an
equal volume of distilled water and 0. 5 ml. of this diluted solution was
used in all subsequent tests, unless otherwise stated.
The rate of hydrolysis of the beta-naphthyl phosphate was
determined by incubating 0. 5 ml. of saliva solution (37 degrees, pH
4. 57) with various concentrations of substrate, ranging from 1. 11 to
17.86 x lo-4 M.
At intervals of 1, 2, 3, 4, 6, 8, and 18 hours after
mixing, triplicate samples were withdrawn and the amount of betanaphthol liberated was measured.
G4
0
18
TIME (HOURS)
Figure 4 . - The kinetics of the enzymatic hydrolysis
of beta-naphthyl phosphate by salivary phosphatase
at 37 degrees using 17.86 (e). 13.38 (0), 11. 15
8 . 92 (D), and 7. 80 .x.lo-4 M (•) solutions of substrate and 0. 50 rnl. of saliva solution. (Co = initial
concentr ~tion of phosphate, C
amount remaining
after time t) .
<•>,
=
63
1.0
0 .8
0 .6
0
*!
0
0 .4
----o
6
18
TIM E (HOURS)
Figure 5 . - Time activity curves fo:r salivary
phosphatase employing 6. 69 (e), 4. 46 (0),
2 . 23
and 1. 12 x 1o- 4M (0) substrate
concentrations . (Go
initial substrate
concentration, C :::.amount remaining).
<•L
=
Figures 4 and 5 present the results of this experiment .
The
reaction rates as obse.rved in Fig. 4 appear to follow zero order when
4
the substrate concentration was sufficiently high {4. 46 to 17. 86 x 1o- M).
The velocity constant K or specific .reaction rate can thus be expressed
by the equation
=
=
K, where CA is the concentra-
tion of beta-naphthyl phosphate and CE equals the concentration of enzyme which is constant .
In such a reaction the velocity is independent of
the time and of the concentration and this behavior is usually due to a
saturation of the enzyme by the reactants.
When the initial substrate
concentration was lowered (1. 11 and 2.2.3 x lo- 4 M) as seen in Fig. 5
there was a deviation fr'o m zero order as the reaction proceeded.
If the initial substrate concentration was further decreased
and increased amounts of enzyme employed (Fig. 6) the -reaction kinetics
tended to follow those of a monomolecular reaction which are first orde-r,
and K = Z. 303 log
t
10
a
a-x
, where tra"· is the initial concentration of
the substrate at the start of the reaction and (a-x) is the amount of sub strate remaining after time lltll.
t
The p!ot of log aax
yielded a straight line whose slope was
9 .-
1.0
~0
0
I! ~
against the time
K
2..3
1/2
_ !(2_ ~
l/2
!12
0 .8
" -8
0 .6
-1.6
%>
0
9
0 .4
0
0 .2
0
1.2
-I
1/ 2
TIME (HOURS)
Figure 6. - Time activity curves for acid phosphatase.
Test conditions : 37 degrees. pH 4. 56, substrate
concentration 0. 89 x 10-4 M Beta-naphthyl phosphate
using 1 . 0 ml. (e) and z.. 0 ml. (0) of saliva as enzyme
sourc-e .
From the results thus obtained it can be seen that when
substrate concentrations ranging from 4. 46 to 17. 86 x 1 o-4 M (Fig. 4)
or 17.84 to 71.44 x 10- 4 M per ml. of saliva were employed the
reaction rate was constant with time, up to at least 18 hours.
With
concentrationE; of I. 11 and 2. 23 x 10- 4 M (Fig. 5) beta-naphthyl
phosphate or 4.44 to 8. 92 x 10- 4 M per ml. of saliva the velocity was
constant only to 8 hours incubation.
the reaction rate.
After this there was a change in
When a lower substrate concentration was used,
0. 89 x 10- 4 M, in conjunction with an increase of enzyme (1. 0 and
4
2. 0 ml. of undiluted saliva) or 0. 89 and 0. 44 x 10- M per ml. of
saliva the order of reaction was changed.
The reaction velocity as
seen in Fig. 6 is now monomolecular and a straight line was obtained
only when the log10 of C/Co was plotted against time.
Furthermore,
it can be noted that even in such a plot the reaction rate was not constant
after certain incubation intervals (4 hours with 1 ml. of saliva and
2 hours with 2 ml. of saliva).
This inhibition of enzyme activity or
substrate hydrolysis may have resulted from a number of factors, the
most likely being due to the pile up of products of the reaction.
TABLE
A
Reaction Velocity and Substrate Concentration
0 . 50 ml. saliva solution, pH 4. 57, 37 degrees.
The
initial substrate concentration was expressed as
M x lo-4, the velocity in meg. beta-naphthyl phosphate
liberated per hour .
Substrate
Concentration
Velocity
(found)
Velocity
(calc.)
1.11
5.30
4.63
6.86
2.23
6.48
4.46
7. 7Z
9.03
6.69
9.44
10.08
7.80
10.99
10.43
8.92
11.45
10. 71
11. 15
11. 98
11. 13
13.38
11. 4 7
11.43
17.86
11.40
11.83
B
Velocity Constant and Enzyme Concentration
1. 00 ml. and 2. 00 ml. of saliva were used as enzyme
source, pH 4. 57, 37 degrees .
calculated from the equation
Substrate Concentration
0.89
Velocity Constant were
K
= 2 . 303
Saliva Volume
log1 0
a
a-x
Velocity K
I0-4 M
1 . 00 ml.
0.2551
o .8 9 x lo-4 M
2.00 ml.
0 . 6863
X
The data shown in Table lA was plotted using the method of
Lineweaver and Burk (154), ~=.!. S
v
+
Km , where Sis the substrate
-v
concentration, vis the observed or calculated velocity, while Vis the
maximum velocity and Km the Michaelis constant.
~
By plotting S against
(Fig. 7), the slope of the resulting straight line is
.
intercept is- Km •
...!.
v
and the ordinate
The maximum velocity calculated in this manner is
v
13. 13 meg. /hr. while the Michaelis constant, Km, has a value of
2. 049 x 1o- 4 M.
8N
~0
~
~0.0
<t
if
gj
:r
Q_
~
>Q_
06
<(5.0
:;;;:
m
04
~
>-
~
0.2
~
0
~o------~5~
. o------~~.~o------~,5D~----~ro~o~-----Lo
SUBSTRATE CONCENlRATION (Mx!0 -4 )
Figure 7. - Determination of optimum substrate
concentration.
Q-0, mean reaction velocity (determined
from Fig. 4 and 5) plotted against sub·s trate concentrationle e, Lineweaver - Burk plot of S against
S/v. Enzyme concentration
0. 50 ml. of diluted
(1:1) saliva. Data plotted as seen in Table IA.
Optimum. substrate concentration noted between
8.92- 11.15 x Io- 4 M.
=
Effect of En.zyme Cone entration:
Figure 8 illustrates the effect .of varying enzym.e concentrations on the reaction velocity under optimal condition.s of pH and substrate
concentration at different incubation temperatures.
The amount of
beta-naphthyl phosphate hydrolyzed was dire:ctly proportional to the
c ·o ncentration of salivary enzyme.
-;:
.<=
.....
3"'
0
w
N
~
0
0::
0
>-
I
w
•
5.o
!;(
I
a.
(/)
0
I
4.0
a.
•
_J
>-
i=
30
I
a.
.0:
zI
<!)_
>f5
f=
2.0
1.0
0
.0:
0 .50
SALIVA VOLUME
0 .75
1.00
(mtl
Figure 8. - Effect of enzyme concentration oti
reaction velocity.
Results are express·ed as the amount of substrate
hydrolyzed per hour at various temperatures, zso(e),
38° (0), and 55°
at pH 4 . 57 from 8. 92 x lo- 4 M
beta-naphthyl phosphate.
<•>·
Effect of Tempe.rature on Enzyme Activity:
By integration of the more exact form of the Arrhenius
-equation as suggested by the theoretical conside.r ations based on
1
statistical me·chani.cs (111) the equation log K:.
-E
(
+C) is
2. 303 R T .
,..;
i
= the
obtained, where K = the velocity constant for the reaction, T
absolute temperature, R =the gas constant and, E =the energy of
activation of the molecules (the kinetic energy which must be possessed
by a molecule before the reaction will occur).
This equation states that if log K is plotted against the
reciprocal of the absolute temperature, the slope of the resulting "best"
-E
calories, thus allowing the determina4.606
tion of the energy of activation from the experimental data. The form
straight line is equal to
for biological reactions is usually written substituting u for E to indicate
that there is no necessary implication of a physical meaning involved.
The energies of activation and inactivation were determined
for salivary acid phosphatase at the pH optimum (4. 57) using varying
amounts of substrate (2. 23, 4.46, 6. 69, and 8. 92 x 10- 4 M).
Enzyme
activity was determined at nine temperatures (25, 30, 35, 38, 41, 44,
4 7, 50 and 55°C).
The results for the plot of log V against ..!_
are
T
recorded in Fig. 9 and the data for values of u, obtained by multiplying
the slopes of these lines (slope from 25° to 4 7°C for u of activation,
and slope from 4 7° to 55°C for u of inactivation) by 4. 606 are shown in
Table II.
TABLE II
Effect of Substrate Concentration on Energy of Activation
u,
Substrate Concentration
cal. /mole
above 300C
below 30°C
2. 23
X
1Q-4 M
6,600
13,600
4.46
X
10-4M
7,300
12,300
6. 69 x 10- 4 M
7,500
12,000
92 x 1o- 4 M
7,600
11,700
a.
Effect of Substrate Concentration on Energy of Inactivation
Substrate Concentration
u,
cal. /mole
10- 4 M
35,900
4.46 x 1o-4M
34,200
6. 6 9 x Io- 4 M
33,400
2. 23
X
8. 92x10
-4
M
32,900
Sizer and Josephson (2.2.0} have observed that the value of
u of activation for enzyme catalyzed reactions was able to change within
a small temperature interval.
In the hydrolysis of tributyrin by .l ipas-e
a sharp transition of the u values was noted at 0 degrees.
A similar
effect was seen at 22 degrees for the decomposition of urea by jack bean
urease .
They suggested that this transition may be due to a shift in the
configuration of the enzyme molecules.
/3
1.4
I ... '
1.3
I
/
P/ /
lI.J
r 1I
1.2
''/ .
0
-...,_
I I P.,.
~
"·a"
" " ' . -Q
'....__
~ '{
l,rl ;~
· ,.
1
~--.::
1r
1.1
flj
>
I/ I
t9
/1/ ~
11,1
,r//
1,,
Ill
0
..J
I
10
· ""
"'-
·
"
'-!!
I
'
~
•,
-
.~"'\:
~
\ '\
"
'\" \
\
'"
0.9
'0..""""'~
Ill
,II
I ll
''\
\~
tJ/
0.8
r/J,'
I
\
.'o
\
I
6/
\
\
0 .7
~
31
32
Vr
33
34
x 10- 4
Figure 9. - E;ffect of temperature on reaction velocity.
Plot of log reaction rate (log v) againS!t the r .e .ci.procal
of absolute temperature (1/ T) for varying sub.strate concentration, 2..23 (eL 4.46 (0), 6. 69(•). and 8. 92 x
10- 4 M (0). Final volume of reaction -mixture :::: 5. 5 ml.,
containing 0. 5 ml. of enzyme solution, 2.. 5 ml. of
S"ubstrate solution, and 2.. 5 ml. of acetate buffe r _pH 4. 57.
In the plot of the data seen in Fig. 9 the.re appeared to be a transition
point at a temperatur-e of 30 degrees centigrade (3.03 A).
The temperature coefficient or biological Q 10 which is the
ratio
Kz
K1
-
velocity T1
velocity T I
substrate concentration.
+ 10
was found to be independent of the
The resultS" are recorded in Table ill.
A
0 10
of 1. 4 to 2.. 0 is usually found in biological reactions indicating that a
ten degree rise .in temperature up to the point where denaturation begins,
resulted in a I. 4 to- 2. fold increase in the reaction velocity .
f~ J
'
TABLE III
Temperature Coefficient (Ql o}
Temperature range 30-40 degrees, pH 4. 57, 0. 5 ml. enzyme solution
010
Substrate Concentration
Catalyzed Substrate Reaction
10~4 M
1. 40
4.46 x Io-4 M
1. 48
6.69 x Io- 4 M
1. 48 .
8 . .92 x Io- 4 M
1. 49
2. 23
X
Results oi the present study indicated that if substrate con-centrations close to optimum (8. 92 x 10-4 M) are employed, constant
values for u were obtained.
When the substrate concentration was
lowered to the point where changes in the reaction order began to occur
(2. 23 x lo-4 M, after 8 hrs.) a different value was obtained for the
energy of activation (Table II) .
Although the energy of inactivation of the enzyme due to
heat denaturation could be calculated by multiplying the slope of the
line obtained in the plot log v again.st
4,
a more accurate measurement
was obtained by inactivating the enzyme before it was added to the
substrate solution.
A
30
60
TIME (min.)
8
0 .9
i
r\\
~03
0
''
'3w
>
0
'
'
\
<!)
\
9
\
''
''
'
''
I
\
0
5
10
20
30
TI ME (min . )
''
60
Figure 10. - Heat denaturation of salivary acid phosphatase.
Results are presented as the amount of beta-naphthyl
phosphate liberated (meg.) per hour with 4. 41 X 1 o- 4 M
substrate at pH 4. 57·, and 37 degrees after previous heating
at various temperatures; 37° (e) , 47° (Q), 50° (•) and 5 5 ° (O) f or
varyi ng time intervals.
Figure 10 shows the extent of inactivation which occurred by
incubating the buffered (pH 4. 53) enzyme solution at different temperatures
for varyin g time periods up to one hour .
The hydrolysis mixture con-
taining the previously heated enzyme was then incubated at 37 degrees
for three hours under standard test conditions.
The observed reaction
velocity, meg. of beta-naphthyl phosphate liberated per hour, was
plotted against the amount of time the saliva was heated at the stated
temperatures..
Since all reactions were of 0 order this is- equivalent to
reaction velocity constants .
The degree of inacti vation was confirmed
by an enzyme sample which had received no previous heating.
It was
7G
noted that temperatures of 37 degrees and less did not result in inactivation of the enzyme.
If the log of the reaction velocity was plotted
against the time, a straight line resulted, as seen in Figure 10 B .
When the log of the slopes of the thus obtained line-s were plotted
against 1 IT (Fig. 11) an accurate measurement of the energy of
-u
Sub4.606
stituting the observed values an inactivation energy of 88, 300 calories
inactivation could be obtained by the equation, slope=
per mo1e was obtained.
This was more than twice the u c alculated by
the previously stated method.
1.0
0.8
06
w
Q_
g
04
(f)
'-"
0
..J
02
00
- 1.8
- 0 .6
30
31
32
1/T X IQ-4
Figure 11. - Determination of the energy of inactivation.
The log of the slope of th.e lines, 470 (0), 50° (•),
and 55° (0) as obtained in Fig. lOA a-re plotted against
the reciprocal of absolutetemperature (1/T) . The
resulting slope equals u/4. 6 06 .
The affinity of enzyme for substrate is measured by the
reciprocal of the Michaelis-Menten constant, Km, which is considered
'
.. 1&1 7
to be the dissociation constant o-f the enzyme - substrate complex.
Ki.ese (140) while studying the carbonic anhydrase system found that the
Km increased with temperature in accordance with the van•t Hoff
equation.
By observing the effect oi substrate C'oncentration on the
reaction velocity at nine different temperatures and plotting the resultant
data according to· the Lineweaver - Burk plot of substrate concentration
against a./velocity, as seen in Figure 12, the effect of temperature on
the maximu:m velocity and the Michaelis constant was determined.
Q'
•
1.1
/
/
/
/
/
1.0
/
/
/
/
0.9
/
/o
/
o.e
/
•
/
/
/
/
0 .7
/
9'
~
/
06
•
/
/
/
/
0.5
/
0.4
0.3
0.2
10
SUBSTRATE CONCENTRATION (Mxi0 - 4 )
Figure 12. - Plot oi S against S/v for the effect of
temperature on the Vmax and Km. The substrate
concentrations ·e mployed were z. 23, 4.46~ 6. 69,
and 8. 92 x lo-4 M. The reaction. velocity was
measure·d at 25o (e), 30o (0), 35o
38° (O),
41°
44° (A), 47° (e), and 55° (Q).
<•L
<•>,
The results presented in Table IV show that the V max
increased with temperature up to the point where the rate of denaturation
was greater than the velocity rate .
However, in c-ontrast to the findings
of Ki.ese there appeared to be little variation of the Km. with heat other
than those that may h ave been introduced by the limits of error of this
experimental procedure .
TABLE IV
Effect of Temperature on the Maximum Velocity
and Mic h aelis - Menten Constant
Temp e ratur e
V max
Km
25
9.48
1. 80
30
13.26
1. 56
35
16.72
1. 78
38
18.05
1. 73
41
19.23
1. 35
44
26.04
2. 15
47
27. 17
2.03
50
21. 37
2. 16
55
8.48
2.51
T he temperature is recorded in degrees
c e n ti g rade. Values for V max are expressed
i n meg. substrate liberated/hr., and Km as
M xl o- 4 .
DISCUSSION
Comparison of the properties of the salivary acid phosphatase
to the phosphatases present in other a n imal tissues is limited due to the
inherent differences present when different substrates and buffer solutions are used.
Classification of the phosphomonoesterases is depen dent
on the optimal pH and various other factors; the one most generally used
being the inhibition or activation produced by the introduction of certain
inorganic ions such as F, divalent cations, sulfhydryl compounds, amino
acids, and alkaline cyanides.
Phosphomonoesterase II, III, and IV (93a)
are generally conceded to have pH optima in the acid range; II is not
effected by Mg, III exhibits inhibition with Mg, and IV shows activation
when this cation is present.
Further characterization depends on the
differences noted in the enzymatic hydrolysis rates of the alpha and
beta- glycerophosphate isomers.
Even with these criteria as a basis for
differentiation a dear cut classification often cannot be made.
The pH optimum of 4. 57 found for the salivary phosphomonoesterase, while lower than the optimum of 6. 0 noted by Roche (202)
for one of the acid phosphatases of erythrocytes was similar to that
found for another erythrocytic phosphatase, by Abul-Fadl and King ( 1),
where the optimum with disodium phenyl phosphate was observed at a
pH of 4. 3 - 4. 8.
Davies (64) and Bamann and Riedel (7) discovered that
the spleen and liver of swine and cattle contained, in addition to an
alkaline phosphatase, a phosphatase with an activity maximum at pH
4. 5 - 5.. 0.
Kutscher and Wolbergs (151) found an acid phosphatase with
a similar pH optimum in the human prostate gland.
In a characterization of enzymes of the rat adrenal cortex,
Gordon (107) observed a phosphomonoesterase which exhibited maximum
activity at pH 4. 8 - 5. 0, with glycerophosphate.
Good proportionality
was obtained for the effects of tissue concentration on the rate of substrate
hydrolysis.
Results of the present investigation (Fig. 8) indicated that in
a similar manner the hydrolysia r.ate of beta-naphthyl phosphate, by the
salivary phosphatase, was directly related to the enzyme concentration.
Lundquist (161) computed the dissociation constant of the
enzyme-substrate complex (Km) from Kutscher and Wornerrs (152)
experiments with beta- glycerophosphate and from his own work with
choline phosphate, phenyl phosphate, and glycerophosphate; using human
semen as the source of prostatic phosphatase.
The data from Kutscher
and Wornerts experiments showed a Km of 2. 8 x lo- 2 Min one case and
l. 5 x 10- 2 Min another.
Lundquist calculated the Km at 37 degrees C,
with citrate ion as the activator, as follows:
choline Km
= 10.27
calcium salt of phosphoryl
x lo-3 M, sodium salt Km
= 21.41
x lo-3 M;
sodium salt of beta-glycerophosphate Km value was 18.52 and 10.49 x
10- 3 M; while the sodium salt of phenyl phosphate exhibited a Km of
9. 38 x lo-4 M.
The measurement of the energy of activation using
these substrates was 11, 300 cal. per mole with beta- glycerophosphate,
11, 800 cal. per mole for phenyl phosphate, and 11, 700 cal. per mole
when phosphoryl choline was employed.
The salivary acid phosphatase
was found to have a Km of 2. 0 x 10- 4 M with the monosodium salt of
beta-naphthyl phosphate and an energy of activation between 6, 600
and 7, 600 cal. per mole.
Schonheyder (213) in studying the acid phosphatase of
human sperm plasma, with phenyl phosphate, noted that the enzymatic
hydrolysis did not follow either zero order or first order kinetics.
The
optimum pH for the hydrolysis was 5. 62 and the value of the Km was
5. 9 x 10- 4 M.
That this intermediate order could have resulted from a
substrate concentration which was insufficient to allow the reaction to
be zero order, but yet was too great to permit first order kinetics, is
illustrated in Figures 5 and 6 where a similar transition in the reaction
order of the salivary phosphatase resulted upon lowering the substrate
concentration.
The erythrocyte enzyme of Abul-Fadl and King (1) was
found to be extremely heat labile and was lost at room temperature in a
few hours.
The salivary phosphatase was also unstable; at a pH of
7. 4 ninety percent of the activity was lost in thirty minutes at 37 degrees
C.
This contrasted to its stability when maintained at an acid pH.
The salivary enzyme would thus tend to fall into the
classification (203) (93a) of phosphomonoesterase III, although studies
on inhibition and substrate isomers are necessary for a more complete
identification.
[-)2
CHAPTER IV
Characterization of Parotid Saliva Total Esterases
Classification of an enzyme as belonging to the group entitled
esterases is dependent upon that enzymets ability to catalyze the reaction
R. COORr
+ HzO =
R· COOH
+ HO ·
R1
•
The chemical nature of the
alcohol and acid or of the radical R and Rt found in the ester causes a
special type of specificity among the esterases.
If simple, common
esters are involved, the enzymes are termed esterases proper, however,
if the alcohol is glycerol and the acid a true fatty acid the designation
lipases is employed.
The hydrolysis of esters containing N- alcohols
and N- substituted alcohols is attributed to the azolesterases which
include the cholinesterases.
Acetylesterase (136) has been recently
separated from the esterase proper group; the former hydrolyzing esters
of acetic acid with particular specificity, while the nature of the alcohol
has little or no effect.
Although the hydrolysis of aliphatic, aromatic,
and amino alcohol acetic acid esters by this enzyme is extremely rapid,
these esters are also subject to hydrolysis by various other esterases
contained in mammalian tissue.
That the relative specificity among the esterases is produced
by the size of the fatty acid moiety has been noted in the esterases
proper of liver, which preferentially split the butyric acid esters and
which exhibit little activity towards fats.
On the other hand, pancreatic
lipase has a pronounced activity for fats and only slight activity for the
simple esters.
The term
11
aliesterases 11 has thus been proposed (199)
to include those esterases which hydrolyze simple aliphatic esters and
glycerides.
S3
Beta naphthyl acetate, a substrate employed for the
demonstration of nonspecific esterases in tissues (180) and serum (198)
has also been used for the colorimetric determination of esterases in
saliva ( Chapt. I).
It has been observed that liver, serum cholinesterase,
human serum, rat brain, and human red cell membrane are capable of
hydrolyzing this substrate (198).
Total inhibition, by physostigmine, of the activity of brain
homogenate, serum cholinesterase, and red cell membranes towards
beta naphthyl acetate indicates ii:Esusceptibility to splitting by acetyl
and pseudocholinesterases ( 198).
Lipase ( 179) obtained from pancreatic
homogenates was also able to hydrolyze this substrate.
In the measurement of the enzymatic hydrolysis of beta
naphthyl acetate by human saliva the term total esterases has been
employed to encompass activity originating from the total group classified as esterases.
The work herein presented is a partial characterization of
the total esterase activity of human parotid saliva.
MATERIALS AND METHODS
Collection of Saliva:
Stimulated human parotid saliva was collected by means of
the Curby parotid cap (63), using the procedure previously described
(Chapt. III).
Estimation of Beta Naphthol:
The beta naphthol liberated through substrate hydrolysis was
estimated from calibration curves prepared under the existing conditions
84
of the assays.
A standard solution was made by dissolving 10.0 mg.
of beta-naphthol in 5. 0 ml. of ethanol and diluting with distilled water
-4
until a final concentration of 1. 4 x 10
M (20. 0 meg. /ml.) was obtained.
Varying amounts of this standard (0. 5 - 2. 5 ml.} were placed in tubes
and brought to a final volume of 2. 5 ml. with distilled water.
Two and
a half ml. of 0. 2M veronal buffer (pH 8. 30) and 3. 0 ml. of parotid
saliva were added to each tube.
0. 1 ml. of 1 M HCl was then placed in
the tubes to lower the pH to the range, 7. 4 - 7. 8, found to be optimal
for the coupling reaction which employed 1. 0 ml. of a 2. 0 mg.
solution of tetrazotized diorthoanisidine (Diazo Blue B).
/mi.
After allowing
3 minutes for complete formation of t h e water insoluble dye complex,
1. 0 ml. of 80o/o trichloroacetic acid was added to arrest the reaction
and precipitate the salivary proteins.
Ethyl acetate (1 0. 0 ml.) was
used as the extraction agent and the color density of 5. 0 ml. of the
organic liquid was measured with a Klett photoelectric colorimeter.
Measurement of Total Esterase Activity:
Parotid saliva total esterases were measured by the method
of Seligman et al. (Chapt. I, II).
acetate,
The substrate used was beta-naphthyl
While the determination of blood serum esterases are normally
measured employing 0. 1 M verona! buffer, a higher molarity (0. 2 M)
was required to maintain the desired pH levels with saliva.
All pH
values reported are those which were measured in the final incubation
mixture, containing buffer, substrate, and saliva.
moval of the assays, after the desired incubation
Following the re-
perio~
0. 5 M HCl
was added to each tube to bring the pH to the optimal range for coupling.
All assays of parotid saliva total esterase activity were
measured in triplicate.
Substrate controls for determining the degree of
spontaneous hydrolysis under the vari ous test conditions were prepared
by incubating buffered substrate in the absence of saliva which was
added just before the coupling with the dye.
Assays were measured
in the Klett-Summerson against the appropriate controls, the difference
in optical density being attributed to enzymic activity.
RESULTS
pH Activity Curve of Parotid Saliva:
Values in the range 6. 52 - 8. 84 were employed for the
determination of the effects of pH on salivary total esterases.
Measure-
ment of enzyme activity at pH values below 7. 0 were made employing
1. 2 M acetate buffer, while 0. 2 M veronal buffer was used for levels
above pH 7. 0.
Comparison of several buffers and molarity showed no
apparent change in enzyme activity.
Adjustment of the final pH, after
incubation, to the desired level for beta-naphthol estimation was made
with the aid of 1. 0 M sodium carbonate and 0. 5 M HCl.
The initial substrate concentration before the addition of
buffer and saliva was 5. 37 x 10
-4
M.
lb
14
'-
~
l3'
12
E:
-
"0
~
e
•
10
"'
@
8
0
.r:
:c
0
(l_
a
6
'
.;=-
4
z
"t
-~
0
<[
2
<$.o
8 .0
6 .0
9 .0
10.0
pH
Figure 1. - pH activity curve of parotid saliva
total esterases .
Incubation at 37 degrees for two hours at
varying pH values. Initial substrate cone.
5. 37 x lo-4 M. Final volume 8. 0 ml . containing 3. 0 ml . saliva, 2. 5 ml. substrate
solution and 2 . 5 ml. buffer. The two determinations A (0) and B (e) were obtained
from the same saliva pool, however testing
was done on successive days.
The results shown in Figure 1 indicate an optimum of
pH 8. 57 for pa.r otid saliva total esterases.
However, since the degree
of spontaneous hydrolysis which occurred .at that pH caused .a severe
decrease in the sensitivity of the measurement of enzyme activity, a pH
of 8. 32
+ 0. 01
was used for all subsequent analysis .
Effect of Substrate Concentration:
The investigation of the effect of substrate concentration was
determined in a series of experiments in which 2. 5 ml . of veronal.
buffe·r (pH 8 . 33) a n.,d 3. 0 ml. of saliva were incubated with 2.. 5 ml. of
4
substrate solutions at varying concentrations {0 . 537 - 21.480 x 10- M)
for two hours at 37 degrees C.
The beta-naphthol liberated enzymatically
was measured and the results recorded as meg. of beta-naphthyl acetate
hydrolyzed per hour.
40 . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ,
...
~35
a-e:
0
-a"30
Q)
N
>0
-6>- 25
1.0
.<::
Q)
:§
20
~
.8
0
=
>-
.<::
a.
s>
.,
15
(f)
0
zI
•
~ 10
::::·s;
2
.....
5
01
•
•
.4
.2
/
I
I
I
0~------~--------~------~--------~------~
0
5
~
~
Substrate Concentration, M
x to-•
w
0
~
Figure 2. - Effect of substrate concentration.
(~) .
is the plot of substrate concentration
vs . reaction velocity; while {e-e), is the same
data plotted according to t h e Lineweaver-Burk
( 154) equation
S/v
=
1/V·S- Km/V
S = substrate concentration; Km = the MichaelisMenten disassociation constant; v = observed
velocity; and V
t h e maximum velocity,
=
A Lineweaver-Burk (154) plot of t h e results obtained in the
above experi ment (Fig . 2) indicates a maximum velocity of 43 . 10 meg. / h r.
and a Michaelis-Menten constant, Krn, of 9. 37 x 10
-4
M.
Effect of Time (Reaction Order) and Substrate Concentration:
Further experiments were carried out to determine the effect
of substrate concentration and length of incubation on the reaction velocity.
Three ml. of saliva and 2. 5 ml. of veronal buffer were incubated for
intervals of 1, 2, 3, 4, 5, 6, and 8 hours with 2. 5 ml. of substrate solu-4
tion (2. 68, 5. 37, 8. 06, 10. 74, 16.11, and 21.48 x 10
M).
110,....------------------,
•
100
90
I
I
~eo
p
12
-5 60
a:;
u
0
•
>.50
_c
:c
o._
0
zr
40
20
0 u__ _ _L2_ _ _ _4L----s~~-~e
0
Time, hours
Figure 3. - Kinetics of parotid saliva total esterases.
Plot of incubation time (hrs.) against rate of
substrate hydrolysis (meg. /hr.} at 37 degrees, using
various substrate concentrations; 2. 68 (e), 5. 37 (0},
8. 06
and 10.74 (C) x 1o- 4 M.
<•>.
25 . - - - - - - - - - - - - - - - - - - . . . . . , 1.0
20
20
Ci>,
/
0
.8
0
.,.."
/
/
/
~15
.6
/
/
-£ 12
_c
Q.
/
/
g~
>-
/
>
........
0/ /
/
~
(j)
/
o-o
/
zI ~>. 10
CQ.2
.. -o
.4
/
>.>.
.~
>
...c
.2
+()
<!
0
o
5
10
Substrate Co ncentration, M x !U - 4
15
°
Figure 4. - Substrate concentration and the reaction
velocity.
(o-o) plot of mean reaction velocity (as determined from Fig. 3) against substrate concentration.
( .....) Lineweaver - Burk plot of above data.
Figure 3 contains plots of enzyme activity, measured as
meg . of beta-naphthyl acetate hydrolyzed per hour, against time of
incubation at 37 degrees C.
When initial substrate concentrations of
2. 68, 5. 37, 8. 06, and 10.74 x 10
-4
M were employed the reaction was
to be zero order up to six hours. For the lowest substrate concentra-4
tion employed (2. 68 x 10
M) the velocity was not constant after six
hours, but showed a decrease .
An extremely high degree of spontaneous
hydrolysis was encountered in the solutions containing 16. 11 and 21.48
4
x 10- M substrate when incubation was greater than two hours. Thus
velocity constants at these latter concentrations could not be calculated.
Figure 4 was plotted using the method of Lineweaver and
90
Burk (154) . . The plot S against S/v, where Sis the substrate concentration and vis the observed velocity, produces a straight line.
of this line is 1/V, while the ordinate intercept is Krn/V.
The slope
The maximum
velocity and Michaelis-Menten constant were calculated to be 47. 62
4
meg. /hr. and 14 . 48 x 10- M respectively.
Reaction Velocity and Enzytne Concentration: .
The effect of varying enzyme concentration on the reaction
4
velocity measured at pH 8. 31 and with 5. 37 x 10- M substrate solution
is presented in Figure 5.
A linear relationship between saliva volume
and the amount of beta-naphthyl acetate hydrolyzed was observed.
20~--------------------------------.
0
0
0
>.r::
:E
Q.
0
zI
5
q
0 ~--~--~--~--~--~--~----L-~
0
2
3
4
So l iva Volume, ml
Figure 5. - Effect of enzyme concentration on the
reaction velocity.
The data a .r e plotted as the reaction velocity
(meg. /hr.) against saliva volume. Incubation at
37 degrees for two hours, pH 8. 31.
9
Temperature and Enzyme Activity:
The activation energy of the salivary total esterases. was
measured by means of the Arrheni u-s
( 1 IT
+ C),
where k
=-u/2. 303R
reaction; T = the
equation log K
=the velocity constant for
the
absolute temperature, R =the gas constant; and u - the energy of activation.
The plot log k vs 1 IT allows the determination of the energy of
activation from the slope of the resulting straight line, which is equal
to -u/4. 606.
1.6
r-----------------------,
I
I
1.4
I
I
I
p
/
/
/
:>
1.2
Cl>
/
/
/
d
0
...J
1.0
o•~---~----~-----~---~---~
h
30
~I
~2
~~
11r ~~o-•
Figure 6. - Effect of temperature on the reaction
velocity.
Plot of log of the reaction velocity against the
reciprocal of the absolute temperature .
An energy of activation of 5, 900 calories/mole was calculated
from the data seen in Figure 6 . Assays were carried out using
-4
5. 37 x 10
M substrate (pH 8 . 33) at 25, 35, 45, 50 , 55, 60, 61, and 6 8
degrees C.
A possible transition point, similar to that which has been
noted with lipase (220), jack bean urease, and salivary acid phosphatase
(Ghapt. III) was observed at 35 degrees.
60 degrees C.
Maximum activity occurred at
The temperature coefficient (0 10 ) of the interval 25-35
degrees C was 1. 69 while a Ql 0 of 1. 38 was observed for the interval
35-45 degrees G.
DISCUSSION
Although the substrate employed in the present investigation
is attacked most readily by the nonspecific esterases of liver (198);
cholinesterase (198) and lipase ( 179) are also able to catalyze its
hydrolysis.
Thus it may be assumed that the cholinesterase and lipase
observed in saliva ( Chapt. I) as well as any nonspecific esterases which
are present contribute to the hydrolysis of beta-naphthyl acetate by
parotid saliva.
In dealing with the multiple effects caused by the simultaneous
action of several enzymes on a single substrate, each enzyme having its
own particular characteristics, certain deviations of the resulting data
from ideal are to be expected.
However, this did not occur with the
total esterase activity of parotid saliva , which acted for all practical
purposes as a single enzyme.
This simplicity of action might be due to
an overwhelming affinity of one of the total esterase components for the
substrate, while the remaining contributors produce only negligible
activity.
While the colorimetric measurement of esterase activity
presents advantages inherent in colorimetry over manometric, titrimetric,
and electrometric methods both histochemically and in the determination
of activity in biologic fluids, the lack of prior investigation on the
kinetic characteristics of mammalian esterases employing beta-naphthyl
acetate as substrate affords no basis for comparison.
Huggins and
Lapides (132) however , have used p-nitrophenyl esters in a colorimetric
determination of the properties of human serum esterase. Their results
-4
indicate a Michaelis-Menten constant of 4. 15 x 10
M for p-nitrophenyl
acetate and proprionate respectively.
The velocity of substrate hydrolysis
was found to be proportional to the enzyme concentration.
Optimal pH
was found to be 8. 4 - 8. 6, which was almost identical with the observations of Glick (103) who recorded an optimum pH of 8.4- 8.5 for the
splitting of acetylcholine by serum.
A temperature optimum of 32 degrees
was observed with a 010 of 1. 59 between 18 - 28 degrees.
The results
of Huggins and Lapides reaction velocity studies did not permit computation of reaction order.
Even though certain similarities in the properties of serum
esterases and salivary esterases are evident, using the work of Huggins
and Lapides for comparison, certain dissimilarities also occur.
The
question of the identity of the salivary total esterases can possibly be
resolved by studies which include the effect of known inhibitory agents
as well as an elaboration on the kinetics of the hydrolysis of beta-naphthyl
acetate by other esterases found in the human body.
xxii
GENERAL DISCUSSION
The data accumulated in the present study indicate that
\
although the parotid secretion is one of the majot contributors to the
admixture termed "whole saliva" certain differences in their composition
exist.
Four of the ten e:nzymes present in whole saliva are absent in
parotid saliva, while of the six enzymes found in both only the cholinesterase, beta- glucuronidase, and lysozyme values found for the parotid
secretion approach the levels noted in whole saliva.
These differences
are assumed to be due, for the most part, to the metabolic processes
of the flora which normally inhabit the oral cavity.
Measurement of the inorganic, organic, and nitrogenous
components of whole and parotid saliva indicated that whole saliva was
higher than parotid saliva in regard to calcium, inorganic phosphorus,
albumins, non protein nitrogen, and hydroxyl ion contents, while the
parotid secretion contained greater amounts of sodium, potassium,
chloride, bicarbonate, organic phosphorus, lactic acid, total proteins,
and globulins.
Analysis of the difference between the means of each
variable in parotid saliva versus its counterpart in whole saliva showed
that with the exception of chloride, inorganic phosphorus, organic
phosphorus, albumins, and lactic acid a significant difference existed
between the mean parotid saliva level and the mean whole saliva level.
Distribution analysis of the salivary components reve.aled
that seven (total esterase, lipase, cholinesterase, total proteins,
globulins, organic phosphorus, and lactic acid) of the twenty-one factors
measured in whole saliva were abnormally distributedi while six (total
esterase, lipase, total proteins, albumins, globulins, and potassium) of
the twenty factors determined in parotid saliva had an abnormal distribution.
The remaining components were considered normally distributed
populations.
xxiii
Phi coefficients of correlation computed for each variabl e
of whole saliva versus its counterpart in parotid saliva showed significant
positive correlations for total esterase, cholinesterase, potassium, and
bicarbonate.
Calcium exhibited and inverse or negative correlation.
When the individual salivary components were compared to
their serum counterparts it was found that only the potassium and
inorganic phosphorus levels of whole and parotid saliva and the calcium
level of whole saliva were greater than normal serum values.
Investigation of the characteristics of parotid saliva enzymes
indicated that the acid phosphatase appeared to be a type III phosphomonoesterase, while the total esterase exhibited properties similar to
those evidenced by the serum total esterase.
xxiv
ABSTRACT
Whole saliva obtained immediately on arising, before brushing
of the teeth, eating, or mouth rinsing was examined for enzyme activity.
The enzymes measured were acid phosphatase, alkaline phosph atase,
total esterase, pseudocholinesterase, lipase, aryl-sulfatase, beta-Dgalactosidase, beta- glucuronidase, hyaluronidase, and lysozyme.
Hyal-
uronidase activities of the saliva samples were determined by a modification of t h e viscosimetric method.
turbidimetically.
Lysozyme activities were measured
The remainin g enzyme activities were determined b y
t h e colorimetric methods of Seligman and his co-workers, modified for
use with saliva.
The measurement of a spectrum of enzymes usin g
various esters and derivatives of a single chromogenic substance,
beta-naphthol, afforded a means of comparing the enzyme activities of
saliva, while the simultaneous determ i nation of ten enzymes on the same
saliva sample permitted evaluation of the possible interrelationship
between individual enzyme systems.
Sterile parotid saliva, obtained by cannulation with a parotid
cap, was shown to contain acid phosphatase, esterase, pseudocholinesterase, lipase, beta-glucuronidase, a n d lysozyme.
The acid phosphatase
activity of parotid saliva was 1 O% of that found in whole saliva.
esterase represented 60% of the activity of whole saliva.
Cholin -
Parotid saliva
contributed about 1% to the whole saliva total esterase activity, while
parotid lipase was approximately 1 O% of the whole saliva lipase titer.
Parotid beta-glucuronidase was from 5-20% of the amount found in whole
saliva.
Only with lysozyme was the activity of parotid saliva higher than
that found in whole saliva.
Broth cultures of whole saliva indicated that all but sulfatase
and lysozyme could be produced by the microorganisms normally
XXV
inhabiting the oral cavity.
These findings indicated that the parotid gland secretion
contributed a part of six of the ten enzymes present in whole saliva, with
the remaining share of these six enzymes apparently derived from the
oral flora, cellular debris, or the other salivary glands.
In order to determine further the part-whole relationship of
the parotid secretion to whole saliva, various inorganic, organic, n i trogenous and enzymic components of stimulated whole and parotid saliva
were measured.
An aqueous mouth rinse was employed to minimize the
effect of oral microorganisms and cellular debris.
One enzyme,
beta- glucuronidase, was determined in whole saliva both before and after
the washing process as an indicator for the effectiveness of the mouth
rinse.
Blood obtained from the test subjects was examined for enzyme
titer so that serum and saliva enzyme levels could be compared using
the same substrate for both, while the saliva levels for calcium, sodium,
potassium, chloride, bicarbonate, phosphorus, lactic acid, and
non-protein nitrogen, total proteins, albumins, and globulins could be
compared to established normal serum values.
In measuring the effect of mouth rinsing on the whole saliva
enzyme levels it was found that the beta- glucuronidase level decreased
40o/o after washing.
Similarly, acid phosphatase values showed a 29o/o
decrease from the values found in the group which did not have the
preliminary mouth washing.
Two tests, the chi square goodness of fit and a plot of the
distribution, of each of the saliva and serum components measured, were
used together in a combined judgment in order to gauge the normality of
the distribution.
Seven (total esterase, lipase, cholinesterase, total
proteins, globulins, organic phosphorus, and lactate) of the twenty-one
factors measured in whole saliva were abnormally distributed; six
xxvi
(total esterase, lipase, total proteins, albumins, globulins, and potassium)
of the twenty factors determined in parotid saliva were found to haye an
abnormal distribution; while one of the serum enzymes, beta-glucuronidase,
proved to be abnormal.
The remaining components for whole saliva,
parotid saliva, and serum could thus be considered normally distributed
populations.
Measurement of the various salivary components indicated
that whole saliva was higher than parotid saliva in regard to the calcium,
inorganic phosphorus, albumins, non-protein nitrogen and hydroxyl ion
contents; wpile the parotid secretion contained greater amounts of sodium,
potassium, chloride, bicarbonate, organic phosphorus, lactic acid, total
proteins and globulins.
An analysis of the difference between the means
of each variable in parotid saliva versus its counterpart in whole saliva
was made.
With the exception of chloride, inorganic phosphorus, organic
phosphorus, albumins, and lactic acid a significant difference existed
between the mean whole saliva level and the mean parotid saliva level.
Human serum normally maintains an anionic and cationic
balance of approximately 155 milli-equivalents per liter, with little variation except during marked acidosis, alkalois, or excessive salt excretion.
Whole saliva had a mean total anion content of 37.7
a mean total cation content of 40.7
+
+
12.8 mEq/liter.
anion content of parotid saliva was 47.4
+ 19. 2
total cation content was 43. 9 + 17. 1 mEq/L.
imately 25% of the ionic content of serum.
12.0 mEq/liter and
The mean total
mEq/liter, while the mean
Saliva thus contained approx-
When the individual salivary
components were compared to their serum counterparts it was found that
only the potassium and inorganic phosphorus levels of whole and _F>arotid
saliva and the calcium level of whole saliva we.re greater than normal
serum values.
It was noted that parotid saliva contained relatively high levels
xxvii
of acid phosphatase and total esterases.
The acid phosphatase levels of
the parotid secretion were found to be similar in titer to those present
in human serum.
It was these findings which led to the study of the
properties of the parotid saliva phosphomonoesterase and total esterase.
C haracterization of these enzymes thus permitted comparison with similar
enzymes present in the other body tissues and aided in their identification.
The pH activity curve for the phosphomonoesterase was determined over the range pH 2. 70-5. 98 in 1. 2 M acetate buffer at a substrate concentration of 8. 9 x lo- 4 M.
Results indicated that the pH optimum for parotid saliva acid
p h osphatase was 4. 57 with beta-naphthyl phosphate as substrate.
The
reaction rate followed zero order when the substrate concentration was
sufficiently high.
When the initial substrate concentration was lowered
there was a deviation from zero order as the reaction proceeded.
If the
initial substrate was further decreased and increased amounts of enzyme
employed the reaction tended to follow those of a monomolecular reaction,
or first order,
Using the method of Lineweaver and Burk a plot of S
(substrate concentration) against S/V (substrate concentration/ velocity of
reaction) the Michaelis constant, Km, was found to have a value of
2 x 1 o-4 M.
The energies of activation and inactivation were determined
for the salivary phosphomonoesterase at the pH optimum.
Enzyme
activity was measured at nine temperatures over 25 and 55° C.
The
energy of activation was between 6, 600 and 7, 600 cal. /mole., while the
energy of inactivation was found to be between 32, 900 and 35,900 cal. /mole.
The temperature coefficient (Q 1 0 ) was 1. 48.
Maximum activity occurred
at 47°C.
Although the substrate employed for the investigation of the
parotid total esterases was attacked most readily by the nonspecific
xxviii
esterases of liver; cholinesterase and lipase were also able to catalyze
its hydrolysis.
Thus it may be assumed that the cholinesterase and
lipase observed in saliva, as well as any nonspecific esterase present
contributed to the hydrolysis of beta-naphthyl acetate by parotid saliva.
In deahng with the multiple effects caused by the simultaneous
action of several -enzymes on a single substrate, each enzyme having its
own particular characteristics, certain deviations of the resulting data
from ideal were to be expected.
However, this did not occur with the
total esterase activity of parotid saliva, which acted as a single enzyme.
Results of the investigation indicated an optimum of pH 8. 57
for parotid saliva total esterases.
When a sufficiently high initial sub-
strate concentration was employed the reaction was zero order up to six
hours.
Longer periods of incubations could not be employed due to the
high degree of spontaneous hydr.olysis encountered.
constant was calculated to be 14.48 x 1 o-4 M.
The Michaelis-Menten
A linear relationship
between saliva volume and the ·amount of beta-naphthyl acetate hydrolyzed
was observed.
The energy of activation was 5, 860 calories/mole.
mum activity occurred at 60°C.
Maxi-
The temperature coefficient (Q 10 ) of the
interval 25-35 degrees C was 1. 69, while a QlO of 1. 38 was observed
for the interval 35-45 degrees C.
.xxix
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·
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xxxii
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XXXV
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liii
Autobiography
Name: Howard H. Chauncey
Date and Place of Birth: May 25, 1927
Boston, Mass.
Father: Albert B. Chauncey
Mother:
Address:
Mollie B. Chauncey
26 Berkshire Avenue, Sharon, Mass.
Educational Background
High School Education: Boston English High, Boston, Mass.
College: Tufts College, Medford, Mass., B.S. (Chemistry) 1949.
Boston University Graduate School of Medical Sciences,
Boston, Mass., A.M. (Biochemistry) 1952.
· Military Service
U.S. Armed Forces- Weather Observer, U.S. Air Corps- Separated
December 21, 1946.
Research Positions
Beth Israel Hospital, Boston, Mass., Research Assistant, Surgical
Research Department.
University of Miami, Coral Gables, Florida, Research Associate,
Division of Biological Research.
Tufts College Dental School, Boston, Mass. Research Associate,
Department of Dental Research.
Bibliography
Effect of Formalin Fixation on the Activity of Five Enzymes of Rat Liver,
Seligman, A. M., Chauncey, H. H., and Nachlas, M. M., Stain
Technology, 26:19, 1951.
liv
The Colorimetric Determination of Phosphatase in Human Serum,
Seligman, A. M., Chauncey, H. H., Nachlas, M. M., Manheimer,
L. H., and Ravin, H. A., J. Biol. Chem., 190:7, 1951.
Histochemical Use of Dithizone, Chauncey, H. H. and Lionetti, F.,
Federation Proceedings, ~:No. 1, March 1952.
The Histochemical Demonstration of Zinc ·in Leucocytes, Chauncey, H. H.,
Thesis submitted for the degree of Master of Arts, 1952.
Hydrolytic Enzymes in Hyaluronidase Preparations, Chauncey, H. H.,
Lionetti, F., and Lisanti, V. F., Science, 118:219, 1953.
Enzymes of Human Saliva, Part I. The Determination, Distribution,
and Origin of Whole Saliva Enzymes, Chauncey, H. H., Lionetti, F.,
Winer, R. A., and Lisanti, V. F., J. Dent. Res. 33:321, 1954.
The Production of ~-Glucuronidase and Hyaluronidase by Streptococcus
mitis, Lorina, P. L., Lisanti, V. F., and Chauncey, H. H., Oral
Surg., Oral Med., and Oral Path., ~:998, 1954.
The Determination of the Enzyme Content of Whole Saliva, Chaup.cey,
H. H. and Lisanti, V. F., presented at International Association
for Dental Research, Philadelphia, March, 1953.
Proteolytic Enzymes of Human Saliva, Chauncey, H. H., Johnson, V. M.,
and Lisanti, V. F., presented at I. A. D. R. Meeting, French Lick,
Indiana, March, 1954.
Parotid Gland Secretions: Flow Rate and Interrelationship of Salivary
Components, Chauncey, H. H. and Lisanti, V. F., to be presented
at I. A. D. R. Meeting, Chicago, illinois, March, 1955.
The Esterolytic and Lipolytic Activity of Oral Microorganisms, Mahler,
I. R., Chauncey, H. H. and Lisanti, V. F., presented at the
I.A.D.R. Meeting, Chicago, illinois, March, 1955.
Societies
Member of the International Association for Dental Research, A. A. A. S.,
and Sigma Xi.