137
078

 

HTHS

A SWQ‘T" fig "E’HE iNFLUENCE 3F
METARY' ANS} G‘fi'iER FACTQRS
ON TEE PRODUCTIGN OF
DEN'ML GAMES {N A
SUSCEPTIBEE STRAIN 0F RATS

Themis {‘m' NW Degree af M S.

MlCHiG-AN $‘E‘ATE QOLLEGE

RObE-i‘? W‘ifliam Si‘ipek
ma

 

,b

n

_ ‘ t' :
‘3: -*

 

 

 

This is to eertifg that the
thesis entitled
A.STUDY OF THE INIIUENGE or DIETARY AND
OTHEB.IAOTORS OR THE PRODUCTION OF
DENIAL CARIES IN A SUSOEPTIBLE STRAIN O! BATS

presented by

Robert V1 1119.!!! Stipek

has been accepted towards fulfillment
of the requirements for

".3; degree in chemistry

Quid.

Major professor 1

Date hungry 19. 19M

 
  

 

-

 

.m- -

 

 

 

I __ .- —_ aim-j I‘Iu -'.

A STUDY OF THE INFLUENCE OF DIETARY AND OTHER FACTORS
ON THE PRODUCTION OF DENTAL CARIES IN A
SUSCEPTIBLE STRAIN OF RATS

By

Robert Hillier: Stipek

A THESIS

Suhaitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Chemistry

19148

-7

’ /
’ L. 1'

N
M

I
ll
; L9

\
k \. ‘ a
\‘\\ \\r\)\ '3
x).
é
I

ACKNOWLEDCMENTS

The author wishes to express his gratitude and
sincere appreciation to Doctor Carl A. Hoppert for
his guidance, interest, and friendship throughout
this study and. his kindly criticism in the prepara-
tion of this manuscript. The author also wishes to
make acknowledgments to Doctor Philip J sy and Doctor
Benjamin H. Pringle for their cooperation and interest.

198864

mmUmIONCOOOOOOOOOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOOOO
HISTme...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0....

A.

B0 Fluorineeeeeeeeeeeeeeeeeeeeeeeeeeoeeeeeeeeeeeeee
Co Amonia-PrOduCing Gunpomdseeeeeeeeeeeeeeeeeeeoo
Do.Miacellan80u3.oo................o............ooo
EXPERIMENTAL...00000000000000e00000000000000000000000000

A.

B.

TABLE OF CONTENTS

Page

Genera m Etiolog000000OOOOOOOOOOOOOOOOOOOOOO

werEIOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0...

10 Animals U86d...............................
20 Care Of the.Aninals........................
3. Observations...............................
he Preparation Of Dietseeeeeeeeeeoeeeeeeeeeeee
50 canpOBition 0f.Dietseeeeeeeeeeeeeeoeeeeeeee
Procedure.......................................
10 Group Ieeeeeeeeeeeeeeeeeeeooeeeeeooeoeoeoee
2. Group IIoeeeeeeeeeeoeeeeooooeeeeeeeeooooeee
30 GrOUP IIIOoeeeoeooooeooooeooooooeeeeoeeeeee
he Group IVOOOOOOOOOoeeooeeeeoeeeeoeeeoeeoonO 16
50 Group Veeeoeeeeeeeeeeeoeeeeoeeeeeeeeeeeoeee 17
60 Graup VIoeoeeeooeeeeooeeeeeeoeeoooooeeeeooo 1?
70 Group VIIeeeeeeoeeeeeeeeoeeoeeeeeeooooeeeee 17
80 Group VIIIeeeeeeooeseeeeeseeeeeeoooooeoeeeo 18
90 Group IXeeeeeeeoeeeeeeeeeeeoeeeeeoooeeeeeeo 18

KEKKGCSif-fififixoqmuww

DATAOOO0.0.0.0....O...OOOOOOOOOOOOOOOOOCOOOOO0.0.0.0.... 19

Table I....0...0.00.0...OOOOOOOOOOOOOOOOOOO00...... 19

Table IIOOOOOOCOOOOOOOOO00.0.0.0....0.0.0.0.0000... 20

Twle IIIOOOOOOOOOOOOOO0.00.000.00.00.0.00.00.00.00 21

Table IVCOCOOCOOCOOOOOOOO0.0.0.000...OOOOOOOOOOOOOO 22

Table VOOOOOOOOOOOOOOOO00.000.000.00.00000000000000 23

T‘blo VIoooeeoeeeooeeeeeoooee00000000000000.0000... 23
Table VIIoeeeeeeeeeeeeeeeeeeeeeoeeeeeeeeeeeeeeeeee. 2h
DISCUSSIONoeeoeeeeeeeeoeeeeoeeeeeeeeoeooeeeeeoeooeoeeoeo 25

1.
2.
3.
h.
5.
6.

7.
8.

Groups I ‘nd IIeeeeeeeeeeeeeeeeeeeeeoeeeeeeeeeee 25
Group IIIeeeeeeeoeeeseesoeooeeeeeeeeoeeeeeeeeoee 26
Group IVeoeeeeeeooeeoooeeoooooooeoeeeeoooeeoeoee 26
GrOUP Veeeeeeoeeeeeeeeeeeoeeeeeeeeeoeeeeeeeeeeee 27
GrOUP VIeeoeeeeeeeeeeeeeoeeeoeeeeeeeeeeeeeeoeeee 28
Group VIIoeeeeeeeoeeeeeeeeeeeeeeeeeeoeeeooeeeoee 28

Grow VIIICOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 29

Group IXeeeeeeeeoeeeeeooeooeooooeoooeeeeeeeeeeee 30

CONCLIJSIONSOOOOOOOOO0.00.00.00.00...OOOOOOOOOOOOOOOOOCOO 32
BBLIWMOOOOO...0.0.0....O0.0...OOOOOOOOOOOOOOOOOOOO 33

INTRODUCTION

The problem of tooth decay is. one which has held the atten-
tion of workers in new fields for a number of years. It is not
a new disease but rather has plagued mankind for centuries. The
fact that it dates back to prehistoric tines is substantiated by
findings in ancient ruins of carious human teeth (1). The
statement has been reads that ”there never was an epoch when the
hman species was not cursed by toothache" (1). It is not easy
to attack this problem since it has been daonstrated by workers
in various fields that diet, heredity, tooth structure, secre-
tions and micro—organisms all play 'a very important part in its
causation. Therefore, it can be seen that it would be impossible
for one person, or a single group of workers, to investigate all
of these contributive factors and arrive at an answer to the prob-
1m as a whole.

Inasmuch as dental caries has been and is at present one of
our chief concerns, it sears desirable to approach the problem
frm the standpoint of prevention as well as to study causative
factors . The availability of a caries susceptible strain of rats
developed by Hunt, Hoppert and m (2) greatly facilitates the
study of causative and preventive factors. In this study the
work was divided into three main parts:

I The Effect of Certain Foods on the Production of

Dental Caries.
In this part of the work sue foods which are nor-
mally a part of man' s diet were fed. The foods selected

.1-

II

III

for special stucbr were two forms of the potato and a
cereal breakfast food, wheat flakes.
The Effect of Amonia Producing Cmpounds and of an
Aloniun Salt on the Prevention of Dental Caries.
This part of the work sought to determine if
urea and urease (alone and in combination) a: dibasic
ammonium phosphate when incorporated in a cariogenie
diet would reduce the incidence of dental caries.
The Effect of Fluorine on Dental Caries in the Rat
When Given in the Drinking Water.
This part of the work was done to deter-ins
whether or not drinking water containing fluorine in

amounts canparable to and greater than those which have
been observed to be caries inhibiting in drinking water
for humans would decrease the incidence of tooth decay

in this susceptible strain of rats.

A.

HISTORICAL

General and Etiology

Dental caries is one of the oldest and camonest diseases
of man. Its existence is known to have gone back as far as
2800 B. C. All through the history of mankind one finds quite
frequent reference to the fact that dental caries existed, re-
gardless of the degree of civilisation, provided that starchy
foods were included in their diet. Early attmpts to find the
basic cause of dental caries net with little success and it
has been only in the last thirty years that real progress has
been made. Marv investigators now think that the basic cause
of dental caries is the fermentation with acid production of
carbohych°ates in the oral cavity (3).

Several. groups in this country and abroad began a series
of studies on the causation of tooth decay in about 1920.
Since that tins great strides have been made in the field and
much has been done in the way of clarifying the causes and
treatnent of the disease. One of the foremost groups of this
field was Bunting, Jay, et.al. (h) of the University of Michigan.
Their contributions have been and are continuing to be most
outstanding. Among others in the field are groups headed by
Doctors Klein and McCollm at Johns Hopkins University, by
Doctor Rosebury and coworkers at Columbia University, and
by Doctor Hanks, et.al. in the Chicago Dental Research Club of
Chicago, Illinois. Outstanding individual workers include
Agnew, Boyd, Bodecker, McBeath, Hunt and Happert, McClure,

-3-

Mellsnby and Armstrong. Each has made his contribution to
the complex problen and it is noteworthy that this study has
brought together people fru the fields of dentistry, phy-
siological chanistry, genetics, bacteriology and nutrition.
The study of tooth decay in animals dates back to 1922
when McCollum, Simone, Kinney and Greives (5) reported find-
ing lesions in the teeth of the albino rat. The gross feat-
ures of decay were pointed out by Greives (6) who observed
that first a penetration of the enamel occurred which was
followed by spread in the dentin with subsequent fracturing.
More recently, Klein and McCollun (7) have attributed the
cause of macroscopic caries in rats to be (a) fracture of
the molar cusps; (b) decay at the bottom of the molar sulei;
( c) interprorinal caries where food impaction has been fre-
quently noted; and (d) a embination of all three factors.
They found that the ingestion or a rachitogenic diet contain-
ing coarse corn meal gave carious lesions, whereas the same
diet containing finely ground corn showed abscence of caries.
The fact that particle size is an etiological agent in
the production of dental caries was first duonstrated by
Hoppert, Webber and Caniff (8) in 1931-32. Rats fed rations
low in calcium and Vitalin D failed to develop cavities,
whereas extensive decay was observed in the control aninals
fed an adequate stock diet. Further investigation by these
workers showed that the stock diet contained coarse corn
meal which was directly concerned with the development of
caries. Other rations, although nutritionally inferior to
.1...

the stock diet, but containing corn meal ground to pass a
60 mesh sieve failed to produce caries.

One factor that must not be overlooked is that of
species difference. Perhaps it will explain the report by
Shaw, Schweigert, McIntire, Elvehjem and Phillips (9) that
the cotton rat is better suited for dental caries studies
due to the fact that there is no difference in the response
of tooth decay when the particle size of the ration is varied.

Particle size etiology has been confirmed, however, by
Klein and McCollum (7), Shelling and Asher (10), Rosebury,
Karshan and Foley (11, 12, 13, 1h). Lilly and Wiley (15) and
King (16).

B. Fluorine

The incorporation of fluorine in the diets and drinking
waters of both man and animal has given a new impetus to the
prevention of dental caries.

The inportance of fluorine was first discovered when it
was observed that eerie s-free individuals were fairly nunerous
in areas in which the domestic water supply contained an appre—
ciable amount of fluorine. Observations made by Dean (17) and
his associates in the United States, by Ockerse (18) in South
Africa, by Ainsworth and Weaver (19) in England, and by others
in different parts of the world substantiate the statement
made by Jay (20) that “the inverse relation betwem fluoride-
bearing domestic water and the prevalence of dental caries in

children has been definitely established”.

.5-

Miller (21) reported in 1938 that the caries-producing
potency of the H-W-C coarse rice ration is greatly rechiced
by the administration of fluorine in the drinking water of
rats. He also found that iodoacetic acid is effective in
accanplishing the same result.

Armstrong (22) reported in 19143 that caries suscepti-
bility of rat molar teeth was decreased by the use of drink-
ing water containing 20 pm. fluorine given simultaneously
with a caries-producing ration.

Arnold and McClure (23, 21;) in 191:1 reported that sub-
cutaneous injections of fluorine in rats which was equiva-
lent to hO-SO p13. fluorine in the drinking water did not
show an decrease in dental caries, the animals being main-
tained on the H—W-C simplified ration. However, they found
that the fluorine content of molar enamel was increased.
This finding, according to Jay (20), night be interpreted
to indicate that the fluorine was adsorbed by the dentinal
aspect of the enamel and hence exerted no antiensymatic or’
antibacterial effect on the oral surface of the teeth. It
was suggested that the enamel is capable of adsorbing
fluorine (25, 26).

It is of interest to note that the rat does not respond
to dosages which are effective in humans. The lowest amount
of fluorine reported effective in rats is 20 ppm. ( 22),
whereas a much naller mount is effective in humans.

Ockerse (27) observed in 109 towns representing 86 districts

--6-

of South Africa, that the caries initiation rate is high if
the fluorine content of drinking water is less than 1 ppm.
but that the caries initiation rate is very low if the
fluorine content is greater than 1 ppm. It appears that the
contact of the teeth with fluorine over a number of years
is essential for inhibiting dental caries as Bibby (28) ob-
served that the use of fluorine-containing dentifrices- over
a period of two years was ineffective.

c. Amenity-Producing Compounds. '
The use of anemia-producing compounds to inhibit tooth decay
is of recent origin. Stephen and Miller (29) reported in
19141; that a 140% solution of urea, when used for about two years
as a dentifrice by a small group of young patients, resulted
in a marked decrease in the incidence of new lesions and also
retarded the progress of decay in lesions already present.
A short time later it was observed by Kesel, O'Donnell, and
Kirch ( 30) that caries imune individuals have enzymes in
their saliva capable of liberating amonia frm several amino
acids although a number of such enzymes were found in the
saliva of persons whose teeth were actually decaying, ensyme
which is capable of deeminising glutamic acid was invariably
lacking. Keyes (31) reported the use of urea in connection
with dental caries in the Syrian hamster. A 50% solution of
urea was topically applied by weekly two-minute treatments.
The animals were fed a H-W-C-sugsr ration. His data showed
even a slight increase in the incidence of dental caries
over that of the controls. These results are surprising

-7-

and may perhaps be attributed to species differences as one
would expect a slight decrease in tooth decay.

Kesel, O'Donnell, Kirch, and tech (32, 33) incorporated
dibasic ammonium phosphate in a mouth wash and a dentifrice
and used these clinically for approximately five months in
persons having active caries lesions. They found that Lacto-
bacillus counts in these subjects were markedly decreased.
They also found that the mania nitrogen that is developed
in hman saliva by natural processes has the ability to inhi-
bit the growth of L. acidophillus, that the micro-organisn
present in the oral flora which sens to be responsible for
much of the ammonia production is B. aerogenes, and that the

inhibition of the L. acidophillus is not due to any alkalinity
developed by the amonia, since the same alkalinity provided

by sodium acetate did not produce the inhibition that mania
acetate did. In vitro experiments (33) indicate that a eu-
bination of 5% dibasic amenium phosphate and 3% urea is more
effective as an antibacterial and antiacidic agent than is
either of these substances alone. Kesel suggests (32) that
the development of aleonia nitrogen in the oral cavity may
be responsible for the abscence of dental caries which sue
persona naturally exhibit.

The finding of Kesel, et. al. that an organism, B. aero-
genes, nay be responsible for the production of mania in
the oral cavity supports the theory advanced by Schsnts and

Scrivener (3h) . They have suggested that the use of mouth

.8-

washes and dentifrices may be instrmental in prmoting tooth
decay, because these products may frequently destroy the deli-
cats organisms that nature has provided as a part of a defen-
sive system against processes destructive to the tooth.

D. Miscellaneous

Sale recent findings that are worth mention are those
of McClure and Hewitt (35). They placed rats on a corn meal
caries-inducing ration containing 75 units of penicillin per
gram and drinldng water containing 75 units per milliliter.
At 125 days the rats receiving penicillin were caries-free
whereas control animals had a 50% incidence of lesions.

Plate counts of L. acidophillus were negative while those
for the controls were greater than 50 colonies per plate.
The ingested penicillin did not produce a measurable concen-
tration in the blood. It was, therefore, assmed to have a
localised action.

Burrill, Calandra, Tilden and Fosdick (36) ministered
synthetic Vitamin K (2-methyl-l,h-napthom1inone-sodim bisul-
phite) to patients in the form of chewing gum. They found
the incidence of new lesions were lowered 60-90% by the use
of this gun. Gm containing calcium carbonate without the
Vitamin K reduced the occurrence of nu cavities approadnately
half as much as the Vitamin K gum.

Turner and Crane (37) found that individuals with exten-
sive cavities produce saliva which Ivdrolyses starch very

rapidly, whereas those caries-free persons produce saliva

.9-

which Wdrolyses starch very slowly. Wach, Kesel, Hine and
O'Donnell (38) have reported that the caries activity of a

patient may be evaluated by determining the pH and the total
titrable acidity.

.10..

EXPERIMENTAL

A. General

1. Animals used

The animals which were used in these experiments were
of the strain produced by Hunt, Hoppert and Erwin (2). This
strain of albino rat was developed by selectively breeding
caries susceptible animals and resulted in the production
of a strain of rats which develops caries in a very short
time and are therefore well adapted for dental caries stud-
ies. The animals used represented the 17th generation of
selectively bred stock.
2. Care of the animals

The breeding stock was kept in suitable cages supplied
with bedding of wood shavings. Each breeding group was com-
posed of animals which were litter nates. The practice of
breeding litter mates was suggested by Doctor Hunt and was
regularly employed in his genetic studies of tooth decay.
It was desirable to have litters containing at least two
females and, as a rule, two males were retained for each
breeding group. Upon showing signs of pregnancy the females
were removed to small isolation cages and were kept on the
stock ration until the young had reached an age of two weeks.
At this time the mother and litter were transferred to a
clean cage and placed on a fermentable carboqudrate-free
rationl' so that caries would not be initiated in the young

1 This ration consisted of casein (h0%), alfalfa

meal (30%), brewer's yeast (9%), fat (lard or
crisco) (20%): and sodium chloride (1%).

.11.

rat before it could be placed on an experimental ration.
At twenty—one days the young were weaned, the mother being
returned to the breeding cage. The use of the fementable
carb ohydrate-free ration was continued until the rats were
placed on the experimental ration.

The animals were housed in metal cages supplied with
wire mesh bottous. Food was kept before the animals at all
times in glass Jar containers and fresh tap water was sup-
plied daily with the exception of one experimental group
(Group II).

3. Observations

Examinations of the lower molars were made at twa week
intervals. A nasal speculum was found to be very convenient
for making the observations. The animals were held by the
nape of the neck and the nasal speculum was used to open the
mouth, and to expose the lower molars to view. A canon
desk lamp with a flexible stand equipped with a 100 watt bulb
supplied adequate light for the examinations. A record of
caries initiation and progression was kept by marking a tooth
map similar to the one designed by Doctor Hunt. When there
was any doubt as to the existence of a carious lesion in a
certain area of a teeth a question mark («a») was recorded
with the date of the observation. When a definite carious
lesion was noted a N" was recorded and the approximate extent
of the lesion indicated on the tooth map. In cases in which a

number of 'doubtfuls" or question marks wowed the date of

.12-

the last one recorded previous to the observance of a positive
lesion-was arbitrarily considered the date of the initiation
of the carious lesion. In these studies it was canon for a
"positive" definite carious lesion to appear two weeks after a
"doubtful" was recorded. A sample of the type of records kept

is herewith shown.

 

In the data tables it may be noticed that, in sane cases, blanks
appear in the "initial”, "moderate", and "severe“ stages. A
blank in the "initial" column means that moderate or severe
caries was found at the initial observation. A blank in the
"moderate" column indicates that the lesions progressed from
the initial to the severe stage between successive examinations,
whereas a blank in the "severe" column indicates that the lesions
in that particular animal did not reach the severe stage before
examinations were discontinued.
1:. Preparation of Diets

The control ration used contained the same ingedients as
the cariogenic ration developed by Hoppert, Iebber and Caniff
(39). However, for nutritional studies on caries prevention

.13-

this ration was found to be too rapid in its initiation and
production of caries. Therefore, the rice for the control
ration used was ground in a Hobart Mill so that only 2% would
be retained by a 20 mesh sieve. When rats of this susceptible
strain are placed on the control ration, caries of the lower
molars will be initiated with 35 to 70 days with slight varia-
tions due to genetic differences in the individual animals.
The dried potatoes and wheat flakes were ground to the same
consistency as the rice. In the case of the dry rations, the
constituents were mixed thoroughly by hand in a large pan.

Fran preliminary experiments it was found desirable to
feed whole boiled potatoes along with a supplauentary ration
to furnish nutrients which are essential for the well being
of the animals. This supplementary ration consisted of
powdered milk, alfalfa meal, and sodium chloride.1 The ani-
mals relished the boiled potatoes and chose them for the
greater part of their daily intake (85%).

The diets containing urease were prepared as follows:
the urease powder (Arlco urease) was taken up in a 30% solu-
tion of ethanol and then mixed with a portion of the dry feed.
Thiswas allowedtodryovernightandthenwas groundina
mall Hobart Mill. If urease alone was to be added to the
ration, this ”pre-mix' was then incorporated directly with

1 This ration was selected because it was damn-

strated previously by Olson to have no caries
producing effects.

a calculated amount of the basal ration. When urea was also

to be added, it was first finely ground in a mortar and added

to the ”pre—mix'. By this method a more even distribution of

the constituents was obtained.

In preparing the rations containing urea only, or those

containing dibasic amonium phosphate, a "pro-mix" was like-

wise prepared with the finely ground canpounds.

5. Composition of Diets

The fine rice ration (Diet I) was used throughout these

studies as a control ration. It was also used as the basal

ration when amonia canpounds and urease powder were fed.

The composition of diets used are listed in the following

 

 

tables.
Diet I Diet II
Fine Rice Ration Dried Potato Ration
Fine rice 66% Ground dried potatoes 79%
Powd. whole milk 30% Alfalfa leaf meal 10%
Alfalfa leaf meal 3% Casein 10%
Sodium chloride 1% Sodium chloride 1%
Diet III Diet IV

Boiled Potato Ration

Wheat Flake Ration

 

Boiled potatoes ad libitum Ground Wheat flakesl 79%

Supplmentary ration " " Alfalfa leaf meal 10%
Powd. whole milk 79% Casein 10%
Alfalfa leaf meal 20% Sodium chloride 1%
Sodium chloride 1%

Diet V Diet VI

Dibasic Ammonium Phosphate Ration

Fine rice ration 99%
Dibasic am. phosphate 1%

Urea - Urease Ration

 

Fine rice ration 98.5%
Urea 1.0%
Urease 0.5%

1 The wheat flakes used were Wheaties, a cannercial

product of General Mills, Inc.
.15.

Diet VII Diet VIII

 

 

Urea Ration Urease Ration
Fine rice ration 99% Fine rice ration 99.5%
Urea 1% Urease 0.5%

The rations were kept in covered metal cans and those con-

taining urease, use or both, were kept in a refrigerator.
B. Procedure

1. Group I

This group consisted of annals which were fed Diet II
(dried potato ration). Five animals were included in this
group. The litters were divided so that control animals
served for both Group I and Group II.
2. Group II

Fourteen animals were included in this group. They re-
ceived the experimental ration listed as Diet III (boiled
potato ration). They represent animals taken fran six litters.
Boiled potatoes were fed daily and the supplasentary ration
was eaten ad libitum. Data for groups I and II are found in
Table I.
3. Group III

Diet IV (wheat flake ration) was the ration fed to this
group. The group included twenty-three animals fran seven
litters of which ten animals were fed the control ration
(Diet I). Date for this group is found in Table II.
it. Group IV

This group of animals was fed a ration containing 99% of

Diet I and 1% of (NHh)2003’ Detailed data are not supplied

.16-

for this group because the experiments were discontinued due
to reasons to be discussed later.
5. Group V

This group represented animals from two couplets litters.
Sixteen animals were used in.this experiment; six of which
‘were placed on the control ration (Diet I), the other ten re-
ceived.Diet V (1% of dibasic ammonium.phosphate incorporated
in the control ration).
6. Group VI

There were actually two lots of’animals included in Group
VI. As may be seen in Table IV of the data, animals having
numbers 33 through 57 had.a.mnch.longer record than those listed
in the remainder of the table. Originally five animals from
four litters were placed on.Diet VI (urea plus urease ration).
When examinations showed.that initiation of caries was not occur-
ring in these animals, an orpansion of the group seamed desirb
able. Therefore, animals from four new litters'were then divided
into four sub-groups and placed on Diets I, VI, VII, and VIII
respectively. Five of these animals were included in the sub-
group which.received.Diet VI and are thus included in this
group.
7. Group VII

Seven animals from the four litters nentioned.above made up
this group. The same animals were used for controls. These
animals were fed the experimental ration listed as Diet VII
(urea ration).

-17-

8. Group VIII

The animals in this group were members of the same
four litters as were those of Group VII. Seven animals are
included and were fed Diet VIII (urease ration). Control
animals are those which also serve for Groups VI and VII.
9. Group IX

This group was canprised of forty-four animals repre-
senting nine litters. These animals were divided into four
groups. The first consisting of fifteen rats was used as a
control group and tap water was supplied for drinking purposes.
Fluorine, in the form of sodium fluoride, was added to the
drinking water of the remaining three groups. The first of
these three groups, containing eleven rats, received water con-
taining two ppn. of fluorine; the second, consisting of eight
animals, received five ppm. of fluorine; and the third group,
comprised of ten rats, received ten ppn. of fluorine. All of
these animus were fed Diet I.

.18-

DATA

TABLE I

EFFECT OF POTATO RATIONS 0N DENTAL GERIES

Litter NO e

hl (12-12)

 

29FCF(12-10)

29FCF(11-27)

143(12-21)

h3(12-30)

M (1-7)

Rat NOe

l

\OODNO‘VLC’UN

10
11

12
13
1h
15
16
17
18
19
20
21
22
23
2h
25
26
27
28

Diet

I
II
II’
I I
I
II
II
III
III
I
II
III
I

I

I
III
III
III
III

III
III

III
III
III
III

Caries time in.Days
Initial. Moderate Severe

 

R L R L R L
55 55 83 - 99 99
Caries-free at 281 days
I I I I I
I I I I I

55 70 83 - 99 99
Caries-free at 281 days
I

h5 70 83 83 99 99
Caries-free at 281 days

h9 h9 - - 77 77
h9 65 - - 93 93
h9 - - - 77 77
Caries-free at 275 days
I I I I I
I I I I I
I I I I I
h3 55 9 - - . 77 77
Caries-free at 269 days
I I I I I
76 '76 (a - 110 110
h6 h6 - - 76 76
Caries-free at Zhh days
I I I I I
I I I I I
s s e e I

 

TABLE II

EFFECT OF IHEAT FLAKE RATION ON DENTAL CARIES

Litter NO e
h2(1-10)

h1(1.10)
32(1-110
h5(1-25)
32(1-16)

h7(1-9)
h2(1-25)

29

Rat NOe D1013

Caries-time in days

 

Initial Moderate Severe
R L R L R L
h6 h6 76 76 110 110
60 60 76 76 110 110

Caries-free at 3h3 days
I I I I I

76 76 - - 110 110
h6 h6 76 76 , 110 110
Cariesefree at 3h3 days
I I I I I
- - 76 76 110 110
- - 76 76 110 110
Caries-free at 3h3 days
I I I I I

- - 62 62 . 96 96
Caries-free at 328 days
I I I I I

I I I I I
h6 h6 76 76 110 110
Caries-free at 3&3 days
65 65 - - 110 110

Caries-free at 3&3 days'
- - 62 62 96 96
- - 62 62 96 96

Caries-free at 328 days

 

-20-

TABLE III

EFFECT OF DIBASIC AMMONIUM PHOSPHATE ON DENTAL CARIES
WHEN INCORPORATED IN THE CONTROL DIET

 

Littu' N00

 

h9(7-16)

Sh(7-16)

M
w
$
70
fl
fl
U
7h
75
M
W

w
W

80,

m
&

Rat NOe Diet

<<<<<<HHH<<<<HHH

Cumsflminflw
Initial ;Moderate Severe

R LR LRL

 

— - 133 133 16h 16h
- - - 16h16h

133 - - - 16h 16h
Caries-free at 200 days
I I I I I
I I I I I
I I I I I
133 - - 133 16h 16h
133 - - 133 16h 16b
133 - - 133 16h 16h
Caubfiuatmoap
I I I I I
I I I I I
I I I I I
I I I I I
u w s s w

 

-21-

um 0F UREA AND UREASE 0N DENTAL CARIES WHEN INCORPORATED

Litter NOe
h1(1-109

32(1-16)
h7(1-9)

h2(1-25)
5h(6-16)
5h(5-28)

hh(6-l)
5h(6-19)

Rat No.

aHaHfifiHH

H<H

HfiH<<H<1
HH H

<
H

TABLEIIV

IN THE CONTROL DIET

Caries time in days
Initial ZModerate Severe

Diet R L R L R L

76 76 - - 110 110
h6 h6 76 76 110 110
Caries-free at 26h days
I I I I I
h6 h6 76 76 110 110
Caries-free at 3h3 days
65 6S - - 110 110
Caries-free at 26h days
- - 62 62 96 96
Caries-free at 250 days
93 115 115 - 1&6 1&6
Caries-free at 162 days
- - 93 115 1&6 1&6
Caries-free at 162 days
I I I I I
- - 93 115 1h6 1h6
Caries-free at 162 days
115 - 162 162 - -
Caries-free at 162 days

H

 

-22-

TABLEV

 

EFFECT OF UREA ON DENTAL CARIES WHEN INCORPORATED IN
THE CONTROL DIET

 

 

Caries time in days
Initial Moderate Severe

 

Litter No. Rat No. S3 Diet R L R L R L
Sh(6-16) 83 M I 93 115 115 - 1146 M
92 M VII Caries-free at 162 days
SHE-28) 85 M I - - 93 115 1116 11:6
93 M VII Caries-free at 162 days
9h F VII 7! I w w w
hh(6-l) 88 M I - - 93 .115. 1146 1146
95 M VII Caries-froe at 162 days
96 F VII 11:6 1&6 162 162 - .-
5h(6-l9) 90 M I 115 - 162 162 - -
97 M VII Caries-free at 162 days
98 F VII 1146 1116 162 162 - -
TABLE VI

EFFECT OF UREASE 0N DENTAL CARIES WHEN INCORPORATED IN
THE CONTROL DIET

Litter No. Rat No. S_e_x Diet

51:05-16)
EMS-28)

hh(6-1)
Sh(6-19)

83

M
M
F
M
M
F
M
M
M
M
F

I
VIII
VIII
I
VIII
VIII
I
VIII
I
VIII
VIII

Caries time in days
Initial Moderate Severe
R L R L R L
93 115 115 - lh6 1&6

Caries-free at 162 days
115 115 1116 11:6 162 -
- - 93 115 1146 11:6

Caries-free at 162 days

I I I I I

-. 7"- 93 115 1146 -1176
Caries-free at 162 days
115 - 162 162 - -
Caries-free at 162 days
115 - 162 - - -

 

TABLE VII

THE EFFECT OF ADDING FLUORINE TO THE DRINKING WATER

Caries time in days
Initial. ‘Moderate Severe

 

 

 

 

Litter No. Rat No. ppm. of F* R L R. L R L
11304-15) 106 None - - 97 97 12h 12k
107 None 12h 12h - - - -
108 2 97 97 - - 12h 121;
109 2 . 97 97 - - - -
110 5 12h 12h - - - -
h1(5-17) 111 None 12h 12h - - - -
112 ' 97 97 - - 12h 12h
113 5 - - - - 97 97
11h 5 97 97 - - 12h 12h
h9(5-2) 115 None 12h 12h - - - -
116 a - — 97 97 12h 121:
117 ' - - 97 97 12h 12h
118 2 Caries-free for 12h days
119 2 - - 97 97 12h 12b
120 5 - - 97 97 12h 12k
121 5 96 - 12h - - -
52(5-8) 122 None a - 97 97 12h 1214
123 2 - - 97 97 12h 12h
12h 2 - - - - 97 97
125 5 97 - 97 - 12h 12h
ENS-10) 126 None - - 97 97 12h 121:
127 2 Caries-free for 12h day
128 5 12h - - - - -
h9(S-25) 129 None 117 - - . . - -
130 2 117 - - 117 - -
131 S - - 117 117 - -
132 10 Caries-free for 117 days
h3(5-25) 133 None 90 90 - - 117 117
13h ' - - 117 117 - -
13S 10 - - 117 117 - ..
136 10 9O - 117 117 - -
137 10 - - - 117 .. -
‘ 138 10 9O 90 - - 117 117
h1(5-21) 139 None 117 - - - - -
Ibo 2 - - 117 117 - -
lhl 10 Caries-free for 117 days
1142 10 117 - - 117 - -
1h3 10 Caries-free for 117 days
lhh 10 117 - - - - -
1126-23) 1115 None 117 - - .. - ..
1116 " 117 - - - - -
1h? 10 - - 90 90 117 117
1118 2 - - 117 117 - -
1&9 2 - 117 117 - -

«111 animals were fed Diet I
.21..

DISCUSSION

1. Groups I and II

Potatoes (White or Irish) were selected for special study
because of their praninence in human diets and because it was felt
that their non-glutenous character might cause that to be non-
cariogenic. They were therefore fed in he forms, rats in Grow I
receiving a diet containing dried, finely ground potatoes and those
in Grow II receiving boiled potatoes swplunented with a non-
cariogenic cmplete feed mixture.

As may be seen in Table I, the control animals (those on
Diet I) showed tooth decay characteristic of the susceptible strain
of rats used in these uperiments, whereas the animals on both
Diets II and III were caries-free when last examined, 281 days
frm the start of the experiment.

The results of these tests may have far-reaching practical
application although it must be recomised that the experimental
procedure uployed here was arbitrary and that no claims can be
made that potatoes would be non-cariogenic in humens. However,
the striking results with the .highly susceptible strain of rats ‘
would make it seen very probable that similar results might be ob-
tained with humans. It would certainly seem logical on the basis
of these results to permit individuals having rampant tooth decay
and restricted by their dentists in the use of carbohydrate foods,
to make liberal use of potatoes. In this connection it is fortui-
tous that the potato lends itself to preparation in so many differ-

.nt “no

2. Group III

The animals included in this grow were fed Diet IV
(wheat flake ration). The control rats were fed the fine
rice cariogenic ration (Diet I).

All of the animals fed Diet IV remained caries-free for
the duration of the experiment, namely 3113 days, whereas the
control rats developed caries in a fairly short time (see
Table II). It is likely that the former would have continued
to remain caries-free indefinitely. That the method of using
the wheat flakes as part of a cqnplete diet was arbitrary must
be admitted. Certainly there is no similarity to the conven-
tional use of breakfast foods by humans. Nevertheless, it is
significant that a highly susceptible strain of rate should
remain caries-free on a diet containing 79% of a ground cm-
mercial breakfast food.

3. Grow IV

Eleven young rats fran the caries-susceptible strain were
divided into two groups, one of which received the cariogenic
diet containing 1% ammonium carbonate and the control grow
the \mswplamented diet. No detailed data is given because
there was no significant difference in the caries-time of the
two pews. However, it had been expected that amenium salts
would inhibit tooth decay so it was apparent that possibly the
volatile nature of amenium carbonate had been ignored.
Although the ration had been stored in the refrigerator, sub-
sequent analysis employing the Van Slyke and Cullen method

~26-

for anmonia disclosed that only traces of ammonia could be
detected in.the ration. This accounts for the apparent fail-
ure of ammonium carbonate to inhibit tooth decay. A positive
inhibition was strikingly demonstrated when dibasic ammonium
phosphate was substituted for ammonium carbonate. The results
of this test are presented in the next section.

h. Group V

The animals comprising the group were fed Diet V, con-
taining 1% dibasic a-mniun phosphate. The results shown in
Table III indicate that dibasic amonim phosphate, when in-
corporated in a cariogenic ration, tends to inhibit tooth
decay in a highly susceptible strain of rats. The animals
were caries-free at 200 days, whereas the control rats showed
initial caries at 130 days.

It is apparent that the initiation time of caries in the
control animals was longer than previously reported to be
characteristic of the susceptible strain of rats. This may
be due to the fact that the rats were somewhat older than
those previously used and had been kept on a fermentable carbo-
hydrate-free ration until the start of the experiment. Never-
theless, the results clearly demonstrat that dibasic almonium
phosphate definitely inhibited the development of caries and
are in substantial agreement with those reported with humans

by KQBCJ.’ Etc .10 (32333).

.27-

5. Grow VI

The animals in this grow received Diet VI containing
urea and urease. To date, all of these animals have renained
caries-free. Part of the group was fed Diet VI for 3143 days
and the rulainder of the grow, which was started later, for
162 days. As may be seen in Table IV, the control animals
showed characteristic susceptibility to tooth decay, although
sue variations in initiation time were observed.

The exact mechanism of the inhibition by urea and urease
is not completely understood, although we can be certain of
the liberation of ammonia by the action of urease on urea when
the food is moistened by saliva. That the ammonia acts merely
as a neutralising agent is questionable since Kesel, et.al.
(32,33) have observed that the L. acidophillus is not inhibited
by a degree of alkalinity swplied by sodim acetate equivalent
to that resulting firm the production of almonia in the oral
cavity of humans. Apparently the ammonium ion swplied either
by amonium salts or by the action of urease on urea will
directly inhibit acidogenic bacteria.

6. Group VII

When the animals fed Diet VI showed an absence of caries
after eating the ration for 180 days, it was decided to deter-
mine whether urea or urease alone would exert a caries-inhibit-
ing action. Accordingly, Grows VII and VIII were set w re-
ceiving the urea and urease supplemented diets VII and VIII re-
spectively. The data for Grow VII is recorded in Table V.

.23-

Most of the animals fed Diet VII were caries-free at 162
days whereas two showed an initiation—time of 1146 days. The
fact that the two positive reactors were females as were two
positive reactors in Group VIII indicates that sex may be a
factor in caries susceptibility. Litter-mate males were caries-
free at the time the last mutations were made. At any rate
the inclusion of 1% urea in an otherwise cariogenic ration of-
fected a definite delay in.the development of caries.

Although Keyes (31) has shown that the topical application
of urea to the molars of the Syrian hampster does not inhibit
dental caries in this species, Stephan (1.0) found that a mouth
wash containing urea did danonstrably inhibit tooth decay in
hunans. Stephan attributed this effect to the denaturation of
bacterial enzymes by the urea solution, although he presented
no direct proof for this view.

It has also been suggested that amonia might be produced
from.urea.by bacterial action. If the latter is true, bacterial
stasis would be caused by the production of amonia by the
bacteria themselves. Until definite proof is supplied the ex-
planation for the inhibiting effect of urea will remain a
matter of speculation.

7. Group VIII

The animals in this group were fed.Diet VIII containing

urease. They were taken free the same litters as those of

Grow VII.

-29-

Here, again, we find sex differences. Two females in
this group showed little evidence of caries inhibition. is may
be seen in Table VI. The rest of the group remained caries-
free.

It would sea, therefore, that urea and urease alone may
exhibit a caries-inhibiting effect when incorporated in a
cariogenic diet. However, the effect is not as striking as
when both urea and urease are present. In the latter case the
production of mania is inevitable, whereas the formation of
mania is less certain in the presence of urea or urease alone.
Whether other modes of inhibition are operative for urea or
urease has not been demonstrated.

8. Group II

The animals used in this experiment were divided into four
groups. IAll were fed the fine rice cariogenic ration. The
first was used as a control and was supplied with tap water.
The other three groups received 2 p.p.m., S p.p.m., and 10 p.p.m.
of fluorine in their drinking water.

In view of the fact that only two animals receiving 2 p.p.m.
of fluorine in the drinking water and three animals receiving
10 p.p.m. of fluorine runained caries-free at 121; days, the
emperiIent was discontinued. The inhibiting effect of fluoride
at these levels was insignificant immuch as the average
initiati on-time in the control animals was 113 days.

It is quite apparent that levels of fluorine in the drink-
ing water up to 10 p.p.m. do not inhibit tooth decay in this

-30.

susceptible strain of rats. These results agree with those of
McClure (23,214) who reported that no reduction of caries
occurred in albino rats fed 10 p.p.m. of fluorine in the drink-
ing water. However, McClure did find levels of 140-50 p.p.m. to
be effective. This represents a much higher concentration than

that found effective in humans viz. l-l.5 p.p.m.

.31...

CONCLUSIONS

The following conclusions may be drawn fran the studies

carried out with a highly susceptible strain of rats:

1.

2.

3.

h.

S.

6.

Potatoes fed separately as boiled potatoes or as finely
ground after cooking and drying proved to be non-cariogenic.
A diet containing 79% of ground commercial wheat flakes

was found to be non-cariogenic.

The addition of 1% of dibasic amoniun phosphate to a cario-
genic diet canpletely inhibited dental caries.

The addition of 1% of urea and .5% of urease (Arlee Urease)
to a cariogenic ration also ccmpletely inhibited dental
caries.

Additions of 1% urea or .S% urease were partially effective
in preventing tooth decay.

The supplying of drinking water containing up to 10 p.p.m.
of fluorine was ineffective in preventing tooth decay in a

caries-susceptible strain of rats.

-32-

1.

2.

3.

h.

S.

6.

7.

8.

9.

10.

BIBLICIERAPHY

Buffer, M.A., Study of Abnormalities and Pathology of
Ancient Egyptian Teeth. Am. J. of Phys. Anthrop., 3;
(1920). Printed in "Studies in the Paleophathogy 01‘

Egypt" 0

Hunt, He He, Hoppert, Ce Ae, and M, We Ge, Inheri-
tance of Susceptibility to Caries in Albino Rats., J.
Dent. Res., 33; 385, (19%)

Dirks, J. J. B., Dental Caries - Etiology and Prophy-
laxis. Nederland Tijdschr. Geneeskundi, 85, 1533-8
(19h1) "

Bunting, R. W., and Jay, P. , Dental‘ Caries, 2nd edition,
76-79 (19h1)

McCollum, E. V., Simonds, N., Kinney, E. M., and Greives,
C. J., The Relation of Nutrition to Tooth Development and
Tooth Preservation. I. A Preliminary Study of Gross
Maxillary and Dental Defects in Two Hundred and Twenty
Rats on Defective and Deficient Diets. Johns Hopkins
Hosp. Bull. _3_3_, 202-215 (1922)

Greives,. C. J., A Preliminary Study of Gross Maxillary
and Dental Defects in Three Hundred Rate an Defective
and Dgficicnt DiBtSe Jo Am. Dante “803e, 9, h67'h9h,
(1922 "

Klein, H., and McCollum, E. V. , The Significance of Food
Particle Size in the Etiology of Maerosc ic Dental Decay
in Ratae Je Dent. R98. E, 69.71., (1933

Hoppert, c. 1., Webber, P. A., and Canniff, 'r. L., The
Production of Dental Caries in Rats Fed an Adequate Diet.
Science 1g, 77-78, (1931)

Shaw, J. H., Schweigert, B. S., HoIntire, J. N., Elveh-
Jen, C. A., and Phillips, P. H., Dental Caries in the
Cotton Rat. I. Methods of Study and Preliminary Nutri-
tional Eacperiments. J. Nutrition fig, 333-316, (19%)

Shelling, D. G., and Asher, D. E., Calcium and Phos-
phorous Studies. VIII. Sane Observations on the
Incidence of Caries-Like Lesions in the Rat. J. Dent.
ROBe 2:23 368-378, (1933)

11.

12.

13.

15.

16.

17.

18.

19.

20.

21.

22.

23.

Rosebury, T. , Karshan, N., and Foley, G. , The Experimental
Production of Typical Dental Caries in Animals, and It's
Value for the Study of Tooth Decay of Teeth in Man. J.
Dent. ReSe 21.—3’, 1’43-1’4’4 (1933)8e

Rosebury, T., Karehan, 16., and Foley, G., Studies, in the
Rat, of Susceptibility to Dental Caries. III. The Experi-
mental Production of Typical Dental Fissure-Caries and
Other Lesions in Rats, and Preliminary Studies of Their
Etiology. J. Dent. Res. 13, 379-398 (l933)b.

Rosebury, T., Karshan, 1a., and Foley, G., Studies in the
Rat of Susceptibility to Dental Caries. IV. Further
Studies of the Etiology of Fissure Caries. J. Am. Dent.
Assoc. _2_1_, 1599-1611, (193h)b.

Rosebury, T., Karehan, N., and Foley, G., Studies in the
Rat of Susceptibility to Dental Caries 3 A Review of Four
Years of Research. J. Am. Dent. Assoc. 2_2_, 98-113, (1935)

Lilly, C. A. , and Wiley, L., Relation Beheen the Physi-
cal Character of Food and Dental Caries in A1me Rats.
J. Nutrition 1, 1.63-1.72 (1931.).

King, J. D. , Dietary Factors in the Promotion of Dental
Disease in Experimental Animals, with Special Reference
to the Rat. Part I. Dental Caries. Brit. Dent. J. :52,
233-2104. 305-316. (1935)- ‘

Dean, H. T. , Domestic Water and Dental Caries. Am. Water
Works. A. J. 3—5.’ 1161’ (19,43).

Ookerse, T. , Endemic Fluorosis in Pretoria District.
S. Afr. Med. J. _1_§_, 261, (19241).

Weaver, R., Fluorosis and Dental Caries on Tyneside.
Brit. Dent. J. 19, 29, (19%).

Jay, P. , Fluorine and Dental Caries. J. Am. Dent. Assoc.
2’ h89‘h95, (19h6)e

Miller, B. F. , Inhibition of Emperinental Dental Caries
in the Rat by Fluorine and Iodoacetic Acid. Proo. Soc.
Exper. Biol. and Med. 32, 389, (1938).

Armstrong, W. D. , Mechanin of Fluorine Inhibition of
Caries in the Rat. J. Dent. Res. 33, 2143-256, (191:3)-

Arnold, F. A. Jr. , and McClure, F. 21., Observations on

Induced Caries in Rats. II. The Effect of Sucutaneous
Injection of Fluoride. J. Dent. Res. _2_(_)_, 239, (19111).

41,-

2h.

25.

26.

27.

28.

29.

30.

31.

32.

33.

31:.

Arnold, F. A. Jr., and McClure, F. J. , Observations on
Induced Caries in Rats. II. The Effect of Subcutan-
eous Injection of Fluoride. J. Dent. Res. _2__O_, 167-1163,
(191:1)-

Pm, Me We, and Armstrong, We De, On the Manner 0:
Acquisition of Fluorine by Mature Teeth. J. Nutrition
_2_1_. 35. (191:1)

Volker, J. F. , Effect of Fluorine on Solubility of
Enmnel and Dentin. Proc. Soc. Exper. Biol. and Med. J.

Ockerse, T., The Relationship of Fluorine Content, Hard-
ness, and pH Values of Drinking Water and the Incidence
of Dental Caries. s. Afr. Med. J. 33, 255-258, (191.14).

Bibby, B. G.,. A Test of the Effect of Fluoride-Containing
Dentifrices on Dental Caries. J. Dent. Res. 21;, 297-303,
(1915 . ""

Stephan, R. M., and Miller, B. F. , Effectiveness of Urea
and of Synthetic Detergents in Reducing Activity of Human
Dental Caries. Proc. Soc. Exptl. Biol. and Med. 55,
101-101., (19kb) . ""

K8861, Re Ge, O'Donnell, Jo Fe, and web, Ee Re, D8-
amination of Amino Acids by the Human Oral Flora; It's
Role in Dental Caries Immunity. Science, 191, 230-231,
(19u5)

Keyes, P. H. , The Effect of Topical Sodium Fluoride
and Urea Applications on Caries Activity. J. Dent. Res.
_2_5-, h69-h75) (19h6)e

Kesel, R. G., O'Donnell, J. F., Kirch, E. R., and Wash,
E. G., The Biological Production of Therapeutic Use of
Ammonia in the Oral Cavity in Relation to Dental Caries
Prevention. J. Am. Dent. Assoc. 23, 695-711;, (19146).

K0861, Re Ge, O'Dmnm, Je Fe, Kirch, Ee Re, and WECh,
E. C., Amonia Production in the Oral Cavity and the
Use of Amonium Salts for the Control of Dental Caries.
he Je W0 and m'flSurge, 22-, 80.101, (19h7)e

Schants, C. N., and Scm-ivener, C. A. , New (beervations
on Inhibitory and Productive Factors in Dental Caries .
J. Am. Dent. Assoc. 33, 180-191, (19146).

35.

36.

37.

38.

39.

’40.

McClure, F. J., and Hewitt, W. L. , The Relation of
Penicillin to Induced Rat Dental Caries and Oral L.
Acidophillus. J. Dent. Res. _2_§, hid-ma, (191.6).

Burrill, De Ye, mandra, Je Ce, Tilden, Ea Be, and
Fosdick, L. S. , The Effect of 2-methyl-l, h-Naphtho-
quinone on the Incidence of Dental Caries. J. Dent.
Res. 3.1.2, 273-282, (1915).

Turner, N. G., and Crane, E. H., The Relationship
Between Dental Caries and Saliva. Science. 99, 262,
(19111:). '—

Wach, E. G., Kesel, R. G., Hine, M; H., and O'Donnell,
J. F., Testing Caries Activity by Acid Production in
Saliva. J. Dent. Res. 23, 1115-1121, (19113).

Hoppert, C. A., Webber, P. A., and Caniff, T. L., The
Production of Dental Caries in Rats Fed an Adequate
Diet. J. Dent. Reszg, 161-173, (1932).

Stephan, R. H., Two Factors of Possible Importance in

Relation to the Etiology and Treatment of Dental Caries
and other Dental Diseases. Science 22, S78, (191:0).

-35-

4- {NJ 1 4 ’48
7m ~ - as
‘ a“: eff

 

1 "gfi. N.,; '. . .
U \ e . I' .
1 . ~ .
f
A; , , - .

p

 

. w-
it L‘V'T-
.

‘

v

 

 

 

0' r -‘
~‘ . -' 11M. . .. . : 2.. .‘ H .
'. .( U . . ._ . .»§_ 4 ‘n I.) . . - . u y ‘ '95,)‘3' .
I f . ‘r y d' ‘ ‘..‘ .' \‘ g ‘ 6‘ ".Y". I. ' I ""‘A.U|. x' .-. he.
° 5 -' I ‘ H. {II-"- r‘ . . . I ' 'I“ t "A". t \ A, -. .-<'.S(J‘-. , ' x. .3“ 'Ix-‘ ‘5 u“): “ ..'.\i,i‘¢}-' ' “a ".3. - . f)
“ e ' ‘ " . k'.‘ " K . ' I .-. ( ““'%O 0".- \; J "‘."CQ‘N.‘ ' l
'5. .‘- I‘ to i . ' . ‘g . ' d " “SJ,~‘ "«.‘.fl/llll? ‘L‘HI I' - ' 1 -.1' "0. "' ' I) 3" If. 4. "5 I ..I
‘ r. r ‘ ' : ... .' ' ' '. ‘0. ' ' ~ ! °._ .. '_ . e 7 ‘r 3.1 - 0" '. 't .. ... “.4
.. ‘: ° .° '4' ' ,' . ( '..-- _.-.~-' .-‘- 1‘,“ .‘kf‘n '.' 1”— we..:5 'i‘w‘» « ‘ .. I. V .; 3'," .‘f’P‘ 4"
. | "~' ' . I. e "" J ‘6 - ‘ a -’| ‘9‘ '9 . .5", 7 . ‘ ”’ '- 'x e
3 n A . .' . .‘ '. ‘ ‘I...e .' ...¢.\( v‘-._ . .‘ .
l . . . .‘. .. : - - '9 / ". O‘. '. 51' y'w~fi.'\":‘14“f'- ’ u--o J): . ‘5 I' "" R3 Y, 9' (‘4‘
‘ 1 e e . ‘ ’ ‘. M‘ t ' ' . ..'- ‘ ‘ 0 ‘.° 1 t ‘ '§' I
. o 1. . ' ‘: ~ ' J- ‘- $ ‘t .)
‘ ‘ . a . ‘ ' .
t), . - ‘ I‘ ‘7 ’
U . '
1 I [I '.- l- "
-l ' I. . 1 .-
a ’ -r .
. ' . - \ ° '
0 .- . e
. .' ‘ 1‘ I‘,‘.
f . . I . . ‘ r.-
I . '
.‘ . ' . AI . .‘ '
L ~ ” . ‘3 \
O ‘ I ' . O
. A] I. V
a '- 0 ' .l' ‘
‘ “37“ _ ‘
"9 . ’ ' .—_ O—a ' .5
I . - - '. . 5 ‘
- I, I I... ‘ .0
.‘e - V ‘ \" -
‘ l . ‘ a ' . I" D
.‘ ° I l ‘
‘0". . ' 0 O J.
. V. I". ' ‘ O.
" ..’ ll.‘ -\.. '*- I t a.”
.. ' r-rh‘ 1}. ' f, - "
k . e ,0
t ‘ .
. I \...' . _ i
7 -, ' , ' ' "
.--.I..-J.-' ,. 'f'v ‘
.' ’ "7 .. '.0 . - H
u 0“ ‘0 , x‘ . r
' 0.. ’
e .‘ .r '

., -' :‘2' 526312.015
_. ‘8859 Stipek

 

     
  

 

I
1 I . M
\‘E‘P' "Fri ' $
I 1'39; ‘2‘“, 'K-l'. “.315“.
I .0" n '

LI. “'5'- .'L. 791‘ ”’5‘“ £32339

, I ‘I I l:
.. ‘V

t] . e .e!

K ‘.
t u,

I II . -..e’.
'fi

I
0:,

 

L)

h
r
em;

.9-

4" ' .‘. .
'5! .- ' .' ’.
£47.. 337
e.“ -.'-'
d
k9

e. O M ;' ""“
r‘ 4 .t' ~I'1'I-I‘ .
. . _ ,1 ,1 . - ~
‘ ‘ . ' T. ‘ . J r.
1’ g Y ." .- 'j“ 5 v . ‘O- - ' . ._ _\
n A . ’ ._ . ' .. . 1. . _ '
)' ' ‘ ' ‘\£'\ '.,(’.‘v ‘ . 4 ‘ #4.. 2*- ‘ a .‘k
' O- I A l 'l . g 5 ... a: x..‘.& 1‘ I
‘~ .. a a- : ~" ..' §. e a -._
T . A :01 .I ‘I ~ ‘\ ' kw'v“ 7_ ’ ’ 1‘s},
‘ ' ‘ . - - ‘ I. I 1 L“? ' £‘w ~23. v‘ .‘:~,~-
u A . ' C. . W . a, N .
e- I .nlI ”If. i I . .k ~ft 0‘23“ 7 l. ._ '
' '5 ' 7 L‘o a ... a! .§"£1..\}~$:n '
7 ' ‘ ' . . ‘
I h . b ‘ l f. V' 1’ ' ’
-‘ -' > m. ‘12. " ~ ’3 . -»‘TI*.."-%‘ "3». '
' I

 

I ... ' '.I J‘ ‘\ ‘I'Q: IN)"
. ,3. 1 ~ 1... .~ ~ 1- e . ~ .
‘ “ ‘ \J 5. l O .1 ' 0| “*0 r 1 J
. U . , . . \I f z I - ,V." yo... ,_, ‘u s 6'.
I ‘ ' I" a I .. . .11 s (
‘_- . [a .‘.,.I '1' 1.1". " ~34 4-1...) .4.) Has?»
‘ e o .- . " ' l ' , " 'fi , 'I ~
I I lo ,t. , .. ‘.'{ .1 , X rt‘l’4 .\ b‘fi9’21‘ ~
. .;..j’ a! ' ' . ..Zzflfi' ' m”. a"
I, . . ° - '.J , ‘ Jen/1:" 7.4:. “3
Q a ' ' 0 - ‘: . .I .‘ 'b p
v - ‘\ ‘ " . ..., ' \II‘ . M ,3
- ' .0 ; 'e -9 ' . "I ' r ' u"
' ’ -'7’ ’é““*1?wu*fiil
o l ‘l ‘ II . ' "
‘ ‘0 ¢ '7 . ‘ . - ‘ , q
.,~ t , ac.¢ - £3 5
‘e I ~ I. 0 \ .v (C' 1‘
- ..- .1 '. e :1 'pr'v. . \
' _ .l . . 394.115;
U \ r . . " . ' ‘3': l'.?t‘.‘l 3" ‘
o ‘ I- I f ." :4‘. . ‘ F“, ..'. ‘i y"; a
i - 'I _;."’ ',. .... #k ‘1
u (. I g . I} ....) ’ . ‘-:’ -~ fip"
. >o ‘ . ‘7', ‘1' ' -" 1’ .t
. : O A n. . ....1‘“._“ ' ‘<' I"
. r 0‘3 "‘0. 1' 3’ \‘r Y ‘ ’
t .- e . I t ', , i 4". - “:"' 11 .,
-I Q ‘3‘ at. \‘ h. . ‘Q - ‘ .. .\‘;” f a 0‘ ' .l l
o . , 7- ..t.\‘ e ' ' fa, .1. . ‘. . I.
I ', A“. W‘s yd F-asr‘ 1'
| 0“ ‘ ' ' a J
"n . Ir. 4'“. ‘ v‘ ‘ d _‘z
o . . ...‘ ' I . ‘ r '
. 2".- ° . . 9‘31)
21.x . -1....~.°...~...IL i

 

 

31293 02446 6850