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INHIBITION OF DENTAL CARIES IN FEMALE OSBORNE MENDEL

RATS BY A FOOD GRADE LIPID, LAURICIDIN R

By

Patricia Lynch

A THESIS

Submitted to
Michigan State University
in partial fulfiilment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1979

ABSTRACT
INHIBITION OF DENTAL CARIES IN FEMALE OSBORNE MENDEL

RATS BY A F000 GRADE LIPID, LAURICIDIN R

By

Patricia Lynch

The present study was undertaken to determine the

R , a generally recog-

cariostatic potential of Lauricidin
nized as safe (GRAS) antimicrobial lipid.
Eighty four female Osborne-Mendel rats were weaned at

l8 days, inoculated with Streptococcus mutans, and fed a

 

cariogenic diet to which 0%, 0.5%, l% and 2% Lauricidin R
had been added. After six weeks the rats were sacrificed,
the jaws were cleaned, and the molar teeth were scored for
dental caries by the method of Keyes (J. Dent. Res. 37:
l088, l958).

Caries scores for rats consuming diets containing 0.5%
Lauricidin R were significantly lower than caries scores
for rats consuming diets containing no Lauricidin R . Caries
scores were 35% lower on the sulcal surfaces, 56% lower on
the proximal surfaces and 90% lower on the buccal/lingual
surfaces of the teeth. Rats consuming diets containing 1%
and 2% Lauricidin R had caries scores on all surfaces which
were not significantly different from scores for rats con-

suming diets containing 0.5% Lauricidin R

ACKNOWLEDGMENTS

I am extremely grateful to Dr. Rachel Schemmel, the
chairman of my committee, for her continual guidance and to
the other members of my committee, Dr. Jon Kabara, Dr. John
Downs, Dr. Stan Miguel and Dr. Wanda Chenoweth for the
constructive criticism which enabled me to complete the
following thesis.

Special thanks is extended to Dr. John Gill of the
Dairy Science Department for his statistical advice.

Appreciation is extended to the National Institutes of
Health Grant #DE 04077 and to Life Savers Incorporated for

financial support.

ii

TABLE OF CONTENTS

LIST OF TABLES.
INTRODUCTION.
REVIEW OF LITERATURE.

The Etiology of Dental Caries
Dietary Carbohydrates
Bacterial Factors
Dental Plaque .
Production of Acid.

Host Factors. .

Control of Dental Caries.
Present Methods
Proposed Solutions. .

The Role of Fat in Dental Caries.
Human Studies
Animal Studies. . .

Enamel Studies (In Vitro)

Antimicrobial Activity of Fats.

 

Dental Caries Inhibition by an Antimicrobial.

Lipid.
MATERIALS AND METHODS

Part I. .
Experimental Design .
Preparation of Diet . .
Experimental Procedures .

Scoring of Carious Lesions.
Analys1s of Data. . .

Part II .
Experimental 06519"
Preparation of Diet .
Experimental Procedure.
Analysis of Data.

RESULTS

Part I .
Body Weights.

Page

Hmduumwwd—aowmm 01

wNN—J—l—l—J—Jd—l

34

Diet Consumption. .

Dental Caries Incidence and Severity.
Right and Left Jaws . .
Maxillary and Mandibular Teeth.
Inoculated and Non- Inoculated Rats.
Lauricidin . .

Part II . .

Body weights.

Diet Consumption. .

Dental Caries Incidence and Severity.
Proximal Surfaces
Sulcal Surfaces . .

Buccal and Lingual Surfaces

DISCUSSION. .
Inhibition of Dental Caries by Lauricidin R on
the Various Surfaces of the Teeth .

Sulcal Caries . . . . .

Interproximal Caries.

Buccal and Lingual Caries . . .
Effect of Various Doses of Lauricidin R . .
Distribution of Caries in the Right and Left

Jaws. . . . . .
Distribution of Caries in the Maxilla and
Mandible. . . . . .
Reduction of Caries by Lauricidin R in the
Maxilla and Mandible.

CONCLUSIONS
BIBLIOGRAPHY.

iv

71
72
73
75
76
76
77
79

8O

LIST OF TABLES

Table Page
l Composition of diet. . . . . . . . . . . . . . . 39

2 Cumulative food intakes and cumulative weight
gains of female 0M rats fed cariogenic diets
with and without Lauricidin R for 6 weeks. . . . 51

3 Severity of dental caries in teeth from the
right and left jaws in female 0M rats fed
cariogenic diets with and without Lauricidin R . 53

4 Severity of dental caries in teeth from the
maxilla and mandible of female 0M rats fed
cariogenic diets with and without Lauricidin . 54

5 Incidence and severity of dental caries in
female 0M rats fed a cariogenic die with and
without the addition of Lauricidin for six
weeks. . . . . . . . . . . . . . . . . . . . . . 57

6 Cumulative food intake and cumulative weight
gains of female 0M rats fed cariogenic diets
with various doses of Lauricidin for six
weeks. . . . . . . . . . . . . . . . . . . . . . 6T

7 Incidence and severity of dental caries on the
proximal surfaces of the teeth ofi female 0M rats
fed various doses of Lauricidin . . . . . 64
8 Incidence and severity of dental caries on the
sulcal surfaces of the teeth of female 0M rats
fed various doses of Lauricidin R . . . . . . . 66

9 Incidence and severity of dental caries on the
buccal and lingual surfaces of the teeth of
female 0M rats fed various doses of Lauri-
cidinR....................68

INTRODUCTION

Dental caries has been called the most prevalent public
health problem in the United States today (Nizel, 1977).
Unlike other infectious diseases in which the incidence has
decreased over the years, the incidence of dental caries has
increased on a national scale (Fitzgerald, 1973). Except in
sections of the population that have been exposed to fluori-
dation of community water supplies, dental caries is a
greater problem today than 50 years ago (Prevention and Oral
Health, 1973). A "massive attack" against this disease has
been urged (Harris, 1970).

Epidemiological studies reveal that almost all devel-
oped countries currently have high rates of dental caries.
Like those decreases seen in the United States, decreases
in other developed countries are seen only where water
fluoridation has been applied (Barmes, 1977). Although
many deve10ping countries have previously demonstrated
low caries rates, the problem of tooth decay is increasing
dramatically in such countries (Barmes, 1977). People
in urban areas are particularly affected. As these popu-
lations take on the ways of modern civilization they also

develop higher levels of dental caries (Greene and Suomi,

1977).

Dental caries is not a life threatening disease but it
can result in pain, infection, facial disfigurement, chewing
impairment, speech impairment, and malnutrition (Nizel,
1977). In 1960-1962, two billion dollars was spent annually
to repair damage due to dental caries (Prevention and Oral
Health, 1973). More recent figures are not available but
obviously have increased due to inflation and increasing
population. It has been said that the financial resources
and manpower necessary to repair the damage due to dental
caries do not presently exist in the United States and are
far less available in developing countries (Navia, 1979).
The increasing life span of people and the increasing
emphasis being placed on the quality of life adds importance
to the control of dental caries (Greene and Suomi, 1977).

It has been mentioned that fluoridation has been effec-
tive in reducing caries in some areas. The potential
effects of water fluoridation are great, however, fluori-
dation of all community water supplies is not politically
possible in this country (Nizel, 1977) and not practical in
developing countries (Greene and Suomi, 1977). Even when
water fluoridation is present, ingestion of water varies
among individuals and among geographic regions (Harris,
1970).

The continuing consumption of sweet, caries-promoting

foods despite attempts by the dental profession to motivate

people to change their dietary habits (Morse, 1976) has

led to the conclusion that cariostatic food additives are
desirable (Harris, 1970). The increasing pattern of fre—
quent snacking in the United States has led to a special
need for modification of the caries promoting properties of
foods consumed as snacks (Navia, 1979). The addition of
cariostatic substances to sugar or flour imported by
developing countries has been recommended as a means of
preventing decay in such areas (Harris, 1970).

In contrast, other researchers have suggested that the
only effective control of dental caries must involve a
means of controlling the microbial vectors (Scherp, 1971).
Antimicrobial agents could be aimed at eliminating the
cause of the disease (Fitzgerald, 1973). An antimicrobial
agent which could be used in foods could combine this
approach with the suggestions made regarding the develop-
ment of cariostatic food additives.

The monoglyceride of lauric acid is an antimicrobial
lipid and a generally recognized as safe (GRAS) substance
which has been shown to inhibit dental caries in rats fed
diets containing 18% skimmed milk and 67% powdered sucrose
among other ingredients (Schemmel gt 11., 1979). The
present studies were conducted to extend the conditions
under which this substance could inhibit dental caries.

Study I was conducted to determine whether Lauricidin R ,

a 90% pure preparation of the monoglyceride of lauric acid,

would inhibit dental caries in rats when included at the 2%
level in a diet containing 57% sucrose, 28% skimmed milk, and
other ingredients identical to those used in previous

studies.

Study II was conducted to compare the effect of a 0.5%

R

and a 1% dosage of Lauricidin with the 2% dosage.

REVIEW OF LITERATURE

The Etiology of Dental Caries

Dental caries has been defined as the localized destruc-
tion of tooth tissue (Gibbons, 1975). The etiology of
dental caries is not fully understood. However, certain
contributing factors are considered essential to the inia-
tion of decay. Fermentable carbohydrates must be in contact
with the tooth surface, and specific bacteria must be
present in the oral cavity (Nizel, 1977). A third factor,
host susceptibility, is equally necessary but much less
understood than the other two factors. Variation in the
incidence and extent of dental caries among individuals
having the same oral bacteria and the same exposure to
carbohydrates has been attributed to many possible host
factors such as tooth structure, tooth composition, saliva

composition and saliva quantity (Shafer t al., 1974).

Dietary Carbohydrates

Carbohydrates must be present in the diet before dental
caries will occur (Shaw, 1954). This was proven by experi-
ments in which the carbohydrate portion of a diet was

administered to rats by stomach tube (Kite gt 1., 1950).

Animals given carbohydrates in this manner did not develop
caries. Rats allowed to eat the same diet in the normal
way developed a considerable number of carious lesions.

There has been much debate and research concerning which
type or types of dietary carbohydrates are necessary for
the initiation and continuation of dental caries. Although
extensive dental caries have been observed in rats fed
cornstarch as the only carbohydrate (Shaw, 1972) starch is
generally considered the least cariogenic carbohydrate
(Nizel, 1969). Sucrose is considered the most cariogenic
(Winter, 1968; Newbrun, 1977). Variable results have been
obtained with disaccharides other than sucrose, and with
monosaccharides. Dental caries have been produced in rats
fed glucose, maltose, lactose, or fructose (Green and
Hartles, 1969). Other researchers have shown significant
reductions in caries when rats were fed diets containing
glucose, fructose, or isomerose (Thompson gt gl., 1976) or
maltose (Guggenheim gt gl., 1966) or lactose (Shaw, 1976)
in comparison with controls fed diets containing sucrose.
Variability in the results of these animal studies could be
due to differences in animal strains, other dietary ingre-
dients, type of microflora, or specific tooth surfaces
examined for caries (Makinen, 1972).

Although sucrose is regarded as the most cariogenic
sugar the cariogenicity of a diet is not only related to

the quantity of sucrose it contains. The frequency of

sucrose consumption and the form in which it is consumed
have been proven to be more important than the actual quan-
tity of sucrose consumed. Larson gt__l, (1962) observed
that rats who ate more frequently had a higher caries inci-
dence than rats who ate less frequently. Konig and
Muhlemann (1967) observed that rats fed a diet containing
powdered sugar developed more caries than rats fed a diet
containing granulated sugar. Gustafsson gt g1. (1954)
demonstrated that both of these factors were also important
in humans. In this study of institutionalized adults the
consumption of stickey sugars resulted in higher caries than
the consumption of non-stickey sugars. The consumption of
sucrose containing foods between meals resulted in higher

caries than consumption of the same amount of sucrose

containing foods with meals.

Bacterial Factors

 

Bacteria are essential for the initiation and continu-
ation of dental caries. Investigators have shown that germ
free rats fed highly cariogenic diets do not develop caries
(Orland gt _l., 1954). Several species of bacteria have
been found capable of causing dental caries when orally
inoculated into gnotobiotic rats (Newbrun, 1977). Trans-
missibility of cariogenic bacteria from caries active to

caries inactive hamsters has been demonstrated (Keyes,

1960) and fulfills one of the requirements that must be met

before a disease can be labeled a bacterial infection
(Newbrun, 1977).

The oral cavity of man and various other animals
cmntainsnumerous bacteria which may be causitive agents of
dental caries when fermentable carbohydrates and susceptible
tooth surfaces are present. At this time all known cario-
genic strains of bacteria are gram positive and with the
exception of a few filamentous organisms all are either
streptococci or lactobacilli (Fitzgerald, 1972). Cario-
genic species that have been used to produce pit and
fissure caries in gnotobiotic rats include the following:

Streptococcus mutans, Strgptococcus salivarius, Strepto-

 

coccus sanguis, Lactobacillus acidophilus, Lactobacillus

 

casei Actinomyces viscosus, Actinomyces naeslundii, and

 

 

Actinomyces israelii (Newbrun, 1977). All of these orga-

 

nisms with the exception of the Lactobacilli and A. israelii
have also been shown to produce root caries. Strgptococcus
mutans is one of the only species capable of causing decay
on smooth enamel surfaces in test animals.

Most of the same organisms used to produce dental
caries in gnotobiotic rats have been found on human
tooth surfaces. Lactobacilli are found both in human saliva
and on human tooth surfaces. Since they constitute only a
small fraction of plaque flora and since their numbers tend
to increase only after decay has already begun they are

thought to be secondary invaders which contribute to the

progression of decay (Gibbons, 1975). Streptococcus mutans

 

has been isolated from almost all human enamel carious
lesions examined (Littleton _t _l., 1970) and has been
reported in a significant proportion of cemental lesions
also (Sumney and Jordan, 1974). g. sanguis and S. mitj§_have
been found on the teeth of humans, however, their pr0por-
tions have been found to correlate inversely with caries
activity (de Stoppelaar t al., 1969). A. viscosus and

A. naeslundii have been found in human plaque and dental

 

calculus in significant numbers (Collins gt_gl., 1973) and
have been isolated from advanced human dentinal lesions

(Loesche and Syed, 1973).

Dental Plaque

 

Dental plaque is a soft bacterial deposit which forms
on the teeth (Loe, 1969). Plaque is 70% microorganisms and
30% other material, mostly polysaccharide (Schroeder and
De Boever, 1970). Plaque can be removed but begins to
reform immediately after cleaning. Initially it is a thin
layer near the gingiva on the smooth surfaces of the teeth.
In the absence of oral hygiene it gradually spreads over
all surfaces of the teeth (Prevention and Oral Health,
1973). Various organisms have been isolated from dental
plaque at different stages of development. Regardless of
the age of the plaque streptococci are the predominant

organisms (Newbrun, 1977). Specific polysaccharides, both

lO

glucans and levans, have been isolated from dental plaque
(Guggenheim, 1970). The glucans are partially insoluble
and may serve as the structural component of the plaque
matrix. The levans and some of the glucans are soluble and
may function as energy reserves for the bacteria (Newbrun,
1977). Enzymes responsible for the synthesis of both
glucans and levans have been isolated and purified from g.
sanguis and g. mutans (Carlsson, 1970; Carlsson gt gl.,
1969). These enzymes are highly specific for sucrose and
will not utilize fructose, glucose, maltose or lactose
(Newbrun, 1977). The enzymes are able to conserve the
high energy link between glucose and fructose that is
found in sucrose and use this high energy to transfer
glucose or fructose units directly to the growing poly-
saccharides. The high energy of hydrolysis of sucrose
makes it much more chemically reactive than other disac-
charides (Nizel, 1969). The polysaccharides in plaque
enable the bacteria to stick to the teeth and therefore
remain in contact with the tooth enamel. Fermentation of
dietary carbohydrates while in this location results in the
release of acid close to the tooth surface and the subse-
quent initiation of a carious lesion (Nizel, 1977).
Mutants of g. mutans that cannot form polysaccharides do

not cause dental caries (Tanzer gt gl., 1974).

11

Production of Acid

 

A drop in plaque pH from 6.5 to 5 following a glucose
or sucrose rinse was first demonstrated with the use of
fine antimony electrodes embedded in plaque (Stephan, 1940).
Modern methods have confirmed the formation of acid by
microorganisms in plaque. A variety of acids have been
found in plaque and in carious lesions (Bibby, 1965).
Lactic acid is the strongest of the acids produced and is
therefore the one most implicated in enamel demineraliza-
tion. If the pH drops below 5.2 in the environment of the
tooth surface then the acid reacts with the hydroxyapatite
crystals of the tooth enamel to form tricalcium phosphate
and dicalcium phosphate. These phosphate compounds are more
soluble than hydroxyapatite. When sufficient dissolution
of the enamel has occurred a carious lesion is initiated

(Nizel, 1972).

Host Factors

 

The occurrence of dental caries in some individuals
who consume very little sugar and the lack of caries in
some individuals who consume large amounts of sugar is a
situation which proves the importance of tooth suscepti-
bility in the cariogenic process (Koulourides gt gl.,
1976). Navia (1979) has summarized the various host
factors which may be involved. They include: the inte-

grity and perfection of the enamel; the morphology of the

12

teeth: the size ratio of teeth to mandible; and the chemi—
cal and immunological composition of saliva and gingival
fluids. Host factors may be genetically inherited or they
may be nutritionally acquired (Shaw, 1959).

The role of saliva in prevention of dental caries has
been verified by experiments on hamsters from which the
salivary glands have been surgically removed (Finn gt gl.,
1955). The desalivated animals developed more than 5 times
the number of caries as the control group of animals that
were not desalivated but were fed the same diet. Increased
caries associated with decreased salivary flow due to various
causes have been reported in humans (Stephen, 1971; Kermiol,
1975). No definite relationship has been found to exist
between caries prevalence and salivary amylase, urea, ammonia,
calcium or pH (Newbrun, 1977). Although saliva is known to
contain various antibacterial factors, salivary content of
lysozymes, lactoperoxidases, or immunoglobulins has not been
correlated with dental caries (Newbrun, 1977).

The chemical composition of the tooth enamel has also
been heavily implicated in dental caries susceptibility. In
several studies no differences were found in the calcium,
phosphorus, magnesium and carbonate contents of enamel from
sound and carious teeth. These same studies reported sig—
nificant differences in the fluoride content of sound and
carious teeth (Shafer _t gl., 1974). The mechanism by which
fluoride confers caries resistance to the enamel is not yet

fully understood (Jenkins, 1967).

13

Control of Dental Caries

Present Methods

 

Knowledge of the most important elements of the dental
caries disease process has led to the promotion of various
solutions. The restriction of dietary sucrose both in
frequency and quantity has long been recommended as a means
of preventing dental caries (Morse, 1976). Oral hygiene
procedures to remove bacterial accumulations from teeth have
been strongly promoted. Fluoridation of community water
supplies to decrease host susceptibility has been advised.
Many experts believe that a conscientious application of
these three solutions could solve the problem of dental
caries (Sherp, 1971; Keyes, 1969; Prevention and Oral Health,
1973). Unfortunately, universal application of these solu-

tions has not been possible.

Total elimination of sucrose from the diet is con-
sidered impossible (Finn and Glass, 1975; Winter, 1968).
Sucrose is the least expensive source of calories since it
requires the least amount of land per million calories
(Stare, 1975). The economic status of a family may limit
its ability to restrict sucrose intake (Shaw, 1959). Many
population groups now require the calories from sugar as a
staple part of their diet and will probably continue to do
so (Winter, 1968). Sucrose is often consumed in combina—

tion with foods which supply other necessary nutrients

l4

(Stare, 1975).

Even when economic or nutritional factors are not
applicable, motivation to change dietary behavior is not
easily achieved (Nizel, 1969). People like sweet foods
and sweet foods are often the most conveniently available
and well advertized foods (Prevention and Oral Health,
1973). Social pressures and individual appetites make the
limitation of frequency of intake especially difficult for
children (Bibby, 1965).

A similar situation has been encountered in the
achievement of effective oral hygiene practices. It is
difficult to achieve the proper motivation and knowledge
required to mechanically control bacterial deposits on the
teeth (Gibbons, 1975). The average time spent in brushing
is one minute according to one study; this is not long
enough to remove plaque from the tooth surfaces (Cohen
_t _l., 1967). Evidence of one researcher indicates that
the technical skill and perseverance required to continu-
ally maintain oral cleanliness exceeds the ability of the
average human being (L6e, 1970).

The third aspect of the present solution to dental
caries is the administration of fluoride to developing
teeth. The most efficient way to achieve this is via water
fluoridation. Emotional arguments and misunderstandings
have limited the adoption of water fluoridation (Gibbons,

1975). As of December 31, 1969 only 43% of the U.S.

15

population had either natural or artificially fluoridated
water (Nizel, 1972). Practical considerations prevent the
adoption of water fluoridation in many developing countries
(Greene and Suomi, 1977).

Although the combination of these three solutions is
potentially very effective it has not been effective at the
public health level. While these solutions are not being
abandoned, the search for additional means of control con-

tinues.

Proposed Solutions

 

One area of research proposed for the solution of
dental caries focuses on the cariogenic properties of foods.
The study of possible modifications in texture and compo-
sition of foods to reduce their cariogenicity has been
encouraged (Navia, 1979). It has been suggested that foods
which are the most appetizing and also the most caries
productive should be processed to make them less cario—
genic (Bibby, 1965). The need for a chemical food additive
that could achieve this goal while retaining the palata-
bility and nutritious content of such foods has been
expressed (Winter, 1968; Finn and Glass, 1975; Nizel,

1969; Navia, 1969). Suggested roles for these food addi-
tives include reduction in the amount of sugar available

to bacterial breakdown, protection of the tooth surface,

and modification of the tooth surface to increase its

resistance to caries.

16

Since dental caries is the result of a bacterial
infection it has been suggested that the most effective way
to control the disease is by controlling the microbial
agents involved (Fitzgerald, 1972). The National Caries
Program of the National Institute for Dental Research has
committed 50% of its total resources to research in com-
batting the microbial agent in dental caries (O'Brien,
1976). The search for antibacterial chemicals which could
be effective against causative bacteria has been encouraged
(Prevention and Oral Health, 1973). Many substances have
been tested, however, the selection of such a chemical
agent is hindered by the long list of requirements that it
must fulfill. The ideal anticaries agent should possess
the following characteristics: a narrow antimicrobial spec-
trum, preferably only cariogenic organisms; a high thera-
peutic index and low order of toxicity; a low potential for
the induction of resistant microorganisms; a low potential
for allergenicity; high stability under storage conditions;
long lasting efficacy with relatively few applications;
lack of general use for other therapeutic applications;
lack of unpleasant organoleptic properties; and low suscep—
tibility to inactivation by substances within the oral
cavity (Fitzgerald, 1972).

A consideration of these requirements in the light of
the suggestions concerning the alteration of food cario-

genicity leads to the conclusion that the absolutely ideal

l7

anticaries agent would be a substance which could reduce
the cariogenicity of foods via antimicrobial effects. The
possibility that such a substance exists is suggested by

research concerning the role of fats in dental caries.

The Role of Fat in Dental Caries

Human Studies

 

Several early investigators observed an association
between high levels of dietary fat in certain cultures and
low incidence of dental caries in those cultures (Volker,
1956). Although these observations are correct the multi-
tude of differences between the observed diets and more
typical caries promoting diets prevent any conclusions
regarding the isolated and specific influence of fat.

Observations of the low dental caries incidence in
diabetic children who were consuming a 7:9:21 caloric ratio
of protein:carbohydrate:fat stimulated Boyd _t_gl. (1929)
to study the possible influence of high dietary fat on
dental caries in such children. They imposed a similar
ratio of protein, carbohydrate, and fat on non-diabetic
children hospitalized in an orthopedic ward. A total
arrest of caries was observed. Caries incidence increased
when the children returned to the ward diet. As part of the
same study another group of normal children in their own homes
were fed controlled diets high in vitamins and minerals and

containing a ratio of protein, carbohydrate and fat that

18

was more balanced than the 7:9:21 ratio consumed by the
diabetic children. Caries were also arrested in these
children. Since an arrest in caries could be achieved by a
more typical diet, strictly supervised, Boyd concluded that
the arrest must have been due to metabolic influences of a
diet high in required nutrients. Since the diabetic chil-
dren were strictly supervised they were more certain to eat
foods containing all needed nutrients than normal children
who were more often allowed to choose foods according to
personal preference. The results of this study do not
negate the influence of fat. They do point out the com-
plexities of the human diet and eating habits which make
it hard to isolate the possible inhibition of caries by fat.
Boyd (1944) continued the investigation of the influ-
' ence of fat on dental caries incidence in diabetic children
whose diet was modified from 70% of the calories from fat
to 35% of the calories from fat. Children were studied for
at least three years. The low incidence of caries was not
changed when the fat level was cut in half even though
carbohydrates were doubled at the same time. Boyd inter-
preted these results as proof that fat did not have a
protective effect. However, the study did not consider
the fact that there may be a level of fat which gives
maximum protection, or conversely a level of dietary

sucrose which does not increase caries incidence.

19

Several studies conducted to determine the influence
of specific supplements on dental caries produced results
which pointed to an inhibitory action due to the fat con-
tained in the supplement. The Committee for the Investiga-
tion of Dental Disease of the National Research Council of
Great Britain administered supplements to children in three
different institutions(Rosebury and Karshan, 1939). Cod
liver oil, olive oil, and treacle (a type of molasses)
were administered. Cod liver oil was found to be the best
protective agent. Olive oil was found to be more protec-
tive than treacle. The sugar content of the molasses
prevented it from being a proper control for the olive
oil. The differences in caries could have been due to
higher sugar intake in the treacle group rather than higher
fat intake in the olive oil group.

McBeath and Verlin (1942) studied the anticaries
action of vitamin D and discovered that the vitamin 0 con—
tained in cod-liver oil had a greater anticaries effect
than similar or even larger quantities of vitamin 0 ad-
ministered as irradiated ergosterol. Although these
authors did not conclude that the differences were due to
differences in fat content of the administration vehicle
another author has suggested that the local action of the
fat in the cod liver oil preparation may have contributed
to the lower caries in individuals who received the vitamin

D in this form (Volker, 1956).

20

There have been no controlled human dietary studies
relating the fat content of a diet to dental caries inci-
dence. Human subjects do not readily volunteer to consume a
strictly controlled diet for the length of time necessary in
the study of dental caries incidence (Newbrun, 1977). Mea-
surements of related parameters other than actual caries have
been used to infer some information about the role of dietary
fat in dental caries. The amount of acid formed due to the
presence of food in the mouth and the amount of a food adher-
ing to the teeth after eating were selected as factors which
might determine the caries promoting ability of a food.
Research was conducted to determine the characteristics of a
food which could influence the retention of the food in the
mouth and the production of acid which resulted from the
presence of the food. A human study conducted by Bibby
_t _l. (1951) showed no consistent differences in acid pro-
duction due to the fat content of the foods tested; however,
the retention of a food in the mouth was significantly re-
duced as the fat content of the food was raised. A further
study of carbohydrate clearance from saliva after consumption
of bread with and without butter indicated faster, but not
significantly faster, oral clearance when fat was consumed
with the bread (Lanke, 1965).

Data relating dietary fat consumption to dental caries

incidence in humans is not extensive and not conclusive.

21

Research with laboratory animals has produced much more

convincing evidence that caries are reduced by dietary fat.

Animal Studies

 

The earliest animal studies relating decreases in
dental caries to increases in dietary fat were not conduc-
ted for that purpose. They were conducted to determine the
effect of calcium, phosphorus and vitamin D on dental
caries incidence in rats (Rosebury gt gt., 1935). A basic
diet containing brown rice and white potato dextrin was
supplemented by weight with 2 and 5% cod liver oil; 2 and
5% corn oil containing a dilution of viosterol adjusted to
have the vitamin D potency of cod liver oil; and 2 and 5%
unsupplemented corn oil. The oils were added to the dry
diets of rats twice a week. Dental caries incidence in
the fissures of the rat teeth was found to be signifi-
cantly reduced by all three supplements. It appeared that
the oil itself was exerting an anticariogenic effect. The
authors made the suggestion that the fat might have exerted
a protective effect in the environment of the teeth rather
than contributing to the mineralization process. This
possibility could not be proven by their data but it could
not be excluded either.

The same authors conducted a second series of experi-
ments to separate the anticaries action of vitamin D from
that of corn oil and to define more precisely the anticaries

action of fats (Rosebury and Karshan, 1939). In the first

22

experiment vitamin D was administered to rats as viosterol
in a minimal amount of corn oil equal to .15% of the diet.
One group of rats received the supplement by mouth. Another
group of rats received a comparable amount of the supplement
mixed with the diet. In the second experiment, 8 groups of
rats were fed either 0%, 0.5%, 2% or 5% corn oil by weight
in either a rice or a cornmeal diet. Five groups of rats
were fed fats other than corn oil at the 5% level by weight
in a rice based diet. The rice diet contained brown rice
which had been ground to pass a sieve having 10 meshes per
linear inch. The corn meal diet contained coarse yellow corn
meal which passed a 20 mesh sieve but was retained on a 30
mesh sieve.

The results of the first experiment indicated that
vitamin D had an additional caries inhibiting effect that

was not dependent on the corn oil in which it was contained.

The results of the second experiment demonstrated that in-
creasing levels of corn oil caused decreasing levels of

dental caries. In the rice diet the reduction was seen at

both the 2% and 5% levels, however, the 5% corn oil in the

cornmeal diet was not found to be more effective than the 2%

level. Caries were inhibited more in the rice diet than in
the cornmeal diet at all levels of corn oil additions. The

amount of total fat was higher in the cornmeal diet due to a

higher level of fat in corn than in rice, therefore, the
reduction in dental caries due to fat was thought to be due

to the free fat in the mouth rather than the physically

23

bound fat. Olive oil, Wesson oil, Crisco R and lard were
found to inhibit caries as well as corn oil did. Paraffin
oil did not cause significant inhibition. Diets used in
this study consisted of coarsely ground particles. The
authors did not consider the fact that the cornmeal diet
was less coarse than the rice diet. The greater inhibition
by fat in the rice diet may have therefore been related to
particle size. The authors suggested that the oil-fat
effect was a local effect exerted in the mouth. They
proposed that the fat may have lubricated the teeth or

may have produced a film on either the food particles or
the tooth surface which may have prevented penetration of
enzymes into the food particles or the penetration of acid
to the tooth surface.

Several early studies demonstrated the caries inhibiting
properties of lard but provided limited information on the
possible mechanisms whereby fat could inhibit dental caries.
Schweigert gt g1. (1946) added 10% lard at the expense of
2.25 grams of carbohydrate per gram of lard to the diets of
cotton rats which were being fed various carbohydrates.
Caries were reduced significantly in all groups; however,
the effect of the reduction of total carbohydrate in the
diet could not be distinguished from the effect of the addi-
tion of fat. Shaw (1950) confirmed the reduction of dental
caries with increasing percentages of lard in the diets of

albino rats. As in the above study, the caries inhibition

24

by fat in this study was also inseparable from the inhibi-
tion effect of a reduced level of carbohydrate in the diet.
The proposed ability of fat to protect the teeth of
cotton rats from the effects of a cariogenic diet was chal-
lenged by Constant gt g1. (1954). In this study the cario-
genic effects of a basal diet of 50% oatmeal, 32% dried
whole milk, and 18% sucrose were compared with the cario-
genic effects of a diet containing 50% oatmeal, 8.5%
vitaminized casein, 18% sucrose, 14.5% dextrin and 9% corn
oil. All ingredients were expressed as percent by weight.
Caries incidence and extent were found to be higher in the
diet containing 9% fat. The authors failed to acknowledge
the additional cariogenic challenge of 26% lower protein
and 23.5% higher carbohydrate that was present in the
second diet. Since the diet containing fat was not compared
with a suitable control the results of this study did not
contradict the possibility of caries inhibition due to fat.
Another series of experiments by the same authors
(Constant gt _l., 1954) compared the effects of natural fat
versus added fat in the diets of cotton rats. In the first
experiment oatmeal was subjected to ether extraction.
Three and a half percent of the oatmeal was extracted. The
remaining, de-fatted oatmeal was fed to cotton rats in a
diet containing 3.5% corn oil and other ingredients.
Dental caries incidence was found to be slightly reduced in

the rats fed the diet with added fat when compared to

25

dental caries incidence in rats fed a diet without added
fat containing a comparable amount of non-extracted oatmeal.

In a second experiment 50% dried whole milk was replaced
with dried skim milk and either 7 or 14% by weight corn oil
or 14% by weight butterfat. A reduction of caries was
observed in the rats fed the diets containing skimmed milk
plus added fats. The 7% level of corn oil was found to
afford maximum protection from caries. The authors conclu-
ded that added fat protected the teeth from dental caries
more than fat found in food naturally. This conclusion
supports previous studies which suggested a local rather
than a systemic influence by fat.

A comparison of the effects of two different fats was
conducted by McClure gt g1. (1956). Thirteen percent

Crisco R

or 13% butterfat by weight were substituted for
cornstarch in the cariogenic diets of white rats. Crisco R
was found to significantly reduce both the incidence and
severity of caries. Butterfat was not found to reduce the
incidence of caries significantly and caused only a margi-
nally significant reduction in the severity of caries. The
different levels of reduction by the two different fats
proved that the effect of the added fat was not only due to
a reduction of carbohydrate in the diet but was probably
due to a characteristic of fat which varied in the two fats

tested. The authors suggested that the reduction of caries

was due to a change in the physical properties of the diet

26

which affected the capacity of the diet to be retained on
the tooth surfaces.

The possibility that the caries inhibiting properties
of various fats could vary depending on the level of unsatu-
ration of the fat was investigated by Gustafson gt g1.
(1953). Seven percent cocoa fat, olive oil, and poppy seed
oil were added by weight to a high sucrose diet and fed to
golden hamsters. The cocoa fat was low in unsaturated
fatty acids, the olive oil had a medium content and the
poppy seed oil was high in unsaturated fatty acids. After
eating the diets for 110 days, the hamsters displayed no
significant differences in dental caries.

Further investigation by the same group of researchers
(Gustafson gt gl., 1955) was conducted to determine whether
a difference in physical properties of fats would correlate
with a difference in caries inhibiting properties of the
fats. Arachis oil and lard both had melting points below
body temperature and were compared to hydrogenated arachis
oil which had a melting point above body temperature. The
fats were added to cariogenic diets at the level of 25% by
weight and the diets were fed to hamsters for 150 days.
There was a large reduction of caries with all three fats,
however, significant differences in the caries inhibiting
properties of the three fats was found. The hydrogenated

arachis oil with the higher melting point caused the

27

greatest inhibition. The arachis oil and the lard caused
very similar reductions in caries. The later two fats have
similar melting points but differ in content of unsaturated
fatty acids. Since they provided practically the same
inhibition of caries the lack of a correlation between
caries inhibition by a fat and the level of unsaturation

of the fat was confirmed. A positive correlation of physi-
cal properties such as melting point of the fat to caries
inhibiting properties of the fat was suggested by the
results.

A simple study on the diets containing the three fats
mentioned above was conducted to investigate a possible
mechanism for caries inhibition by dietary fat. It had
been suggested that the fat added to a cariogenic diet
forms a film of fat around the food particles and protects
them from penetration by mouth fluids (McCollum, 1939).

The solubility of sucrose in the diets containing the

three fats was determined by measuring the amount of
sucrose dissolved from each diet in 15 seconds following
the addition of water. Whereas 86.7% of the sucrose was
dissolved from the fat free diet, significantly less
sucrose was dissolved from the fat containing diets. The
solubility did not correlate with the relative caries
inhibitory properties of the fats. There is some implica-
tion that fats may reduce the availability of sugar in food

mixtures by affecting solubility but a correlation with the

28

amount of caries inhibition was not found in this study.

Alam gt gt. (1973) proposed that a higher content of
linoleic acid in diets with increased content of certain
fats was contributing to lower caries susceptibility by
altering the composition of the teeth. Soybean oil, cotton-
seed oil, Crisco R , and butter were each tested at the 3%
and 9% level in the diet of rats. The increase to 9% was
at the expense of cellulose. The cumulative caries score
was lower in rats consuming 9% than for rats consuming 3%
of each fat tested. Cottonseed oil was the only fat for
which the reduction was significant. Although analysis of
the fatty acid composition of the teeth revealed a higher
content of linoleic acid in the teeth of rats fed diets
containing a higher content of linoleic acid the reduction
in caries could not be attributed to this change in tooth
composition.

Several studies have appeared to contradict the theory
that dietary fat inhibits dental caries in rats, however,
results of such studies were not actually conclusive.
Harris _t _l. (1965) reported that the addition of 5.19%
hydrogenated lard by weight to a diet which was already
cariogenic significantly increased the cariogenicity of
the diet. The authors suggested that the particles of lard
became impacted in the pits and fissures of the teeth and
therefore increased the caries in those areas. Results of

the experiment do not indicate whether there were any

29

changes in caries on the buccal or lingual or proximal
surfaces of the teeth. The authors conclude that fats could
not be cariostatic agents. These results should not have
been applied to all fats. There may be differences which
make individual fats more or less cariostatic.

Osborn gt g1. (1966) reported that fat in the diet of
rats had no influence on either the development or the
prevention of dental caries. Ten percent and 25% fat by
weight were added to the diets of rats and no change in
dental caries was observed. The particular kind of fat
used in this study and the additional ingredients other than
casein and gluten were not reported, therefore, interpreta-

tion of these data are difficult.

Enamel Studies (In Vitro)

 

Most of the above animal studies implied that dietary
fat had a role in the inhibition of dental caries. Several
mechanisms were proposed. The suggestion was made that fat
could be creating a film on the teeth and preventing the
dissolution of the enamel (McCollum gt _l., 1939). Several
investigators have tested this possibility in vitro with
both dietary and non-dietary fats. Walsh gt g1. (1950) con-
ducted a study to determine to what extent an intact tooth
surface already wet with saliva could be covered with a

protective film. Various fats were applied to human teeth

that had been presoaked in saliva. After application of

30

the fat the teeth were again soaked in saliva for a set
time and the amount of calcium in the saliva was measured as
an index of decalcification. Olive oil, paraffin oil, cetyl
alcohol in paraffin and 0.1% aqueous sodium lauryl sulphate
were found to have no significant influence on the rate of
decalcification. Butter, lard, and petroleum jelly gave a
small level of protection. Oleic acid gave significant
protection. Cetylamine at the 1% level in paraffin oil gave
the greatest protection. At a pH of 4, cetylamine was found
to reduce decalcification as much as 50% over a 20 hour
period. The authors concluded that the cetylamine had a
strong affinity for the tooth surface. Since cetylalcohol
was ineffective, the authors suggested that the NH2 group
was probably adsorbed on the enamel allowing the hydrocar-
bons of cetylamine to form a bridge to paraffin oil. The
paraffin oil then formed a film on the tooth. It was
assumed that the affinity between the enamel and the amine
caused the displacement of saliva and the establishment of
the more viscous oils on the tooth surface. Since dietary
fats such as butter and lard did not have a very strong
effect it was assumed that their slight action was due to
mechanical properties and not due to the formation of films.
A more recent study on the dissolution of hydroxyapa-
tite crystals by medium chain fatty acids was conducted by
Dirksen gt g1. (1973). Octanoic, decanoic, lauric, and

myristic acids were tested for their ability to inhibit

31

dissolution of hydroxyapatite crystals in an acetate buffer.
Fatty acids were prepared in 5% and 15% ethanol. None of
the fatty acids reduced dissolution levels in the 5%
ethanol. Lauric acid reduced dissolution in the 15% ethanol.
The above enamel dissolution experiments are difficult
to apply to a dietary situation. They do imply that inhibi-
tion of dental caries by fat may be due to multiple mech-

anisms.

Antimicrobial Activity of Fats

 

Derivatives of fat have long been known to exhibit
bactericidal properties (Kabara, 1979) . Soaps, the salts
of long chain fatty acids, are believed to exert an inhibi-
tory action on bacteria by suppressing the surface tension
in the bacterial medium. Bacteria are dependent on surface
tension for many processes including the accumulation of
nutrients on their surface membranes (Pelczar, 1965). A
depression of surface tension often leads to bacterial cell
death.

Research on the antimicrobial activity of fatty acids
other than soaps has also been conducted. The search for
an appropriate culture medium for lactic acid bacteria led
Williams gt g1. (1947) to discover that oleic acid at
certain concentrations is an essential growth factor for
several lactic acid bacteria. At other concentrations oleic

acid was found to inhibit growth. Other fatty acids,

32

especially unsaturated ones, were also found to have a toxic
effect on the bacteria.

The ability of a series of fatty acids to support growth
of oral microorganisms evaluated by Stephen (1950). The
fatty acids evaluated were oleic, linoleic, undecylenic,
and the short chain fatty acids from C2 to 018' The micro-
organisms tested were 25 strains of lactobacilli, several
strains of staphlococci and streptococci and mixed oral
flora of plaques and saliva. All of the microorganisms
showed some susceptibility to the inhibitory effects of C6
to C12 acids. Slight stimulatory effects were observed for
some of the lactobacilli with oleic and linoleic acids.
Fatty acids of carbon lengths 14-18 gave small inhibition or
none at all.

These early studies indicated that antimicrobial acti-
vity of fatty acids did exist and varied in intensity
depending on the chain length and degree of unsaturation of
the fatty acid. More recent studies confirmed these results
and provided more information on the relationship of the
chemical structure of fatty acids and derivatives to their
antimicrobial action. Kabara gt g1. (1972) tested the bac-
terial inhibition of 30 straight chain fatty acids and
derivatives. Twenty microorganisms were studied. Many of
the compounds tested were inhibitory. The minimum inhibi-
tory concentration (MIC) for saturatedfatty acids decreased

from a maximum for the C6 and C20 fatty acids to a minimum

33

for the 012 fatty acid. The greatest bacteriostatic activity
against gram (+) microorganisms was found in lauric acid
(012). Unsaturated fatty acids tested were C14, C16’ and
C18. The bacteriostatic activity of these acids was
increased by the addition of a cis double bond in a delta 9
position. The addition of a second double bond further
increased the bacteriostatic action of C18. Since lauric
acid (C12) was found to have the greatest bactericidal
activity of the saturated fatty acids various derivatives
of lauric acid were also tested. The alcohol and aldehyde
were found to be similar in activity to the acid form
against most organisms. The 1,3-dilaurin and trilaurin
derivatives were found to be less active than lauric acid
but the monolaurin proved to be more active than the free
acid.

Kabara _t__l. (1977) confirmed these findings in a
further study of 40 lipophilic substances, both natural
and synthetic. Many of the substances exhibited antimicro-
bial effects. In general lower chain fatty acids were
biologically more active when esterified to glycerol. C11
and C13 fatty acids as monoglycerides were less active
than C12 as a monoglyceride. Lauric esters of other poly-
hydric alcohols were also active. Monolaurin was again the
most active bactericidal compound screened.

The confirmation that fatty acids and derivatives did

inhibit bacterial growth and that certain fatty acids and

34

derivatives were more effective than others implied that
the more active compounds might be specifically applied to
various situations where antimicrobial agents were

required.

Dental Caries Inhibition By an Antimicrobial Lipid

 

The possible role of fat in dental caries inhibition
has been previously discussed. Several mechanisms were
proposed for the inhibition. Fat may have increased oral
clearance of carbohydrates from the mouth, it may have
decreased the solubility of sugars in the mouth, or it may
have coated the enamel with a protective film. None of
these mechanisms was proven. Knowledge of the antimicrobial
properties of specific fats implied that inhibition by
dietary fat could be due to a fourth mechanism. The content
of antimicrobial fats in the dietary fats tested could be
causing the inhibition. It was possible that a known anti-
microbial fat could provide maximum inhibition of caries by
exerting more than one mechanism.

Most cariogenic bacteria are gram (+) microorganisms.
The monoglyceride of lauric acid had previously been shown
to be the most effective antimicrobial lipid against gram
(+) organisms. Since monolaurin was considered a generally
recognized as safe substance (GRAS) by the Food and Drug
Administration, monolaurin was studied for its anticario-

genic properties.

35

Kabara gt gt. (1979) conducted an it vitro study of

the activity of Lauricidin R

which was a 90% pure prepara-
tion of the monoglyceride of lauric acid. Various oral
microorganisms were tested. Lauricidin R had strong
antibacterial effects on gram positive microorganisms.

Streptococcus mutans was inhibited to the greatest extent

 

of all the organisms tested.
The ability of monolaurin to inhibit the growth of

Streptococcus mutans in the oral cavity of rats was studied

 

by Whitlock gt _1. (1978) in conjunction with an analysis

of dental caries incidence in the same rats. Inhibitory
action of monolaurin was compared to the action of two other
fats; Crisco R which was considered non-bactericidal and
sucrose ester (SSE) which was considered slightly bacteri-
cidal. The fats were incorporated at the 2% level by

weight into diets containing 67% refined sucrose, 18% skim
milk powder and several other ingredients. Rats were fed
chow type rations for two days after weaning and then were
inoculated with Streptococcus mutans and fed the test diets
for four weeks. Streptococcus mutans colony forming units
(CFU) in the oral cavity of rats fed the diet containing
monolaurin were significantly lower than for rats fed either
of the other two fats. A 59% reduction in dental caries in
the group of rats fed monolaurin compared to the group fed

R

Crisco was not significant.due to the large individual

differences within each group.

36

Feeding studies on rats were also conducted by Kabara
gt gt. (1979). Lauricidin R and Crisco R were compared
at the 2% level in the diet of rats. Animals in this study
were weaned and immediately fed the test diets to insure
adequate development of caries. After four weeks the animals
were sacrificed and the microorganisms present in the oral
cavity were counted. Wide variation between animals resul-
ted in no statistical differences between the control
and monolaurin groups for any of the groups of microorga-
nisms studied. Teeth from these rats were analyzed for
dental caries by Schemmel gt gt. (1979). A statistically
significant reduction in smooth surface dental caries was
found in the rats fed monolaurin (Lauricidin R ).
The previous experiment was repeated by the same group
of investigators (Kabara gt gt., 1979). Most conditions
were the same. Animals were sacrificed and analyzed for
oral microorganisms and dental caries at the end of two,
four, and six weeks. Since liver powder, an essential in-
gredient of the previous diets, was not added until the
fifth week analysis of data from the two and four week experi-
mental periods was difficult. Stregtococcus mutans CFU,
total CFU, and smooth surface caries were all significantly
lower in rats fed monolaurin for six weeks than in control
rats which did not receive monolaurin.

The action of monolaurin as an anticaries agent was

promising but was in need of further verification under

37

various dietary challenges. Various dosages needed to be
investigated to determine the lowest concentration that
could be added to a food product to reduce its cariogeni-

city.

MATERIALS AND METHODS

Part I

Experimental Design

 

Thirty six female Osborne-Mendel (O.M.) rats were
distributed by weight at 18 days of age among several
treatment groups so that the combined weight of the rats
in each treatment group was approximately the same. The
rats were bred in the laboratory of the Department of Food
Science and Human Nutrition (FSHN). The original O.M. stock
came from the National Institutes of Health (NIH). The
original NIH colony of Osborne-Mendel rats was Caesarian
delivered and then infected withfour organisms. These were
rodent strains oftwo lactobacilli, an enterococcus and a
bacteroid (Fitzgerald and Larson, 1971). Presumably, the
animals in the Department of FSHN carried these same orga-
nisms since the colony is maintained under conditions which
minimize entry of extraneous bacteria.

Three experimental groups of 12 rats each were fed
diets containing 2% of a test fat. All other ingredients
of the diets were identical (Table 1). Two of the groups

were infected with Streptococcus mutans. One of these groups

 

received 2%Lauricidinizand one received 2% hydrogenated

38

39

Table 1. Composition of diet

 

 

Ingredients Percent by weight
Skimmed milk powderR 28
Sucrose (6x)b 57
Brewer'syeastc 4
Alfalfad 3
Liver, dessicated, defattede 1
Sodium Chloride 2
Corn oilf 3
Test fat9 2

 

aHigh heat, spray processed non-fat dry milk purchased
from Valley Lea Dairies Inc., South Bend, Indiana.
bBig Chief 6x powdered sugar (starch content not over 3%)

purchased from Monitor Sugar Co., Bay City, Michigan.

cBrewer's yeast purchased from Teklad Test Diets, Madison,
Wisconsin. (Type ZOO-B; 54.7% protein; containing the
following per gram: 200 mcg pantothenic acid, 30 mcg
pyridoxine, 0.5 mcg biotin, 3600 mcg choline, 4000 mcg
inositol, 20 mcg folic acid, 500 mcg niacin, 70 mcg ribo-
flavin, 600 mcg thiamine; plus the other 8 complex factors
naturally present in yeast.

dAlfalfa purchased from Ohio Blenders, Toledo, Ohio. (Com-
posed of not less than 17% crude protein, not less than
1.5% crude fat, not less than 27% crude fiber.)

eLiver, dessicated, defatted purchased from U.S. Biochemi-
cals Corporation, Cleveland, Ohio.

fCorn oil purchased from Nugget Distributors, Stockton,
California.

gTest fat was either Crisco R purchased from Proctor and
Gamble, Cincinnati, Ohio, or Lauricidin R which was a

90% pure preparation of the monoglyceride of lauric acid
and which was generously supplied by Medchem Laboratories,
Monroe, Michigan.

4O

vegetable shortening. The third group of rats was not

inoculated and was fed 2% hydrogenated vegetable shortening.

Preparation of Diet

 

The diet is a modified form of the high sucrose NIH
diet 2000 (Keyes, 1959). The diet was prepared a few days
before the start of the experiment and again at four weeks.
All ingredients were placed in a 5 kg. capacity Hobart mixer
and allowed to mix for at least 30 minutes. Diet was stored

under refrigeration at all times during the experiment.

Experimental Procedure

 

Immediately after all rats were assigned to treatment

groups the rats of treatment groups 1 and 2 were inoculated

with a pure culture of Streptococcus mutans 6715]. Approx-

 

imately 0.2 m1 of the 30 hour working culture was placed in

the mouth of each rat. Streptococcus mutans 6715 cells for

 

inoculation were obtained from an 18-24 hour Trypticase soy
broth culture and standardized to 25% transmittance at 550 nm
with a spectronic 202. The rats were also given 5% sucrose
water with 10 ml standardized culture per liter for 48 hours.

After 48 hours drinking water was changed to pure distilled

 

1The original culture was obtained by Drs. Mickelsen
and Schemmel, through the generosity of Dr. Rachel Larson,
NIDR. It has been carried through several generations in
Dr. J.J. Kabara's laboratory.

2Bausch and Lomb.

41

and rats were given it gg libitum for the duration of the
experiment.

Rats were housed in pairs in metal screen suspended
cages. The right ear of the even numbered rats was clipped
for identification. Rats in treatment group three which were
not inoculated with t. mutans were housed on a rack other
than the one used for the other two groups to avoid cross
infection. The environment was controlled with 12 hours of
light of each 24 hours with a temperature maintained at 23 i
l C. Rats were weighed in the afternoon after the first 48
hours and once a week thereafter.

Diet was available gg libitum for a period of 44 or 45
days. Food dishes for all rats were refilled every two or
three days or whenever the level of food for any pair of rats
was below one half cup. Porcelain cups were weighed when
full and when taken from the cage for re-filling. Spillage
was collected on newspaper beneath each cage. Spillage was
separated from feces, weighed, and added to the weight of
the cup when taken from the cage. Food intake for each
feeding period was determined by subtracting the weight of
the cup, remaining contents, and spillage from the original
weight of the full cup. Food intakes were totaled for each
week. Clean cups and water bottles were provided weekly.

After being fed the diet for 44 or 45 days, rats were
anesthetized by ether inhalation and then decapitated.Six rats

from each group were sacrificed on day 44. The remaining six

42

rats were sacrificed on day 45. After decapitation, the
heads were divided along the sagital plane and the molar
teeth from the left mandibular and maxillary jaws were evalu-
ated for both microorganisms and dental caries. The teeth
from the right jaws were evaluated for dental caries only.
The incisors were not studied because they are generally

not considered to be involved in dental caries.

In order to quantitate the microorganisms from the left
molars the teeth were aseptically removed from the jawbone
retaining enough bone to keep the 3 molars together. The
jaw section containing the teeth was transferred to 5 ml of
reduced transport fluid (RTF). After sonication the teeth
were removed from the fluid and air dried before being
scored for dental caries.

To insure that the microbiological procedures were not
damaging the left molars a different method of soft tissue
removal was used on the right jaws. The right halves of the
heads were placed in the MSU dermestid beetle colony. After
removal from the beetle colony, the jaws were soaked in a 2%
solution of ammonium hydroxide for 30 minutes, soaked in tap

water for 30 minutes and allowed to air dry for 24-48 hours.

Scoring Carious Lesions
Jaws were coded before scoring to avoid possible bias
during scoring due to knowledge of which treatment group

the teeth were from. Three molars in each jaw quadrant

43

were scored for caries by the method of Keyes (1958). This
method specifies four separate surfaces of each tooth to be
examined. The buccal surface is the surface closest to the
cheek; the lingual surface is the surface closest to the
tongue. The buccal and lingual surfaces are the surfaces
Keyes refers to as smooth surfaces. The proximal surfaces
are those surfaces between the teeth where the teeth are
touching or nearly touching. The sulcal surfaces are the
inner surfaces sometimes called occlusal or fissure sur-
faces. The three molars in each quadrant are different sizes.
Keyes has defined a basic linear unit and defined the number
of these units on each surface of each tooth. The number

of sulcal units was determined by viewing the sulci of
sagitally sliced teeth. The depth of decay has been

defined according to four categories. When water is allowed
to evaporate off a tooth surface any decalcified enamel
dehydrates more rapidly than sound enamel and appears as a
chalky white area. If the surface is chalky but unbroken
the caries involvement is said to be E. If the surface is
slightly loose and flaky the involvement is 05. If a probe
can be inserted into the lesion the involvement is Dm. If
the probe can be extended all the way to the pulp chamber
the involvement is Dx. Each surface of each tooth is given
a score which represents the number of linear units in-
volved in E decay, Ds decay, Dm decay, and 0x decay. The

total caries score for that surface of that tooth is the

44

sum of all the linear units involved at any depth. It is
equivalent to a 2 dimentional (length vs. depth) score.
Using this method it is possible to compare total caries
per surface area per rat. It is also possible to compare
enamel caries or 05 caries separately. Totals for buccal
and lingual surfaces are usually added together and re-
ferred to as total smooth surface areas. Proximal surfaces
have been included in smooth surface areas by many
researchers but this study considered proximal areas
separately. Smooth, proximal and sulcal areas are never
added together.

Each jaw quadrant was soaked in water for a few
minutes and then examined under aEMusch and Lomb dissecting
microscope under low magnification (20x). As the water
evaporated the buccal and lingual surfaces of the teeth
were scored for caries. The typical buccal or lingual
lesion is a long narrow horizontal area. Sulcal caries
that have spread into the smooth areas were carefully
excluded from the scores for the smooth areas.

After all the teeth were scored for buccal and
linqual caries theyvmre allowed to soak in a murexide3
stain for 24 hours. After 24 hours the stainvms removed
and the teethvmre allowed to air dry for several days.

The buccal and lingual scoresvmre then verified under

 

3Murexide (ammonium purpurate) purchased from Sigma
Chemical Co., St. Louis, Mo. (60 mg murexide dissolved in
75 ml distilled water and 175 ml absolute alcohol and
filtered through a vacuum).

45

magnification.

In order to examine the sulcal areas of the teeth each
jaw quadrantvms slicedRinto equal halves at an angle
parallel to the buccal and lingual surfaces. Before sli-
cing, the teethwere coated with clear nail polish to avoid
loss of teeth during the slicing process. While being
sliced the teeth wem3held with a small hemostat beneath a
slow drip of water. The sliced teeth were air dried for 24
hours and then examined for sulcal and proximal caries.

The depth of the sulcal and proximal lesions was
determined by the amount of penetration of the stain. For
these surfaces the border between the enamel and the
dentin can be seen. If the pink stain has not penetrated
this border then the lesion is E only. If the stain is
1/4 way into the dentin then the lesion is 05. If the
stain is 1/4 to 3/4 of the way into the dentin then the
lesion is Dm. If the dentin is totally stained then the
lesion is Dx.

Proximal surfaces were scored in the same manner as
sulcal, however, if no proximal lesions were visible a
probe was used to separate the lst molar from the 2nd
molar and the 2nd molar from the 3rd molar to expose the
entire proximal surface. This insured that proximal
caries were not overlooked due to uneven slicing of the

teeth.

 

4A steel disc saw; 0.004 inch thickness and 0.75 inch

in diameter was mounted on a mandrel held in a straight
handpiece of a standard dental engine.

46

Caries were recorded on the score sheet designed by

Keyes. Morsel caries were not evaluated in this study.

Analysis of Data

 

Means and standard errors were determined for cumula-
tive weight gains and cumulative food intakes for each
group of rats. Means and standard errors were determined
for the number of carious teeth per rat and for the total
caries score per rat for each of the three tooth surfaces for
each group of rats. Analysis of variance was performed by
the CDC 6500 computer, Michigan State University.

Significant differences between mean values for caries
scores was assessed by Dunnett's test (Dunnett, 1955) using
treatment 1 (inoculated rats fed 0% Lauricidin R ) as the
control. The Student's paired t-test (Navia, 1977) was
used for comparison of mean values of total caries scores
on the right and left side of the jaws for each group of
rats. Student's paired t-test was also used to compare
mean values of total caries scores on the maxilla and

mandible of the teeth from each group of rats.

Part II

Exgerimental Design

 

Forty eight male Osborne-Mendel rats were distributed
by weight at 18 days of age among six treatments so that the

combined weight of the rats in each treatment group was

47

approximately the same. Rats were bred from the same stock
as those used in Part I. Six experimental groups of eight
rats each were fed diets containing 2% of a test fat combi-
nation in one of the following ratios: 2% Lauricidin R /O%

R R, 0.5% Lauricidin R /

Crisco , 1% Lauricidin R /1% Crisco
1.5% Crisco R and 0% Lauricidin R/2% Crisco R . Four of

the groups were inoculated with Streptococcus mutans and

 

each of these groups were fed one of the four fat ratios.
Two of the groups were not inoculated. Rats in these groups
were started on the experiment one day later than rats in
the other four groups. One of the groups that was not
inoculated was fed 2% Lauricidin R and 0% Crisco R . The
other non-inoculated group was fed 0% Lauricidin R and 2%
Crisco. All other ingredients in the diet were identical

to those used in Part IR.

Preparation of Diet
To insure uniform mixing of the fat with the remaining
ingredients, diets were prepared in the following way:
1. All ingredients except corn oil and the test fat
were measured and placed in a 5000 gram mixer.

They were mixed at the lowest speed for 10 minutes.

 

5The source of purchase of Brewer's yeast was not the
same as Part I. Brewer's yeast for Part II was purchased
from Schiff Bio-Food Products, Inc. Moonachie.N.J. (25 call
7 grams: 45 to 48% protein, all essential amino acids,
fortified with 812; containing the following per 02.:
4.28 mg B], 1.14 mg 82, 11.40 mg niacin, 1.14 mg 85, 1.42
pantothen1c acid, 2 mcg cobolamin, 90 mcg folic ac1d, 25
mcg biotin, 140 mcg PABA, 100 mcg choline, 100 mcg inosi-
tol, 540 mg potassium, 60 mg calcium, 2.5 mg iron, plus the
rest of the B vitamins natural to yeast.)

48

Corn oil and the proper ratio of test fat were
measured and blended in a bowl.

Portions of the pre-mixed ingredients from step 1
were added to the fat mixture and creamed to-
gether by hand. Dry ingredients continued to be
added until the fat mixture was too stiff to
blend.

The fat mixture was added to the rest of the
ingredients in the 5000 gram mixer and mixed for

30 minutes.

Experimental Procedure

 

Identical to Part I except for the following:

1.

Small metal food savers were placed in the food
dishes to limit the amount of spillage.

Only the six teeth from the left sides of the jaws
were scored.

The right sides of the rat heads were not de-
fleshed by the beetle colony. They were auto-
claved at 121-123 C for 5 minutes. Soft tissues
were removed with instruments. The jaws were

then soaked in a 2% solution of ammonium hydroxide
for 5 minutes.

The smooth surfaces were examined under a stream
of forced air.

The murexide stain was left on the teeth for 7

hours instead of 24 hours.

49

Analysis of Data

 

Means and standard errors were determined for the
cumulative weight gains, the cumulative food intakes and
the total caries scores on each of the three tooth surfaces
for each group of rats. Analysis of variance was per-
formed by the CDC 6500 computer, Michigan State University.

A Scheffé procedure (Gill, 1978) was used to analyze
all possible comparisons among treatment means for cumula-
tive food intakes. A Bonferroni-t statistic (Gill, 1978)
was used to compare specific contrasts between treatment
means for total caries scores on each of three tooth surfaces.
The following contrasts were performed: 1 vs. 4, 2 vs. 4,

3 vs. 4, 5 vs. 6, 1 vs. 3, 1-4 vs. 5-6.

RESULTS

Part I
Body Weights

 

Mean body weight gains from 18 to 62 (63 days of age)
are presented in Table 2. One animal in treatment group 2
died after five days on the experiment. Means in treatment
group 2 were computed for the remaining 11 rats. The re-
maining animals in each of the treatment groups increased
in weight over the six week experimental period. Final
weights ranged from 169 i 3 to 174 i 4. Rats which were

not inoculated with Streptococcus mutans and not fed

 

Lauricidin R gained a mean weight of 137 i 3 g. Rats

which were inoculated and not fed Lauricidin R

gained a
mean weight of 134 i 2 g. Rats which were inoculated and
fed 2% Lauricidin R gained a mean weight of 136 s 7 9.

Differences between the groups were not significant.

Diet Consumption

 

Over the six week feeding period from age 18 to 62
days rats which were inoculated and not fed Lauricidin
ate a mean of 1223 i 29 9 (Table 2). Rats which were
inoculated and fed Lauricidin R consumed a mean of 1152 i
53 g. Rats which were not inoculated and not fed Lauri-
cidin R consumed a mean of 1215 i 23 9. There were no

50

51

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com mums so oposoc co mcwom pcmwmz w>PpoP=s=o new moxoucw coo» m>wpopasau .N wpnoh

52

significant differences in mean food intake among rats in

the three treatment groups.

Dental Caries Incidence and Severity

 

Right and Left Jaws. Means, standard errors, and

 

statistical analysis of dental caries severity in the right
and left jaws of rats in each treatment group are presented
in Table 3. In all treatment groups on all three tooth sur—
faces there were no significant differences between the mean
caries scores on the left sides of the jaws and the mean

caries scores on the right side of the jaws.

Maxillary and Mandibular Teeth. Means, standard errors,

 

and statistical analysis of dental caries severity in teeth
from the maxilla and mandible of rats in each treatment group
are presented in Table 4. Rats which were not inoculated did
not develop caries on the buccal/lingual surfaces in the
maxilla. Rats which were inoculated and not fed Lauricidin R
had a mean caries score of 42.5 i 10.6 on the buccal/lingual
surfaces of the teeth in the mandible and a mean caries score
of 2.0 i 0.7 on the buccal/lingual surfaces of the teeth in
the maxilla. The differences in caries scores on the maxilla
and mandible was statistically significant (p<0.005). Percent
of buccal/lingual caries in the maxilla was only 4.5.

Rats which were inoculated and fed 2% Lauricidin
had a mean caries score of 19.6 i 6.1 on the buccal/lin-

gual surfaces of the mandible and a mean caries score

53

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54

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.w «Posh

55

of 3.0 i 0.8 on the buccal/lingual surfaces of the maxilla.
The difference in caries scores on the maxilla and mandible
was statistically significant (p<0.01). The percent of
caries found in the maxilla was 13.3.

There were no significant differences between caries
scores on the proximal surfaces in the maxilla and mandible

for either of the two groups that were not fed Lauricidin R .

R had a mean

Rats which were inoculated and fed Lauricidin
caries score of 8.1 i 0.6 on the proximal surfaces of the
mandible and a mean caries score of 11.6 i 0.7 on the proxi-
mal surfaces of the maxilla. The percent of proximal caries
found in the proximal surfaces of the maxilla was 58.9. This
was statistically significant (p<0.005).

Mean caries scores on the sulcal surfaces of the teeth
in the maxilla were significantly lower for rats in each of
the treatment groups. Rats which were not inoculated and
not fed Lauricidin R had a mean caries score of 37.0 s 1.6
on the sulcal surfaces of the teeth of the maxilla and a
mean caries score of 62.6 i 1.8 on the sulcal surfaces of
the teeth of the mandible. The percent of the sulcal caries
which were found in the maxilla was 37.2 (p<0.005).

Rats which were inoculated and not fed Lauricidin R

4.

had a mean caries score of 36.5 _ 1.5 on the sulcal surfaces
of the teeth of the maxilla and a mean caries score of 61.3 i
2.8 on the sulcal surfaces of the teeth of the mandible.

The percent of sulcal caries which were found in the maxilla

56

was 37.3 (p<0.01).

Rats which were inoculated and fed 2% Lauricidin R
had a mean caries score of 32.6 i 1.3 on the sulcal sur-
faces of the teeth of the maxilla and a mean caries score
of 46.7 i 2.3 on the sulcal surfaces of the teeth of the
mandible. The percent of caries found on the maxilla was
41.1 (p<0.005).

Inoculated and Non-Inoculated Rats. Means,standard
errors and statistical analysis of dental caries incidence
and total dental caries scores for all three treatment
groups are presented in Table 5. One of the rat jaws from
treatment group 2 was lost during preparation for scoring.
Data for treatment group 2 are presented for the remaining
ten rats. Data for treatment groups 1 and 3 are presented
for 12 rats in each group.

92% of the rats which were not inoculated with Strep-
tococcus mutans and not fed Lauricidin R developed caries
on the buccal/lingual surfaces of the teeth. The mean
number of carious teeth per rat in this group was 1.6 i
0.3. This means that for any one rat an average of 1.6
out of the 12 teeth scored was decayed. The location of
the decayed tooth was similar for most rats in the group.
84.2% of the decayed teeth were mandibular 3rd molars.
15.7% of the decayed teeth were mandibular 2nd molars.

100% of the rats which were inoculated and not fed

Lauricidin R developed caries on the buccal/lingual

57

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58

surfaces of the teeth. The mean number of carious teeth
per rat was 5.4 t 0.7. This was a statistically signifi-
cant increase from the non-inoculated group (p<0.01).

Rats which were not inoculated and not fed Lauricidin
had a mean caries score of 4.0 i 0.7 on the buccal/lingual
surfaces of the teeth. Rats which were inoculated and not
fed Lauricidin R had a mean caries score of 44.5 i 10.9
on the buccal/lingual surfaces of the teeth. The increase
in caries on the buccal/lingual surfaces due to inoculation
was 1112.5% (p<0.01).

100% of the rats which were not inoculated and not fed

Lauricidin R

developed caries on the sulcal surfaces of
the teeth. The mean number of carious teeth per rat was
11.9 i 0.3. The mean caries score per rat was 99.6 i 3.4.
100% of the rats which were inoculated and not fed
Lauricidin R developed caries on the sulcal surfaces of
the teeth. The mean number of carious teeth per rat was
11.9 i 0.1. The mean caries score per rat was 97.8 s
3.5. Differences in mean number of carious teeth per
rat and differences in mean caries score per rat between
the inoculated and non-inoculated rats was not signifi-
cant.
100% of the rats which were not inoculated and not fed
Lauricidin R developed caries on the proximal surfaces of

the teeth. The mean number of carious teeth per rat was

5.4 i 0.7. The mean caries score per rat was 8.1 i 1.3.

59

100% of the rats which were inoculated and not fed
Lauricidin R developed caries on the proximal surfaces of
the teeth. The mean number of carious teeth per rat was
11.0 i 0.4 and the mean caries score per rat was 28.3 1
2.3. On the proximal surfaces inoculation with g. mutans
resulted in a significant increase (p<0.01) in mean number
of decayed teeth per rat and a significant increase (p<0.01)
in mean caries score per rat.

Lauricidin R . 90% of the rats which were inoculated

 

and fed diets containing 2% Lauricidin R deve10ped caries
on the buccal/lingual surfaces of the teeth (Table 5). The
mean number of carious teeth per rat was 4.7 i 0.9, and

was not significantly different statistically from the

mean number of carious teeth in the inoculated rats which

R

were not fed Lauricidin The mean caries score per rat

R

in the rats fed Lauricidin was 22.6 i 6.1. This is a

50% reduction from the mean caries score per rat in the

group of rats which were inoculated but not fed Lauricidin R ,
but it is not statistically significant due to the wide
variation in caries severity seen in both groups of rats.

The caries scores for inoculated rats which were not fed

R

Lauricidin range from 2 to 115 units. The caries scores

for the inoculated rats which were fed 2% Lauricidin R
range from O to 62.
100% of the inoculated rats fed diets containing 2%

Lauricidin R deve10ped caries on the sulcal surfaces. The

60

mean number of carious teeth per rat was 11.9 i 0.1 and

was not significantly different from the mean number of
carious teeth per rat in the group of inoculated rats which
were not fed Lauricidin R . The mean caries score per rat
for the group of rats fed Lauricidin R was 79.3 i 2.7.

The mean caries score per rat for the inoculated rats which
were not fed Lauricidin R was 97.8 i 3.5. The 19% reduc-
tion in sulcal caries due to 2% Lauricidin R in the diet
was statistically significant (p<0.01).

100% of the inoculated rats fed diets containing 2%
Lauricidin R developed caries on the proximal surfaces.
The mean number of carious teeth per rat was 10.0 i 0.3 and
was not statistically different from the mean number of
carious teeth per rat in the group of inoculated rats which
were not fed Lauricidin R . The mean caries score per rat

R was 19.7 11.1.

in the group of rats fed Lauricidin
The mean caries score per rat for the inoculated rats which
were not fed Lauricidin R was 28.3 i 2.3. The 30% reduc-
tion in proximal caries due to 2% Lauricidin in the diet

was statistically significant (p<0.01).

Part 11
Body Weights
All of the rats in the six treatment groups gained
weight. Mean body weight gains and standard errors from

18 to 61-63 days of age are presented in Table 6. The

61

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62

range in mean body weight gain was from 128 i 6 g in

group 4 to 143 i 4 g in group 6. Rats in group 4 were
inoculated and fed diets containing no Lauricidin R . Rats
in group 6 were not inoculated and were fed diets containing
R

no Lauricidin One way analysis of variance indicated

no significant differences among the treatment groups.

Diet Consumption

Since rats were housed two per cage and food intake was
recorded per cage the mean cumulative food intake reflects
the diet consumed by pairs of rats housed together. It is
assumed that each rat consumed half of the food which dis—
appeared. Mean cumulative food intakes and standard errors
ranged from 1046 i 30 for rats in treatment group 6 which
werernot inoculated and were fed diets without Lauricidin R
to 1227 i: 27 for rats in treatment group 4 which were
iruaculated and were fed diets without Lauricidin R (Table 6).
One way analysis of variance indicated a statistically
Significant difference among treatment groups (p<0.001). A
Scheffé procedure was used to analyze all possible compari-
sons between means. Significant differences were found
between mean food intake for rats in treatment group 4

R

which were inoculated and not fed Lauricidin and mean

food intake for rats in treatment group 5 which were not

inoculated and were fed 2% Lauricidin R . Significant dif-

ferences were also found between mean food intake for rats

63

in treatment group 4 and mean food intake for rats in
treatment group 6 which were not inoculated and were fed
diets containing 0% Lauricidin R . All other comparisons

were not significant.

Dental Caries Incidence and Severity

 

Proximal Surfaces. 100% of the rats in each of the six

 

treatment groups developed caries on the proximal surfaces.
The mean number of carious teeth per rat ranged from 3.3 to
5.8 (Table 7). Inoculated rats fed each of the three doses

R

of Lauricidin had significantly fewer carious teeth per

rat than inoculated rats that were not fed Lauricidin R
All other differences in number of carious teeth were
insignificant (Table 7).

The mean caries scores per rat for rats inoculated
with g. mutans were 13.3 i 0.7 for rats fed diets without
Lauricidin R , 6.3 i 0.7 for rats fed diets containing 2%

Lauricidin R , 7.8 i 0.9 for rats fed diets containing 1%

R , and 5.8
R

H-

Lauricidin 1.1 for rats fed diets containing

0.5% Lauricidin Mean caries scores for rats fed diets
containing each of the three doses of Lauricidin R were
found to be significantly different from mean caries scores
for rats fed diets not containing Lauricidin R (p<0.005).
The mean caries score for rats fed diets containing 2%

R

Lauricidin was not significantly different from the mean

caries scores for rats fed diets containing 0.5% Lauricidin

R

64

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A.o A m.0 0m.o A 0.0 00A 0.N A
0000A0000H
A0uzv
A00e\muwc0v 0sz 00; 000 A00 A00 00000
000m 000000 0000p 0000000 0000 0000000 0 0000000000 000500000

 

.0 0000000000 00 00000 m0omc0> 000 000; so 0A0500 00
00000 000 00 00000000 A0EAx000 0:0 :0 000000 A00000 00 00000>0m 0:0 00000000H .N 0A00».

65

The mean proximal caries scores for rats which were not
inoculated with s. mutans were 3.3 i 0.3 in the group of

R and 5.6 e 0.9 in

rats fed diets containing 2% Lauricidin
the group of rats fed diets not containing Lauricidin

The difference in mean caries scores in these two treatment
groups was not statistically significant.

The reduction in mean proximal caries score for inocu-
lated rats fed diet containing 2% as compared to 0% Lauri-
cidin R was contrasted with the reduction in mean proximal
caries scores for non-inoculated rats fed diets containing
2% compared to 0% Lauricidin R . This contrast was statis-
tically significant (p<0.025).

Sulcal Surfaces. One hundred percent of the rats in

 

each of the six treatment groups developed caries on the
sulcal surfaces (Table 8). All of the six teeth which were
scored in treatment groups 1,2,3, and 4 had caries on the
sulcal surfaces. The mean number of carious teeth per rat
was 5.6 i 0.7 in treatment group 5 in which the rats were
not inoculated and were fed diets containing 2% Lauricidin R ,
and 5.9 i 0.4 in treatment group 6 in which the rats were
inoculated and fed no Lauricidin R

The mean sulcal caries score for inoculated rats fed
diets not containing Lauricidin R was 40.1 i 1.5. The
mean caries scores for inoculated rats fed diets containing

2%, 1%, 0.5% Lauricidin R were 30.3 i 2.3, 28.3 i 4.1,

and 25.9 i 2.5 respectively. The mean caries scores for

66

.AmN0.0v0v 00 000 0000.0v0v 00.me0.0v0v N0.0w0.0v0v 00 0002 000000000
00 .Aoimv .0> 00-00 n 00 00 .0> 0 u 0 00 .0> m u 0 00 .0> 0 u 00 we .0> N
00 .0> n 00 0000000000 000300000 000 0000000 00 0000 003 00000 .000uv >.0
.N\0.0000 0: A.0\xwom.\00 u 00 000000000i0 0000000000 000 .0000.0v0v 000000 000000000

000 00000 0000000000 00000000000 0000000000000 000000000 0000000> 00 00000000 00: 0000

 

.me H cmmZu

.000000 000
00 00000 000 00003 000 0000000 0000 00000 00003 00000 00000000000 00 000000000 00000>0

.000 000 000000 00000 x000

 

 

 

 

00.0 0 0.00 0.0 0 0.0 000 0.0 0
0.N 0 0.0N 0.0 0 0.0 000 0.N 0
0000000000 002
00.0 0 0.00 0 000 0.0 0
0.N 0 0.0N 0 000 0.0 0
0.0 0 0.0N 0 000 0.0 N
00.N 0 0.00 0 000 0.N 0
0000000000
0E:
00000\00000v 0020 000 000 00v 000 00000
00000 000000 00000 0000000 0000 0000000 0 0000000000 000000000

 

. 0000000000 00 00000 000000> 000 0000 :0 000000
00 00000 000 00 00000000 00000m 000 00 000000 000000 00 00000>00 000 000000000 .0 00000

67

rats fed diets containing each of the three doses of Lauri-
cidin R were found to be significantly less than the mean
sulcal caries score for rats fed diets containing 0% Lauri-

R (p<0.05 for 2% vs. 0%, p<0.025 for 1% vs. 0%,

cidin
and p<0.005 for 0.5% vs. 0%). The mean caries score for
rats fed diets containing 2% Lauricidin R was not signifi-
cantly different from the mean caries score for rats fed
diets containing 0.5% Lauricidin R .

The mean sulcal caries score for rats which were not
inoculated with §. mutans was 4l.0 i 3.3 for rats fed diets
not containing Lauricidin R and 29.4 i 2.4 for rats fed
diets containing 2% Lauricidin R . The 28% reduction
between these two groups was found to be statistically sig-
nificant (p<0.025).

The reduction in mean sulcal caries scores for inocu-
lated rats fed diets containing 2% as opposed to 0% Lauri-
cidin R was contrasted with the reduction in mean caries
scores for non inoculated rats fed diets containing 2% as
opposed to 0% Lauricidin R . This contrast was not found
to be statistically significant.

Buccal and Lingual Surfaces. All of the inoculated
R

 

rats which were not fed Lauricidin developed caries on

the buccal/lingual surfaces (Table 9). The addition of

R to the diets of the

the various doses of Lauricidin
inoculated rats resulted in a marked reduction in the

number of rats which developed caries on the buccal/lingual

68

.0000.0v0v 00 000 0000.0v0v N0 .0000.0v00 00 0003 000000000 00000000000

.0 00000 .0 00000000 00 00 00000000 0003 000000000 0000000000 .000000 000000000 000 00000
00000000000 00000000000 0000000000000 000000000 0000000> 00 00000000 003 000 < .00000000
00000000000 000000 00000 000000 0000 00 00000 003 00 00 000000 < .0000 000 00 0000

-0000000000 000000000000 000N 0003 00003 000000 000000 00 000000 00000 0 00 00000000 0000

.200 0 00020

.000000
000 00 00000 000 00003 000 0000000 0000 00000 00003 00000 00000000000 00 000000000 00000>0

1.0m..— Lwa UwLoum gamma. xwmm

 

0 0 0.0

0000000000 002

0.0 + 0.N 0.0 0 0.0 0.N0 0.0 0
0.N 0

 

 

 

0N 0 m 0 00 0 0 m 0.N 0.000 0.0 0
N.0 H 0.0 0.0 H 0.0 0.0N 0.0 0
0.0 H 0.0 0.0 H 0.0 0.N0 0.0 N
00.N + 0.0 00.0 + 0.0 0.00 0.N 0
0000000000
00",;
0000\000000 0020 000 000 000 000 00000
@0000 000000 00000 0000000 0000 0000000 0 0000000000 000000000

 

0 0000000000 00 00000 000000> 000 0000 20 000000 00 00000 000
00 00000000 0000000 000 000000 000 00 000000 000000 00 00000>00 000 000000000 .0 00000

69

surfaces. Only 25% of the rats fed diets containing 0.5%
Lauricidin R developed caries. Only l2.5% of the rats fed
diets containing l% Lauricidin R developed caries, and
only 37.5% of the rats fed diets containing 2% Lauricidin R
developed caries. The mean caries scores for inoculated
rats fed 2%, 1%, and 0.5% Lauricidin were 3.5 i 2.4, 1.8 i
l.8, and l.5 0 1.2 respectively. This was a highly signifi-
cant (p<0.005) reduction of 78% to 90% from the mean caries
scores of l6.4 i 4.2 for inoculated rats which were not fed
Lauricidin

Caries were present on the buccal/lingual surfaces in
62.5% of the non-inoculated rats which were not fed Lauri-
cidin R . There were no buccal/lingual caries in any of
the non-inoculated rats which were fed Lauricidin R . The
reduction due to Lauricidin R in the non-inoculated rats
was not statistically significant due to the very low caries

scores of the non-inoculated rats that were not fed Lauri-

cidin

DISCUSSION

Lauricidin R caused significant reductions in inci-
dence and severity of dental caries in rats inoculated with
Streptococcus mutans 67l5 and in rats which were not inocu-
lated. Since Streptococcus mutans is one of the bacteria
highly implicated in human dental caries, caries which
resulted after inoculation with this organism were assumed
to be similar to human caries. The ability of Lauricidin
to inhibit dental caries which occured after inoculation in
rats implied an ability to inhibit dental caries in humans,

R would have to be tested in a human

however, Lauricidin
population for conclusive evidence.

It was previously demonstrated that several dietary
fats inhibited the production of dental caries (Rosebury
and Karshan, l939; Gustafson gt al., 1955). In the present

R

studies diets containing Lauricidin were compared with

diets containing equivalent amounts of Crisco R . The
greater ability of Lauricidin R to inhibit dental caries
production showed that Lauricidin R had a specific inhibi-
ting action beyond that of another fat. It is possible
that Lauricidin R inhibited the growth of S. mutans and

therefore inhibited dental caries that result from infection

70

71

with S. mutans. This will only be determined after the
microbiological data collected in this study have been
analyzed.

Reduction of dental caries in rats has been associated
with a reduction in the frequency of food intake (Larson gt
al., l962). In the present studies frequency of food intake
was not recorded or controlled. Since the only significant
differences in cumulative food intake between treatment
groups were between groups which were not compared for den-
tal caries and since there were no significant differences
in cumulative weight gains among the treatment groups it is
assumed that the rats in the various groups experienced equal

exposure to the cariogenic diet.

Inhibition of Dental Caries by Lauricidin R

on the Various Surfaces of the Teeth

Incidence and severity of carious lesions were recorded
and totaled separately for three types of surfaces found
on the teeth, namely, buccal/lingual surfaces, interproxi-
mal surfaces and sulcal surfaces. Smooth surface lesions
are caries found on the buccal, lingual and interproximal
surfaces of the rat molars. Buccal and lingual caries are
those found on the surfaces of the teeth exposed to the
cheek and tongue respectively. Interproximal lesions are
caries which are found at or below the contact points

between two molars. Sulcal lesions are caries found on the

fissures of the occlusal surfaces of the rat molars.

72

Proximal and sulcal sites differ from buccal/lingual sites
because they are less exposed to the oral cavity, they are
more retentive sites for food debris, and they may form a
special ecological niche for development of specific micro-
organisms (Navia, l977). Proximal and buccal/lingual
sites, however, are more dependent on the development of
plaque than sulcal caries are (Shafer gt_gl., 1974).
Because of these important differences in etiology a
separate discussion of caries activity in the proximal,

buccal/lingual, and sulcal sites is essential.

Sulcal Caries

 

In both Study I and Study 11 rats which were not
inoculated with S. mutans had mean caries scores in sulcal
sites which were not statistically different from the mean
caries scores in the same sites in inoculated rats. This
is consistent with the observation that the normal flora
of Osborne-Mendel rats cause some fissure caries (Newbrun,
1977). Factors responsible for caries in the occlusal
sulci of the rat molars were neither hindered nor stimu-
lated by the addition of S. mutans. The added S. mutans
was able to effect a symbiotic relationship with the native
oral flora of the rats (Newbrun, 1977).

The addition of Lauricidin R to the diet of uninocu-
lated rats in Study II caused a significant reduction in

R

sulcal caries. This proved that Lauricidin could

73

inhibit caries which were due to factors other than S.

R to the diets of

mutans. The addition of Lauricidin
inoculated rats in both Study I and Study II caused a sig-
nificant reduction in sulcal caries. The inhibition in
this case, however, was not greater than the inhibition
seen in non-inoculated rats so any specific action of

R .
Lauricidin on caries that were the result of inoculat1on

could not be identified.

Interproximal Caries

 

In both Study I and Study 11 rats which were not inoc-
ulated developed proximal caries. In both studies mean
caries scores for proximal caries due to inoculation with
S. mutans were approximately double the scores for rats
that had not been inoculated. Although proximal caries
are similar to sulcal caries because both types of caries
may be partially caused by the trapping of food particles,
proximal caries are also stimulated by the presence of
dental plaque (Shafer gt_gl., l974). S. mutans has been
frequently isolated from the plaque of caries active popu-
lation (Hoerman gt 11., 1972) and is therefore suspected
of being one of the plaque forming organisms in humans.

The fact that there was an insignificant reduction in
proximal caries when various doses of Lauricidin R were

fed to uninoculated rats could be due to the very low mean

74

caries scores of rats in the group that was not inoculated.
The levels of caries may already have been so low that
addition of an inhibitory agent could not produce a sig-
nificant effect.

The addition of Lauricidin R to the diets of inocu-
lated rats caused significant reductions in mean caries
scores on the proximal surfaces. A specific inhibition of
those proximal caries which were caused by the presence of

S. mutans was implicated.

Buccal and Lingual Caries

 

Rats which were not inoculated with_S: mutans developed
very few buccal/lingual lesions in Study I and developed
no carious lesions in Study II. Inoculation with S.
mutans resulted in large increases in buccal/lingual caries
in rats in both studies. The occurrence of caries on
buccal/lingual surfaces is similar to the occurrence of
caries on proximal surfaces. Both are smooth surfaces and
caries on smooth surfaces are known to be dependent on
plaque formation (McDonald, l977; Newbrun, 1977). S. mutans
is one of the only bacteria that has been successfully used
to produce smooth surface caries in animals (Newbrun, 1977).
Although rats do not develop grossly visible amounts of
plaque when infected with S. mutans, it is assumed that
caries that result due to the presence of S. mutans are due

to the initiation of plaque formation (Navia, l977).

75

The addition of Lauricidin R to the diet of inoculated
rats in Study I caused a 50% reduction in buccal/lingual
caries. The reduction was not significant due to wide
variation in the extent of caries in both Lauricidin R fed
rats and control rats. A large coefficient of variation
frequently occurs in rats fed cariogenic diets and inocu-
lated with S. mutans but to be sure that it was not due to
the possible unequal exposure of rats to Lauricidin R , in
Study II special attention was paid to the mixing of all
diet ingredients to be sure that both the Lauricidin R
and the control fat were well distributed throughout the
diets. The addition of Lauricidin R to the diets of inocu-
lated rats in Study II caused from a 62% to an 87% decrease
in the number of rats which developed buccal/lingual caries.
The mean caries score per rat was 78% to 90% lower and all
reductions were statistically significant (p<0.005).

Effect of Various Doses of Lauricidin R

 

Reduction in mean caries scores was found to be as

great in rats fed a diet containing 0.5% Lauricidin R as

in rats fed the same diet containing 2% Lauricidin R .

This was true on the proximal, buccal/lingual, and sulcal

surfaces of the teeth. The fact that a diet containing

. . . R . . . .
0.5% Laur1c1d1n was as effect1ve 1n reduc1ng caries as a

76

diet containing 2% Lauricidin R may be due to the existence
of a critical dosage similar to that demonstrated by Kabara
_t _l. (l979) in an jg tittg_study of the inhibition of

S. mutans by Lauricidin RR. In that study it was found

that the killing index of Lauricidin R was more dependent
on time of exposure to Lauricidin R than on concentration

. . . . . . . R
of Laur1c1d1n when the concentrat1on of Laur1c1d1n was

increased beyond a critical level.

Distribution of Caries in the Right and Left Jaws

The fact that no significant differences were found
between mean caries scores on the right and left sides of
the jaws of rats in each of the treatment groups in Study I
confirmed the data of Schemmel gt g1. (1979). This finding
is consistent with human studies (Shafer gt_gl,, 1974) and
it indicated that the sonication procedure used on the jaws
did not damage the teeth or lead to an alteration in

detection of caries.

Distribution of Caries in the Maxilla and Mandible

The fact that rats in all 3 treatment groups had
significantly more buccal/lingual and sulcal caries in the
mandible than in the maxilla is consistent with previous
rat studies (Shafer gt gt., 1974). Although humans usually
have a greater number of carious teeth in the maxilla

(Shafer gt gt., l974), the mandibular molars develop

77

carious lesions sooner than the maxillary molars and attain
maximum caries development by age 20 more often than
maxillary molars (Rowe gt gt., l976). The fewer number of
teeth with caries in the mandible is due to the relative
immunity to caries of the human mandibular incisors (Shafer
_t _l., 1974). Since the present study was concerned only
with the molar teeth of the rats, the higher percentage of
buccal/lingual and sulcal caries in the mandible was indi-
cative of a similarity between dental caries production in
the rats in this study and dental caries development in
humans.

Since the occurrence of proximal caries in the maxilla
was not significantly different from the occurence of
proximal caries in the mandible in either of the groups of
rats which were not fed Lauricidin R , proximal caries are

probably caused by a combination of factors which are

equally present in the maxilla and mandible.

Reduction of Caries by Lauricidin R in the

Maxilla and Mandible

 

Reduction in buccal/lingual caries due to the addition

R

of 2% Lauricidin to the diet occurred exclusively in

the mandible. Reduction of sulcal caries due to the addi-

tion of 2% Lauricidin R

to the diet occurred predominantly
in the mandible. Reduction of proximal caries due to the

addition of 2% Lauricidin R to the diet was greater in the

78

mandible although there was a significant reduction in both
the maxilla and mandible.

Although previous studies with Lauricidin R have not
demonstrated this tendency, Lauricidin R showed a definite
tendency in the present study to inhibit caries on all 3
surfaces more in the mandible than in the maxilla. It is
possible that other factors were already exerting a maximum
inhibitory action in the maxilla and therefore Lauricidin R

was ineffective as an addition to those factors.

CONCLUSIONS

A food grade lipid, Lauricidin R , inhibited the
severity of dental caries by 25% to 35% on the sulcal
surfaces, by 42% to 56% on the proximal surfaces, and by
78% to 90% on the buccal/lingual surfaces of the teeth when
included in a cariogenic diet fed to rats that had been

R also

inoculated with Strgptococcus mutans. Lauricidin
inhibited the severity of dental caries on the sulcal but
not the smooth surfaces of the teeth of rats which were

not inoculated with Strgptococcus mutans. The inhibitory

effect of Lauricidin R

at a dietary level of 0.5% was not
significantly different from that at the 2% level.
Conclusions regarding the inhibition of dental caries

R in humans cannot be made. However, the

by Lauricidin
present studies suggest that its potential for inhibition

of caries in humans should be tested.

79

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