Effect of Dietary stearic Acid On The Daily Growth
Of Mouse Mammary Tumors
An Honors Thesis (HONRS 499)
by
Chris Frasier
Thesis Advisor
Dr. Alice Bennett
Ball state University
Muncie, Indiana
June, 1996
Graduated July, 1996
i
Table of Contents
List of Tables . •
ii
Introduction
1
Review of Related Literature
2
Materials and Methods
4
Results
6
I.
II.
III.
IV.
V.
Discussion
11
VI.
References
14
ii
List of Tables
Table
1.
WEIGHTS OF TUMORS TWO WEEKS AFTER IMPLANTS
.7
2.
WEIGHTS AND DAYS OF REMOVAL OF TUMOR INJECTIONS
.9
3.
TUMOR GROWTH PER DAY AND AVERAGES IN mg • • • •
10
1
I.
Introduction
Breast cancer is one of the most prevalent forms of cancer
in women and is a great source of concern.
Although huge amounts
of research have been done about breast cancer, there is still
no consensus on what causes it or how to effectively treat it.
To date, the only way to reduce the risk of breast cancer is
to alter the amount of dietary fat consumed.
studies on this
are inconclusive, but it is generally thought that reducing
the amount of certain fats will reduce the risk of breast cancer.
The purpose of this study will be to study the effects of
different dietary fats on daily tumor growth and time to tumor.
It is presumed that diets that contain stearic acid, as their
fatty acid, will have a slower daily growth and a longer time
to tumor.
II.
Review of Related Literature
In 1974 saturated fat, in terms of nutrient labeling, was
defined as the sum of lauric, myristic, palmitic and stearic
acids (16).
This definition was adequate for that time because
nutrient information was voluntary except for when a special
claim was made.
In 1990 the Nutrition Labeling and Education
Act (NLEA) kept the same definition of a saturated fat but
required all foods to display nutritional information (16).
The 1974 definition of a saturated fat is not adequate for this
day and age because not all saturated fats behave the same,
and the nutritional information can be misleading.
Stearic
acid is one such fatty acid that does not behave in the same
way as other fatty acids.
When stearic acid is the major source
of fatty acids in an animals diet, there is a decrease in tumor
incidence and an increase in time to tumor as compared to animals
whose fatty acids are obtained from other sources.
Bennett
showed that mice fed a high fat diet, in which stearic acid
is the primary fatty acid, have a longer time to initial
development of spontaneous mammary adenocarcinomas than do mice
fed a low fat diet (1).
Tinsley also showed that stearic acid
was associated with decreased tumor incidence and increased
time to tumor (16).
Stearic acid is also unique from other
saturated fatty acids in that it does not raise cholesterol
levels (9).
In fact, Bonanome has shown that stearic acid
reduces cholesterol levels by as much as 14 percent as compared
to other saturated fats (2). This only happens when stearic
is ingested alone and therein lies the problem.
Most foods
that contain stearic acid also contain the saturated fatty acids
that are most people try to avoid.
To combat the intake of
saturated fats, people usually decrease their total intake of
fats.
This is not always a good thing.
When a person reduces
their fat intake greatly, they might be increasing their risk
of other complications.
In order to combat this, and breast
cancer, effectively, a procedure must be devised where a person
can increase their stearic acid intake without substantially
increasing or decreasing their overall,non-stearic, saturated
fat intake.
4
III.
Materials and Methods
Strain A/St mice were maintained in our laboratory by
brother sister inbreeding. The mice were kept in a controlled
enviroment of 21-24 C and a 12 h light: 12 h dark cycle and
fed a 4.5% fat stock diet of Purina Lab Chow (Ralston Purina,
st. Louis, MOl and water ad libitum upon weaning.
Originally,
10mg portions were cut from a 400 to 500mg perpetuated mouse
tumor.
These portions were then surgically implanted into the
right number 4 mammary gland.
The incision was then closed
by two surgical steel staples and an ear tag was placed in their
left ear.
The mice were then sacrificed two weeks later, and
the resulting tumors were removed and weighed.
After the first
attempt at this surgery, the procedure was altered slightly.
Instead of implanting 10mg portions, a set of 50mg portions
and a set of 20mg portions were implanted in the right number
4 mammary glands of mice.
These mice were then sacrificed two
weeks later, and the tumors were removed and weighed.
After receiving no clear data, a new approach was tried.
The mice were randomly separated into four groups of five.
These groups were then feed one of the following diets after
they were injected with a tumor suspension: SF containing 15%
safflower oil, SF-1 containing 1% safflower oil, PA containing
or SA-1 containing 14% stearic acid and 1% safflower oil.
5
A mouse with a perpetuated tumor of about 500mg was sacrificed
and the tumor was placed in a RPM I solution.
masserated until the solution was homogenous.
was then filtered through nylon mesh.
cells pass through.
The tumor was
The solution
This allows only single
A 1:10 single cell suspension to trypan
blue solution was made and counted on the hemocytometer. The
resulting solution was diluted so that an injection of .2 ml
of solution contained 600,000 single cancer cells.
Twenty mice
that were two months old were injected with .2 ml of the cell
suspension.
The mice were checked daily, and when the resulting
tumor reached the size of approximately 1-2mm across, or about
150-200mg, they were removed and weighed.
6
IV.
Results
The results from the original 10 mg implants showed that
there was nodirect link in the size of tumor implanted and
resulting size of the final tumor. Final weights ranged from
140 mg to 780 mg.
This was too broad of a range to draw any
conclusive results.
The results from the altered procedure also showed that
no link can be drawn from the tumor implant size to the final
tumor size.
The tumors removed from the
two mice that had
approximately 50 mg implants different by 1383 mg and, the tumors
from the 20 mg implants were different by 2786 mg (Table 1).
Both trials showed no consistency in the size of the tumor
implant and the time amount of concurrent growth.
An interesting
phenomenon was observed in the two mice that received the 50
mg implants.
When the tumors were removed, they had a hard
capsule covering almost like that of a cyst.
When the capsule
was pierced and removed, there was a build up of fluid observed
within the capsule.
It was almost as though the mice's bodies
had responded to the large 50 mg implants as a foreign object
rather than a tumor.
After all the tumors were removed from the mice with the
600,000 cell injection, the mg of growth per day were calculated
(Table 3).
The averages of daily growth for each diet were
7
Table 1: WEIGHTS OF TUMORS TWO WEEKS AFTER IMPLANTS
Implant weight
Age of Mouse
Tumor weight
(mg)
(months)
(mg)
18
7
4795
53
7
4287
20
5
2009
47
6
2904
TABLE 2: WEIGHTS AND DAYS OF REMOVAL OF TUMOR INJECTIONS
Diet
SF
SF-1
SA-1
PA
299mg 206mg
95mg
202mg
237mg
Days
18
110mg
21
89mg
23
25
110mg
100mg
128mg
136mg 147mg
26
29
30
105mg 54mg
225mg
137mg 194mg
161mg
184mg
then found (Table 3).
the expected.
9
The averages were very different from
The SA-l, or stearic acid rich, diet was found
to have the highest average daily growth.
There was also a
different result than was expected in the unsaturated fatty
acid diets.
The diet that held only the minimum daily
requirements of fatty aCids had a higher average growth rate
than the high fat diet.
10
Table 3: TUMOR GROWTH PER DAY AND AVERAGES IN mg
Tumor 1
Tumor 2
Tumor 3
Tumor 4
Tumor 5
Average
Diets
,
L.
SF
6.11
4.24
4.00
5.37
SF-1
5.24
9.78
5.12
4.72
6.69
SA-1
16.60
9.78
12.48
5.23
5.65
6.31 I
10.26,
5.28
11 .29
7.17
2.35
6.1 3
6.44
PA
4.93!
I
1
11
V.
Discussion
According to the existing literature, stearic acid causes
the least detrimental effects of all saturated fatty acids.
This is not what we found, and in fact, we found the opposite
to be true in our experiment.
If this type of experiment was
to be repeated, there should be more than 5 mice in each of
the groups, and the entire experiment should be repeated more
than once so that consistency could be proven.
It has been
shown many times in results from different research labs that
stearic acid has different effects than other saturated fatty
acids.
According to our results stearic acid is no different
from other fatty acids in its effects on tumor growth.
Although we were trying to find a relationship between
different dietary fats and daily growth of tumors, no data was
found that showed us that one type of fatty acid is better for
a person over another.
Our tumor implanting technique was not
successful at producing reproducible results.
This inconsistent
data may be the result of many avenues of error.
First of all,
we found that the size of the implant is crucial to its growth.
If too big a piece of a tumor is implanted, the body acts
strangely to it.
The body encases the tumor in a cyst-like
capsule and this probably impedes the true growth of the tumor.
Second, we didn't use enough test subjects to get a very accurate
12
reading.
If more test subjects were used, there might have
been consistent results for the tumor growth per day.
Also
if we had put tumor implanted mice on the different fat diets,
this might have gave us better results than the stock diets.
For future reference, large amounts of test subjects and tumor
implants under 50 mg should be used for this type of
experimentation.
We did find one interesting thing about the
whole procedure.
The tumors in the older mice, ones that were
four months old, seemed to grow faster than the tumors in the
younger mice, ones of two months old.
The final tumor size
in each of the age groups were about the same no matter what
size of tumor was implanted.
This could
be an area that would
be a source of good information.
Even after changing our procedure, we still came out the
experiment without any data that specifically showed that one
fatty acid is better than any other.
per day was far from what we expected.
The average tumor growth
If the results had gone
as expected, the PA diet would have yielded the highest average
growth per day.
As it turned out, PA yielded the second highest
growth per day behind the SA-1 diet which should have yielded
the lowest growth per day out of all of the diets.
Not only
were the saturated fats not as expected, but the results of
the unsaturated diets was not as expected either.
The high
percentage saturated fat diet resulted in a lower daily growth
than the diet that contained only the minimum daily requirements
of fat.
This data may not give consistent results because the
a small group of each diet was used.
This leaves a lot of room
13
for deviation from the normal.
14
VII.
1. Bennett, Alice S.
References
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15
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16
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I
17
I'
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.I
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