F ELECTRlCAL STRESS AND P
ACTWlTY ON BLOOD CHOLESTEROL LEVELS

WHOLE BLOC?!) COAGULATTON TTME AND
RAL ORGAN WETGHTS- OS ADULT MALE RATS ‘

THE EFFECTS O

SEVE

This: for the Dag!» of Ph 0.:

MLCHIGAN STATE ‘UQNNERSITY
Mishaei G; Maksud 7
1965

Hvsxcm. _ ' ' '

THESIS 'JLIBDARY

Michib .11 State
Uni» ersity

L.

l
s
i
i

       
   

This is to certify that the

thesis entitled

The Effects of Electrical Stress and Physical
Activity on Serum Cholesterol, Coagulation Time,

Organ Weights, and Voluntary Activity of Adult
Male Rats

presented by

Michael George Maksud

has been accepted towards fulfillment
of the requirements for

Ph.D. Physical Education

degree in

[/fl/u /%%\

Major professor

 

Date August 1+, I965

0-169

ABSTRACT

THE EFFECTS OF ELECTRICAL STRESS AND PHYSICAL
ACTIVITY ON SERUM CHOLESTEROL LEVELS,
WHOLE BLOOD COAGULATION TIME, AND
ORGAN WEIGHTS OF ADULT MALE
RATS

by Michael G. Maksud

The purpose of the study was to investigate the
effects of electrical stress and physical activity on the
serum cholesterol levels, whole blood coagulation time and
several organ weights of adult male rats. In addition,
variations in food consumption, total body weight and
voluntary activity were observed.

Ninety adult male rats (Sprague-Dawley Strain) were
divided into five equal groups on the basis of initial serum
cholesterol and blood coagulation determinations.

Each group was randomly assigned to one of five experi—
mental regimens. Group 1, which served as the control, was
housed in sedentary cages and received no stress; Group 2
was housed in sedentary cages and received electrical
stress; Group 3 received no stress and was housed in
voluntary activity cages; Group A was housed in voluntary
activity cages and received electrical stress; Group 5
was housed in sedentary cages and received both electrical

stress and forced exercise.

Michael G. Maksud

The electrical stress consisted of daily one-half
hour periods of confinement to compartmentalized "Stress
Cages" with each compartment measuring 6 1/2 x 6 1/2 x 6 1/4
inches. During the daily confinement the animals were
subjected to 15 milliamperes (60 v.) of direct current at
fifteen second intervals; the duration of each impulse
was approximately one—half second. The investigation was
conducted for ten weeKs.

The forced swimming program consisted of one-half
hour of swimming daily with 2 % of the animals body weight,
in the form of lead sinkers, attached to the base of the
tail. The swimming was conducted in individual compartments
measuring 12 X 12 X 30 inches with water temperatures
maintained between 35 and 37 degrees centigrade.

During the "non-stress" periods each animal was
housed in either a standard activity cage with rotating
drum or a sedentary housing cage which measures 10 x 8 x 7
inches. All animals were maintained on a standard ground
meal diet and were fed ad libitum. Drinking water was
always available.

The results indicated a significant difference between
group means in food consumption with the sedentary group
having the greatest food consumption.

Analysis of organ weights, based upon percentage of
body weight, indicated no significant differences between

groups for kidney, liver and spleen weights. However,

Michael G. Maksud

analysis of adrenal, heart and tests weights reflected
significant differences between group means at the one per
cent level; the stressed animals had the larger organ
weights.

Significant differences were found in final body
weight, with the stressed groups having the lower body
weights while the sedentary group had the highest body
weight.

Analysis of variance of the differences between
initial and final cholesterol values indicated no signifi-
cant differences between group means.

Statistically significant differences were not found
in whole blood coagulation time nor in voluntary activity.
The results suggest the following conclusions:

1. The stress conditions of swimming and/or electri-
cal stress had a significant effect upon food
consumption-~the stressed animals consuming less
food.

2. The experimental conditions of physical activity
and/or electrical stress had a significant effect
upon total body weight-~the stressed animals had
lower total body weights.

3. The stress conditions of physical activity, both
voluntary and forced, and electrical shock had
no significant effect on relative spleen, liver
and kidney weight. The stress conditions resulted

in significantly larger adrenal weights.

Michael G. Maksud

The "double stress" of electrical shock and forced
swimming resulted in significantly larger hearts
as determined by relative organ weight.

The experimental conditions reflected no signifi-
cant differences in whole blood coagulation time
or serum cholesterol levels.

Electrical stress had no significant effect upon

voluntary activity of adult male albino rats.

THE EFFECTS OF ELECTRICAL STRESS AND PHYSICAL
ACTIVITY ON BLOOD CHOLESTEROL LEVELS,
WHOLE BLOOD COAGULATION TIME, AND
SEVERAL ORGAN WEIGHTS OF ADULT

MALE RATS

A}
Michael Gel Maksud

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the reguirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Health,
Physical Education and Recreation

1965

ACKNOWLEDGMENTS

The writer wishes to express his sincere gratitude
to Dr. Wayne Van Huss for his guidance and assistance,
without which this study could not have been conducted.
The writer is also indebted to Dr. William Huesner, Mr.
David Anderson, Mr. Rex Carrow, Mr. Kenneth Coutts, and
Mr. Robert Kertzer for their assistance throughout the

conduct of the study.

Dedicated to my wife, Ann

and my daughters, Dawn and Michelle

CHAPTER

I.

TABLE OF CONTENTS

INTRODUCTION TO THE PROBLEM AND DEFINITION

OF TERMS.
Purpose of the Study. . . . . . .
Definitions of Terms.
Stress.
Diet
Voluntary Activity.
Limitations of the Study

References

II. REVIEW OF RELATED LITERATURE.

III.

Animal Studies and Atherosclerosis
Diet and Cholesterol. .

Activity and Cholesterol Levels

Cholesterol Levels in Different Societies.

The Effects of Stress

Stress and Coagulation . . . . .
Stress and Adrenal Histology .
Cholesterol Levels and Heredity

References

EXPERIMENTAL METHOD. . . . . . . .

Material and Equipment

Design of the Experiment

PAGE

I:

\ONUWUIUT

ll

1
1A
18

23
32

38
39
A6
A6
1+9

CHAPTER
Statistical Procedures . .
References
IV. RESULTS AND DISCUSSION.
Results .
Food Consumption
Organ Weights
Total Body Weight
Cholesterol Values. . . . . . .
Coagulation Time
Voluntary Activity.
Discussion of Results
Food Consumption
Organ Weights . . . . .
Body Weight . . . . . . . . .
Cholesterol Values.
Coagulation Time
Voluntary Activity.
References
V. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS.
Summary . .
Conclusions.
Recommendations
BIBLIOGRAPHY

APPENDICES . . . . . . . . . .

TABLE

II.
III.

IV.

VI.
VII.
VIII.

IX.

XI.

XII.

XIII.

XIV.

XVIII.

ANALYSIS OF
CORRELATION
ANALYSIS OF
ANALYSIS OF
ANALYSIS OF
ANALYSIS OF
GROUP MEANS
ANALYSIS OF
ANALYSIS OF
ANALYSIS OF

ANALYSIS OF

DUNCAN MULTIPLE RANGE

TREATMENT
WEIGHTS
ANALYSIS OF
ANALYSIS OF
ANALYSIS OF

ANALYSIS OF

DUNCAN MULTIPLE RANGE TEST-~COMPARISON

LIST OF TABLES

VARIANCE—-FOOD CONSUMPTION .

OF BODY WEIGHT AND ORGAN WEIGHTS
VARIANCE-~RELATIVE LIVER WEIGHTS
VARIANCE-~RELATIVE KIDNEY WEIGHTS.
VARIANCE-—RELATIVE SPLEEN WEIGHTS.
VARIANCE-—RAW SPLEEN WEIGHTS
FOR SPLEEN AND TESTES--RAW DATA
VARIANCE—~RELATIVE ADRENAL WEIGHTS
VARIANCE-~RELATIVE HEART WEIGHTS .
VARIANCEm-RELATIVE TESTES WEIGHTS.
VARIANCE--RAW TESTES WEIGHTS
TEST-~COMPARISON OF

MEANS OF RAW SPLEEN AND TESTES

VARIANCE——INITIAL BODY WEIGHT .
VARIANCE-—FINAL BODY WEIGHT.

VARIANCE-wFINAL CHOLESTEROL VALUES
VARIANCE--FINAL COAGULATION TIME

OF

TREATMENT MEANS .

ANALYSIS OF VARIANCE——VOLUNTARY ACTIVITY

PAGE

O‘\.
OO

74
75

LIST OF APPENDICES

APPENDIX PAGE

A.

'IJLTJUOUJ

C121 Q

l—l

L—‘WC—l

..<‘..’

s.

t(JO 2

TOTAL BODY WEIGHT--INITIAL READING (Grams) . . 97
TOTAL BODY WEIGHT--FINAL READING (Grams). . . 98
TOTAL FOOD CONSUMPTION (Grams) . . . . . . 99
ADRENAL WEIGHTS . . . . . . . . . . . 100
HEART WEIOHIS. . . . . . . . . . . . 101

LIVER WEIGHTS. . . . . . . . . . . . 102

KIDNEY WEIGHTS . . . . . . . . . . . 103
SPLEEN WEIGHTS . . . . . . . . . . . 10A
TESTES WEIGHTS . . . . . . . . . . . 105

SERUM CHOLESTEROL VALUES-~1NITIAL . . . . . 106
SERUM CHOLESTEROL-~FINAL VALUES. . . . . . 107
WHOLE BLOOD COAGULATION TIME-~INITIAL VALUE. . 108
WHOLE BLOOD COAGULATION IIME--EINAL VALUE . . 109
TOTAL VOLUNTARY ACTIVITY (Revolutions) . . . 110
M-I RAT RATION . . . . . . . . . . . 111

STRESS CAGES AND STIMULATOR . . . . . . . 112

CHAPTER I

INTRODUCTION TO IRE PROBLEM AND DEFINITION

OF TERMS USED

One of the most widely discussed subjects in both
medical and lay circles is the probable relationship between
blood cholesterol levels and atherosclerosis. Athero— .
clerosis is generally considered to be the major disease
of this era. Its consequences in the form of occlusions
and plaques in the coronary, cerebral, and peripheral
arteries are responsible for more deaths and disability than
any other disease.

While often considered to be one of the degenerative
diseases of old age, atheroclerosis is quite prevalent among
younger men. The not-too—rare occurence of coronary artery
occlusions in young men from 20 to A0 years of age testifies
to the fallacy of the idea that atherosclerosis is a
problem of the aged or senile. The absence of the disease
at autopsy in many persons who have survived to be
octogenarians reflects the need to approach atherosclerosis
as a disease manifestation and not as an inevitable
consequence of aging.

Although a definite cause-effect relationship between

elevated blood—cholesterol levels and atherosclerosis has

not been established, it has been widely implicated. The
extent of this implication is a matter of debate. In
addition, a variety of conditions which influence the level
of blood cholesterol have been studied. Among these are
diet, heredity, cultural background, and stress including
both physical exercise and states of anxiety.

The dietary relationship is generally manifested in
terms of elevated cholesterol levels. The results of
several studies (A, 5, 6, 9, 14) support the hypothesis
that dietary intake of fat influences the level of serum
cholesterol, particularly that of the B-lipoproteins, and
may be one of the major factors influencing the pathogenesis
iof coronary heart disease. Cholesterol, in both free and
esterified forms, is present in higher quantities in
atheromatous arteries than in normal ones. Pertinent data
of this type have been obtained from coronary arteries of
patients dying of myocardial infarction in which four times
as much cholesterol was found as in those of patients dying
from other causes. (13)

Hereditary implications have also come under scrutiny
as a possible cause of atherosclerosis. (15) While the
absolute relationship of heredity and atherosclerosis is
not clear, there is indicated a pre—disposition to elevated
cholesterol levels if this condition exists in the family

history.

The effects of cultural conditions on serum choles—
terol levels have also come under intensive study. (1, 8,
10, 11) Generally speaking, elevated cholesterol levels
are more prevalent among sedentary individuals, those
engaged in occupations involving mental-emotional stress,
and those enjoying a high standard of living.

The effects of stress are difficult to quantitate in
terms of a casual relationship to atherosclerosis. Physical
stress induced by physical activity has been shown to reduce
cholesterol levels. (7, 12)

The effects of emotional stress are not well docu—
mented, although some studies indicate elevated cholesterol
levels during periods of anxiety. (2, 3) A possible dif~
ference in cholesterol levels resulting from the various
forms of stress is suggested by the metabolic pathways for
the synthesis and oxidation of cholesterol.

It is generally accepted that the stress syndrome
involves the adrenal medulla which in turn is an extension
of the sympathetic nervous system. Among other responses,
the stress syndrome elicits the release of epinephrine and
nor-epinephrine from the chromaffin cells of the medulla.

A major target tissue of these adrenal hormones is adipose
tissue resulting in the release of free fatty acids during
conditions of stress. Free fatty acids serve as precursors
in the synthesis of acetyl—CoA which, in turn, is a major

component in the Krebs—tricarboxylic acid cycle.

It is assumed that under conditions of physical
activity the synthesis of acetyl—CoA serves to meet the
metabolic demands of the activity, minimizing accumulation
of acetyl-COA.

However, when stress is induced while physical
activity is curtailed, the metabolic needs of the organism
do not correspond to the elevated levels of circulating free
fatty acids and acetyl-COA. Since the latter is also a
precursor in the shythesis of cholesterol, the hypothesis is
that the excess fatty acids are shunted toward the formation
of cholesterol resulting in elevated blood cholesterol

levels.

Purpose of the Study

 

The purpose of the experiment was to study the effects
of physical activity and electrical shock on the serum
cholesterol levels of adult male albino rats, In addition,
variations in organ weights, whole blood clotting time,
food consumption, weight gain and voluntary activity were

observed.

Definition of Terms

 

Stress.--The stressors utilized in the study were of

two forms:
1. Forced physical activity which involved swimming
daily for one-half hour with two per cent of the

animal's body weight attached to the base of the

tail. The animals were placed in separate
cubicles during the swimming program.
Electrical shock consisted of 15 milliamperes
(60 v.) of direct current administered every
fifteen seconds for one-half hour daily, during
which the animals were confined to the stress
cages. The electrical shock impulses were

approximately one-half second in duration.

Diet.—-This consisted of a standard ground meal diet

as defined in Appendis P.

Voluntary Activity.—~This consisted of spontaneous

 

activity as measured by the number of revolutions in a

standard activity cage.

Limitations of the Study

 

1.

While the total N of ninety is respectable, the
group n of eighteen might be considered
minimal.

The experimentation involved adult male albino
rats; therefore, interpretations can be assumed
to apply only to animals of similar strain under
similar conditionS.

The experimental period of ten weeks may not be
adequate for the establishment of significant
patterns.

While food consumption was measured, no attempt

was made to equalize food consumption.

KN

Constant temperature and humidity conditions
were not maintained in the animal quarters.
Owing to laboratory limitations, the experimental

design did not include all possible control groups.

10.

11.

References

 

B. Bronte- Stewart, A. Keys, and J. F. Brock, HSerum
Cholesterol. Diet and Coronary Heart Disease, an Inter—
Racial Survey in Cape Peninsula," Lancet, 2:11O3,
1955. I

M. Friedman, and R. H. Rosenman, ”Association of
Specific Overt Behavior Pattern with Blood and
Cardiovascular Findings,” Journal of American
Medical Association, 169:1285, 1959.

 

 

M. Friedman, R. H. Rosenman, and V. Carroll, ”Changes
in Serum Cholesterol and Blood Clotting Time in
Men Subjected to Cyclic Variation of Occupational
Stress,” Circulation, 17:852-861, May, 1958.

 

J. W. Gofman, ”The Role of Lipids and Lipoprotein in
Atheroclerosis," _§iience, 111 160, 1950.

J. W. Gofman,

t al. ”Blood Lipids and Human Athero-
clerosis," 1r

e
C'r :7Jlat ion, 5:119-134, 1952.

 

H. Gordon, et al., ”Dietary Fat and Cholesterol
Metabolism, Lancet, 2:1299, I95 .

D. Gsell, ”Serum Cholesterol and Its Increase with
Age in a Population with High Fat Consumption and
High Physical Activity,H Gerontologica C liniCa,
3-Azi9A, 1961-62. ”“” “

 

M. J. Karvonen, et al., ”Diet and Serum Cholesterol

of LumberjacijWuiBritish Journal of Nutrition,
15:157, 1901.

 

 

A. Keys, "Human Atherosclerosis and the Diet,"

Circulation, 5:115-118, 1952.

 

A. Keys, et al. "Studies on Serum Cholesterol and
Other Characteristics of (Wlini ally Healthy Men in
Naples,” Archives of Internal Medicine, 93: 328,

1954.

A. Keys, M. J. Karvonen, and F. Fidanza, HSerum -
Cholesterol Studies in Finland,H Lan et, 2:175—178,
1958.

 

12. H. J. Montoye, et al., HThe Effects of Exercise on
Blood Cholesterol in Middle-Aged Men,” Journal of
Clinical Nutrition, 7:139, 1959.

 

 

13. L. M. Morrison, and K. D. Johnson, ”Cholesterol Content
of Coronary Arteries and Blood in Acute Coronary
Artery Thrombosis,” American Heart Journal, 39:31—3M,
January, 1950.

 

14. W. J. Walker, ”Relationship of Adiposity to Serum
Cholesterol and Lipoprotein Levels and Their
Modification by Dietary Means,” Annals of Internal
Medicine, 39:705, 1953.

 

 

15. C. F. Wilkinson, E. A. Hand, and N. T. Fleigelman,
"Essential Familial Hypercholesterolemia,” Annals
of Internal Medicine, 29:671, 1948.

 

CHAPTER II

REVIEW OF RELATED LITERATURE

In reviewing the literature relative to degenerative
cardiovascular disease, one is immediately impressed by the
extent and scope of the experimental work conducted. Inten—
sive animal research involving stress and serum cholesterol
levels has been conducted. The effects of activity and
cultural background on the levels of serum cholesterol have
been widely studied. Considerable research has been done
in the area of mental-emotional stress and its implications
on heart disease and the coagulation mechanism. Finally,
there has been an attempt to correlate genetic factors
with elevated serum cholesterol levels.

Atherosclerosis in all probability has no single
cause. t results, most likely, from a combination of
factors. Among the factors presently implicated are
heredity, diet, morphologic and chemical anatomy of the
blood vessel wall, arterial blood pressure, blood coag-
ulability, lipid content of the blood, sex, and stress.
Experimental work in all areas has been conducted on
animals as well as humans, with various theories being

proposed as to the mechanism of development.

lO

Pollack (55) suggests a mechanism for atheroclerosis
based on the hydropic swelling of the intimal endothelial
cells found in all subjects exposed to shock. He suggests
that (l) the physiochemical disturbances of plasma colloids
in shock initiate hydropic swelling of the intimal
endothelial cells followed by increased permeability,

(2) seepage of plasma through the defective endothelium
causes edema and hyalinemucoid changes of the subintima,
and (3) after the break~down of lipo-proteins and
reabsorption of most of the foreign material, liquids,
namely cholesterol, remain in the subintima where they act
as irritants and initiate the alterations we call athero-
sclerosis.

Enos and his co-workers (13) studying lesions in
coronary arteries indicate that the ”eddying" about the
bifurcation of the coronary arteries, occurring during the
diastolic recoil of the dilated coronary arteries, is a
dynamic factor in the mechanism of plaque formation.

A triangular scheme for the pathogenesis of
atherosclerosis is suggested by Miasmikov (47): (1) the
dietary factor, (2) the nervous impairment of vasomotor
regulation and exchange, and (3) the endocrine disturbance
of regulation of the lipid (especially cholesterol)
metabolism subordinate to it. While these ”theories” do
not explain the essence of the pathological process, they

do depict its most important aspects.

11

Animal Studies and Atherosclerosis

A relationship between the existence of modern man
and the caged wild animal is suggested by Ratcliffe and
Cronin. (61) Studying animals at the Philadelphia Zool-
ogical Garden, the authors found arterioclerosis, at
autopsy, in about two per cent of the mammals and birds
between 1916 and 1931. From 1931 to 1956 the frequency
of the disease increased by about ten-fold in mammals
and by about twentymfold in birds.

The most recent increases in the frequency of the
disease apparently have been independent of age and diets
but associated with rises in population densities. These
observations suggest that social pressure, through an
imbalance in adrenal secretions, has become a major factor
in the frequency of arteriosclerosis in the animals.

The relationship between domestication and athero—
sclerosis is also suggested by Wolffe,_et_al. (77) These
studies of the spontaneous occurence of atheromatosis and
atherohepatosis in wild ducks revealed the percentage to
be extremely small as compared with domesticated ducks and
geese. Domesticated Norway rats have been found to have
much smaller and less active adrenal glands. (79) As a
result of the atrophy of these organs, the rats were less
resistant to stress.

Considerable animal research has dealt with the

effects of physical exercise on blood lipids. Montoye,

l2

_g£_§1. (50), found animals subjected to swimming had
significantly lower total and free blood serum cholesterol
than inactive animals. The exercised animals were also
leaner (Specific gravity) with larger hearts and adrenal
glands.

A possible correlation between stress, serum choles-
terol levels, and atherosclerosis has received intensive
study in animal research. The forms of stress have been
variable as have been the results. Lyons, (Al) subjecting
cats to emotional situations, reports a transient hyper-
cholesterolemia. The increase is represented by a peak
rise in about half an hour, with a return to normal about
an hour after the excitation. The emotional hypercholes-
terolemia did not occur in sympathectomized animals. The
emotional situation involved tying the animal to a cat
board and rotating the animal and placement in a cage
near barking dogs.

However, Mann, (43) studying the effects of inanition
and cold, proposes reduction of total serum cholesterol as
a physiological response to stress. The plasma cholesterol
variations were shown to be associated with parallel
alterations in the size and cholesterol content of the
adrenal glands. The experimental work was done on rats
with the reactions mediated through the adrenal gland, but

not through the action of cortisone.

13

Diet and Cholesterol

 

The effect of various dietary regimens, with and
without physical activity, on the blood cholesterol levels
also provide some understanding of the atherosclerotic
syndrome. Kobernick, et a1. (UO), using rabbits, fed the
animals 28 grams of cholesterol for 60 days. During the
experimental period, one-half of the animals were exercised
(on a rotating drum), the other half remained sedentary.
The results indicate a significant diminution of athero~
sclerosis in the exercised animals as determined by visual
grading and by chemical analysis of the lipid content of
the aorta.

I

Uhley and Friedman (69) subjected animals to the
stress of electric shock while maintained on a diet high

in fat and cholesterol. Such rats, at the end of 10 months,
exhibited a greater degree of hypercholesterolemia and

hyperlipemia than the control animals which were not

-1

subjected to the stress of electrical shock. In addition,
considerably more coronary atherosclerosis was observed
in the experimental than in the control group. Blood
clotting times were also markedly faster in the experi~
mental group.

Fillios, et a1. (17), indicate gross athersclerosis
was produced in rats by feeding purified diets containing
cholesterol, sodium cholate, and thiouracil for periods

I‘

up to 363 days. The results indicate marked increases 01

14

serum cholesterol and beta lipoproteins with gross lesions
visible on the intimal surfaces of the vessels of the
animals observed. The lesions were most prominent in the
heart valves and the aortic arch.

Choline deficient diets have also been found to
result in sclerotic aortic changes. (73) The aortic
lesions consisted of intimal and medial deposits of lipids.
Vessels of choline-supplemented rats were found to be
entirely normal. The authors suggest that the vascular
lesions are primarily due to a relative lack of lipotropic
substances in the diet.

Studying rabbits, Miasmikov, (A8) found that ascorbic
acid reduced development of hypercholesterolemia and
lessens and retards the development of experimental
lipidosis in the aorta.

Mann, et_a1. (44), using a diet high in cholesterol
but poor in amino acids containing sulfur, report atherc-
sclerotic changes in monkeys. The deposit of lipids was
focused chiefly in the ascending aorta and carotid arteries.
In addition they indicated a rise in the cholesterol
concentration from an average value of 125 m. g. per cent

to 300-800 mg. per cent.

Activity and Cholesterol Levels
The effect of activity on the serum cholesterol levels
in man has received ccnsiderable study. McDonald (A6)

suggests the hypothesis that the incidence of coronary-

15

artery disease is inversely related to the general level
of physical activity and high fat diets. He found a
shortened recalcified plasma-clotting time (R. P. C.)
following ingestion of a high fat meal. In contrast, the
R. P. C. was lengthened following physical activity.
If shortening of the R. P. C. time can be

regarded as indicating a greater tendency to

fibrin formation in vivo, and if intravascular

fibrin formations is the important factor leading

to atheroma, it follows that any factor which

operates over lcng periods and which can be

shcwn to shcrten the R. P. C. time is likely to be

causally related to the development of atheroma.

In our opinion two of these factors are high

intake of fat and low level of physical

activity. (A6)

Taylor (67) studied death rates among clerks,
switchmen, and secticn-men employed by the railroad
industry for the purpose of obtaining information on the
relationship of physical activity and coronary heart
disease. He concludes that the men in sedentary occupations
have more coronary heart disease than those in occupations
requiring moderate to heavy physical activity.

Wolffe (76) studied 300 athletes actively engaged in
vigorous physical activity and 300 executives, all past
middle age, for evidence of atherosclerosis. His results
indicate definite or suggestive evidence of atherosclerosis
in a surprisingly high number of the executive group, with
curtailed physical activity. The athletes engaged in

vigorous physical activity showed little evidence of

atheroclerosis.

16

Keys, g§_§l, (36) found that in the 40-49 age range
the men in occupations demanding heavy physical labor have
lower cholesterol levels than men in lighter work, but
at other ages the cholesterol level appeared to be un-
related to habitual physical activity.

Golding (25) found total serum cholesterol levels
were significantly lowered by 25 weeks of endurance and
strength training. On the other hand, Johnson and Wong
(32) did not find a significant decrease in plasma choles-
terol following a competitive training program for
varsity swimmers. However, the results are complicated
by a low initial cholesterol value (187 mg. per cent) and
various interruptions in the training program (Christmas-
New Year Vacation and semester examination period).

Mann, g§_al. (#4) have shown a rather striking effect
of severe exercise on serum lipoproteins and cholesterol
levels in young adult males who consumed a diet high in
fat, both in total 053-174 gm.) and in animal fat, with a
daily caloric intake of approximately 6000. The serum
lipids were not increased as long as their caloric expend-
iture was great enough to prevent any appreciable weight
gain. As soon as forced exercise was stopped, there was a
gain in weight and an increase in serum lipid levels.

Montoye (51) has examined the effects of exercise on
blood cholesterol levels in middle aged men. His results

indicate that supervised exercise over a three month period

17

resulted in a fall in the lipid fraction in the blood, but
the change appeared to be related most significantly to the
weight reduction that occurred simultaneously.

Rochelle (62) determined cholesterol values before
and after a training period and before and immediately
after exercise. His results indicate a significant
decrease in mean cholesterol values between the pre-training
and post-training levels. He also found a significant
rise in cholesterol immediately following exercise. The
activity consisted of daily two mile runs for time.

Employing subjects between 30 and 60 years of age,
Chailley-Bert (9) concludes that ”actifs” subjects have a
lower cholesterol level than sedentary subjects. In a
second series of experiments three initially sedentary
subjects reflected a decrease in cholesterol levels -
following a period of physical training.

Bortz (4) suggests that exercise allays anxiety with
resultant relaxation; exercise acting as natureis sedative
for the tense, harassed and tired business man. He
indicates that psychological factors frequently outweigh
physical stress in alteration of heart rate, blood pressure,
clotting time and plasma lipids.

In contrast to these findings Keys, et_al. (37),
as part of their epidemiologic studies, have estimated the
physical activity of the groups of men they have studied

in several countries and have concluded that ”differences

18

in physical activity do not explain the large differences
in serum cholesterol which are found when groups with

different dietary habits are compared."

Cholesterol Levels in Different Societies

 

While atherosclerosis afflicts members of all
societies, there is considerable evidence that the incidence
varies with different cultural groups. Bronte-Stewart (5),
studied 383 men aged 40-58 residing in the Cape Peninsula
area. Three major ethnic groups--Europeans, Bantu, and
Cape Colored--were studied in terms of serum—cholesterol
and serum lipoprotein levels. Highly significant differ—
ences in the mean total cholesterol levels, and particularly
in the cholesterol level in the beta-lipoprotein fraction
of the serum were found. The Europeans had the highest
cholesterol level and the Bantus the lowest. These differ~
ences may reflect the variations in average diets, with the
Europeans consuming more than twice the fat that is consumed
by the Bantus.

Keys, et_al. (39) studied two populations of Spanish
men~—one group from the professional class, the second
consisting of underprivileged workers. The cholesterol
data of the study conform to the theory that total dietary
fat is a primary determinant of the serum cholesterol
concentration in men beyond their twenties.

The results of these studies support the hypothesis

that dietary intake of fat influences the level of serum

\O

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cholesterol, particularily that of the beta—lipoproteins,
and in turn may be one of the major factors influencing the
pathogenesis of coronary heart-disease.

Gordon, et_al. (26) qualify this hypothesis suggesting
the saturation of the fat is responsible for the cholesterol-
producing effects. His conclusion reflects the decreased
cholesterol levels he found upon ingestion of unsaturated
fat and sunflower seed oil; while hydrogenated coconut fat,
fed under similar conditions, produced a sustained rise of
the serum cholesterol level.

Karvonen, §§_al. (33), studying lumberjacks in
Finland, found that in spite of an unusually large con~
sumption of fat, the serum cholesterol levels of the lumber—
jacks were similar to those of other males in the local
population. That the serum cholesterol level was no higher
was probably due to a depression of the serum cholesterol
content by heavy work. However, many of the lumberjacks
did have cholesterol values in the range where the risk of
atherosclerosis is thought to be increased (260 mg./100 ml.)

Gsell and Mayer (30) studied the serum Cholesterol
level of a population from a Swiss Alpine village and a
working class of similar ethnic origin living in an urban
area. Despite their high caloric intake and the high
saturated fat content of their diet, the villagers showed
a low serum cholesterol level, compared to that observed

in poor, underprivileged populations on low fat diets.

20

The differences cannot be explained by differences in
weight, adiposity, altitude and climate, smoking habits,
or serum magnesium. It is suggested that the striking
difference in physical activity may be reSponsible for the
difference in cholesterol level.

The nutritional status, serum-cholesterol values, and
incidence of atherosclerosis were investigated in recent
and early Yemenite immigrants in Israel. (68) Serum-
cholesterol and total lipid levels were significantly
lower among the recent immigrants. 0n the other hand,
the atherosclerosis mortality rate was four times higher,
for the same age and sex, in the early Yemenite immigrants
and approached the rate for European Jews.

Brunner and Manelis (6) studied the influence of
physical exercise on the incidence of myocardial infarction
in communal settlements in Israel. Eighty—five hundred
people were employed in the study. The incidence of
myocardial infarction and mortality rate was three times
greater in the sedentary workers than among the ”active”
workers. He concludes that "these findings underline the
importance of physical exercise as a factor in the pre-
vention of myocardial infarction.H

Studying Japanese in Japan, Hawaii and California,
Keys, gt_al. (38) found that the incidence of coronary
heart disease is related to the proportion of calories in

the diet provided by fats, particularly the common saturated

fats. The serum cholesterol concentration showed a linear
relationship to the percentage of calories provided by
fats in the diet. He concludes that these differences
could not be accounted for by differences in climate, rel-
ative obesity, physical activity, the use of alcohol and
tobacco, the concentration of protein in the diet, or the
intake of ”essential” fatty acids.

Several investigators have studied the effects of
diet on serum lipoproteins other than cholesterol.

Walker (70) following the Sf 12-20 and Sf 20-100
lipoprotein fractions and serum cholesterol found a direct
relationship between state of nutrition and the level of
Sf 12-20 and serum cholesterol levels. While not claiming
to exclude dietary fat or cholesterol as important causal
factors in the genesis of atherosclerosis, he suggests that,
in humans, total caloric intake may be a much more important
factor.

The relationship of total caloric intake and increased
Sf 12-20 lipoprotein fraction is significant in light of
studies conducted by Gofman and his co-workers. (2M) They
conclude that Sf 12-20 lipoproteins have at least a two-
fold, and up to a possible tenfold, higher relationship
with atheroclerosis than does the total serum cholesterol.
The Sf 30-100 lipoproteins also indicate a strong

association with atherosclerosis.

R)
R)

Groen, e£_al. (27) in a particularly interesting study,
investigated the comparative influence of nutrition and
”way of life" on the serum cholesterol, prevalence of
hypertension, atherosclerotic (coronary) heart disease,

nd angina pectoris among 181 Trappist monks who lived
on a vegetarian diet and 168 Benedict he monks who lived on
a "Western” diet.

The results indicate the average blood cholesterol
level was significantly higher among the Benedictine monks
than among the Trappist monks. However, there was no
difference in prevalence of myocardial infarcts, angina
pectoris, hypertension, and electrocardiographic signs of
atherosclerotic (ischemic) heart disease between the two
groups. These results support tne hypothesis that acute
occlusion of a major coronary artery is less related to
nutrition and blood cholesterol than to certain psychosocial
factors (stress) common to the western way of life, against
which both the Benedictine and Trappist monks are protected.

That hypercholesterolemia is not always observed in

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atherosclerosis is indicated by Paterson and Cornish.
They cite the results of 50 autopsies on patients with
atheroclerosis, in whom blood cholesterol was not elevated.
They indicate no relationship between blood cholesterol
levels and the degree of atherosclerotic development.

Keys (35) also concludes that a substantial measure

of control of the development of atherosclerosis in man

23

may be achieved by control of the intake of calories and

of all kinds of fats, with no special attention to the

 

cholesterol intake. He feels that fats, rather than choles-
terol, are correlated with the high incidence of athero-
sclerosis.

Finally, Wilkinson (73) concludes tnat the absolute
amount of carbohydrate, fat, protein, and cholesterol in
the diet had no demonstrable relation to the level of total

serum cholesterol.

The Effects of Stress

 

The etiology of atherosclerosis is a highly complex
and, at best, poorly understood phenomena. Investigators
have implicated dietary intake, physical inactivity, faulty
metabolic mechanisms, and several other factors as respon—
sible for its manifestation. Also implicated is the stress
of modern societies‘ highly technical, dateline oriented
existence.

The opinion of many present-day investigators is
reflected in the following editorial comment of Oliver and

Boyd in the British Heart Journal:

 

The remarkable social and economic changes of the
century are undoubtedly stressful to some people.
Increasing stress might favor the development of
clinical coronary disease by disturbing normal
endocrine balance and thus influencing both cholesterol
metabolism and the coagulation and fibrinolytic
systems. Similarly, long, continued environmental
stress might disturb the autonomic control of the
coronary arteries. (53)

Selye (66) also suggests that hypertensive cardio-
vascular disease may be caused by stress. The line of
reasoning may be summarized as follows: (1) stress is
known to increase the secretion of adrenal corticoids;

(2) corticoids, when administered in excess (as in Cushing‘s
disease, or primary aldosteronism), cause hypertensive
cardiovascular disease; hence, manifestation of the disease
through stress.

Russek, (64) in a discussion of the etiology of
coronary artery disease, suggests that sympathetic adren-
ergic effects on vascular tissue metabolism as a result of
stress are factors in the develOpment of atherogenesis.

He indicates that the deposition of cholesterol in the
intima is accelerated and intensified by epinephrine and
that catecholamine-phospholipid compounds seem to possess
a particular affinity toward arterial tissue. The
relatively large quantities of catecholamines found in
arterial tissue may, by injuring the intima, enhance
subsequent lipid deposition. In addition catecholamines
by diminishing myocardial efficiency and by wasting oxygen
are capable of inducing severe potentially necrotizing
myocardial hypoxia. The evidence strongly suggests that
the maintenance of normal cholesterol metabolism and of
the coagulation-fibrinolysis system could be disturbed by

an alteration of the physiological endocrine balance

resulting from emotional stress.

He concludes that;

emotional stress acting through the cerebral cortex
and hypothalmus via neurohumoral and humoral
mechanisms may be responsible for (1) an increase
in the lipid receptiveness of the vascular walls,
(2) an elevation of blood cholesterol, and (3)
an acceleration of blood clotting time.

Rabb (60) indicates that the catecholamines,
norepinephrine and epinephrine, occupy a prominent position
in the "dynamic, metabolic and structural pathogenic
mechanisms” of the various cardiovascular disorders. While
the catecholamines are essential under normal physiologic
conditions their potentially oxygen wasting, necrotizing
and vasoconstrictor properties may be accentuated by
certain conditions including deterioration of sympatho-
inhibitory and cholinergic counter regulatory mechanisms.
He suggests that this balance may be maintained by
augmenting vagal tone through physical exercise of an
endurance nature.

Investigating the thesis that sympatho-adrenergic
over-activity in the heart muscle results in a Hloafers
heart," Babb, (57, 58) studied 360 men with widely varying
exercise habits. The parameters under investigation were
resting heart rate and length of the isometric period
of the left ventricle. The latter measure serves as a
sensitive indicator of neurovegetative influences, in that
a shortening of the isometric period occurs under sympa—

thetic adrenergic action, a lengthening under cholinergic

and/or sympatho-inhibitory influence. The results

26

indicated a proportionate relationship between the degree

of habitual physical exercise and the neurovegetative

status of the heart. With increasing exercise habits, there
was a linear decrease of sympathetic tone (diminution of
heart rate and len thening of the isometric period) and

vice versa. The shifts in the neurovegetative equilibrium
were shown to be reversible by physical training and by
prolonged lack of exercise. Smoking habits did not appear
to have any effective on the cardiac neurovegetative

status.

Haab (59) has also found a higher incidence and

degree of myocardial necroses in wild rats, than among
domesticated animals, when exposed to various stresses.

He suggests that in the wild rats the motor and cardiac
"fight and flightH reflexes are less blunted than in the
domest cated white rats, so that the suppression of the
musculomotor outlet in isolation cages may have rendered
more toxic the concomitant adrenergic effects on the heart
muscle. This lack of activity may contribute to the
ugmented myocardial vulnerability of civilized, immobilized
man under modern conditions of emotional frustrations and
stresses.

The prophylactic effect or exercise has been suggested

by recent research. Selye (65) has advanced a theory of
cross-resistance in stress related cardiopathies.

Basically, he suggests that stress, if applied prior to

27

certain cardiotoxic agents, can protect against the latter.
Animal experimentation indicates that pretreatment with
moderate amounts of well tolerated stressors, such as

cold baths or muscular exercise, are the most effective

and most generally applicable cardioprotective agents 0

U)

far revealed.

Studying periodic mental stress, Grundy and Griffin
(:9) observed the effects of quarterly final examinations
on serum cholesterol levels in two large groups of freshman
medical students. During the two examination periods
studied (winter term final and spring quarter final) the
mean cholesterol levels were elevated significantly over
control periods of relative relaxation. The two groups of
50 and 47 male students showed a 16.5 per cent and an ll
per cent increase in serum cholesterol levels during winter
and Spring term quarter examinations respectively.

In another similar study Grundy {28} found the
cholesterol levels increased 23 and 27 per cert during the
examination periods as compared with contro
relative relaxation. The changes in standard Svedberg
flotation unit (Sf) O~l2 lipoprotein classes were not signi-
ficant but the standard Sf 12-400 classes increased 51 and
56 per cent for the two groups. Gofman has found increased
levels of this lipoprotein class to have a higher statis—
tical relationship to the occurrence of coronary artery

disease than those of any other lipoprotein class. {2b}

Wertlawe,.et al, {71; also report a significant
increase in mean value for serum cholesterol accompanying
the mental and emotional stress of examination week in a
group of an apparently ne"l-ny, male medital students,

The mean value for seium cholesterol was found to ircrease
to 238 mg. per cent which represehted and ll per cent
increase over the control mear value of Elh mg, per cert

Since there is a -lose relaiio 5 ip bet een rervo.:
stimuli and endo;-ine gland fanTio . emotional stress
actifmg'througgi'tre cemwecral (”urtex arri'tyootm21uwis may
exert important effects .po the end; rine system. Cor-
siderable evidence ttests t; the Ia t “n"t tie mainteo: e
of normal cholesterol metehoTism ard of t*e ,oagulation—
fibrinolysis system ,an be distu’ter by SlfCSS-inOaiea
alteratior of the ptvsiological erdo‘ri e tale; e. {t3l
Of particular interest is tte effett of adrenal se refio,s
and tre ’3 ldEK,€ of ”lteziitemia.

"Powers.€fy‘; fougml t-at aqhéevale izrrized (£255 mair—
tained on desoxvcortirost.ro1e acetate mafifest a :‘o-
gressive depletion of plasma phbsiVOllpld agd roleS‘t ‘1.
The daily addition of molestercl to {he Eirjha c: w-
prevented the hypoligeni; :esrorse and resulted i. sig'i-
ficant elevation in the coh‘emtratio' of plasma holest- l.

The feedin of ar identi al amount of armlesteSCJ
to dogs with intact adre1als was not a357_ia“ed wit a
alteration in plasma lipid leve s. A signifi*a,t

1);)

29

contribution of the adrenal gland to the regulation of
plasma lipid metabolism in normal and cholesterol-fed dogs
is indicated.

Russek (63) suggests that various forms of stress
stimulate the anterior pituitary to discharge ACTH, the
latter stimulating the adrenal cortex to discharge
glucocorticoids; the corticoids then stimulate the pos-
terior pituitary to release a "lipid~mobilizer” substance
which acts on the fat depots to rel“ase neutral fats into
the portal circulation. If the liver is not capable of
handling this fat load, neutral fat is discharged into
the peripheral circulation, in association with choles-
terol, resulting in a generalized hyperlipemia. it appears
possible, therefore, that the metabolism of lipid from the
fat depots by this mechanism may be an important cause of
hypercholesteremia and coronary atherosclerosis,

LBall and Jungas (3) offer the following scheme in
explanation of accelerated release of fatty acids from
adipose tissue and increased oxygen consumption by certain

hormones including adrenalin;

 

 

Glucose I

 

Glycogen Cell
rGlucose-6~P04 --+Glycerol-~-PO —______, FatH
O I
R
AMP + ACYL, COA M

Adipose Tissue

 

 

 

O I

2 ADP

 

+
til-(7'

/,2
/ \ fin n . a '
.Fotty Aold + Glycerol

 

 

 

 

 

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BLOOD STREAM

Under normal conditions a continual hydrolysis of
fat to fatty acids and glycerol is suggested which is bal—
anced by a re—esterfication of the fatty acids to form new
triglycerides" In the presence of adrenalin an acceleration
of the lipolysis of fat to fatty acids and glycerol takes
place. As the fatty acids begin to accumulate in the
tissue, two pathways are open to them; they may leave the

tissue to enter the blood stream or they may undergo

U)
r

re-esterification at an increased rateu A third
possibility exists--they may proceed through acetyl CoA

to steps leading to their oxidation, Under conditions of
physical activity and the increased demand for energy, this
third possibility is probable,

Ellis, (11) in reviewing the literature related to
the metabolic effects of epinephrine, indicates that epi'
nephrine appears to control the cellular concentration
of active phosphorylase, the enzyme which catalyzes the
reversible reaction glycogen + phosphate : glucose—l-
phosphate, Since the first step in glycogenesis is the
slowest, and thus the rate-limiting reaction, epi~
nephrine, by increasing the level of active phoSphorylase,
causes an increased glycogenolysis, which, in turn, causes
an increase in the cellular concentration of glucose~l—

phOSphate and glucose~6~phosphate, in the liver the

U)

increased concentration of glucose~6-phOSphate acceleratc
the rate of glucose liberation and thus causes hyperglycemia“
While normally epinephrine depresses glucose utili-
zation, under conditions of physical exercise epinephrine
increases glucose consumption, (23) Just as the effect of
insulin on glucose utilization is magnified by muscular
exercise the effect of epinephrine in reducing glucose
utilization may be overcome by exercise. If this is the
case, epinephrine hyperglycemia and muscular exercise
would be acting synergistically to increase carbohydrate

metabolism.

32

The Specificity of the adrenal medullary secretions
to various types of stress has been investigated recently.
Funkenstein (23) suggests that the secretion of adrenalin
or nor-adrenalin is specific to the form of stress. Anger
directed "outward" is associated with secretion of nor-
adrenalin, while depression and anxiety are associated with
the secretion of adrenalin. There is some evidence that
the specificity of the secretions is controlled by centers
in the hypothalmus leading to production of nor-adrenalin
in the first case and adrenalin in the second.

This concept was also investigated by Elmodjian and
his co-workers. (12) The excretion of epinephrine and nor-
epinephrine was determined in athletes (hockey players and
boxers), normal subjects in anticipatory states, and
psychiatric subjects. The results support the hypothesis
that active, aggressive emotional displays are related to
increased secretion of norepinephrine, with or without
increased excretion of epinephrine, whereas tense, anxious
but passive emotional displays are related to increased
excretion of epinephrine in association with normal

excretion of norephinephrine.

 

Stress and Coagulation

The precise mechanism responsible for the coagulation
of blood is unknown, Basically the coagulability of blood
depends upon an intricate balance of the ”procoagulants”

and "anticoagulants” focused in the blood and related tissue.

33
In general the coagulation process follows three
sequential steps as suggested by Guyton. (31)

First, a substance called thromboplastin is released
from the injured tissue area. Second, the
thromboplastin initiates a series of chemical re-
actions in the plasma which eventually converts pro-
thrombin into thrombin. Third, thrombin then acts
as an enzyme to convert fibrinogen into fibrin threads
that enmesh red blood cells, platelets, and plasma
to form the clot itself.

Among the various precipitants of the coagulatory
phenomena is emotional stress. The hastened coagulation

of blood as a result of emotional reactions is believed by

CI)

Cannon and Mandenhall (8) to be mediated by planchnic
stimulation of the adrenal gland. The discharge of epine-
phrine by the latter organ (adrenal) is believed to alter
or be altered by some liver function which produces the
observed changes in coagulation.

Several studies are reported in the literature
investigating the relationship of emotional states and the
coagulability of blood. Friedman, §t_aln (21) studied
accountants selectively chosen as a self-controlled group
for studying the effects of cyclo—occupational stress upon
serum cholesterol and blood clotting time, since their
routine work schedule is interrupted by urgent tax dead—
lines, associated with severe occupational stress. Marked
acceleration of blood clotting time consistently occurred at
the time of maximal occupational stress, in contrast to
normal blood clotting during periods of respite.- The results

could not be ascribed to any changes of weight, exercise or

diet.

3A

Katz and Zweibel (3A) studied sixteen psychotic
patients employing electroshock therapy as the stressor.
Using three groups, the subjects were subjected to the
following regimen: Group I was subje ted to ele2tr oshoek
therapy accordih to normal hospital routine (announced);
Group II was handled in iden :L al fashion with the excep
that no electric current was applied; and Group III comm
sist ed of subjects who were suddenly subjected to EST
without anticipating it. Blood sugar levels were Cerermined
lmefore and after the electro-shosk therapy. The results

indicate a marked shortenin~

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an elevation of blood sugar pre~eeding and following n31,
which was most marked immediately before and after electro-
shock therapy. The de viaoio‘s of the blood coagulation
time and blood sugar levels ihdi ate some adrenomedullary
stimulation which is irdepende t of EST, but rather results

from anticipation and anxie H

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t1).dyL ng tne effects of behavior patterns. Friedman
a:hd Rosenman (2 0; found clini al coio orary disease wcs
seven times more prevalent in the group ctaracterized by
intense, sustained drives for achievement and as being
continually involved in rort HtiOh and deadlines. A
hastening of blood clotting time was not observed in all

of the men of this group but only in those exhibiting the

’"Vx‘

most fully developed form of the behavior patt e

35

De Long, e§_al. (lo) subjected rats to a form of stress
involving a sense of urgency and apprehension by periodically
and alternately charging half of the floor of a specially
constructed cage, forcing migration to the uncharged area.
Three groups of rats were placed in the cage for l, 3 and
6 hours respectively. At the end of the stress period the
clotting time of the experimental animals was taken and
compared to controls. The results indicate that in
addition to marked behavior differences, the stressed rats
exhibited significant hastening of the coagulation time.

Employing a similar stress situation, Friedman and
Uhley (18), found blood cholesterol and phospholipid levels
were similar in the experimental and control animals; both
being high since they were on an atherogenic diet.

However, there was a striking difference in blood clotting
time as determined in capillary glass tubes. The average
blood clotting time for the experimental group was 64
seconds; that of the control group averaged 111 secondso

While many investigations implicate the adrenals in
the coagulatory process, some doubt is raised by Friedman
and Uhley. (19) While they observed a marked fall in
adrenal cholesterol content during a period of stress, the
hastening of blood coagulation after exposure to stress was
not altered by removal of the adrenal glands. It was con-
cluded, therefore, that the coagulation phenomenon observed

was independent of adrenal activity.

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Ox

Monkhouse (49), indicates that lipids, especially the
phosphatides, play an important part in the reactions which
result in the formation of a blood clot. Several other
investigators have shown that lipemic blood may hasten
coagulation in vitro (7, 22), or shorten plasma clotting
time induced by an incomplete thromboplastin, (42) The
effect of fat ingestion on coagulation is possibly asso-
ciated with changes in circulating phosphatidyl-ethanolamine
(2, 52), which may act as a thromboplastin,

Although it is unlikely that any changes in the
coagulability of blood as a result of variations in lipid
level are important under normal blood flow rates and
healthy endothelium, there may be a relationship when blood
flow is impaired and degenerative changes in the vessel

wall exist,

Stress and Adrenal Histology

 

That the stress of physical activity results in
enlarged adrenals has been demonstrated by Van Euss and his
co~workers. (78)

Fiske and Lambert (16), studied the effects of light
on adrenal weight of the rat. Using intact and gonadecto-
mized rats they found that exposure to continuous light
had no effect upon adrenal weight of the intact male animal
but did result in a marked reduction in size in the intact

females.

37

On the other hand, castration induced an increase in

adrenal weight irrespective of the amount of light to which

Cf.

he animals were exposed. Following spaying, the adrenals
were decreased in size in both the rats under continuous
light and their day-night controls.

Eranko and Harkonen (14), using albino mice found
that swimming to exhaustion daily (e days a week) for 30
days resulted in differences in adreralin and nor—adrenalin
concentration. Swimming was found to cause an increase in
the volume of the nor—adrenalin—containing medullary
parenchyma cells without affecting tte volume of the
adrenalin-storing cells.

Absolute volume of the medulla did not differ signie
ficantly from the control group” However, when the
medullary volume was expressed in terms of body weight ratio,

there was an increase of about 20 p r cent in the mean

(D

volume of the medulla of the group killed immedia*ely 55*6T

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swimming. After one month there were no '

’1

-igrj Ii a?fit dii‘fer-
ences between any of the variables measured.

Studying rats, Eranko and his co—workers (lbl, found
that a two month swimming program resulted in a significant

increase in the adrenal weight as well as the volume of tr

(D

adrenal medulla. Both the Hnor-adrenalin containing” and

+

the ”adrenalin containing” medulla . tissue had increased

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volumes with the adrenalin containing tissue reflecting

the greatest increase.

Cholesterol Levels

As indicated previously,

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References

 

David Aldersberg,_et al., Hincidence of Hereditary
Hyper—Cholesterolemia,” Circulation, 2 A75, 1950.

 

 

wied, "Fhospholipids
and Inhibitor s o f

P. Backhan, M. J. Newlands, a;; .
r S
LL, 1956»,

from Brain Tissue as Accele
H

d
to

r F
a r
Lancet, 2: 20

Blood Coagulation,

E. G. Ball, and R, L. Jungas, HOn the Action of
Hormones which Accelerate the Fate of Ox;ygen Consump—
tion and Fatty Acid Release in Fat Adipose Tissue
in Vitro,' Proc edings of Latioral Arademy of S ierce

(47: 932- 924.1, 1961.

 

 

E. L. Bortz, 'Exercise, Fitness and Ag C,ing,'i CollQQiim

____—.

on the Scientific Aspects of Exercise and Fitness _,
University of IlllIlOlE, De ember, 1959.

 

B. Bronte-Stewart, A. Kevs, and J. F. Brock, HSerum
Cholesterol, Diet, and Coronary Heart Disease, An
Inter-Racial Survey in Cape Peninsula, Bagggj.
2:1103, 1955 H

D. Brunner, and G. Manelis, ”Myocardial Infarction
Among Members of Communal Settlements in Israel,”
Lancet ll, lOA9—1050, l9o0.

 

-~ _.o ,_ ”W." .1, e- . ‘ .20 ,, ,1. “J...
F. BruZitia, au:<j i. reys, tlooo Loagula lOe H1 c:

a Fat IEa 1. ' ..ircui3:io: lA:th, l950.
M. B. Cannon, and W, L. Menderhall, ”The ":astenirg of
o 'x c .0 J ‘3,” _ 7,. A o _ T"..\-., . . _‘ '1
Coagulation by Stimulating tie .tia ::iiz nerves,

if

American Journal of L“”°‘ologx, 3A ijA3, l9l

a,

 

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E. De Long, H. N. Uhley, and M, Friedman, ”Changes in
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l96zA29, 1959.

S. Ellis, ”The Metabolic Effects of Epinephrine a;r d
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A0

12. F. Elmadjian, J. M. Hope, and E. T. Lamson, "Excretion

 

of Adrenalin and Various Emotional States," Journal
of Clinical Endocrinology and Metabolism, 17:608-620,
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13. W. F. Enos, R. H. Holmes and J. C. Beyer, ”Coronary
Disease Among United States Soldiers Killed in Action
in Korea,” Journal of the American Medical Association,

15221090, 1953.

 

1A. O. Eranko, M. Karvonen, ”Long-Term Effects of Muscular
Work on the Adrenal Medulla of the Mouse,” Endocrinol-
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15. O. Eranko, M. J. Karvonen, and L. Raisanen, ”Long-Term
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the Rat," ACTA Endocrinology, 39:285-7, February,
1962.

 

16. V. M. Fiske, and H. H. Lambert, ”Effect of Light on
the Weight of the Adrenal in the Rat,” Endocrinology,
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17. L. Fillios, et al., ”Experimental Production of Cross
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18. M. Friedman, and H. Uhley, ”Experimental Stress, Blood
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Academic Press, Inc., 1959.

 

19. M. Friedman, and H. N. Uhley, "Role of the Adrenal in .
Hastening Blood Coagulation After Exposure to Stress,”
American Journal of Physiology, 197 (l): 205, July,
1959.

20. M. Friedman, and R. H. Rosenman, "Association of
Specific Overt Behavior Pattern with Blood and
Cardiovascular Findings,H Journal of the American
Medical Association, 169:1286, 1959

 

 

 

21. M. Friedman, R. H. Rosenman, and V. Carroll, "Changes
in Serum Cholesterol and Blood Clotting Time in Men
Subjected to Cyclic Variations of Occupational Stress,”
Circulation, 173852—861, May, 1958.

 

2A.

25.

30.

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32.

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H. W. Fullerton, W. J. Davie, and G. Anastosopaulos,
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D. H. Funkenstein, "Physiology of Fear and Anger,"
Scientific American, 192:7A~81, May, 1955.

 

J. w. Gofman, et al., ”Blood Lipids and Human Athero-
sclerosis,” Circulation, 5:119—13A, 1952.

 

L. A. Golding, "The Effect of Physical Training Upon
Total Serum Cholesterol Levels,“ Research Quarterly,
32:A99, December, 1961.

 

H. Gordon, et al., ”Dietary Fat and Cholesterol
Metabolism,i Lancet, 221299, 1957.

J. J. Groen, et al., ”The Influence of Nutrition and
Ways of Life on Blood Cholesterol and Prevalence of
Hypertension and Coronary Heart Disease Among Trappist
and Benedictine Monks,“ American Journal of Clinical
Nutrition, lOzA56, 1962.

 

 

S. M. Grundy, and A. C. Griffin, “Relationships of
Periodic Mental Stress to Serum Lipoproteins and
Cholesterol Levels,“ Journal of the American Medical
Association, 1713179A~6, November 28, 1959.

 

 

s. M. Grundy, and A. c. Griffin, "Effects of Periodic
Mental Stress on Serum Cholesterol Levels,” Circula-
tion, 19:A96:98, April, 1959.

 

D. Gsell, and J. Mayer, Y"Low Blood Cholesterol Asso—
ciated with High Calorie, High Saturated Fat Intakes
in a Swiss Alpine Village Population,” American
Journal of Clinical Nutrition, 10:171, 1962.

 

 

Arthur C. Guyton, Textbook of Medical Physiology,
Philadelphia: W. B. Saunders Co., 1961, p. 202.

 

T. F. Johnson, and H. C. Wong, ”The Effect of Exercise
on Plasma Cholesterol and Phospholipids in College
Swimmers," Research Quarterly, 32:51A, December,

1961.

 

M. J. Karvonen, et al., ”Diet and Serum Cholesterol
of Lumberjacks,L British Journal of Nutrition,
152157, 1961. '

 

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34. E. Katz, and A. Zwiebel, ”Changes in B1ood Clotting
Time and Blood Sugar Levels in Relation to Electro-
shock Therapy,” Psychosomatic Medicine, 16:33A, 195A.

 

35° A. Keys, "Human Atherosclerosis and the Diet,"
Circulation, 5:115~118, 1952,

 

36. A. Keys, M. J. Karvonen, and F. Fidanza, "Serum Choles-
terol Studies in Finland," Lancet, 175-178, 1958.

37. A. Keys, et al., ”Physical Activity and Diet in
Populations Differing in Serum Cholesterol,fi
Journal of Clinical Nutrition, 35:1173, 1956.

 

38. A. Keys, et al., ”Lessons from Serum Cholesterol Studies
in Japan, Hawaii and Los Angeles,” Annals of Internal
Medicine, A8283, 1958.

 

 

39. A. Keys, et al., ”Studies on the Diet, Body Fatness
and Serum Cholesterol in Madrid, Spain,” Metabolism,
~3:195-212, 1954.

A0. S. D. Kobernick, 3. Nivayama, and A. C. Zuchlowski,
"Effect of Physical Activity on Cholesterol Athero-
sclerosis in Rabbits,” Proceedingswof the Society
of Experimental Biology and Medicine, 96:623,

1957.

A1. C. Lyons, ”Emotional Hypercholesterolemia,“ American
Journal of Physiology, 98 156—162, 1931.

 

 

 

 

 

 

A2. N. F. MacLogan, and J. D. Billimaria, ”Food Lipids
and Blood Coagulation," Lancet, 2:235, 1956.

A3. G. V. Mann, and H. S. White "influence of Stress on
Plasma Cholesterol Levels,‘ Metabolism, 2:47, 1953.

 

AA. G. V. Mann, et al., ”Exercise in the Disposition of
Dietary Calories, Regulation of Serum Lipoproteins
and Cholesterol Levels in Human Subjects]' Mew
England Journal of Medicine, 253:39A, 1955.

 

A5. G. V. Mann, S. B. Andrus, and A. McNally, "Experimental
Atherosclerosis in Cebus Monkeys,” Journal of
Experimental Medicine, 98:195-218, 1953.

 

 

A6. G. A. MCDonald, and H. W. Fullerton, "Effect of Physical
Activity on Increased Coagulability of Blood After
Inggstion of a High Fat Meal,“ Lancet, 2:600—l,

195 .

247.

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53.

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55.

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A. L. Miasmikov, "Atherosclerosis; Occurence, Clinical
Forms, and Therapy,” U. S. Government Printing

Office, Washington, D. C., 1962. Translation Sponsored
by National Health Institute-~Mational Institute of
Health, Bethesda, Maryland.

A. L. Miasmikov, "Influence of Some Factors on Develop-
ment of Experimental Cholesterol Atherosclerosis,
Circulation, 17:99, 1958.

 

F. C. Monkhouse, “Blood Coagulation and Lipid
Metabolism,” American Journal of Clinical Nutrition,
8:1—6, JanuaryeFebruary, 196C.

 

f Exercise and Jllk
s terol ir Ra ts,
’ l9t2.

H. J. Montoye, 9:191. Effe

Consumption on Blood Serum Siol
Research Quarterly, 33 A30, Oil.

“r
V'Q"

(DO

.1
o
5
V—\

 

_ _ its of Exercise on
Blood Cholesterol in Middle Men,‘ Journal of

H. J. Montoye, et al., "Tne Eff
as
Clinical Nutrition, 7:139, 2’

 

5.42
J"

 

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J. R. O‘Brien, ”Effects of a al of Eggs and Different
Fats on Blood Coagllat1113y, Lagge:, 2:232, 1956.
M. F. Oliver, and C. S. Boyd, “Some T
the Artiology of Coronary Artery Dis

Heart Journal, 19:582, 1957.

 

J. C. Paterson, B. R. Cornish, and E. C. Armstrong,
”The Serum Lipids in Human Atherosclerosis,
Circulation. 13 (2): 22 -23A, 195h.

 

O. J. Pollack, "An Etiologic Concept of Atherosclerosis
Based on Study of Irfi Ef‘fi‘ 1 Alterations After Shock,
Circulation, 5:539, 1952.

 

B. S. Powers, and N. J. DiLuZio, "Dietary Jholesterol
. —. - 9 H
and Adrenal Regulation of Plasma Lipids, Ameri a:r.

Jourr al of Physi ologL 195:166-179, October, 1953

 

W. Raab, ”Metabolic Protection and Reconditioning of

the Heart Muscle Through Habitual Physical Exercise,"
Annals of Internal Medicine, 53; 87-105, 196C.

 

W. Raab, et al., ”Adrenergic Preponderenc e Increases
with Decreasing Exercise Habits,“ American Journa

of Cardiology, 5: 300, 1960.

 

W. Raab, J. P. Chaplin, and E. Bajusz, Myocardi lal
Necrosis in Wild Rats Produced by Anxiety, '
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61.

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H. C. Ratcliffe, and M. T. Cronin, "Changing Frequency
of Atherosclerosis in Mammals and Birds in the
Philadelphia Zoological Garden," Circulation,
18:41-52, 1958:

 

R. H. Rochelle, "Blood Plasma Cholesterol Changes
During a Physical Training Program," Journal of
Sports Medicine and Physical Fitness, 1:63, September,
1961.

 

 

H. I. Russek, "Role of Heredity, Diet, and Emotional
Stress in Coronary Heart Disease,” Journal of American
Medical Association, 171:503-8, October, 1959.

 

 

H. I. Russek, "Emotional Stress and the Etiology of
Coronary Artery Disease,” American Journal of
Cardiology, 2:129, 1958.

 

Hans Selye, The Pluricausal Cardiopatnies, Springfield;
Charles C. Thomas, 1961, p. 3A8.

 

Hans Selye, The Physiology and_Pathology of Exposure
to Stress, Montreal: Acta, 1950.

 

 

H. L. Taylor, §§_a1., ”Death Rates Among Physically
Active and Sedentary Employees of the Railroad
Industry,” American Journal of Public Health,
52:1697, October, 1962.

 

M. Toor, et al., “Serum Lipids and Atherosclerosis
Among Yemenite Immigrants in Israel,7 .Lancet,
2:12.70-3, 19570

H. N. Uhley, and M. Friedman, HBlood Lipids, Clotting
and Coronary Atherosclerosis in Rats Exposed to

a Particular Form of Stress," American Journal of
Physiology, 197:396-8, August, 1959.

 

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71.

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P. T. Wertlake, et al., Welationship of Mental and
Emotional Stress to Serum Cholesterol Levels,”
Proceedings of Society of Experimental Biology and
Medicine, 97:163-165, January, 1959.

 

 

G. F. Wilgram, W. S. Harcroft, and C H. Best,

"Abnormal Lipid in Coronary Arteries and Aortic
Sclerosis in Young Rats Fed a Choline Deficien tDiet,
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There a Relationship Between Diet and Blood Choles—
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1950.

C F. Wilkinson, E. A Hand, and N J. Fliegelman,
"Essential Familial Hypercholest erolemia," Annals of
Internal Medicine, 29: 671, l9u8

 

 

 

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tionship Between Dietary Composition and the Blood
Cholesterol Level,” American Heart Journal, 36:A72,

1948.

J. B. Wolffe, "Continual Vigorous Physical Activity
as a Possible Factor in the Prevention of Athero—
sclerosis," Circulation, 16 517, 1957.

 

 

J. B. Wolffe, et a1. ”Experimental Atheromatosis and
Ather- -Hepatosis in Ducks and Geese; Its Reversability
and Its Clinical Implications,” American Heart
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, ”Domesticated Adrer als,” Scientific American,

185:52, December, 1951.

______J "Prevention of Heart Disease: A Summary,”
The American Heart, April—June, 1951,

 

CHAPTER III

EXPERIMENTAL METHOD

The study was conducted to determine the effects of
electrical stress and physical activity on several
parameters including serum cholesterol, whole blood
coagulation time and several organ weights, In addition,
variations in food consumption, total body weight and

voluntary activity were recordedo

Material and Eguipment

 

The experimental animals used in the study were adult
male albino rats of the Sprague-Dawley strain received at
100 days of age. Ninety animals were subjected to the
experimental regimen with final data collected on all but
two of the initial group. One animal died of unknown causes
during a basal metabolism determination while a second
animal drowned during the training period. The animals were
maintained on a standard ground meal diet placed in
porcelain food cups. A cover and food follower were pro-
vided to prevent food loss. Sixty grams of food were
placed in the food cups on alternate days with care being
taken to keep the water and food supply adequate at all
times. Records of food intake were maintained and computed

on the basis of average food intake per animal per WEEK?

1+7

The sedentary animals, which included three groups
of eighteen animals each, were housed in individual cages
of quarter inch wire mesh which were suspended over litter
pans lined with newspaper. The sedentary cages measured
10 x 8 x 7 inches and were arranged on wheeled racks
containing 30 cages on each side. The position of the
rack was rotated daily. All cages were thoroughly cleaned
and soaked in a solution of quanternary ammonium prior to
initiation of the experiment.

The "voluntary animals" which consisted of two groups
of 18 animals each, were housed in spontaneous activity
cages consisting of a sedentary cage measuring 12 x 6 x 5
inches and a rotating drum 14 inches in diameter and
five inches wide. A counter was attached to the shaft of
the rotating drum for recording revolutions. A daily
record of activity was maintained for each animal.
(Appendix N.) The activity cages were placed on racks
consisting of four shelves. Four activity cages were
placed on each shelf. The position of the activity cages
was maintained constant throughout the study.

The forced swimming was conducted in a specially
constructed tank. One-half of the original tank, measuring
eight feet long by three feet wide and 30 inches deep, was
employed during the swimming periods. The tank was divided
into separate cubicles measuring one foot square by 1/3

inCh thick transparent sheet plastic. Each animal was

48

placed in a separate cubicle eliminating interference with
swimming by other animals. During the swimming period the
water was maintained between 35 and 37 degrees Centigrade.
The animals swam in two shifts of nine animals each

for one-half hour daily. The sequence was rotated daily
with all swimming done between 12:00 noon and 2:00 p. m.
The animals swam daily except during two basal metabolism
determinations when activity was eliminated for 18 hours
prior to the metabolism measurements.

Upon termination of each swim, the animals were
placed in a large holding cage whichwas placed approximately
18 inches below a bank of two 250 watt infra-red heat lamps.
The animals remained in the holding cage for approximately
25 minutes whereupon they were placed in their normal cages.

There was no temperature or humidity control of the
animal room. The temperature in the animal quarters
ranged between 7H and 8d degrees fahrenheit. The mean
temperature was 80.6 degrees.

During one-half hour daily, the "stress animals"
were placed in Specially constructed ”stress” cages. The
cages were constructed of one-fourth inch clear plastic
with a stainless steel rod grid serving as the flooring.
(Appendix Q.) The stress cages were wired to a specially
constructed stimulator capable of delivering various
intensities of direct current with a variable time delay.

For the purpose of the experiment the stimulator was

49

adjusted to deliver 15 milliamperes (60 v.) of current at
approximately 15 second intervals. Each impulse was
approximately one-half second in duration. A stop watch
was utilized to terminate the daily one-half hour of
stimulation.

During the stress period three cages with a capacity
of six animals each were employed. Since each stress
group consisted of 18 animals the entire group was ”shocked"
simultaneously. The order of stress for each group was
rotated daily. All stimulation was conducted between 9:00
a. m. and 12:00 noon.

The animals were wighed weekly utilizing a Toledo
scale at approximately the same time each week. Records
of weight variations were maintained and plotted.

All animals were numbered on the tail using a
waterproof marker pen for identification. In addition,

each animal was handled daily.

Design of the Experiment

 

The animals were received in the laboratory at 100
days of age. After two days of sedentary housing a blood
sample was taken employing the technique developed by
Stone. (3) At the same time whole blood clotting time was
determined using a modified capillary tube technique. Blood
was allowed to flow from the ”orbital sinus” into a one
millimeter bore non-heparinized capillary tube until full

at which time a stop watch was started. At 15 second

intervals, a small segment of the tube was broken and
observed for fibrin threads. Formation of an observable
fibrin thread was used to estimate clotting time.

Cholesterol values were determined employing a
simplified spectrophotometric technique developed by
Huang. (1)

The animals were divided into five groups of 18
animals each, on the basis of the initial cholesterol and
clotting time values. The groups were then randomly
assigned to one of five experimental regimens. The regimens
were as follows:

Group l.--Sedentary animals which received no stress

Group 2.~~Sedentary animals which received electrical

stress

Group 3.--Voluntary activity animals which received

no electrical stress

Group A.~-Voluntary activity animals which received

the electrical stress

Group 5.-—Sedentary animals which received the elec~

trical stress and forced swimming

The experimental program was initiated ten days after
the animals were received. The stressed animals (Groups 2,
A, 5) were subjected to the following program: daily
periods of electrical stress of one-half hour duration
conducted between 9:00 a.m. and 12:00 noon. Group 5

received, in addition, one-half hour of swimming daily.

51

On the first day of training the animals swam for 15 minutes
without addition of weight. 0n the second day the aninals
were forced to swim for one-half hour with no addition of
weight. On the third day of training 1 per cent of the
animal's body weight was added to the base of the animalis
tail with adhesive tape and the animal forced to swim for

15 minutes. From the fourth day on the animals were forced
to swim for one-half hour daily (with the exception noted
earlier for BMR) with two per cent of body weight, in the
form of lead sinkers, attached to the base of the tail. A11
swimming was conducted between 12 noon and 2:00 p.m.

All animals were initially capable of swimming for
the one-half hour with the additional two per cent of
body weight. However, during the final weeks of the experi-
ment, several animals had to be removed due to exhaustion.
An animal was considered to have reached exhaustion when
it became obvious that he could not regain the surface and
was in danger of drowning.

After ten weeks of experimentation, the animals were
sacrificed on two successive days. All animals in the
stressed groups were sacrificed on the same day while the
non-stressed animals were sacrificed on the following day.
Following ether anesthesia, a blood sample was drawn off
and centrifuged for a minimum of 15 minutes. The serum
was pipetted off and frozen for future cholesterol deter—

mination. Clotting time was determined at the same time

\Fl
H)

employing the previously described method. The animals
were then exsanguinated via the femoral artery (the
brachial artery was used initially). Following exsan-
guination the abdominal cavity was opened and several
organs removed.

The adrenal glands were removed first, cleaned of
all connective tissue and weighed.

Immediately after removal of the adrenals, the heart
was removed and also trimmed of excess connective tissue
and weighed. Other organs which were removed, cleaned and
weighed were kidneys, spleen, liver and testes. A

Mettler balance was used to weigh all organs.

Statistical Procedures

 

To analyze the data tre Analysis of Variance-«
randomized block designm-was employed. This design is used
when the experimental units can be meaningfully grouped,

In this study the grouping was based upon initial serum
cholesterol values and whole blood coagulation time. The
object of grouping is to have the units in a block as

uniform as possible so that observed differences will be

largely due to treatments, During the course of the experi»
ment all units in a block (animals in each group} must be
t other

treated as uniformily as possible in every respecu

than treatment.

53

The randomized complete-block design has many
advantages over other designs. (2) This design permits
grouping the experimental units so that more precision is
obtained than with the completely random design; in addition,
there is no restriction on the number of treatments.
Unusable data from a complete block or for certain treatments
may be omitted without complicating the analysis, while
missing data can be estimated.

The chief disadvantage of the design is that when
the variation among experimental units within a block is
large, there will be a large error term.

The Null hypothesis tested in the analysis was that
of equal means (u1 = u2 . . . = um). The alpha level was
set at .01 while the beta was set at the .05 level. It
was decided that a difference of .2 standard deviations
between groups should be detected. To meet the above
criteria would require an N of 515 cases. Since the group
N was only 18, the following critical regions were estab-
lished:

F > 3.59~~reject Ho at the 99 per cent level of

confidence

F < 1.36--accept H0 at the 95 per cent level of

confidence

l.36_: F.i 3.59—-reserve judgement

To determine differences between group means, the

Duncan Multiple Range Test (2) was used. Since the Duncan

is a "range" test, a five per cent level of confidence was
employed in order to minimize a Type II error.

The analysis of organ weights was based on percentage
of body weight for those organs that had a significant
correlation (alpha = .05) with total body weight. The organs
which did not reflect a significant correlation with total
body weight were analyzed using both raw and relative organ

weights.

L.

2.

3.

References

 

’-

 

 

 

 

W c. Huang, v, Welfer, and A. Raftery, ”A Simplified
Spectrophotometric Method for Determination of Total
and Esterified Cholesterol with Tomatine," Aralytical
Chemistry, 35:1757-8, October, 1953.

Robert G. D. Steel, and James R. Torrie, Principals
and Procedures of Statistils with Stefiel Re-e’eiTe to
the Biological Sciences, New York: Graw- ill book

 

Company, 19b0, p. 107.

S. H. Stone, ”Method for Obtairi g Ver Ous

H

the Orbital Sinus of the Rat or house, Siienc _,

119 100, iggu.

~lood from

CHAPTER IV

RESULTS AND DISCUSSION

Results

The results of the study were analyzed employing a
randomized block model for the analysis of variance. To
determine differences between the various group means, the
Duncan Multiple Range Test was utilized. The parameters
analyzed included food consumption, organ weights, body
weight, cholesterol level, whole blood coagulation time, and
voluntary activity.

Food Consumption.--The animals were fed a standard

 

ground meal diet placed in porcelain cups. The weeKly
differences in food ccnsumption are graphed in Figure 1.
Mean values for total food consumption are granre in
Figure 5.

To test the hypothesis of equal means, analysis of
variance (randomized block) was employed, As indicated in
Table I, the F value of 19.25 is significant at the one per
cent level of confidence, indicating rejection of the
hypothesis of equal means.

To determine differences between the group means, the
Duncan Multiple Range Test (3) was used. The results o

the anal sis between means are indicated in Table XVII.
y

57

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TABLE I

ANALYSIS OF VARIANCE--FO0D CONSUMPTION

 

 

 

Source of Variance DF Sum of Sq. Mean Sq. F Test
GPOUp A 316296 00 79074.00 19.25%
W. Group 83 340884.00 A107.03
Total 87 657180.00

 

*Significant at the one per cent level of confidence.

Organ Weights.—-At sacrifice, several organs, including

 

adrenals, heart, liver, Kidneys, spleen and testes, were
excised following exsanguinatioh via the femoral artery.
Using a Mettler balance, the organs were weighed to deter-
mine the effects of the experimental conditions upon organ
weight. Appendices D through I list the raw data obtained
for the various experimental groups.

The analysis of organ weights was based on percentage

of body weight for those organs tnat rad a significant

l._1
rj‘

correlation (alpha = .05) with tota .odv weight. he orgars

L)

which did not reflect a significant correlation with tot l

o . 7
Cliff? 01‘

£1)

body weight were analyzed using both raw and rel

m

weights. The correlations between organ weight and body

weight of the control animals are indicated in Table II.

TABLE II

CORRELATION OF BODY WEIGHT AND ORGAN WEIGHT

__—
‘:—

 

Organ Value of r
Adrenals .70*
Heart “44*
Kidneys . 044*
Liver .A7*
Spleen .lO
Testes .27

 

*Significant at the five per cent level of confidence.

On the basis of these significant correlations, the
adrenals, hearts, kidneys, and liver were analyzed using
the percentage of body weight. Spleen and testes, which
did not indicate a significant correlation with body weight,
were analyzed using both the raw data and the relative body
weight. The lack of correlation between body weight and
the organ weight of the spleen and testes is not readily
explained. It is suggested that the small sample may
account for the low correlation since the addition of 12
values from another study resulted in a higher correlation
(r = .27) between body weight and spleen weight.

Tables III, IV, and V reveal an F value which supports

the Null hypothesis of no real differences among the

60

TABLE III

ANALYSIS OF VARIANCE-RELATIVE LIVER WEIGHTS

w

 

Source of Variance DF Sum of Sq. Mean Sq. F Test
Group 4 .220320 .055080 .6152*
W. Group 83 .74308A .089528
Total 87 .765116

 

*No significant difference.

TABLE IV

ANALYSIS OF VARIANCE-RELATIVE KIDNEY WEIGHTS

 

 

Group M .029559 .007389 1.9105*
W. Group 83 .3210A3 .003867
Total 87 .350603

 

*No significant difference.

TABLE V

ANALYSIS OF VARIANCE—RELATIVE SPLEEN WEIGHTS

 

 

 

Source of Variance DF Sum of Sq. Mean Sq. F Test
Group A .0008277 .0002069 .A933*
W. Group 83 .03A8I6A .OOOAIQA
Total 87 .0356AA2

 

*No significant difference.

treatment means, at the one per cent leve

 

 

 

 

 

, o
Kidney and spleen weights using the relative organ weigrt.
However, analysis of raw spleen weights ”Table VI! indicates
a significant difference between group means
TABLE VI
ANALYSIS OF VARIANCE—~RAW SFLEEX WEIu.TS

Source of Variance D” Sum of 5;. lean Sq F Zest

Group a 189355 ,cu73ul ; g.7y

W. Group 83 .COlAl? ,cc79n8

Total 87 .850782
‘tSignifica-n:zit tte onc tru' _erf level cfi“c¢m fide' e.

The Duncan Multiple Range lest reelits are irg- ten
in Table XII. Significant differenres are fou'd at e
sedentary and voluntary-ciewt is; senwytarj i «r? r —
electric plus forced exercise; and Sedcytary «i ece i ~
electric. Ir add-:i: t ::e s ..g ifl a; 7‘ e ‘
voluntary group and the voluntary-elc trio. T;a “T4
means for raw spleen weights are Ifld7’1:€fl i :at-e ”1;.

Figure 2, indicates the group leans fo‘ the l; 2;,
Kidney, and Spleen weights following tre exp rimental ta-iad
The values are based on ’ie percentage of to al b dy veig *
Figure 2 also includes the results of two ta al m.ta*: j~~
determinations, which were conducted as part of a sage 2*:

investigation.

TABLE VII

GROUP MEANS FOR SPLEEN AND TESTES-—RAW DATA

m.

HQ

 

 

Group Spleen

 

Sedentary .79A7
Voluntary .7375
Sedentary-electric .70 7
Voluntary—electric .Ofby
Sedentary—electric

plus
Forced Exercise ,67@

\C )

 

Anal sis of adreral weights indicates re ec
o u

h’ull hypothesis of no diffe ere ce ir treatments follow“

r‘r—
—<

the experimental regimen.
of 32.67 which is significant at the one per cent

confidence.

 

 

i
/ \
O
m
1
,IL.
1 D
‘1
r
#4

Source of Variance :r Sum

 

, -3, a ~»-~-...-
Gr 011p H a \. \J ‘J h-- :1 ‘ ‘V "x/ ‘v‘ R.) : ~—:— ‘vii/

I, , ‘ I L. , A ”\r’fl 1,
W. Group 83 .00C 537 .bOqujis

Total 8? .0534111

 

*Significant at the one per cent level of confidenr.

Spleen Weighi (%Body Wt.)

Liver Weight (°/o Body Wt.)

63

 

A

 

65 -
JG“ 2 64 ~ 335?:
.l67 3 11‘»
>‘ ; :
,I66 ’8 63 - ~:-\
CD 7, L
.l65 g ’ ' ‘
,l64 z 62» ”f
o - . -
.l63 '5 , *
3 _: ‘
.52 >~ 6' ’ " L:
Q) , « 7 <
.l6l E, ', 1‘
.l60 3‘ 60- {2'35
: : :1
m xlfii

 

 

 

 

 

 

 

 

 

 

 

 

 

Sedentary
Voluntary
Sedentary Electric

Voluntary Electric

Sedentary Electric +
Forced Exercise

MDIHD

Fig. 2.—?Relative Spleen, Kidney, and Liver Weights

A study of Figure 3 indicates a correlation between
stress and mean values for adrenal weight, with group 5,
subjected to both forced swimming and electrical stress,
having the largest adrenalso Groups 1 and 3, which were not
subjected to stress, reflect the smallest adrenals,

Employing the Duncan range test, significant differ—
ences were found between means in all but one case, as
indicated in Table XVII, at the five per cent level of
confidenceo It is interesting to note that significant
differences were found between the sedentary group and the
group given voluntary activitya

Mean heart weight values we;, also analyzed employing
the analysis of variance methodo lqe results (Table IX)
of the analysis indicate an F test of 6072 which is signi-
ficant at the one per cent levelo The Duncan Range lest
indicates mean differences between the group sub
both forced swimming and electrical stress (Group 5) and

the other experimental groups“

TABLE IX

ANALYSIS OF VARIANCE-RELATIVE HEART WEIGHTS

 

 

Source of Variance DF Sum of Sq, Mean sq, F Test

 

Group A ,01673o “EVAlSAl 6.7ao3t
W. Group 83 ,051612 ,CCGCZlS
Total 87 .0683A8

 

*Significant at the one per cent level of confidencec

65

   
 

Heart Weight l%Body Wt.)

A i5.0
”’2

x

g I40

>~

U

o

aJBO ......
2% 3327222
size ‘
Q)

; iiéiéii;
a no ”11;;11
:, ......
E.’

'6

< I0.0

 

BASAL METABOLIC RATE

 
   
        
  
  
 
 

Testes Weight (% Body Wt.)

‘; (Difference 34th-53rd day)
0
8m Q3
...... E
..... 0"
......... E
25222352 5
S
.330 §§§§§§§é§§ 4:
:::t::::‘: C.
:5;:::::; E
”I'LL, 5’,
BIG "XII” S
.::I: U
.::::' ::: N
......... o
790 112:: ::: C
a»
::;:::::‘: 0"
.m :::::::::: s
XVII: .5
......... U

 

D Sedentary Voluntary I Sedentary Electric

55?: Voluntary Electric % Sedentary Electric + Forced Exercise

 

Fig. 3.—~Basal Metabolism and Relative Heart,
Adrenal, and Testes Weights

{L

The mean values for heart weight are graphed in
Figure 30 The results again reflect a gereral trend in t1at
the stressed animals show larger hearts on the basis of
percentage of body wei ht

Since testes weights did not indicate a significant
correlation with body weight, two analyses were indicated)
Analysis of variance of mean values, for relat ve organ
weight, reflected an F value of E,cu which is significan
at the one per cent level of confidence as indicated in
Table X, The means for the experimen al groups are graphed

in Figure 33

i ‘i
:1:
Lil
r 1
{Tl

ANALYSIS OF MAhiAnon~ FELAIIWE QESL S WEIGEIS

 

 

" "- c I’ ~» “i 3... ~ mg Q r: The...
source of Variarce nF gum of Sqr atafi sq i i321
' ‘ — r“ ‘ .i. .. /
.. .- i — ’jf'x ," (“l f. ‘: fl,“ /.\. {'1 I‘ ,7 _.".-V-
Group a ”ifcjuu ,o3o;j o én_un,~
W Gr 3 p :3 ~-trjl Legit 3
IT. .- 1 L“ E '1 .-. z' ,
“OTal $7 0 Jgfifgg

 

 

 

*Significant at the one per cent level of confiden;en

est, Table XVll, indica
fic ant mean differer3es between toinn ary~ele3tric and

L

sedentary; voluntary—electric and voluntary; sedentary-
electric plus forced exercise and se H69 tary; and sedenaety-
electric and sedentary at the five per cent level of

confidence»

57

The analysis of variance of the raw data for testes is
indicated in Table XI. The group mean values for testes
weights are indicated in Table VII, The Duncan Range Test
(Table XII) indicated significant differences between the
group subjected to electric stress and forced exercise and

the other treatments,

TABLE XI

ANALYSIS OF VARIANCE—-RAW TESTES WEIGHTS

 

 

 

Source of Variance DF Sum of Sq, Mean Sq. F Test
Group A .75378 .188445 2,9856*
W, Group 83 5.23872 ,063117
Total 87 5.99925

 

*No significant difference,

Total Body Weighto—-To determine the effects of the

 

experimental conditions on body weight, the animals were
weighed weekly beginning with the first day of the study,
Readings were taken to the nearest gram with all recordings
made at approximately the same time each week. While the
initial groupings were based on cholesterol values and
coagulation time, study of Table XIII reveals that there
were no significant differences between mean weight values
for the five experimental groups at initiation of the study.

The initial mean weight values are indicated in Figure A.

TABLE XII

DUNCAN MULTIPLE RANGE TEST—-COMPARISON OF TREATMENT
MEANS OF RAW SPLEEN AND TESTES WEIGHTS

 

 

 

Comparison of Treatment Spleen Weight Testes Weight
Means (Raw Data) (Raw Data)

Sedentary vs. Voluntary Nb N
Sedentary vs. Sed-electric S N
Sedentary vs. Vol-electric S N
Sedentary vs. Sed—electric

and swimming S S
Voluntary vs. Sed-electric N N
Voluntary vs. Vol—electric S N
Voluntary vs. Sed—electric

and swimming N S
Sed-electric vs. Vol-electric N N
Sed—electric vs. Sed—electric

and swimming N S
Vol—electric vs. Sed-electric

and swimming N S

 

bN indicates no significance, S indicates statistical

significance, .05 or better.

TABLE XIII

ANALYSIS OF VARIANCE--INITIAL BODY

 

 

 

Source of Variance DF Sum of Sq. Mean Sq. F
Group 4 1112.20 .05 l.
W. Group 85 _ lélAl.80 90
Total 89 1725A.00

 

*No significant differences.

69

 

2:85 .. C253 0' . .lo

:25; oillo
€25ch OIIIO

 

 

 

 

 

H A T _ _ 4 q A q q u _ a q
8.23m 82°“.
+ u_=u~_m-:2=o8m ol. '0 \0/ 252m r3283 0| ..|o
r8598 olllo \o\ recouuw old
\.\o.| ‘o\
\O/ \\ \O
‘v\ /o\\01

 

 

 

 

233 .5353

Gram s

7O

0n the final day of experimentation, body weights of
all animals were recorded. Figure 5 indicates the mean
values for total body weight at termination of the experi-
mental procedure. Employing an analysis of variance, signi-
ficant differences, at the one per cent level, were found
between the groups. The analysis of variance and F values

are indicated in Table XIV.

TABLE XIV

ANALYSIS OF VARIANCE—~FINAL BODY WEIGHT

 

 

 

Source of Variance DF Sum of Sq. Mean Sq. F Test
Group A 47999.70 11999.92 20.5889*
w. Group 83 48375.30 582.83
Total 87 96375.00

 

*Significant at the one per cent level of confidence.

For a comparison between groups, the Duncan Range
Test was again employed. Table XVII indicates significant
differences between all possible pairs with the exception
of the second and fourth groups, and fourth and fifth
groups. It is obvious that the stressed groups have the
lower body weights, whereas the completely sedentary group

reflects the highest body weight.

Differences, Initiol to Final Tests (sec)

Food Consumption Totol (gms.)

 

 

71

COAGULATION llME SERUM CHOLESTEROL-TOTAL

Differences. lnitiol to Final Tests lmg°/o)

 

Body Weight, Final Test lgmsl

 

E] Sedentary E Voluntary . Sedentary Electric
E] Voluntary Electric g Sedentary Electric + Forced Exercise

Fig. 5.—-Total Food Consumption, Final Mean
Body Weight, and Differences in Serum Cholesterol
and Coagulation Time.

Cholesterol Values.——Initial and final serum choles—

 

terol values were determined employing the simplified
Spectrophotometric technique developed by Huang. (1) The
initial cholesterol values, which served as the basis for
grouping, are tabled in Appendix J. Final cholesterol values
are indicated in Appendix K. Analysis of cholesterol values
are based on differences between initial and final values.
The analysis of differences, Table XV, indicates no signifi-

cant differences between group means.

TABLE XV

ANALYSIS OF VARIANCE--FINAL CHOLESTEROL VALUES

 

 

 

Source of Variance DF Sum Of Sq. Mean Sq. F Test
Group A 205.306 51.3265 “2907*
W. Group 83 1A652.8AA 176.5A02
Total 87 1A858.l50

 

*No significant differences.

Coagulation Time.——Employing a capillary tube technique,

 

whole blood coagulation time was determined prior to and

tion time

Q)

following the experimental regimen. The coagul
data are indicated in Appendices L and M. Analysis of
variance was based on differences between initial and final

values and is indicated in Table XVI. Study of the analysis

\J
LU

indicates no significant differences between means
(F = .2306). However, it is interesting to note (Figure 5)
that the non—stressed groups reflect the slower mean

coagulation values.

TABLE XVI

ANALY SI S OE VARI AlX E —- — El??? [:1 ‘7'} Oil: {"3 Il—iTIOZ‘x." TI 1le

 

 

Source of Variance DF Sum of Sq. M3

l)
(1')
73
U)

Q
’11
m
(D
U“

l

 

Group A 1193.78

l\ )

\\C\

J:— CD
" if
I:-

U1

i\ )

9’

l\

k

to
C)

W. Group 83 107A03.67 1

n3
KO

Total 87 l08597.A5

 

*No significant differences.

Voluntary Activity.~eVoluntary activity of the animinal;

 

. - lr . . . . ii .,\5 -3.
housed in the voluntary activity cages was folloced Uciiy.
the results being tabled in Appendix N. Study of tre val es
indicates considerable individual varian e within the two

groups.

Analysis of variance of voluntary activity, Table ITII’

indicates no significant differences although the mean value
for the two groups reflect considerable difference.
(Figure 5.) This lack of significant difference may be

attributed to the large individual variance.

74

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75
TABLE XVIII

ANALYSIS OF VARIANCE--VOLUNTARY ACTIVITY

 

 

Source of Variance DF Sum of Sq. Mean Sq. F Test
Group 1 7568A0000 7568A0000 .200A*
w. Group 34 128383190000 3775976100
Total 35 1291A0030000

 

*No significant difference.

0n five occasions during the swimming program an animal
had to be removed from the water in an exhausted state. This
occurred during the eighth and nineth week of the study and
involved three animals; one animal accounted for three of
the recoveries. It is difficult to determine whether this
reflected a circulatory failure or a transient metabolic
deficiency.

The findings generally reflect a relationship between
the intensity of stress and most of the parameters investi-
gated. The adrenal weights show the treatments to be pro-
gressively more intensive in the order: sedentary, voluntary,
sedentary with electrical stress, voluntary with electrical
stress, and sedentary with both electrical stress and forced
swimming.

The same pattern is reflected by the heart weights
and relative testes weights with the exception of the group

subjected to voluntary activity and electrical stress.

The food intake was generally lower with greater
intensity of stress, with the exception of the group
subjected to both electrical stress and swimming. Although
the food intake was greater in the double stress group than
in the single stress groups, the mean body weight was less,
in direct relation to the intensity of the stress.

Mean serum cholesterol levels reflected greater mean
decreases with progressively more intensive stress
although the group differences were not statistically
significant.

The coagulation time values and relative liver, kidney

and spleen weights were not statistically significant.

Discussion of Results

 

Food Consumption.--The data on food consumption reflect

 

a negative relationship between food consumption and stress.
The two groups (1 and 3) which were excluded from the "stress
programs" had the highest food consumption, whereas the
sedentary group subjected to electrical stress had the lowesr
food consumption. Group 5, which received both electrical
stress and forced swimming, maintained an intermediate
position in food consumption, probably reflecting the ih~
creased metabolic demands of the swimming program. The
general trend of increased food consumption under sedentary

~conditions has been suggested by Meyer. (2)

77

Organ Weights.--The analysis of organ weights gener-

 

ally followed expected patterns. The relative increase in
adrenal weight under conditions of stress was in accord
with Selye's work. While it would be of considerable
interest to isolate the adrenal area responsible for the
increase, this facet was not investigated at this time.
Study of the mean heart weights was again in accord
with expected patterns. The group subjected to both
electrical stress and forced swimming had significantly
larger hearts than the other four groups. That this increase
in heart size is a function of the swimming program was
suggested by the lack of significant differences when the
group subjected only to the electrical stress component was
compared to the other groups. Cardiac hypertrophy is today
generally considered a positive response to the increased
circulatory demands of physical activity. More specifically,
the stimulus to cardiac hypertrophy is the anaerobic
resynthesis (phosphorylation) of adenosinetriphosphate. (4)
The Duncan Multiple Range Tests, on relative organ
weight, indicate significant mean differences between the
stressed groups and the sedentary group in testes weight.
The mechanism may be explained as correlative to the adreno-
hypophyseal axis which is known to respond to stress.
Hypophysectomy, which results in marked gonadal atrophy of
both interstitial cells and tubular structures, indicates

pituitary involvement in gonadal responses. It is

78

postulated that stress stimulates the pituitary to secrete
interstitial cell—stimulating hormone (ICSH)--in a response
analogous with pituitary secretion of ACTH and its effect
on adrenal tissue—-which results in Leydig cell hypertrophy
as a function of the stress. The Leydig cells are known to
be under the direct control of the hypophysial interstitial
cell-stimulating hormone. (5)

The significant differences found when the raw spleen
data were analyzed cannot be explained except to suggest
that the differences reflect body weight and that the lack
of correlation between organ weight and body weight is due
to the small sample size.

Body Weight.--Study of Figure 5 indicates a relation-

 

ship between food intake and final body weight. Groups 1

and 3, both nonstressed, have the highest food intake
consumption as well as the highest total body weight. Group
5 has the intermediate position in food consumption while
reflecting the lowest body weight. This in all probability
is a reflection of the increased metabolic requirements
induced by the swimming program. That the stress of
electrical shock affects food consumption, which is reflected
in body weight, seems apparent. The mechanism cannot be
explained at this time.

Cholesterol Values.-—The lack of significance in mean

 

cholesterol values following the experimental regimen was

not expected. The results may be explained on the basis of

79

large individual variances within groups which reflect
individual differences in responses to stress. The results
may also reflect the content of the diet, which consisted
of a standard ground meal mixture, with no attempt to
increase the lipid content. Feeding a high fat diet under
similar conditions of stress might result in a different
pattern of cholesterol values.

Coagulation Time.--While the analysis of variance

 

indicates no significant differences between groups following
the experimental regimen, a pattern does seem to exist. The
sedentary group indicates the least change during the ten
week study while the group subjected to voluntary activity
indicates the next lowest change. Subjecting animals to
electrical stress along with a sedentary existence appears

to result in a faster coagulation time. Under conditions

of vascular damage the increased coagulation time may enhance
the possibility of thrombus development.

Contrarily, groups 4 and 5, subjected to the electrical
stress in conjunction with a program of physical activity,
indicate an intermediate position in coagulation time. It
is suggested that physical activity minimizes the effect of
the electrical stress via a mechanism which at this time
cannot be explained——perhaps by increasing the circulating
levels of anticoagulants.

The lack of significant differences between groups

might be attributed to the capillary tubing and time

80

intervals employed in determining coagulation time. The
technique involved breaking off a segment of the capillary
tube at 15 second intervals. It is suggested that owing

to the small bore of the capillary tube, the 15 second
interval is too gross a measure, thereby clouding the actual
differences suggested by the general trend of the group
means.

Voluntary Activity.--Analysis of voluntary activity

 

indicates no significant differences between the two group
means. However, these results may be clouded by the
unusually great activity of several animals. One animal
accounts for 25 per cent of the total activity in Group A
while three animals account for 55 per cent of the total

activity in Group 3.

References

T. C. Huang, V. Wefler, and A. Raftery, ”A Simplified
Spectrophotometric Method For Determination of Total
and Esterified Cholesterol with Tomatine,” Analytical

Chemistry: 35:1757-8, October, 1963.

2. Jean Mayer, ”Exercise and Weight Control,"
Fitness, The Athletic Institute, 1959, p.

1.

Exercise and
110.

3. Robert G. D. Steel, and James H. Torrie, Principals and
Procedures of Statistics, with Special Reference to

the Biological Sciences, New York: McGraw—Hill
Book Company, 1960, p. 107.

4. Arthur H:.Steinhaus, An unpublished review of the work
of S. P. Letunov of the USSR’s Central Physical Culture
Research Institute.

5. Jay Tepperman, Metabolic and Endocrine Physiology, Chicago:
Year Book Medical Publishers Incorporated, 196?,

p. 49.

CHAPTER V
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Summary

The purpose of the study was to investigate the
effects of electrical stress and physical activity on
serum cholesterol levels, whole blood coagulation time, and
several organ weights of adult male rats. In addition,
variations in food consumption, total body weight, and
voluntary activity were observed.

Ninety adult male albino rats (Sprague-Dawley Strain)
were divided into five groups on the basis of initial serum
cholesterol and blood coagulation values. Each group was
randomly assigned to one of the five experimental regimens.
Group 1, which served as the control, was housed in
sedentary cages and received no stress; Group 2 was housed
in sedentary cages and received electrical stress; Group 3
received no stress and was housed in voluntary activity cages;
Group A was housed in voluntary activity cages and received
electrical stress; Group 5 was housed in sedentary cages
and received both electrical stress and forced exercise.

The study was conducted for ten weeks with the
electrical stress consisting of daily one—half hour perioos

of confinement to the ”stress cages.” During this period

83

the animals were subjected to 15 ma (60 v.) of direct
current at 15 second intervals. The duration of each
impulse was approximately one-half second.

The forced swimming was conducted in individual com-
partments measuring 12 x 12 x 30 inches with water tempera-
ture maintained between 35 and 37 degrees centigrade. The
animals were subjected to one—half hour of swimming daily
with two per cent of each animal‘s body weight, in the form
of lead sinkers, attached to the base of the animal's tail.

The ”voluntary activity” cages consisted of standard
activity cages with a rotating drum. The ”sedentary cages”
are the standard housing cages measuring 10 x 8 x 7 inches.

All animals were maintained on a standard ground meal

diet and were fed ad libitum. Drinking water was always

 

available.

Employing the analysis of variance technique
(randomized block) the several variables were analyzed. The
results indicated a significant difference between group
means in food consumption with the sedentary group reflecting
the greatest food consumption.

The analysis of organ weights was based upon the
percentage of body weight for those organs which reflected
a correlation with body weight. For those organs which did
not reflect a correlation with body weight two analysis were
conducted-—relative organ weight and raw data.

Analysis of organ weights, based on percentage of body

weight, indicated no significant differences between groups

8A

for kidney, liver and spleen weights. However, analysis of
adrenal weights reflected significant differences between
group means at the one per cent level. The data suggest a
correlation between stress and adrenal weight with the group
subjected to both electrical stress and swimming (Group 5)
having the largest mean adrenal weight value. The groups
which were not subjected to stress had the smallest mean
adrenal value.

Analysis of mean heart values and testes weights also
indicated significant differences between group means.

The raw data analysis of spleen and testes indicated
significant differences in Spleen weight while no signifi-
cance was found in testes weights.

Study of the final body weight also indicated signifi-
cant mean differences with the stressed groups having the
lower body weights, while the non‘stressed sedentary group
had the highest body weight.

Analysis of differences between initial and final
cholesterol values indicated no significant differences
between group means.

Statistical analysis of mean whole blood coagulation
time also reflected no significant differences between the
groups. However, the non—stressed groups did have the slower
coagulation time values.

No significant differences were found in voluntary

activity between the two groups housed in voluntary cages.

85

Conclusions

 

The results of the study suggest the following con—

clusions:

l.

The stress conditions of swimming and/or
electrical stress had a significant effect upon
food consumption.

The experimental conditions of physical activity
and/or electrical stress had a significant effect
upon total body weight.

The stress conditions of physical activity, both
voluntary and forced, and electrical stress had a
significant effect upon relative adrenal weight
while the effect on relative liver, spleen, and
kidney weight was not significant. Analysis of
raw spleen weights indicated significant group
differences.

The ”double stress” of electrical shock and forced
swimming resulted in significantly larger hearts
as determined by relative organ weight.

The experimental conditions reflected no signifi—
cant differences in whole blood coagulation time
or serum cholesterol levels.

Electrical stress had no significant effect upon

the voluntary activity of the animals studied.

86

Recommendations

 

la

The experimental procedure should be repeated
under conditions designed to equate food consump-
tion.

Studies of adrenal and cardiac histology should
be made to determine the effects of the experi-
mental stress on these tissues.

The experimental design should be conducted
under controlled temperature and humidity.
Forced activity, other than swimming, should be
studied under a similar experimental design.
Studies should be designed to collect data on
nocturnal animals during their i7active” periods,
rather than following the current practice of
collecting data during the day.

Anesthetics other than ether should be studied
in an attempt to study the effects of ”anesthetic
stress” on various parameters.

Studies of varying duration and intensities of
stress should be conducted to establish a base—
line of stress effects.

Studies which provide a control for each form
and intensity of stress should be conducted.
Additional parameters, such as blood glucose
levels, should be observed to determine the
effects of the various stress regimens on these

parameters.

V

 

“

'v—vV

IO.

11.

BIBLIOGRAPHY

Aldersberg, David, Louis E. Shaefer, Stanley R. Drachman,
and Rhoda Dritch. HIncidence of Heredity iner—
Cholesterolemia,” Circulation, 2: A75, 1950

 

Backhan, P., M. J. Newlands, and F. Wied. ”Phospholipids
From Brain Tissue as Accelerators and Inhibitors
of Blood Coagulation,” ‘Lancet, 2:204, 1950.

Ball, E. 0., and R. L. Jungas, "0n the Action of Hormones
Which Accelerate the Rate of Oxygen Consumption and
Fatty Acid Release in Rat Adipose Tissue in Vitro,"
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19 l

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1.- - -, 1‘ "1 * ' ~ 7 ' .. ' .,. - . ’1'” .. ., -. ..~
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.— } , {21111e1, iliai,l..

 

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APPENDICES

TOTAL BODY WEIGHT—-INITIAL READING

APPENDIX A

97

 

 

 

(Grams)

Animal # l —g%222§7' 3 4 5
1 388 401 366 377 364
2 380 377 358 389 379
3 386 403 349 382 408
4 378 382 370 363 364
5 384 384 392 380 384
6 379 364 388 364 400
7 375 387 368 382 387
8 388 397 376 406 394
9 403 392 391 385 403

lo 389 390 393 '371 406
11 415 381 381 379 380
12 391 386 379 393 375
13 372 395 378 380 400
14 401 380 400 374 384
15 362 393 381 380 397
15 369 375 403 377 379
17 337 389 367 372 359
18 404 379 369 369 395

 

TOTAL BODY WEIGHT——FINAL READING

APPENDIX B

98

 

 

 

(Grams)

Animal # l —%£22E§—' 3 4 5
l 495 471 478 400 403
2 460 433 473 431 39-
3 468 438 455 428 404
4 159 132 445 412 385
5 438 443 487 4:6 423
6 486 427 460 400 432
7 497 415 437 444 ---
8 479 434 444 448 405
9 486 426 468 464 407

10 462 446 455 418 455
11 500 433 447 324 438
12 483 436 333 456 339
13 463 442 431 488 393
14 490 442 493 —-- 409
15 455 462 448 4:4 434
16 469 407 474 404 390
17 462 398 453 414 375
18 468 418 460 395 416

 

APPENDIX C

TOTAL FOOD CONSUMPTION

99

 

 

 

(Grams)
Groups

Animal # 1 2 3 4 5
1 1493 1385 1498 1145 1245
2 1380 1295 1370 1254 1258
3 1418 1219 1432 1221 1270
4 1350 1264 1363 1264 1250
5 1295 1271 1434 1358 1406
6 1479 1279 1493 1250 1348
7 1488 1233 1404 1305 1325*
8 1399 1273 1278 1299 1322
9 1438 1211 1459 1408 1287
10. 1377 1259 1302 1275 1386
11 1527 1262 1320 1305 1405
12 1463 1279 1382 1406 1309
13 1453 1371 1404 1389 1270
14 1458 1273 1435 1252* 1348
15 1334 1281 1295 1212 1332
16 1375 1252 1447 1234 1289
17 1419 1143 1274 1273 1305
18 1411 1216 1303 1178 1316

 

*Includes mean values substituted for missing values.

100

APPENDIX D

ADRENAL WEIGHTS*

 

 

 

 

Groups
Animal # l 2 3 4 5

1 0.0524 0.0524 0.0473 0.0610 0.0487
2 0.0430 0.0460 0.0532 0.0523 0.0670

3 0.0438 0.0527 0.0557 0.0532 0.0496

4 0.0463 0.0491 0.0484 0.0474 0.0554

5 0.0401 0.0476 0.0517 0.0563 0.0496
6 0.0509 0.0563 0.0542 0.0599 0.0630
7 0.0450? 0.0540 0.0484 0.0441 ---

8 0.0476 0.0558 0.0626 0 0552 0.0599

9 0.0476 0.0601 0.0546 0.0576 0.0601
10 0.0481 0.0437 0.0452 0.0497 0 0610
11 0.0466 0.0488 0.0480 0.0592 0,0525
12 0.0481 0.0507 0.0458 0.0545 0.0487
13 0.0410 0.0557 0.0450 0.0684 0.0626
14 0.0466 0.0550 0.0497 —-— 0.0531
15 0.0385 0.0537 0.0432 0.0565 0.0727
16 0.0422 0.0426 0 0452 0.0503 0.0631
17 0.0454 0.0526 0.0551 0.0538 0.0552
18 0.0383 0.0579 0.0503 0.0534 0.0713
*Note——All readings are in grams.

lOl

APPENDIX E

HEART WEIGHTS*

 

 

 

Groups
Animal # 1 2 3 4 5

1 .5734 1.3821 5058 1.1668 1.2047
2 .3077 1.3654 .3675 1.3523 1.4181
3 .4074 1.3390 3446 1.2255 1.3101
4 .4226 1.4336 .3607 1.2981 1.2917
5 .5116 1.4278 .3834 1.3617 1.3681
6 .5060 1.3588 .4310 1.3261 1.4569
7 .4573 1.2732 3546 1.3636 ——-

8 .2950 1.3192 .5741 1.2320 1.6008
9 .4270 1.3123 3596 1.4187 1.4134
10 .4403 1.3148 .3358 1.1717 1.3985
11 .5303 1.3368 3058 1.3985 1.3240
12 .3020 1.3548 .2691 1.3649 1.4157
13 .4431 1.4710 .3512 1.4079 1 3088
14 .6646 1.3777 .4525 -—- 1.4217
15 .2197 1.3091 .3481 1.2941 1.3912
16 .3167 1.3350 4766 1.1690 1.2717
17 .1271 1.2743 2236 1.2835 1.3916
18 .1910 1.2786 .3791 1.2524 1.3494

 

*Note——All readings are in grams.

APPENDIX E

LIVER WEIGHTS*

lO2

 

 

 

 

Groups
Animal # l 2 3 4 5

1 17.7947 15.6626 16.1535 14.6136 13.9664
2 16.6824 14.6436 14.9107 15.0767 13.2778
3 15.3137 14.7848 14.5026 14.3718 14.6220
4 14.0419 15 6538 15 8146 13.9905 13.6227
5 15.9925 15.5051 16.6930 16.2098 13.5877
6 16.7955 14.5716 18.7620 13.9172 14.5100
7 16.1137 15 5605 15.0641 14.3270 -—-

8 14.3464 14.2719 11.0038 14.0125 14.3472
9 16.3955 12.7202 14.6107 13.3568 13.7476
10 14.2962 13.3033 16.0865 12.5849 14.0784
11 15.1007 14.5740 14.2206 14.9157 15.5820
12 17.1545 12.6350 14.4212 15.0417 12.7244
13 14.0546 14.1382 14.1904 13.7135 14.0825
14 15.8232 13.5792 16.3488 --- 13 8844
15 13.3277 15.1331 16.0137 13.1333 13.3305
16 14.9235 13.3972 15.8952 13.2335 12 5067
17 12.9338 10.8647 13.3623 12.8251 12 4009
18 14.7167 13.8851 15.1694 11.9260 13.2824

 

*Note--All readings are in grams.

103

APPENDIX G

KIDNEY WEIGHTS*

 

 

 

 

 

Groups
Animal # t l 2 3 4 5

1 2.8148 2.5628 3.0432 2.6881 2.7341
2 2.5587 2.6269 2 9068 3.1084 2.7321
3 3.0881 2.6886 2.8461 2.7181 2.5895
4 2.6608 I 2.9295 2 9279 2.7341 '2.6003
5 2.7969 2.9338 2.9342 3.1505 2.5171
6 3.2373 2.9116 2.9400 3.1253 2.6138
7 2.8047 2.8166 3.0123 3.0219 ---

8 2.9145 3.0234 2.6484 2.6241 2.7395
9 2.9932 2.8525 3.0481 2.6869 2.2983
10 2.5858 2.6612 2.5550 2.1867 2.8817
11 2.7774 2.5394 2.7622 3.1029 2.8407
12 3.0568 2.6420 2.8067 3.0631 2 6706
13 2.7354 3.0929 2.9923 2 5589 2.4713
14 3.2274 2.7259 2.9223 —-- 2.3987
15 2.6127 2.7977 2.8660 2.4486 2.8952
16 2.6198 2.6848 3.3554 2.2698 2.2817
17 2.5546 2.4002 2.7122 2.4634 2.3294
18 3.1148 2.8078 3.3282 2.3371 2.4436

 

*Note--All readings are in grams,

104

APPENDIX H

SPLEEN WEIGHTS*

 

 

 

Groups

Animal # 1 2 3 4 5
1 1.0410 0.8009 0.7448 0.5002 0.6215
2 0.8891 0.5966 0.7324 0.7300 0.5508
3 0.8468 0.7410 0.7805 0.6670 0.5172
4 0.7698 0.7974 0.8666 0.7152 0.6002
5 0.9501 0.7169 0.8204 0.6941 0.6636
6 0.8742 0.6568 0.7214 0.6262 0.6926

7 0.7839 0.6370 0.5645 0.7985 ——-
8 0.8022 0.6190 0.5496 0.6269 0 7698
9 0.6828 0.8100 0.7504 0.7694 0.7096
10 0.7564 0.6681 0.8448 0.6085 0 7221
11 0.8608 0.6264 0.7012 0.6721 0.6922
12 0.8078 0.7072 0.6724 0.7756 0.7311
13 0.7451 0.7846 0.6656 0.6281 0.7299
14 0.6410 0.7039 0.8110 --- 0.6279
15 0.8168 0.8608 0.7626 0.7201 0.6785
16 0.8048 0.6238 0.6998 0.5962 0.7736
17 0.6035 0.6139 0.7775 0.4923 0.7645
18 0.6288 0.7028 0.8098 0.7170 0.6634

 

*Note--All readings are in grams.

105

APPENDIX I

TESTES WEIGHTS*

 

 

 

 

Groups
Animal # 1 2 3 4 5

1 3.8588 3.8837 4.0784 3.4002 3.2819
2 3.4766 3.4940 3.5416 3.4120 3.1594
3 3.7586 3.8013 3.7082 3.6048 3.2924
4 3 7771 4.1348 3.3874 3.5438 3.4102
5 3.3142 3.2198 3.5300 3.6360 3.2886
6 3.6714 3.5448 3.5880 3.5893 3.7406
7 3.4053 3.3273 3.6191 4.1958 ---

8 3.5432 3.3142 3.0496 3.6516 3.2947
9 3.3624 3.5895 3.7104 3.5032 3.4280
10 3.3677 3.6239 3.8359 3.4528 3.5050
11 3.3342 3.3078 3.8152 3.8982 3.5976
12 3.5280 3.8963 3.4330 4.1362 3.1222
13 3.4778 3.5343 4.0143 3.5734 3 2434
14 4.5428 3.5035 3.5349 --- 3.3009
15 3.5487 3.6845 3.7875 3.2339 3.505
16 3.6387 3.3404 3.7906 3.6776 3.5267
17 3.6572 3.5281 3.3101 3.5396 3.3981
18 3.4170 3 3291 3.0479 3.7781 3.2031

 

*Note--All readings are in grams.

SERUM CHOLESTEROL VALUES*——INITIAL

APPENDIX J

106

 

 

 

 

Groups

Animal # l 2 3 4 5
1 96 68 80 89 73
2 81 81 68 87 80
3 84 84 75 84 82
4 73 77 78 73 83
5 87 78 82 81 79
6 81 99 74 67 81
7 77 90 75 96 75
8 80 87 86 78 88
9 68 80 70 81 95
10 78 73 85 69 67
11 65 84 71 80 85
12 75 80 79 77 77
13 84 81 83 75 87
14 80 67 92 72 72
15 72 75 74 84 70
16 69 72 94 80 78
17 89 64 81 75 67
18 75 75 67 66 75

 

*Note--All readings are in Mg. per cent.

107

APPENDIX K

SERUM CHOLESTEROL--FINAL VALUES*

 

 

 

Groups
Animal # l 2 3 4 5

1 69j7 79.6 82.9 82.6 58.0
2 58.0 69.0 66.7 71.4 60.4
3 85.0 77.0 61.0 58.0 74.6
4 68.6 67.7 81.0 68.0 92.5
5 75.5 78.7 68.0 81.0 64.8
6 84.9 85.8 61.0 82.1 65.5
7 77.5 133.1 63.2 62.4 --
8 69.4 71.3 92.8 81.8 73.3
9 31.7 82.1 77.7 82.9 89.2
10 80.8 68.4 74.4 71.8 65.6
11 68.4. 70.8 56.5 97.6 68.0
12 65.5 64.1 61.2 77.7 85.6
13 82.6 73.3 68.0 68.6 63.1
14 100.1 49.7 68.6 -— 2.3
15 76.6 79.0 79.8 61.0 69.5
16 78.5 68.6 90.0 60.6 67.8
17 100.1 59.3 103.7 52.9 71.6
18 73.6 53.8 84.3 71.4 72.5

 

*Note--All readings are in Mg. per cent.

108

APPENDIX L

WHOLE BLOOD COAGULATION TIME*—-INITIAL VALUE

 

 

 

 

Groups

Animal # l 2 3 4 5
l 60 45 30 120 30
2 45 30 60 30 60
3 45 90 30 30 30
4 30 30 45 30 45
5 30 30 30 60 30
6 30 30 45 75 45
7 45 30 30 30 30
'8 45 75 45 45 45
9 30 60 75 45 75
10 135 30 45 45 45
11 60 30 30 60 30
12 30 30 45 30 45
13 45 30 30 30 30
14 60 135 45 3O 45
15 45 60 30 30 30
16 75 60 45 30 45
17 30 60 30 45 30
18 90 60 90 120 90

 

*Note—«All readings are in seconds.

WHOLE BLOOD COAGULATION TIME*-—FINAL VALUE

APPENDIX M

109

 

 

 

 

Groups

Animal # 1 2 3 4 5
1 150 15 45 45 45
2 75 45 45 45 3O
3 75 30 45 105 l5
4 15 30 6O 30 30
5 45 15 3O 45 45
5 45 15 60 75 30
7 15 15 15 30 --
8 15 90 -- 30 30
9 45 15 30 45 15
10 15 15 45 30 15
11 45 30 30 105 15
12 15 30 30 15 15
13 135 105 60 15 15
14 30 75 15 —— 90
15 45 30 30 45 60
16 30 30 30 15 60
17 30 60 15 15 105
18 45 15 30 30 60

 

*Note—-All readings are in seconds.

110

APPENDIX N

TOTAL VOLUNTARY ACTIVITY

 

 

 

 

(Revolutions)
Groups
Animal # 3 V 4
1 35622 27067
2 20874 29823
3 36342 19985
4 28367 43049
5 13052 45856
6 156759 81436
7 33924 31585
8 24078 49618
9 36228 21883
10 22177 40424
11 57663 51647
12 311716 31475
13 116889 226564
14 18732 36947
15 38938 27579
16 63232 47209
17 16544 17585
18 29641 65981

 

APPENDIX 0

 

 

M-I Rat Ration
(500 lbs.)

 

Ground Corn—~1/4” Screen
Soybean 011 Mea1——50%
Alfalfa Leaf Meal

Fish Meal

Whey

Limestone (CaCo3)

Dicalcium Phosphate (CaHPou)
NaCL (lodized)

Vitamin Mix (see below)

000 T. M. Premix 5% Zinc

Vitamin A Pfizer 10?

303.
140.

Vitamin D 9f, Fleischman Irridated Yeast (Dry)

Pro-Strep-—20

Pro-Gen--20

lbs.
lbs.
10. lbs.
12. lbs.
lbs.

5

O

O

5

12.5 lbs.

0

751bs.
5

[00000

lbs.

113 grams

 

Vitamin Mix for 500 lbs.

M—I Ration

 

Choline Chloride
Ca Pantothenate
Riboflavin
Niacin

Vitamin B12 (.1% Mannitol

Refrig. Trituration)

Alpha Tocopherol (liquid)
Menadione

D. L. Methionine

159.0 grams

1.25 grams

.750 grams

7.5 grams
1.5 grams

1.0 grams
.5 grams

113.5 grams

111

112

APPENDIX P

STRESS CAGES AND STIMULATOR