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RELATIONSHIP OF CONTROLLED
ENVERONMENTAL STRESS AND PROTEEN

iNTAKE TO GROWTH AND OTHER
PHYSIOLOGICAL MANIFES'FATEONS
EN YOUNG RATS

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Thesis {or Hue Degree of M. S.
MECHIGAN STATE UNIVERSITY
Helen Louise Bishop
1963

LIBRARY

Michigan State
University

 

 

ABSTRACT

RELATIONSHIP OF CONTROLLED ENVIRONMENTAL STRESS
AND PROTEIN INTAKE TO GROWTH AND OTHER
PHYSIOLOGICAL MANIFESTATIONS IN YOUNG RATS

by Helen Louise Bishop

The object of this experiment was to study the response
of male albino rats to the stress of bright, flashing lights
when the diet contained varying levels of protein. Forty
rats were divided into four groups of ten rats each (Groups
A, B, C, and D). Two groups (A and B) were fed a low-
protein diet (9% casein) and one of these groups (B) was
placed in a stressful situation by being exposed to bright,
flashing lights. Two groups (C and D) received a high-
protein diet (20% casein) and one of these groups (D) was
also exposed to the lights.

On the 28th day, blood samples were taken from all ani-
mals for cholesterol determination and the animals were
sacrificed. Portions of liver were taken for the determina-
tion of glutathione levels, and adrenal glands were removed
for weighing.

In both groups subjected to the stress of the flashing
lights there was a marked increase in liver glutathione
levels when compared with the controls. This was interpreted
as evidence that the lights were causing physiological upsets

in the animals. Adrenal glands - on the basis of weight per

Helen Louise Bishop

100 g. body weight - were found to be largest in the
stressed, low-protein group and smallest in the non-stressed,
high-protein group. Both high—protein groups showed highly
satisfactory weight gains. The stressed, low-protein group
(B) gained significantly less weight than didebup.A despite
the relatively similar levels of food intake in both groups,
indicating that the stress might have introduced an addition-
al requirement for protein which was met at the expense of
the animals' growth. Total cholesterol levels did not vary
significantly between A and B, nor between C and D. This is
in accordance with the findings of other authors that a long
period of time is needed for marked differences in cholester-
ol levels to appear in rats subjected to stress. There was
no significant difference between the total cholesterol blood
levels of Groups A and C - possibly because the difference in
protein intake of these two groups was not extreme enough.
There was, however, a difference in the total blood choles-
terol level between Groups D and B, significant at the 5%
level, suggesting that the combination of the stress of the
flashing lights with the 11% difference in protein intake

was sufficient to differentiate the cholesterolemic responses.
Group B showed significantly higher levels of free serum
cholesterol and Group D significantly lower levels than
either Group A or Group C. It is suggested that the differ-
ence in available protein for extra cholesterol mobilization

in the form of lipoprotein is the reason for this finding.

RELATIONSHIP OF CONTROLLED ENVIRONMENTAL STRESS
AND PROTEIN INTAKE TO GROWTH AND OTHER
PHYSIOLOGICAL MANIFESTATIONS IN YOUNG RATS

by

Helen Louise Bishop

A THESIS

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

MASTER OF SCIENCE

Department of Foods and Nutrition

1963

ACKNOWLEDGEMENT

The author wishes to express her grateful appre-
ciation to Dr. Theodore F. Irmiter for his advice and
guidance throughout the course of this research, to the
faculty and staff of the department of Foods and Nutrition
for their help and encouragement, and to Michigan State
University for making the research possible.

Special thanks to Sister St. Catherine Maureen,

C.N.D., for her inspiration and counsel.

To My Parents

TABLE OF CONTENTS

Page
INTRODUCTIONOOOOOOOOOOO0.0000000000000000000000000000 1
REVIEW OF LITERATUREOOOOOO0.00000000000000.0000.000.. 4
A. General Effects of Stress..................... 4
B. Relation of Stress and Protein Requirements... 10
C. Effect of Protein on Serum Cholesterol
concentrationOOooooooOOOOOOOOOOOOOOOOOOOO00°00 lo
“‘“EXPERIMENTALOOOO00000000000000000000.000000000000000. 14
A. Diets and Experimental Design................. 14
B. Chemical Analyses............................. 16
C. Statistical Analyses.......................... 2O
RESULTSOOOOOOOOOOOOOOOOO00000000000000000000000000... 21
A. Effects of Stress with Varying Levels of
ProteinOOOOOOOOOOOOOO00.0.0000000000000000000. 21
B. Effect of Varying Protein Levels.............. 23
C. Graphical Representation of Results........... 25

DISCUSSIONOOOOOOOOOOIOOOOOOOOOOOOOOOOOOOOIO0.000.000. 32
A. Effects of Stress with Varying Levels of

Protein....................................... 32
1. Influence of stress on growth........... 32
2. Influence of stress on nitrogen

retention............................... 34
3. Influence of stress on liver

glutathione levels...................... 35
4. Influence of stress on adrenal gland

weights................................. 35
5. Influence of stress on serum

cholesterol............................. 36
6. Influence of stress on liver weights.... 39

B. Effect of Varying Protein Levels.............. 39
SUGGESTIONS FOR CONTINUATION OF THIS STUDY........... 40
SUMMARY AND CONCLUSIONS.............................. 41
REFERENCES........................................... 43

INTRODUCTION

Ershoff (8) in a paper entitled "Nutrition and Stress",
states that any factor that interferes with the digestion,'
absorption, or utilization of nutrients, or increases their
destruction or excretion may result in malnutrition despite
the apparent adequacy of the diet fed. Body requirements may
furthermore be significantly increased for purposes of de-
toxification or by factors such as physical exertion, fever,
burns, major surgical procedures, abnormal environmental con-
ditions and others. The net result of such factors may be an
increase in body requirements beyond the usual or average
range and the precipitation of nutritional deficiencies on
diets that would otherwise be adequate.

It would be erroneous to generalize by stating that
"stress" per se increases nutritional requirements, for, as
Ershoff explains, "stress" is not a specific, unvarying and
distinct entity. ”Stress" is a state of the organism, the
result of those processes an organism employs in attempting
to maintain homeostasis under unfavorable alterations in the
environment. Furthermore, since life is a process in which
the organism attempts to maintain itself in a changing envi-
ronment, "stress" is inherent in life. Ershoff maintains
that increased nutritional requirements following exposure to
stressor agents result not as a consequence of "stress" per

se butzether from the increased requirement for specific

1

nutrients due to the physiologic effect of the particular
stressor agent involved. It has been demonstrated by H.
Selye (35), however, that prolonged exposure to a wide vari-
ety of stressor agents results in a stereotyped response such
as increased adrenaline and cholesterol levels in the
stressed animals. Hence, in the present study, which employs
bright, flashing lights as a stressor agent, the general
usage of the term "stress" to convey the influence of the
lights upon the experimental animals is thought to be a
legitimate one.

Considerable data - of which Selye's are of great im-
port - are available [Adams et a1. (1), Baur and Filer (2),
Denenberg and Karas (7), Ershoff (8), and Hartel (l7£] in-
dicating that diet per se and the animals nutritive state
are intimately related in adaptation to stress. Varying
levels of dietary protein induce biochemical changes similar
to those elicited by various stressor agents [éones and
Huffman (22) and Moyer et a1. (29)]. The present study
explores the possibility of an association between stress
and protein requirements.
Ob ectives
To ascertain whether:

1. Stress affects protein utilization.

2. The amount of protein in the diet has any influence

on response to stress.

Hypothesis:

The effects of stress are in part a function of a pro-
tein deficiency brought about by an induced increase in
protein requirements.

The recommended allowances for protein may be rendered

inadequate by stressful situations.

REVIEW OF LITERATURE

A. General Effects of Stress

At about the middle of the nineteenth century the French
physiologist Claude Bernard (3) had already recognized that
to maintain life, the constancy of the "milieu interieur"
must be preserved in spite of changes in the environment.
Subsequently the American investigator Walter Cannon (5) in-
troduced the term "homeostasis" to designate the maintenance
of this steady state and directed attention to the role of
adrenaline and of the autonomic nervous system in bringing
about the internal adjustments upon which homeostasis de-
pends. He listed a number of Specific reactions which are
important in maintaining the constancy of the ”milieu in-
terieur" during certain "emergencies" such as result from
muscular work, nervous irritation and variations in the
surrounding temperature.

According to Selye (35) man has had some idea, albeit
subconscious, of the stress concept ever since the word "dis-
ease" has been used. The very fact that a single term can
be used to denote a great variety of individual maladies,
clearly indicates these maladies have something in common,
possessing some nonspecific features which permit the disease
state to be distinguished from the condition of health. Yet,
precisely because these manifestations are not characteristic

4

of any one disease, they have received little attention in
comparison with the specific ones. Through a series of ex-
periments on animals, however, Ingle (21) demonstrated that
the organism responds in a stereotyped. manner to a variety
of widely different disturbances such as: infections, intoxi-
cations, trauma, nervous strain, heat, cold or muscle fatigue.
The only common feature was that they placed the body in a
state of general (systemic) stress. It was concluded, there-
fore, that the chemical responses characteristic of all epe-
cific stresses are indicative of the general effect elicited
by nonspecific systemic stress.

Selye (35) defined stress as "the sum of all nonspecific
changes caused by function or damage." He reported that the
first and most outstanding manifestation of this response to
stress was adrenocortical enlargement.

Jurtshuk, Weltman, and Sackler (23) explored alterations
in certain biochemical parameters which were measured in rats
subjected to repeated, prolonged, high-intensity auditory
stimulation. Specifically, levels of blood glutathione,
adrenal ascorbic acid, and total adrenal cholesterol were
reported. They used blood glutathione index as an indicator
of "generalized" stress since Selye (35) had found that both
cortisone and adrenocorticotropic hormone (ACTH) injections
cause a transitory drop in the glutathione content in blood
of rats and humans. The studies of Jurtshuk et al. tend to

indicate that the glutathione index may be a significant

metabolic parameter in the investigation of emotional dis-
turbances. It was the finding of the authors that severe
auditory stimulation caused a marked reduction in glutathione
levels in the blood of female rats. The frequency of the
auditory stimulation was related inversely to the recovery
rate after the stimulation. An increase in adrenal weights
and adrenal ascorbic acid, as well as a decrease in total
adrenal cholesterol were noted. Jurtshuk's work extended
the analysis of physiologic changes reported by other work-
ers in the field of audiogenic stress. Hurder and Sanders
(19) had indicated that animals with larger adrenals were
more susceptible to audiogenic seizures than animals with
smaller adrenals.

Although diverse types of systemic stress have long been
known to modify the immunological responses of mammals (Selye,
35) precise experimental investigations have only recently
elucidated the effect of such stress on particular immune
reactions. Thus, mice subjected to a standardized avoidance-
learning type of stress show an increased susceptibility to
herpes simplex virus infection, as well as a decreased sus-
ceptibility to passive anaphylaxis, and a depressed colloid-
clearing capacity of the reticuloendothelial system (Rasmus-
sin, 34).

Denenberg and Karas (7) explored the effects of differ-
ential infantile handling upon weight gain and mortality in

the rat and mouse. Complete lack of handling or prolonged

handling both acted to reduce survival time; those animals
receiving moderate handling survived a significantly longer
time than either of the other two groups. The effect of in-
fantile handling was to stress the organism and this stress
acted to reduce the animals' responsiveness to other stressor
agents later in life. Seymour Levine (26), who also studied
infantile handling, found that non-handled animals were more
profoundly affected by stress - both psychological such as
is induced by bell ringing, light flashing, and confinement,
and physiological as produced by burns, infection and muscle
fatigue.
Stress and cholesterol

It has been frequently demonstrated that certain forms
of stress can elevate serum cholesterol. In a study of 55
male subjects, Kuhl et al. (25) found brief immersion in cold
water was followed by an immediate and significant increase
in serum cholesterol. Wertlake et al. (37) studied 44 male
students during periods in which they were free of examina-
tions as well as during an examination week. The purpose of
this study was to determine what possible effect mental and
emotional stress might have on the level of serum cholesterol.
A significant increase in mean value for serum cholesterol
accompanied the stress of the examinations. These data co-
incide with changes which Selye (35) described in connection
with the "resistance phase" of the general adaptive response

to various forms of systemic stress.

Uhley and Friedman (36) conducted a study in which a
group of rats fed a diet high in fat and cholesterol was ex-
posed to a particular form of stress induced by periodic
electric charging of their cages. During the first four
months the average serum cholesterol and phOSpholipids of
the rats exposed to the electrically charged cage were not
significantly different from those of the control rats. The
blood samples obtained from the animals at the tenth and
final month of the experiment, however, were found to have
considerable differences in lipid concentrations, with the
average cholesterol content of blood from the experimental
rats 47% greater than that of the control rats. The conclu-
sion was that with a diet designed to produce atherosclerosis,
chronic stress aggravated the process.

The high incidences of atherosclerosis among business
executives and others under constant pressure has led many
persons to Speculate that emotional stress may be an impor-
tant factor in atherogenesis. Only recently, however, has
there been any acceptable evidence to support this concept.
Friedman and co-workers (13) reported increases in serum
cholesterol levels in men subjected to occupational stress.

Workers have consistently found periods of undue stress
result in elevation of serum cholesterol levels, but the
mechanism of this elevation remains unexplained.

Stress and lipoproteins
Grundy and Griffen (16) used students subjected to the

stress of final exams and found together with the increase in
serum cholesterol levels, a significant (50%) increase in the
SF 12-400 class of lipoproteins.) .

It is generally agreed [Friedman et al. (13), Gero et al.
(14), Gofman et al. (15), Jurtshuk et al. (23)] that the
characteristic serum-lipid alteration in atherosclerosis is
an increase (both relative and absolute) in the level of the
low-density B-lipoprotein fraction. According to the "fil-
tration" theory of atherogenesis, deposition of cholesterol
in the arterial wall is enhanced by plasma-lipoprotein alter-
ations and by changes in the subendothelial ground substances.

Gero and his co-workers (14) attempted to influence the
course of atherosclerosis, induced by cholesterol feeding, by
immunization with B-lipoprotein. Ten cocks and ten rabbits
were given weekly intramuscular injections of an alum pre-
cipitated antigen containing 22.5 mg protein. When the im-
munization schedule was finished, the immunized group of
rabbits and an equal number of non-immunized rabbits were
fed 1 g cholesterol daily for sixteen weeks, the cooks re-
ceived 2 g daily. During this time the immunized group
received two more antigen injections. The immunization peri-
od itself was followed by a rise in plasma cholesterol; but
the level after cholesterol feeding showed marked differences
between the immunized and non-immunized groups - the former
having significantly lower cholesterol levels than the latter.

Immunization with B-lipoprotein was found to inhibit the

10

development of experimental atherosclerosis in both species

of experimental animals.

B. Relation of Stress and Protein Requirements

Although concerned with purely physiological stresses
(starvation and thirst), the work of Baur and Filer (2) lends
some support to the hypotheses put forth in the present ex-
periment. They found that the ability to survive stress in
the form of starvation, thirst, or both, is influenced by
body composition and prior plane of protein nutrition.
Furthermore, under all three types of stress the rate of
protein catabolism was two to three times greater for those
animals fed a high protein diet than for the animals fed a
low protein diet. The authors concluded the body cataboli-
cally accustomed to a higher protein intake is unable to gear

down its metabolic processes when suddenly faced with a di-

etary emergency.

C. Effect of Protein on Serum Cholesterol Concentration

The first demonstration of an anti-atherogenic influence
of dietary protein came as a result of the work of Katz,
Stamler and Pick (24). Their experiments showed that high-
protein supplementation suppressed hypercholesterolemia and
atherogenesis in cockerels ingesting either a purified or
commercial ration high in cholesterol and fat. In studies on
the mechanism of this phenomenon, it was shown that vitamin

J‘

supplementation enhanced the cholesterol reducing activity

ll

of the increased protein, but vitamin supplementation alone
was without influence. On the other hand, high-protein alone
apparently suppressed coronary atherogenesis in a manner simi-
lar to the high-protein, high-vitamin combination. However,
high-protein diet alone apparently had less definitive and
clear cut effects than the combined high-protein diet with
vitamin supplementation.

There have been several reports [Moyer et al. (29),
Mann et al. (27), Jones and Huffman (22E) concerning effects
of dietary protein on serum cholesterol concentration and
recently the subject has begun to attract great attention.
In 1958, Nath, Harper, and Elvehjem (30) undertook a study
of the effects of various dietary levels of some proteins of
both animal and vegetable origin on serum cholesterol con-
centration in the rat. A comparison of the effects of casein
and wheat gluten indicated both the level and nature of the
dietary protein influenced serum cholesterol in this species.
The results showed with casein as the protein source, serum
cholesterol varied inversely with increasing protein levels
up to a level of 40% casein in the diet. Increasing the
casein level beyond 40%, however, led to a corresponding in-
crease in serum cholesterol levels.

When wheat gluten replaced casein as the dietary protein,
the relationship between the level of protein and serum cho-
lesterol concentration was somewhat different. In contrast

to the trend with higher levels of casein, each increase in

12

the level of wheat gluten from 10% to 68.5% of the diet
caused a progressive lowering of serum cholesterol levels.
Also, serum cholesterol values for rats fed the diets con—
taining wheat gluten were lower than values for rats fed the
diets containing casein at each level of dietary protein at
which the comparison was made.

The relationship of a dietary amino acid deficiency to
defects of cholesterol metabolism was suggested to Fillios
and Mann (27) by the preventive action of either Dmeethionine
or L-cystine supplementation to sulfur amino acid-deficient
diets made with soybean protein and by the curative action of
DL-methionine supplements given to monkeys with established
hypercholesterolemia. In a study exploring the effect of diets
made with soybean protein on cholesterolemia in the mouse and
rat, Fillios and Mann (10) found when a protein deficient in
sulfur amino acids was substituted for casein in the diet,
hypercholesterolemia was demonstrated.

Further investigation by Fillios, Andrus, Mann and Stare
(ll) lent support to the theory of an existing relationship
between dietary protein and serum cholesterol. They found
the hypercholesterolemic response among rats varied according
to the dietary protein level, with the lowest response ob-
served among those animals receiving the highest level of
dietary protein. However, there was no significant differ-
ence in cholesterolemic response between groups in which the

protein intake did not vary greatly. Thus, the animals

13

receiving 10% casein in the diet did not show significantly
different levels of serum cholesterol from the animals on
the 20% casein diet.

Nishida, Ueno, and Kummerow (31) showed the serum cho-
lesterol level in chicks kept on a low protein diet was sig-
nificantly elevated as compared with those on a high protein
diet. They proposed in a later study (32) that a low dietary
protein level may impair directly or indirectly the enzymatic
systems involved in cholesterol catabolism due to a combina-
tion of the following five possibilities: (1) a low protein
diet may partially impair some enzyme system involved in
cholesterol catabolism, (2) a low protein diet may inhibit
the production of some coenzymes or cofactors necessary for
cholesterol catabolism, (3) if cholesterol is assumed to be
mobilized for catabolism in the form of lipoproteins, a low
protein diet may produce less protein than necessary for
mobilization, which may result in lowered cholesterol catab-
olism, (4) low protein may produce less taurine which is
necessary to conjugate free bile acids, and, (5) as the
metabolism of essential fatty acids seems to be correlated
with cholesterol catabolism in vivo, a low-protein diet may
impair the metabolism of essential fatty acids in liver,

which may indirectly decrease the rate of cholesterol

catabolism.

EXPERIMENTAL

A. Diets and Experimental Design

Forty male, weanling, albino rats of the SpraguemDawley
strain were divided into four groups of ten animals each for
the experiment. The four groups of animals were treated as
follows: (1) two groups were subjected to the flashing of
16-60 watt lights at irregular intervals for 12 hours each
night; one of these groups was fed a 20% casein diet (Group
D), and the other a 9% casein diet (Group B); (2) the other
two groups were likewise differentiated by the level of pro-
tein intake - Group A, 9% casein and Group C, 20% casein.
The experimental groups (exposed to flashing light) were in
one section of the room with their cages enclosed by a wooden
frame to which a heavy canvas covering was nailed. The can-
vas was rolled up when the lights were not in operation, and
let down when the lights were flashing. The control animals,
in another section of the room, were in a similar canvas
enclosure so that both experimental and control animals were
subjected to a 2 degree rise in temperature during the night.
The control animals were thus shielded from the lights by
two thicknesses of canvas and the distance between the two

enclosures.

The diets provided the following nutrients expressed

per 100 gm diet:

14

15

Low Protein Dietl

Casein

Salt Mixture W2
Choline

Corn Oil

Sucrose 8
DL-Methionine
DL-Tryptophan
DL-Threonine
Vitamin Mix

0

MWHUCDOHOO

UIONOOJE‘

O O 0
U1

OOOOOU‘IO-b'Ko

0 O O 0 0

The vitamin mixl

100 gm diet:

Vitamin A Powder (20,000 IU/gm)
Niacin

Calciferol

Thiamine HCl

Riboflavin

Pyridoxine

Calcium Pantothenate

Inositol

Folic Acid

Vitamin 812 (0.1% trituration with mannitol)

Biotin
Para-aminobenzoic Acid
Menadione

Sucrose

The initial average weight of any one group of rats did

High Protein Diet

Casein

Salt Mixture W2
Choline

Corn Oil
Sucrose

Vitamin Mix

20.0
4.0
0.15
5.0

70.60
0.25

contained the following in milligrams per

H
HOMOOI’DOOOOHO

(D

O O O O

OOOOOOMU‘IUli-JOO
U‘l

H

O o O
|-’ h.)

0

O

0.38
222.16

not vary from that of any other group of rats by more than

one gram. The animals were housed individually in metabolism

cages. Food and water were provided ad libitum to all ani-

mals and records were kept of each animal's food intake and

weight gain. Urine and feces were collected during the last

 

1. Harper, A.E. 1959 Sequence in which amino acids of
casein are limiting for rat growth.
2. Wesson modification of Osborne and Mendel salt mixture.
Science, Zfifi339, 1932. Obtained from Nutritional Bio-

chemicals Corp.

J. Nutrition,§1:109.

16

two weeks of the experiment to be used in the calculation of
nitrogen balance. On the 23th day, the animals were anes-
thetized and blood samples obtained from tail snips. There»
after, they were decapitated and their livers and adrenal
glands removed for purposes of the chemical analyses to be
described below.

B. Chemical Analyses

Serum Cholesterol Determinations; Total and free cho-
lesterol in the blood sera were determined by a modified
SchoenheimermSperry methodl. Serum samples of 0.2 ml were
pipetted into a 5 ml volumetric flask containing about 3 m1
of 1:1 acetone-alcohol. The mixture was heated, agitated,
cooled and made to volume with acetone-alcohol. The protein,
denatured by the solvent and heat treatment, was removed by
filtration through fluted, fatwfree filter paper. One ml of
the filtrate was pipetted directly into 5 ml centrifuge tubes
for the total cholesterol; 2 ml of this filtrate were pipetted
directly into 5 m1 centrifuge tubes for the free cholesterol,
and 50% KOH was added to each tube for saponification and
consequent release of cholesterol from its esters. For both
total and free cholesterol, 2 ml of a solution of 0.5%
alcoholic digitonin were added to the acidified extracts
(alcoholic acetic acid). The precipitated digitonide was
allowed to flocculate in a water bath at 600 C for about

30 minutes, and was then stored in the dark at room

 

l. Schoenheimer, R. and W.M. Sperry 1934 A micro deters
mination of free and combined cholesterol. J. Biol.

Chem., 106:745.

17

temperature for 48 hours. The tubes containing the cholesa
terol digitonide were centrifuged for 30 minutes at about
1500 r.p.m. and the supernatant was decanted. The precipi-
tate was washed again with 1:1 acetone-ether, recentrifuged
for 15 minutes, and the supernatant again decanted. The
washing process was repeated two more times with anhydrous
ether. The centrifuge tubes were placed in a sand bath in an
oven at 110-1150 C for 30 minutes and each precipitate was
then dissolved in 1 ml of glacial acetic acid while still
hot. After cooling, 2 ml of cold Liebermananurchard reagent
(1:20 sulfuric acid and acetic anhydride) were added to each
tube. After allowing 27 minutes for the development of color,
the optical densities of the cholesterol solutions were read
in a Beckman DB Spectr0photometer at 620 mu. Cholesterol
concentrations were determined by comparison with a standard
curve.

The mg of total serum cholesterol per 100 cc of blood
was obtained by multiplying the reading on the standard
curve by a factor of 2500,and a factor of 1250 was used for
determining mg of free serum cholesterol per 100 cc of blood.
The factors were obtained thus:

factor for total = 5 ml (total vol. of extract) x 100 : 2500
0.2 ml (vol. serum) 1 ml
(sample)

factor for free : 5 m1 (total vol. of extract x 100 = 1250

0.2 ml (vol. serum) 2 ml
(sample)

18

sample calculation:
Al - Beckman reading: total, .044; free, .021

standard curve reading for total cholesterol: .029

standard curve reading for free cholesterol: .014

total mg cholesterol % : .029 x 2500 : 72.5

free mg cholesterol % = .014 x 1250 : 17.5

Liver Glutathione: Method of Woodward and Fry (39). The

livers, which had been stored frozen, were thawed at room
temperature for about 2 hours. A 5 gm portion was homogenized
with 5 ml of 4% sulfosalicylic acid in a Waring homogenizer.
The contents were decanted into a 50 m1 volumetric flask and
the residue was again homogenized with 10 m1 of 2% sulfosali-
cylic acid. The contents were quantitatively transferred to
the 50 ml volumetric flask containing the original extract.
The volumetric flask was then filled to volume with 2% sulfo-
salicylic acid. The mixture was shaken and allowed to stand
for 30 minutes whereupon an extract was obtained by centri-
fuging and decanting the supernatant. A 10 ml aliquot of
the sulfosalicylic acid extract was placed in a 50 ml Erlen-
meyer flask. To this sample were added 2.5 ml of 4% sulfo-
salicylic acid, 2.5 m1 of 5% potassium iodide, and 2 dropaof
starch solution. A blank was prepared in a second 50 ml
Erlenmeyer flask by adding the same reagents to 10 ml of 2%
sulfosalicylic acid. The flask containing the sample was
placed against a white background in a water bath containing
ice and water. A microburet was used to add 0.001 N potas-
sium iodate to the sample and the end point identified when

there was a persistent blue color. The blank was treated in

a similar manner. In order to determine the ascorbic acid

19

content of the samples, a second titration series was carried
out. A 10 ml aliquot of the sulfosalicylic acid extract was
placed in a 50 ml Erlenmeyer flask, while a blank consisting
of 10 ml of 2% sulfosalicylic acid was prepared in a second
flask. The sample, blank, and 10 m1 of standard ascorbic
acid solution were titrated with 2,6-dichlorophenolindophenol
reagent using a microburet. The end point was reached when
there was a faint pink color that persisted for 2 minutes or
more.

The uncorrected glutathione concentration for 10 ml of
sulfosalicylic acid filtrate (in milligrams per 100 gm) was
obtained by dividing 100 timaathe volume (ml) of 0.001 N
potassium iodate (sample minue blank) used by 3.26 which is
the theoretical titer for 1 mg of glutathione. Then 3.5
times the ascorbic acid concentration (100 times the volume
in ml of 2,6-dichlorophenolindophenol - sample minus blank-
used, divided by the titration volume for the same amount of
standard ascorbic acid solution) was subtracted from the un-
corrected glutathione value in order to obtain the corrected
glutathione concentration.
sample calculation:

Al - weight of liver sample: 5.00 gm

titer of K104: 4.14 ml

titer of 2,6-dichlor0phenolindophenol: 1.73 ml

blank titer of K104: .05 m1

blank titer of 2,6-dichlorophenolindophenol: .04 ml

titration of standard ascorbic acid with
2,6~dichlorophenolindophenol: 10.50 ml

100 x (4.14-.05) - 3.5 x (loo x (1.73-.04); = 69.11 mg

3.26_ 10.50 glutathione

per 100 ml
extract

20

Nitrogen Determination: Urinary and fecal nitrogen were
determined by the macro Kjeldahl technique.

C. Statistical Analysis

Standard error of the means were calculated on all data

and Student's "t" test was used as a measure of significance.

RESULTS

A. Effects of Stress with Varying Levels of Protein
The flashing of 16-60 watt bulbs into the cages of male,
albino rats from a distance of two and one half feet at ir-
regular intervals, while not affecting food intake signifia
cantly as is seen by comparison with the food intake of the
control animals (Groups A and C), had a marked effect on the
growth of the low-protein, stressed animals (Group B). The
growth of the high-protein stressed animals (Group D) was
not affected (table 1, figure 1).
Table 1. Influence offlashing light stress on growth and

food intake in albino rats receiving high or low
protein diets.

 

Food Intake1 Wt. Gain2 Wt. Gain

 

Group _:§tress _2iet ngweek gm/week ng4 weeks
A No 9% Casein 8112* 30:1 123:4
B Yes 9% Casein 75t2 2511 98:3
C No 20% Casein 9010 4310 17012
D Yes 20% Casein 9010 4310 173:2

 

* Standard error of the mean

1 Level of statistical significance: A vs B, none; C vs D,
none; A vs C, 1%; B vs D, 1%.

2 Level of statistical significance: A vs 8, 1%; C vs D,
none; A Vs C, 1%; B vs D, 1%.

 

All groups were found to be in positive nitrogen balance

21

22

(table 2, figure 2); liver glutathione was found to increase
with protein level and stress (table 3, figure 3). Total
serum cholesterol levels did not differ significantly among
any of the groups (table 3, figure 4) but free serum Choles-
terol was found to be highest in the low~protein, stressed
animals, and lowest in the high-protein, stressed animals
(table 3, figure 5).

Table 2. Nitrogen retention in the albino rat as affected

by the stress of flashing lights with high and
low protein diets.

 

 

 

Group Stress Diet Mg Nitrogen1 retainedlday
A No 9% Casein 169t4*
B Yes 9% Casein 15815
C No 20% Casein 26016
D Yes 20% Casein 306:6
*

Standard error of the mean
1 Level of statistical significance: A vs B, none; C vs D,
1%; A vs C, 1%; B vs D, 1%

 

Table 3. Influence of flashing light stress on cholesterol
and glutathione levels in albino rats on high and
low protein diets.

 

 

Liver Total Serum Free Serum

Glutathione1 Cholesterol Cholesterol

Group Stress Diet mg/lOO gm mg/lOO 002 mg/100 003
No 9% Casein 55.7:4.0* 83.3:5.3 22.211.l

Yes 9% Casein 104.112.0 94.314.0 30.011.7
No 20% Casein 134.2i6.7 94.815.4 21.010.5
Yes 20% Casein 160.915.3 81.8:4.o 14.2t0.2

UOwb

 

*

Standard error of the mean

1 Level of statistical significance: A vs B, 1%; C vs D, 1%;
A vs C, 1%; B vs D, 1%

Level of statistical significance: A vs B, none; C vs D,
none; A vs 0, none; B vs D, 5%

Level of statistical significance: A vs B, 1%; C vs D, 1%;
A vs C, none; B vs D, 1%

WM

The low-protein, stressed animals had the largest adrenal
glands (weight per 100 g of body weight); the nonwstressed,
low-protein group had the next largest, and the highuprotein
groups had the smallest (table 4, figure 6). The low-protein,
stressed animals had the smallest livers per 100 g of body
weight; the livers of the other groups did not differ sig-
nificantly (table 4, figure 7).

Table 4. Adrenal gland weights in albino rats subject and

not subject to the stress of flashing lights while
fed high or low protein diets.

 

Adrenal Gland Wt.1 Liver Wt.2

 

 

 

Group Stress Diet gm/100_gm of body weight
A No 9% Casein 18.1io.8*- 5.010.1
B Yes 9% Casein 22.4xo.9 4.110.l
C No 20% Casein 14.9il.0 5.2:0.1
D Yes 20% Casein 15.410.6 5.0i0.1
*

Standard error of the mean

1 Level of statistical significance: A vs B, 1%; C vs D,
none; A vs C, none; B vs D, 1%

2 Level of statistical significance: A vs B, 1%; C vs D,
none; A vs C, none; B vs D, 1%

 

B. Effect of Varying Protein Levels

When the high and low protein control groups were com-
pared with each other (Groups C and A), the expected results
were noted. Rats fed the 20% protein diet consumed signifi-
cantly greater amounts of food and gained a significantly

greater amount of weight (table 1). The rats fed the 20%

24

protein diet retained more nitrogen (table 2) and had higher
levels of liver glutathione than did those animals fed 9%
casein (table 2). Total and free serum cholesterol (table 3)
and adrenal gland weights (table 4) did not vary significantly
between the two groups. Liver weights per 100 gm body weight
also did not vary between the two groups (table 4).

When the high and low protein stressed groups were com-
pared with each other (Groups D and B), weight differences
were again noted; the high protein group weighed considerably
more than the low protein group (table 1). The stressed
rats fed the high protein diet retained more nitrogen (table
2) - almost twice as much m and had higher levels of liver
glutathione (table 3) than did the animals receiving the low
protein diet. Unlike the comparison of the controls, however,
the comparison of the stressedggbups revealed the low~protein
stressed rats (Group B) had significantly higher total and
free serum cholesterol levels than the high-protein stressed
rats (Group D, table 3). Group B also had larger adrenal

glands and smaller livers than group D (table 4).

Weight in Grams

225

210

195

180

165

150

135

120

105

90

75

6O

45

30

15

25

C. - Graphical representation of the results

Figure 1. Influence of stress on growth of male, albino rats fed

verying levels of protein (casein) in the diet. {Weaniing
rats sacrificed after 28 days on experimentfifi

 

 

 

 

A - low (9%) protein diet, no stress o—o
.. B - low (9%) protein diet, STRESS 1:1-«E1
C - high (20%) protein diet, no stress A—sA A
__ D - high (20%) protein diet, STRESS em.
"- >
C
11-
-.. q
, O
I
.1- e
9
—u— :/
ii 1 l i: i: 1411.1
2 4 6 8 10 12 14 l6 18 20 22 24 26 28

Mg Nitrogen Retained

310

,-

20

Figure 2. Influence of stress on nitrogen retention of male, albino

rats fed varying levels of protein (casein) in the diet.
(Weanling rats sacrificed after 28 days on the experiment.)

‘_

 

300.._

290-—

280-4—

270....

260__,_

250—-—

U0033>

240-1—-

230-..

220—

210—

200-—

190—.

180..

170—

iso—r

 

150

 

low (9%) protein diet, no stress
low (9%) protein diet, STRESS
high (20%) protein diet, no stress
high (20%) protein diet, STRESS

 

 

 

 

 

 

 

 

 

 

 

 

27

Figure 3. Influence of stress on liver glutathione levels of male,

albino rats fed varying levels of protein (casein) in the diet.
(Weanling rats sacrificed after 28 days on experiment.)

 

170
160 -—
150 __
140 ~—
130 ——

120 -+—

1104-

o x] 00 \o 5
o o o o o
1 l l l L
I 1 I I 1

Mg of Glutathione per 100 gm. Liver
Ln
0
l
1

30 L—

10....

 

UOtD3>

low (9%) protein diet, no stress
low (9%) protein diet, STRESS
high (20%) protein diet, no stress
high (20%) protein diet, STRESS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

:r
all

.1. .
.x‘iv

Blood

Mg Cholesterol per 100 C-o‘Co

28

Figure 4. Influence of stress on total serum cholesterol levels of male,
albino rats fed varying levels of protein (casein) in the diet.
(Weanling rats sacrificed after 28 days on the experiment.)

 

100 . .
low (9%) protein diet, no stress

low (9%) protein diet, STRESS
high (20%) protein diet, no stress .
high (20%) protein diet, STRESS

95——

 

U0w>
I

 

94+.

93-1.

0) CO 0) oo (3
O‘ x) 00 \o O
L 1.. l l l 1
l I l l I

oo
_ U1

1

I

00
w:-

_____L
1

 

83-5—

82—h.-

 

81 ..'...

 

 

 

 

 

 

 

 

 

80‘ —~

 

 

Mg. Free Cholesterol per 100 c. c. Blood

Figure 5.

29

Influence of stress on free serum cholesterol levels of male,
albino rats fed varying levels of protein (casein) in the diet.
(Weanling rats sacrificed after 28 days on the experiment.)

 

32

3o ——

28 ._.._

26 1—

24 ——

N
N
l
I

N
o
L
I

55
I
I

’5
J
I

p—a
g:

H
N
l
l

H

O
I
r

 

‘-

low (9%) protein diet, no stress
low (9%) protein diet, STRESS
high (20%) protein diet, no stress
high (20%) protein diet, STRESS

 

UOU53>
I

 

 

 

 

 

 

 

 

 

 

 

 

Weight of Adrenal Glands-grams per 100 g's of Body Weight

23

22

21

20

l9

18

17

16

15

14

13

12

11

10

30

Figure 6. Influence of stress on adrenal gland weights of male, albino
rats fed varying levels of protein (casein) in the diet.
(Weanling rats sacrificed after 28 days on the experiment.)

 

 

 

 

 

A - low (9%) protein diet, no stress

B - low (9%) protein diet, STRESS
'T' C - high (20%) protein diet, no stress

D - high (20%) protein diet, STRESS
AI—

 

 

 

 

 

 

 

 

 

 

 

- g. per 100 g. of Body Weight

Liver Wt.

5g.

4g.

3g.

2g.

31

Figure 7. Influence of flashing lights on liver weights of male,

 

 

UOUJIP-

albino rats fed varying levels of protein (casein) in
the diet. (Weanling rats sacrificed after 28 days on
experiment.)

low (9%) protein diet, no stress
low (9%) protein diet, STRESS
high (20%) protein diet, no stress
high (20%) protein diet, STRESS

 

i
l
I
!
i
!

 

 

 

 

 

 

 

 

 

 

DISCUSSION

A. Effects of Stress with Varying Levels of Protein
1. Influence of stress on growth

Introducing the stress of flashing lights to rats re-
ceiving a diet containing 9% casein as the protein source
markedly reduced the growth of makaalbino rats as is evi-
denced by comparison of the growth of these animals with the
growth of rats receiving the same diet but not subjected to
the stress. This growth reduction occurred despite the fact
that there was no significant difference in food intake be-
tween the two groups. It should be noted, however, that the
difference in food intake and difference in growth between
the two groups are of the same magnitude. The fact that the
food intake differences are not statistically significant is
undoubtedly influenced by the difficulties in obtaining exact
measurements of food intake. It should be considered, there-
fore, that the reduced food intake on the part of the stressed,
low-protein animals had some bearing on the reduced growth
rate. The low level of protein alaa influenced the restric-
tion of growth as is evidenced by the fact that the low-
protein, non-stressed group gained significantly less weight
than did the high-protein, non-stressed group. However, in
this latter case there is also a difference in food consump-
tion to be considered, for the low protein group ate con-
siderably less food than did the high protein group.

32

33

An examination of data in the two high-protein groups
reveahsthat the introduction of the lights had no influence
on the growth rate of the rats - both stressed and non-
stressed groups attaining the same level of growth.

In view of the reports by Chaloupka et al. (6) indi-
cating preferential use of limiting amounts of amino acids,
it seems in this study the preferential use of amino acids
might be an important factor in the reduced growth rate of
the stressed animals receiving the low protein diet.
Chaloupka reported the first requirement met by amino acids
was for the maintenance of nitrogen balance and growth re-
quirements were fulfilled after this. Assuming protein ca-
tabolism to be increased by stress, then Group B (low-protein,
stress) would have a greater requirement for amino acids to
maintain positive nitrogen balance (since these were growing
animals) than would Group A (low-protein, non-stress).

Hence the supply of amino acids for growth in Group B would
be decreased, resulting in the observed decreased growth
rates in these animals. Also, the increased levels of the
nitrogen-containing compound, glutathione, in the liver in-
dicate further increase in nitrogen requirements which would
limit the supply of nitrogen available for growth.

Ample supplies of amino acids were available to animals
on the high protein diets to satisfy the requirements for

both maintenance of positive nitrogen balance and growth.

34

2. Influence of stress on nitrogen retention

While all groups remained in positive nitrogen balance,
as is mandatory with the growing animal, it can be seen that
the low-protein, stressed animals showed the lowest degree
of positive balance, and the high-protein, stressed animals
the highest. Chaloupka's theory on preferential distribution
of amino acids offers a possible explanation for the varia-
tion in nitrogen retention among the four groups. The non-
stressed group (A) apparently had sufficient protein to keep
them in positive nitrogen balance and to promote growth at a
moderate, if somewhat limited, rate. The stressed group (B)
on this diet, while able to maintain positive nitrogen bal-
ance, had markedly reduced growth rates.

Group C - the animals receiving the 20% casein diet and
not subjected to the light - apparently was receiving more
than adequate supplies of essential amino acids. These ani-
mals were kept in a state of positive nitrogen balance, grew
at a healthy rate, and excreted the excess nitrogen. Group D,
however, receiving the same diet but subjected to the lights,
while maintaining positive nitrogen balance and growing Just
as well as its non-stressed counterpart, retained more nitro-
gen than did Group C and it is suggested that this difference
in nitrogen retention reflects an increased need for amino
acids established by the stress of the flashing lights. The
mechanism whereby the stress caused an increased amino acid

requirement was not elucidated in this particular study.

35

3. Influence of stress on liver glutathione levels

Although no confirmation of this theory can be found in
the literature, it would not seem unreasonable to suggest
liver glutathione levels increase with an increase in dietary
protein. Jurtshuk et al. (23) found blood glutathione levels
dropped significantly when the animal was exposed to intense
auditory stimulation - but whether an inverse relationship
exists between blood glutathione levels and liver glutathione
levels has not been established. The stressed animals in
this experiment showed significantly increased levels of
liver glutathione. It is a possibility that stress inhibits
mobilization of glutathione out of the liver.

4. Influence of stress on adrenal gland weights

The animals with the heaviest adrenal glands per 100 gm
of body weight were those stressed animals receiving the low
protein diet. Evidence that the low protein nature of the
diet also contributed to the general stress of the animals
is offered by the fact that the adrenal glands of the non-
stressed animals on the low protein diet were considerably
larger than those of the non-stressed animals on the high-
protein diet. Moreover, the differences in adrenal size
between the two non-stressed groups were not significant
while the differences between the two stressed groups were
significant at the 1% level, indicating the combination of
low protein plus the stress of the lights had a greater ef-

fect on adrenal gland size than did the stress of the low

36

protein diet alone.
5. Influence of stress on serum cholesterol levels

The total serum cholesterol levels of the rats subjected
to the stress of the flashing lights and fed a diet containing
9% casein were not significantly higher than those of animals
fed the same diet but not exposed to the lights. There was
likewise no significant difference between total serum cho-
lesterol levels of the two high protein groups - stressed and
non-stressed. These findings are in keeping with those of
Uhley and Friedman (36) who found that at four months, rats
subjected to the stress of electric shock had total serum
cholesterol levels comparable to those of animals not re-
ceiving the shock, but at ten months the stressed rats had
significantly higher levels of serum cholesterol. Again,
in keeping with the findings of Fillios, Andrews, Mann and
Stare (11) who found raising the level of dietary protein
waseffective in lowering serum cholesterol only after flfgy-
six‘ggyg on the diet and there had to be a great difference
in dietary protein levels to show a significant difference
in cholesterolemic response, the two non-stressed groups -
one fed 9% casein, the other fed 20% casein, did not show
significant differences in total serum cholesterol levels.
The two stressed groups, however, one on a high protein diet,
the other on a low protein diet, showed a difference in
cholesterolemic reSponse significant at the 5% level, indi-
cating the stress of the blinking lights coupled with the

37

low protein nature of the diet tended to bring about an in-
crease in the level of total serum cholesterol that was not
evident when the only difference between groups was an 11%
difference in the level of dietary protein.

While the differences are not statistically significant,
there is a trend for the high-protein, non-stressed group to
have higher total serum cholesterol levels than the high-
protein, stressed group. This might possibly be explained
on the basis of efficiency of cholesterol mobilization and
catabolism: the animals in Group D (high protein) as a result
of the stress to which they were subjected, were operating
at a greater level of efficiency, as evidenced by the fact
of greater nitrogen retention and lower serum cholesterol
levels.

Some interesting results were obtained with reSpect to
the levels of free serum cholesterol: the low-protein,
stressed animals had free serum cholesterol levels signifi-
cantly higher than those of any of the other groups, while
the high-protein, stressed animals had free serum cholesterol
levels significantly lower than those of any of the other
groups. In reviewing the reports of other workers,frequent
suggestions are made of an association between B-lipoprotein
levels and cholesterol leVels. Gero et a1. (14) found that
rats injected with B-lipoproteins showed significantly lower
cholesterol levels than those not receiving the injection,

and Nishida et al. (31) stateithat if cholesterol is assumed

38

to be mobilized for catabolism in the form of lipoproteins,
a low protein diet may produce less protein than required
for mobilization which could result in lowered cholesterol
catabolism.. In the light of these findings certain sugges-
tions in regard to the results obtained in the present study
seem appropriate.

In the first place, it is possible that the reason for
the higher level of free serum cholesterol in the stressed,
low-protein group is that there was insufficient protein
available for cholesterol binding. The possibility exists
that the resulting inefficiency in mobilization of the cho-
lesterol for catabolism might eventually result in the depo-
sition of the free cholesterol on the arterial walls and
this possibility is in need of further investigation.

A second possibility is that Group D, stressed and re-
ceiving a diet high in protein, produced more cholesterol as
a result of the stress and by retaining more nitrogen was
able to facilitate mobilization and eventual catabolism of
the cholesterol. In other words, Group D produced more
cholesterol, as did Group B, however, unlike Group B, had
sufficient protein (as a result of increased nitrogen reten-
tion) to mobilize the cholesterol in the form of lipoproteins
for catabolism, resulting in a final total serum cholesterol
level comparable to that of Group C (high-protein, non-
stressed) and a free serum cholesterol level lower than that

of Group C because of increased effeciency in cholesterol

39

binding.
6. Influence of stress on liver weights

Of the two stressed groups, only the one fed a low pro-
tein diet exhibited significantly smaller livers on the basis
of weight per 100 gm of body weight than did its dietary
counterpart. The fact that this stressed group had smaller
livers than the nonmstressed group could perhaps be explained
on the basis of a reduction of the protein availability by
the introduction of the stress. That is, the stress possibly
introduced increased requirements for amino acids,thereby
reducing the supply available for the synthesis of liver
tissue.

B. Effect of Varying Protein Levels

When the high and low protein groups are compared with
each other (Groups C and A) the results are as expected
(tables 1-4). However, none of the differences are as great
as those seen when the stressed groups are compared (tables
1-4). This indicates differences resulting from a variation
in protein intake are augmented and compounded by the intro-
duction of stress. In the nonmstressed animals there is no
significant difference in the total cholesterol levels of
the low and high protein groups; in the stressed animals

this difference is significant.

SUGGESTIONS FOR CONTINUATION OF THIS STUDY

Several questions have not been fully answered by the
present study and several points have been raised. After5
verifying the results obtained in this experiment, an at-
tempt should be made to clarify these points.

It would be of some value to use brighter lights and to
try increased exposure to the lights. Other levels of can
sein in the diet should be used as well as other sources of
protein, also at various levels.

The relationship between stress, protein, and cholesterol
need further elucidation. Continuation of the experiment over
a longer period of time might result in more significant re=
ductions in blood cholesterol levels. Examination of the
arterial walls for evidences of cholesterol deposition might
reveal the extent of the influence of the highmprotein diet.

It is suggested that adult animals be used in the study,
and blood samples for cholesterol determination be taken at
the beginning of the experiment as well as at the end. The
reason for the increased nitrogen retention on the part of
the stressed, high-protein animals should be explored. Also
there should be a determination of B-lipoprotein (SF 12~4OO
type) level in both the stressed and non-stressed animals at
the beginning and end of the experiment. At some date the
mechanism through which stress affects blood cholesterol

levels should be explored. 4
O

SUMMARY AND CONCLUSIONS

The object of this study was to determine the influence
of varying levels of protein on the effect of the stress of
bright flashing lights on male, albino rats. Forty rats were
divided into four groups of ten animals each. The four groups
were housed in a non-temperature controlled, non-air-condi-
tioned room where two of the groups were subjected to the ir-
regular flashing of 16-60 watt bulbs at a distance of two
and one half feet for twelve hours each day. Ten of these
"stressed" rats were fed a diet containing 20% casein and
ten received a diet containing 9% casein. The other two
groups in the same room were likewise divided according to
diet, but were not subjected to the stress of the flashing
lights. The animals were sacrificed after 28 days and liver
and blood samples were taken for analysis.

Both of the stressed groups showed larger adrenal
glands than their dietary counterparts and also had higher
levels of liver glutathione. The low-protein, stressed ani-
mals gained less weight and had higher free serum choles-
terol levels than did their controls, while the high-protein,
stressed group had lower free serum cholesterol levels and
higher nitrogen retention than did its control group.

It has been postulated that the results obtained re-
garding growth and nitrogen retention lend support to the
hypothesis that stress alters protein requirements: in the

41

42

low-protein, stressed group there was only sufficient pro-
tein to maintain positive nitrogen balance - not to support
the rapid growth that was evident in the animals receiving
the same diet but not subjected to the flashing lights;
perhaps the stress was causing a need for extra protein to
meet a Specific but as yet unknown requirement. The high-
protein, stressed group, however, had enough protein to pro-
mote growth, maintain nitrogen balance and meet the unknown
requirements.

Lack of protein for cholesterol binding is believed to
be the reason for the high level of free serum cholesterol
in the stressed, low-protein animals. At the same time, the
high degree of nitrogen retention in the stressed, high-
protein animals is believed to account for the cholesterol
situation in this group - the stress caused them to produce
more cholesterol,additional nitrogen was retained to facili-
tate cholesterol binding and mobilization which resulted in
less free serum cholesterol.

It is concluded the stress induced in rats by bright,
flashing lights has a definite effect on the protein re-

quirements of the male albino rat.

10.

ll.

12.

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