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This is to certify that the

thesis entitled

CHANGES IN TOTAL SERUM CHOLESTEROL IN RESPONSE TO A DECREASE
IN DIETARY CHOLESTEROL AND MODIFICATION OF THE AMOUNT AND
TYPE OF DIETARY FAT: A CONTROLLED DIETARY STUDY

presented by

D. Margaret Ullmann

has been accepted towards fulfillment
of the requirements for

M. S. degree in Nutrigign

 

 

Date 2/19/80

0-7839

OVERDUE FINE§z
25¢ per do per it.
RETUMIIG LIBRARY MATERIALS:

Place in book return to remove
chum fro- circulation records

 

 

 

 

CHANGES IN TOTAL SERUM CHOLESTEROL IN RESPONSE TO A DECREASE
IN DIETARY CHOLESTEROL AND MODIFICATION OF THE AMOUNT AND
TYPE OF DIETARY FAT: A CONTROLLED DIETARY STUDY

By

D. Margaret Ullmann

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1979

ABSTRACT
CHANGES IN TOTAL SERUM CHOLESTEROL IN RESPONSE TO A DECREASE

IN DIETARY CHOLESTEROL AND MODIFICATION OF THE AMOUNT AND
TYPE OF DIETARY FAT: A CONTROLLED DIETARY STUDY

By

D. Margaret Ullmann

Thirty-two healthy men (mean age = 24.8 years) were
fed a control diet containing 42 to 45% of total calories
from fat, a P/S ratio of 0.3 to 0.5, and two eggs/day for
ten days. During the next eight weeks, 16 subjects received
each of the following diets for four weeks in a crossover
design: (l) Diet C-E, the control diet (with two eggs/day
or (2) Diet C-ES, the control diet with eggs replaced by a
cholesterol-free egg substitute. The remaining 16 subjects
received each of the following diets in a similar crossover
design: (l) Diet MF-E, a modified fat diet containing 35% of
total calories from fat, a P/S ratio 21.0, and two eggs/day
or (2) Diet MF-ES, the modified fat diet with eggs replaced
by a cholesterol-free egg substitute. Diets for each
subject were adjusted to maintain body weight. Average
serum cholesterol values during the control period ranged
from 195 to 288 mg/lOO ml. A change from the control period
to the modified fat diet produced a decrease in serum
cholesterol concentration in 15 of the 16 subjects. In
addition, comparison of the mean serum cholesterol con-

centrations during the control period with average weekly

D. Margaret Ullmann
serum cholesterol values during the experimental diet
periods showed a statistically significant decrease (p<0.5)

when subjects consumed egg substitute versus two eggs/day.

ACKNOWLEDGMENTS

I wish to express my sincere appreciation to the many
people who helped to make this study possible. To:

Esperanza Briones for her assistance in menu planning
and daily diet modification calculations.

Shirley Ann Mellen and Patricia Smith Brown for their
assistance in performing the laboratory analyses.

Dr. Charles Rhoades for the time he contributed
generously in serving as the physician for the study.

Dr. J. Gill for statistical advice and assistance.

The men who participated as subjects in the study for
their cooperation and compliance.

The members of my thesis committee - Dr. Jenny Bond,

Dr. Gilbert Leveille, and Dr. David Rovner for their

expertise in research and thesis development.

Dr. Wanda Chenoweth, my major professor, for her

guidance, expertise, patience, kindness, criticism,

and words of encouragement.

I wish to express gratitude to Standard Brands, Inc.,
for their support of this project, and particularly to
Dr. Ron Simpson.

And last, but not least, I would like to acknowledge
my father, Alexander Ullmann, my sister, Sabrina, and my

friends for their love and support throughout my graduate

school career.

ii

TABLE OF CONTENTS

LIST OF TABLES.

LIST OF FIGURES
INTRODUCTION.

REVIEW OF LITERATURE.

Historical Approach.
Dietary Cholesterol. .
Compensatory mechanisms for serum choles-
terol homeostasis.
Dietary Fat. . . .
Level of dietary fat . . .
Degree of saturation of fat.
Chain length of fatty acids. .
Proposed mechanisms for cholesterol lowering
effect of polyunsaturated fats
Summary. . .

METHODS AND PROCEDURES.

Subject Recruitment.
Screening Procedures
Subject Characteristics.
Orientation. .
Informed Consent . .
Experimental Diet Plan
Menus.

Meals. .
Food Preparation . .
Food Intake Records.
Blood Tests.
Statistical Design

RESULTS .

Body Weights
Nutrient Intake. . .
Total Serum Cholesterol.

DISCUSSION.
The Design of the Study.
Subject Selection.
Subject Compliance .
Body Weight. .
Alcohol Consumption. .
Changes in Serum Cholesterol
CONCLUSIONS .
SUGGESTIONS FOR FURTHER RESEARCH.
APPENDICES.

LIST OF REFERENCES.

iv

106

LIST OF TABLES

Table Page
1 Summary table of subject characteristics. . . . 35
2 Experimental diet plan. . . . . . . . . . . . . 40

3 Comparison of the nutrient composition of two
large whole eggs and an equivalent serving
portion of the cholesterol-free egg substitute
used in this study. . . . . . . . . . . . . . . 43

4 Schedule of blood tests . . . . . . . . . . . . 48

5 Average chan e in body weight within the con-
trol period weeks 1-2) and each of the experi-
mental diet periods (weeks 4-6, 7-10) . . . . . 51

6 Dietary intake of protein, fat and cholesterol. 54

7 Concentration of total serum cholesterol and
percentage change in subjects who continued on
the control diet with 2 eggs/day during the
first 4-week experimental diet period, followed
by egg substitute during the last 4-week
experimental diet period. . . . . . . . . . . . 57

8 Concentration of total serum cholesterol and
percentage change in subjects who consumed the
control diet with egg substitute during the
first 4-week experimental diet period, followed
by eggs during the last 4-week experimental
diet period . . . . . . . . . . . . . . . . . . 58

9 Concentration of total serum cholesterol and
percentage change in subjects who consumed the
modified fat diet with 2 eggs/day during the
first 4- week experimental diet period, followed
by egg substitute during the last 4- week
experimental diet period. . . . . . . 59

Table Page

lO Concentration of total serum cholesterol and
percentage change in subjects who consumed the
modified fat diet with egg substitute during
the first 4- week experimental diet period,
followed by eggs during the last 4- week
experimental diet period. . . . . . . . . 60

ll Analysis of variance of the data for total
serum cholesterol . . . . . . . . . . . . . . . 67

vi

LIST OF FIGURES

Figure Page

l Changes in total serum cholesterol (mg/lOO ml)
during each experimental diet period relative
to the control period for each of the four
diet groups. . . . . . . . . . . . . . . . . . . 65

vii

INTRODUCTION

Coronary heart disease (CHD) is the major cause of
death in the United States today (Gotto, l979). There is
much documentation in the literature of an association
between high serum cholesterol concentration and athero-
sclerosis (Keys, 1970; Kannel et al., l97l; Carlson and
Bottiger, l972). Epidemiological studies consistently have
shown higher serum cholesterol levels and a higher incidence
of CHD among p0pulations consuming diets high in cholesterol
and fats, particularly saturated fats (Jolliffe and Archer,
l959; Keys, 1970). Although the etiology of CHD is known
to be multifactorial, elevated levels of blood cholesterol
have been identified as a major risk factor in the devel-
opment of the disease (Connor and Connor, 1972). Numerous
studies in experimental animals and humans have demonstrated
a direct influence of diet on serum cholesterol concentra-
tions. Although the studies reported in the literature
fail to provide incontrovertible evidence of a causal rela-
tionship of serum cholesterol concentration to CH0, collec-
tively they support the theory that dietary manipulations
designed to lower serum cholesterol concentrations may be

effective in reducing the risk of CHD.

The effects of dietary cholesterol and fats on serum
cholesterol concentration have been studied extensively in
humans using formula diets as well as diets containing
natural foods; the effects of each have been studied inde—
pendently as well as together. There is general consensus
among researchers that reduced intake of saturated fat,
increased intake of polyunsaturated fat, and a decrease in
total dietary fat will reduce serum cholesterol concentra-
tion. There is, however, much controversy regarding the
extent to which an increase or decrease in dietary choles-
terol may alter serum cholesterol concentration in man.

Recently several studies have been reported indicating
that healthy subjects show no change in serum cholesterol
in response to ingestion of cholesterol in amounts equiva-
lent to one or two eggs/day (Slater et al., 1976; Porter
et al., 1976). Kummerow et a1. (1977) and Flynn et a1.
(1979) reported similar findings. Although the results of
these studies suggest that consumption of egg cholesterol
in regular meals does not significantly increase plasma or
serum cholesterol in man it would seem inappropriate to
conclude from them that changes in dietary cholesterol have
no effect upon serum cholesterol. The intake of foods
other than eggs by these subjects was not controlled. It
is possible that variation in cholesterol intake from foods
other than eggs or variations in the amount or type of fat

may have been sufficient to mask the effect of the amount

of cholesterol provided by one or two eggs/day.

In the experiment described herein, the effects of
the restriction of dietary cholesterol and modification of
amount and type of fat on serum cholesterol were studied
in free-living adult male subjects whose serum cholesterol
levels were in the upper normal range. This population
group was selected because of the belief that young men
with moderately elevated serum cholesterol levels would be
likely to benefit from preventive dietary measures designed
to lower serum cholesterol levels.

During the winter and spring of 1978 a two-part con-
trolled human feeding study was conducted at Michigan State
University to determine the effect on total serum choles-
terol of

1) replacing two eggs/day in a "typical" American diet

with a cholesterol-free egg substitute.

2) feeding a modified fat diet containing either two

eggs/day or an egg substitute.

The major difference between the present study and the
previous experiments of the effects on serum cholesterol
of adding or deleting one or two eggs/day to the diets of
young men in a free-living population is the degree of

control over food intake.

REVIEW OF LITERATURE

Historical Approach

The discovery over a century ago that cholesterol is
a prominent constituent of atherosclerotic plaque (Vogel,
1847) and that this same substance is also present in the
blood has stimulated interest in pursuing a better under-
standing of the interrelationship between diet, serum
cholesterol, atherogenesis and coronary heart disease
(CHO). Although it constitutes only a small fraction of
total body cholesterol, plasma cholesterol is the only
pool of cholesterol incriminated in CHD (Sodhi and Mason,
1977). Initially it was thought that the lipid deposits
found within the atheromatous arteries were produced by
local synthesis within the arterial walls. It has been
demonstrated conclusively, however, through the use of
radio-labelled cholesterol that cholesterol from the blood
stream does enter into the intimal atheromas and that much
of the labelled cholesterol recovered from the athero-
sclerotic plaque was that which was fed in the diet
(Chobanian and Hollander, 1962; Connor and Jackson, 1963).

It has been shown that hypercholesterolemia induced

in a variety of ways results in the development of

atherosclerotic lesions in a number of animal species.
Anitschkow (1933) produced hypercholesterolemia and the
characteristic lesions in the aorta and coronary arteries
in rabbits by feeding them 0.5% cholesterol dissolved in
highly unsaturated sunflower seed oil. Other investigators
have shown that the feeding of a cholesterol-vegetable

oil mixture or foods rich in cholesterol and fat induced
hypercholesterolemia and atherosclerosis in the guinea

pig, chicken, rat, hypothyroid dog, pig and monkey (Katz

et al., 1958).

Much criticism has been generated about the appli-
cability of the results from animal studies to humans
because of the inherent differences in the susceptibility
of various animals to CH0 and because of the differences
in their serum lipid responses to dietary modifications.
Frequently the level of cholesterol fed in animal studies
was enormously high in comparison with levels normally
consumed by humans. Cholesterol levels in the diet fed
to induce atherosclerosis in animals tend to range from
0.5 to 5.0% by weight of dry food, which would be equiva-
lent to feeding 1000 to 10,000 mg of cholesterol/1000 kcal
daily (Keys, 1952).

Several longitudina1,prospective studies have demon-
strated in a variety of population samples that risk of

CHD was directly related to the antecedent serum cholesterol

levels (Keys et al., 1963; Chapman and Massey, 1964;

 

Rosenman et al., 1970; Carlson and Bottiger, 1972). Data
from the Framingham study (Kannel et al., 1971) revealed
a distinct and Striking increment in the risk of CHD pro-
portional to the antecedent serum cholesterol concentration.
These data showed that serum cholesterol levels of 260 mg/
100 ml or more resulted in a five-times greater incidence
of CHD than a level below 200 mg/100 ml in men aged 30-49
years, and that plasma cholesterol concentration was pre-
dictive of risk in the development of heart disease.
Indirect evidence linking atherosclerosis to serum choles-
terol concentration is found in the diseases that are
associated with both hypercholesterolemia and premature
atherosclerosis, such as diabetes mellitus, nephrosis, and
hypothyroidism. Persons with inborn errors of cholesterol
metabolism, such as familial hypercholesterolemia and
hypercholesterolemia xanthomas, are particularly susceptible
to early development of atherosclerotic disease (Stanbury
et al., 1972). A parallel relationship also has been shown
between the age trend in serum cholesterol concentrations
and the age trend in development of atherosclerosis (Keys,
1952).

The theory that diet has a powerful effect on serum
cholesterol concentration and on the incidence of CHD has
been supported by convincing evidence from several world-

wide epidemiologic studies (Keys et al., 1957a;Jolliffe and

Archer, 1959; Toor et al., 1960; Connor and Connor, 1972)

 

which revealed a strong relationship between populations
consuming diets containing large quantities of animal
products and a high incidence of CHD. As early as 1916
De Langen reported that plasma cholesterol levels in
healthy Javanese men were much lower than in the Nether-
lands, France, or Germany. He observed that CHD was rare
among the Javanese population and suggested that this was
because their diet was low in cholesterol and other lipids.
Toor et a1. (1960) reported that Israeli immigrants from
Yemen had much lower serum cholesterol levels and were
far less prone to CH0 than either the eastern European
Israeli immigrants who consumed diets that were much higher
in dietary fat and cholesterol, or the Yemenites who had
immigrated to Israel earlier and had already adopted a
higher fat diet. Keys (1970) studied 18 population samples
of 40-59 year old men from seven different countries and
found that serum cholesterol levels correlated with the
consumption of cholesterol and saturated fat and with the
incidence of CHD. He concluded that 80% of the serum cho-
lesterol variability among these groups could be explained
by differences in the diet.

Other lines of epidemiological evidence linking diet
to CHO include the effects of wartime privations (Helwig
et al., 1952), geographic migrations (Toor et al., 1960),
and social class differences (Logan, 1952), all of which

are known to have profound effects on dietary patterns.

That the differences in the incidence of CHD seen in
the various population groups studied epidemiologically
were not simply racial or genetic variables has been demon-
strated in migrants of the same race who changed from a
low to a high cholesterol-high fat diet and experienced a
corresponding change in their serum cholesterol levels
together with a change in their propensity to CHO (Keys
et al., 1958). This point is clearly illustrated in a
study by Toor et a1. (1960) who showed that Yemenites gradu-
ally 1ost their relative protection from CHD as they
adopted the dietary pattern of European Jews. Similarly,
De Langen (1916) observed the Javanese stewards on Dutch
passenger ships who ate Dutch food developed similar blood
cholesterol levels to the Dutchmen and developed a higher
incidence of CHD. Bersohn and Wayburne (1956) also provided
evidence in support of the claim that serum cholesterol
concentration is dependent upon the composition of the diet
and is not the result of racial or genetic differences.
They demonstrated that the cholesterol concentration of
blood drawn from the umbilical cord of newborn infants of
different populations throughout the world showed no
dissimilarity due to race or class and was about 80 mg/

100 m1.

Epidemiologic studies have made it possible to survey

the effects of many different types of diets on serum cho—

lesterol levels in relatively large population samples.

The use of epidemiologic evidence, however, is often
belittled because of the arguments that (1) mortality and
CHO are influenced by a lifelong exposure to diet while
data of this kind often reflect dietary habits over a
relatively short period of time, (2) the changes in dietary
patterns are usually accompanied by changes in lifestyle
and environmental factors, such as stress, level of exer-
cise, calorie intake, smoking habits, sanitation and the
incidence of other diseases, and (3) food consumption data
are often unreliable in terms of describing individual
food consumption.

Although there is much documentation in the literature
of an association between high plasma cholesterol levels
and atherosclerosis, an etiologic association is more dif-
ficult to prove. An association is considered likely to
be "causal" if it a) is a consistently strong association
and b) precedes the disease by an appropriate period of
time. Serum cholesterol concentration meets both of these
criteria for causality. Although causality has yet to be
proven, it is evident that plasma cholesterol plays a sig-
nificant, albeit, undefined role in the development of CHD

and atherosclerosis.

Dietary Cholesterol
Extensive research has been conducted over the past
half-century in the hope of defining the relationship of

dietary cholesterol to serum levels in man. The results

10

of many of the studies have yielded conflicting, question-
able, and often inconclusive data (Ahrens, 1976). Instead
of providing answers, the results of these studies have
generated more confusion and controversy.

The effect of added cholesterol has been studied
using formula diets (Kinsell et al., 1952; Beveridge et a1.
1960; Erickson et al., 1964; Mattson et al., 1972). Diets
containing conventional foods also have been used to study
the effects of dietary cholesterol on serum cholesterol
concentrations (Messinger et al., 1950; Connor et al.,
1964; Grande et al., 1965; Anderson et al., 1976). In some
experiments comparisons were made between nearly choles-
terol-free diets and diets containing very large quantities
of cholesterol (Beveridge et al., 1960; Connor et al.,
1961). Purified cholesterol was added to the diet in some
studies (Kinsell et al., 1952; Beveridge et al., 1960)
while in others frozen egg yolk or whole fresh eggs were
used (Messinger et al., 1950; Hegsted et al., 1965;
Kummerow et al., 1977). Studies also varied in length
of dietary periods from 8 days (Beveridge et al., 1960) to
3 months (Porter et al., 1977). In addition, some studies
were conducted under strictly controlled conditions (Connor
et al., 1964; Mattson et al., 1972) whereas in others the
subjects were asked only to keep food records (Hildreth
et al., 1951; Shorey et al., 1974; Porter et al., 1977;

Simons et al., 1978). Furthermore, some studies were

11

conducted using healthy subjects (Hegsted et al., 1965;
Slater et al., 1976) while others used hypercholesterolemic
and convalescent patients (Steiner et al., 1962; Kummerow
et al., 1977). This tremendous variation in experimental
design has made interpretation of the often contradictory
results nearly impossible.

The results of a number of human studies conducted
in the 1950's led several investigators (Keys et al.,
1955; 1956; Kinsell et al., 1952; Ahrens et al., 1957)
to conclude that restriction of dietary cholesterol was
unnecessary and unwarranted because its ingestion failed
to alter significantly serum cholesterol levels in man.
These investigators believed that the relative homeostasis
of serum cholesterol concentration was maintained despite
even large increases in cholesterol intake because of
negative feedback on cholesterol synthesis in the body.
This conclusion was reached by each investigator after
feeding crystalline cholesterol in the amounts of 0.5 to
60 gm in special formula diets under strict metabolic
conditions. The results of these studies are in agreement
with a report from an earlier study (Steiner and Domanski,
1941) in which 10 gm of crystalline cholesterol in 400 cc
of milk added to the daily diet of a group of patients for
six weeks failed to induce significant elevations in serum
cholesterol levels. In a later study, however, Steiner

et al. (1962) discovered that the addition of 3 gm of

12

crystalline cholesterol to a cholesterol-free formula diet
containing 95 gm of fat produced a significant elevation

in serum cholesterol concentration in a group of hospital-
ized men and women. Beveridge et a1. (1960) demonstrated
an increase in serum cholesterol when supplements of highly
purified cholesterol in the amounts of 0-1600 mg/950 kcal
were added to a fat-free formula diet for a period of 8
days following an 8-day period in which a cholesterol-
free, fat-free formula diet was fed. The results of this
study have received much criticism because of the lack of
fat in the diet, which is known to be essential for choles-
terol absorption (Keys et al., 1965b).

Experiments designed to study the effects of added
cholesterol in the form of egg yolk and egg yolk powder
similarly produced conflicting results. Messinger et a1.
(1950) reported that the addition of 150 gm of egg yolk
powder produced a significant increase in serum cholesterol
concentration. Mattson et a1. (1972) utilized a formula
diet to which various levels of dry egg yolk cholesterol
were added. They reported a linear increase in plasma
cholesterol within the range of 0 to 317 mg cholesterol/
1000 kcal. Connor et a1. (1961) tested the effects of
adding either egg yolk cholesterol or crystalline choles-
terol to formula diets at various levels and found that egg
yolk powder produced greater serum cholesterol elevations

than did much larger amounts of purified cholesterol, with

13

or without added fat. Hegsted et a1. (1965) similarly
showed that addition of egg yolk powder to a diet of ordi-
nary foods under metabolic conditions produced a signifi-
cant increase in serum cholesterol levels. Mayer et a1.
(1954), in contrast, found that the addition of approxi-
mately 800 mg of cholesterol/day in the form of egg yolk
to a diet low in fat and low in cholesterol produced no
demonstrablewchange in plasma cholesterol levels of five
healthy male subjects after a period of one week. A lack
of serum cholesterol response to increased intake of egg
yolk cholesterol was also reported by Keys et a1. (1950),
who concluded that the serum cholesterol intake of "normal"
men is not significantly related to differences in the
habitual cholesterol intake over a range of approximately
250-800 mg/day.

Interpretation of many of the early studies in which
egg yolk was used as the source of dietary cholesterol is
complicated by the fact that the addition of egg yolk also
increased the fat content of the diet. The addition of
150 gm of egg yolk powder, for example, would add approxi-
mately 95 gm of fat/day to the diet (Mayer et al., 1954).

Several investigators have studied the effects of vari-

ous levels of dietary cholesterol added to formula and conven-
tional foods diets under strictly controlled conditions
in an attempt to describe the relationship of dietary

cholesterol to serum cholesterol concentration over a wide

14

range of intake. From the results of a series of experi-
ments covering a two year period in which schizophrenic
patients were fed a natural foods diet to which egg yolk
cholesterol was added in amounts ranging from 100-700 mg/
day, Hegsted et a1. (1965) concluded that serum cholesterol
was linearly related to dietary cholesterol. They predicted
that an increase of 100 mg of cholesterol would produce a
rise in serum cholesterol of approximately 5 mg/100 ml.
Similar results were reported by Mattson et al. (1972) who
compared the serum cholesterol response of four groups of
prisoners to the change from zero dietary cholesterol to
106, 212, 317 mg of egg yolk cholesterol/1000 kcal/day.

They concluded that the changes in serum cholesterol were
essentially linearly related to intake and were approximately
12 mg for each '100 mg cholesterol/1000 kcal.

Keys and Parlin (1966), in contrast, found that serum
and dietary cholesterol were not linearly related, but that
the change in serum cholesterol was proportional to the
square root of cholesterol intake. These conclusions were
based on the results of a series of studies in which
schizophrenic patients were fed a natural foods diet con-
taining cholesterol supplements ranging from 125-540 mg/
1000 kcal. When the results from this study were analyzed
together with those by four other investigators the follow—
ing prediction formula was derived:

ACholesterol (mg/100 ml) = 1.5 (Z2 - Z1)

15

where Z is equal to the square root of dietary cholesterol
measured as mg/lOOO kcal. Keys and Parlin emphasized that
this equation applies to groups of people and not to indi-
vidual subjects.

Several investigators have reported that once a certain
level of dietary cholesterol was reached, further increments
in intake had little additional effect on serum cholesterol
concentration. Beveridge et a1. (1960) reported finding
good correlation between serum cholesterol concentrations
and dietary increments of cholesterol up to intakes of
approximately 650 mg/day; dietary cholesterol beyond that
level failed to produce additional increases in serum
cholesterol. In a similar study Connor et a1. (1964) found
that the addition of 475 mg cholesterol/day to the diet
produced a significant increase in serum cholesterol, but
intakes of twice that amount did not cause a greater
increase in serum cholesterol.

The results of several recent studies on the effects
of added cholesterol in amounts normally consumed in the
American diet disagree with the findings of Hegsted et a1.
(1965), Mattson et a1. (1972) and Keys and Parlin (1966).
In a study by Slater et a1. (1976) three groups of young
and middle aged men in free-living populations were asked
to eat one or two eggs/day in addition to their usual
diets, or to eliminate all eggs. No significant changes

in the plasma cholesterol values were observed after the

16

6-8 week periodson extra eggs compared to periods in which
eggs were absent from the diet. Porter et a1. (1977) and
Flynn et a1. (1979) similarly failed to find a significant
association between dietary and serum cholesterol levels
with the addition or elimination of one egg daily in the
customary diets of healthy male subjects. In another study
Kummerow et a1. (1977) studied the effects of adding two
whole fresh eggs incorporated into either a custard or a
milk shake to the diets of hospitalized men and women in
three separate hospitals. No significant change in serum
cholesterol concentration was found in the majority of
patients after 54 days of continued consumption of two
eggs/day.

Although statistical analyses of the data reported by
these investigators support the conclusion that dietary
cholesterol in amounts equal to two eggs/day does not sig-
nificantly influence serum cholesterol concentration,
examination of the data for individual subjects revealed
a marked variation in individual response of plasma choles-
terol to dietary cholesterol challenges in man. It has
been shown that some subjects may not show any change;
some others may even demonstrate a decrease in their
plasma cholesterol when dietary cholesterol is increased
(Quintao et al., 1971; Nestel and Poyser, 1976). It has
been suggested that the variability in responsiveness of

plasma cholesterol to changes in the diet that is shown

17

to exist among individuals consuming the same diet may
reflect changes in other dietary components that necessarily
accompany the adjustment of the diet to meet individual
needs. Another possible explanation for differences in
responsiveness is the existence of genetically determined
compensatory mechanisms.

Unlike many animals such as the monkey and the rabbit,
which may develop serum cholesterol levels of 600-2000 mg/
100 ml following a high cholesterol diet, ingestion of very
large quantities by man results in a much smaller increase
in serum cholesterol concentration (Connor et al., 1961,
1964). Even when cholesterol intake exceeds the normal
turnover rate in man, the rise in serum cholesterol almost
never exceeds 100 mg/100 ml (Steiner et al., 1962). Despite
the extensive research conducted in the area of cholesterol
metabolism over the last few decades, the mechanisms res-
ponsible for the relative homeostasis of plasma cholesterol
in man have not been clearly defined (Sodhi and Mason,
1977). It is believed that total body cholesterol is regu-
lated by a complex, dynamic interplay between absorption,
synthesis and excretion. The following compensatory mecha-
nisms have been proposed to account for this homeostatic
phenomenon:

1) Limited absorption of dietary cholesterol. In a
recent study, Connor and Lin (1974) demonstrated that the

amount of cholesterol absorbed was roughly linear for

18

different quantities of dietary cholesterol between 110 and
610 mg and that the percentage absorption of cholesterol
remained relatively constant at 45% despite the increased
intake. Wilson and Lindsey (1965) discovered a maximum
daily absorption of approximately 300 mg when 3 g of
cholesterol wenafed and they concluded that the human
intestine has a limited ability to absorb dietary choles-
terol. Quintao et a1. (1971), on the other hand, demon-
strated that absorption is proportional to the amount
ingested and that much greater amounts can be absorbed if
the intake is high enough. At intakes up to 1 g/day,
about half is absorbed; at higher intakes the percent
absorbed becomes less.

Several factors are known to limit cholesterol absorp-
tion. The requirement for exogenous fat to assist the
absorption of dietary cholesterol has been clearly demon-
strated (Keys et al., 1965b). Quintao et a1. (1971) have
shown that the proportion of a large single dose of choles-
terol that may be absorbed depends upon the previous intake
of cholesterol. When cholesterol intake has been low, a
larger fraction of any single dose is absorbed than when
the intake has been high. This finding suggests a possible
role of the degree of saturation of the intestinal mucosa
in controlling absorption of dietary cholesterol. It also

has been demonstrated that the amount of absorption varies

with the source of dietary cholesterol, with egg yolk

19

cholesterol being more effectively absorbed than crystal-
line cholesterol (Cook et al., 1956; Connor et al., 1961).

2) Inhibition of cholesterol biosynthesis. Isotopic
studies in man under steady state conditions have shown
that biosynthesis of cholesterol in the liver remains
relatively constant and that cholesterol biosynthesis is
not usually altered by various quantities of dietary cho-
lesterol. Wilson and Lindsey(l965) using a double isotopic
steady state technique showed that dietary cholesterol,
whether fed in the form of eggs or in a purified state,
had little effect on the total rate of endogenous choles-
terol synthesis. Quintao et al. (1971), on the other hand,
found that increased absorption of cholesterol resulted
in a decrease in total body synthesis in five of six
patients; however, the amount of suppression in synthesis
was extremely variable from person to person.

3) Increased excretion of acidic sterols. Although
balance studies carried out in rats by Wilson (1964)
indicated that a significant amount of absorbed dietary
cholesterol was excreted through the enhanced formation
of bile acids, enhanced absorption of dietary cholesterol
has not been shown to produce a significant increase in
excretion of acidic sterols in man (Quintao et al., 1971).
They have shown, however, an increased excretion of endo-
genous neutral sterols with cholesterol feeding which may

have been due to inhibition in reabsorption of endogenous

20

cholesterol by exogenous cholesterol.

Dietary Fat

The effects of dietary fat on blood cholesterol con-
centration and its relationship to CHO have been studied
extensively in experimental animals and in epidemiological,
clinical, and pathological studies in humans using a three-
pronged approach: (1) level of dietary fat, (2) degree of
saturation of fatty acids, (3) the chain length of the
fatty acids in dietary triglycerides.

Level of dietary fat. It has been nearly a quarter of
a century since Keys et a1. (1955) demonstrated a statisti-
cally significant relationship between death rate from CHO,
plasma cholesterol, and the proportion of total calories
derived from fat. Keys (1952, 1970) and Jolliffe and Archer
(1959) have shown that in those countries where death rate
from CHO is low, the p0pulations exhibit low serum choles-
terol levels and tend to consume less fat than population
groups with a higher incidence of CHD.

Although the conclusions from epidemiological studies
have generally supported the theory that a change in the
level of consumption of dietary fat would produce a concom-
mitant change in serum cholesterol concentration, this has
not been consistently demonstrated in controlled human
studies. From the results of a study in which groups of

schizophrenic patients were fed low-fat diets to which

21

various test oils were added, Hegsted et a1. (1965) reported
that the amount of dietary fat, tested at levels equal to
22 to 40 percent of total calories, appeared to be without
influence on the level of serum cholesterol. They found no
effect on serum cholesterol when various levels of dif-
ferent fats were tested, as long as a constant polyunsatu-
rated:saturated (P/S) fat ratio was maintained. Similar
results were attained by Grande et a1. (1972) which showed
that varying the amount of fat had no effect on serum
cholesterol when the composition of dietary fat was such
that 2 S- P = 0, where S and P represent, respectively,

the percent of total calories from saturated and polyun-
saturated fatty acids. Keys et a1. (1950), Hildreth

et a1. (1951) and Mayer et a1. (1954), on the other hand,
reported that an increase in dietary fat, whether of animal
or vegetable origin, led uniformly to higher plasma choles—
terol levels. Other investigators (Kinsell et al., 1953;
Ahrens et al., 1954: Beveridge et al., 1955) reported
increased plasma cholesterol levels following increased
intakes of animal fat; similar amounts of vegetable fat,
however, produced decreases in serum cholesterol levels.
Malmros and Wigand (1957) found, in contrast, no essential
difference between vegetable and animal fat. Most of the
vegetable oils that they studied produced cholesterol-
depressing effects, but coconut oil did not. Milk fat was

shown to raise serum cholesterol, but whale oil depressed

22

it. They concluded that the cholesterol depressing effect
of some of the oils was due to their unsaturated fatty
acids.

Additional evidence refuting the idea that the level
of dietary fat is important irrespective of source is
supplied by Hardinge et a1. (1962), who demonstrated that
strict vegetarians exhibited lower serum cholesterol levels
than either lacto-ovo-vegetarians or non-vegetarians in
spite of a very liberal intake of vegetable fat. Simons
et a1. (1978) confirmed reduced plasma cholesterol levels
in vegetarians.

Degree of saturation of dietary fat. Numerous inves-
tigations have shown reductions in serum cholesterol con-
centrations in man following the dietary substitution of
many vegetable oils for common animal fats or certain
tropical oils such as coconut oil (Kinsell et al., 1952,
1953; Ahrens et al., 1954, 1955; Bronte-Stewart et al.,
1966; Beveridge et al., 1955, 1956). The relatively high
degree of saturation of fats of animal origin and coconut
oil had been associated with their cholesterol elevating
effect and has led to the development of the concept that
saturated fatty acids are hypercholesterolemic and that
polyunsaturated fatty acids are hypocholesterolemic
(Jolliffe, 1961).

The relationship of the degree of saturation to serum

cholesterol concentration has been studied and characterized

23

according to (l) the iodine number, (2) the P/S ratio,
(3) the percent of total calories from saturated and poly-
unsaturated fatty acids.

Iodine number, a measure of total unsaturation of
dietary fats, has been used by some investigators to
quantify the effects of dietary fat modifications on serum
cholesterol concentrations. Ahrens et a1. (1957) demon-
strated a rough inverse relationship between serum choles-
terol concentration and the iodine number of various oils.
They concluded that serum cholesterol response to changes
in dietary fat composition was proportional to the average
net unsaturation of the fatty acids, and that monosaturated
fatty acids were half as effective as linoleic acid, a
diene, in reducing serum cholesterol levels. Gunning et
a1. (1964) found that the plasma cholesterol levels of the
four patients they studied were highly correlated with
the square root of the iodine number of the total fat in
the diet. They concluded that net unsaturation correlated
better with plasma cholesterol levels than did either the
P/S ratio or the ZS-P formula. Keys et al. (1965a) concurred
that cholesterol level does correlate with the iodine
value, or its square root, when total dietary fat is con-
stant, provided that the fats contained no significant
amounts of fatty acids more unsaturated than linoleic acid
and that the proportion of monosaturated fatty acids did

not vary widely. They found that changes in serum

24

cholesterol could be predicted with far greater accuracy
from the amounts of saturated and polyunsaturated fatty
acids than from total degree of unsaturation because the
effects of some fatty acids on serum cholesterol were
discrepant from what would be expected from a simple direct
iodine number function. It was shown, for example, that
the high degree of unsaturation beyond the dienes in fish
oils was not reflected in the change in serum cholesterol
concentration using iodine number as a predictor (Keys

et al., 1965a).

Jolliffe (1961) proposed that the effect of dietary
fats on serum cholesterol concentration was related to
their P/S ratio rather than to the net unsaturation of the
dietary fatty acids, and that the P/S ratio could be used
to predict the effect on serum cholesterol of modifying
dietary fat. Keys et a1. (1957) also showed that quanti-
tatively the P/S ratio was more significant than the
quantity of dietary cholesterol in predicting serum cho-
lesterol changes. Other investigators have found that P/S
ratio did not always predict serum cholesterol changes
accurately. Stamler (1960) observed that when the amount
of saturated fat in the diet was controlled, the P/S
ratio played a lessened role, particularly when the dietary
cholesterol levels were below 400 mg/day. Connor et a1.
(1964) showed that changing the P/S ratio from 0.2 to 2.6

had no effect on blood cholesterol when subjects consumed

25

a cholesterol-free diet. Erickson et a1. (1964) demon-
strated that plasma cholesterol level was unaffected by
variations in the P/S ratio between 0.1 and 1.6. Similar
findings were reported by Bierenbaum et a1. (1961) who
found no significant differences in serum cholesterol
levels when subjects were fed diets containing 28% of
total calories from fat, less than 400 mg cholesterol/day,
and a P/S ratio of either 0.34 or 2.6.

Keys et a1. (l965d)demonstrated that the relationship
between dietary fat and serum cholesterol concentration
could be described quantitatively from the percent of total
calories as saturated and polyunsaturated fatty acids.

From a comprehensive study in which groups of schizophrenic
patients were fed a series of 40 different diets under
metabolic conditions it was shown that saturated fatty
acids had a cholesterol-raising effect that was twice the
cholesterol-lowering effect of polyunsaturated fatty acids.
It was also shown that monosaturated fatty acids were
essentially neutral in their effect on serum cholesterol.

Multiple regression equations were developed to relate
the change in serum cholesterol to dietary content of
saturated (S), monosaturated (M), and polyunsaturated (P)
fatty acids and the following prediction equation was
derived.

ACholesterol (mg/100 ml) = -l.68-+ 2.76 AS + 0.05 AM -

1.35 AP in which AS, AM, and AP were the change in the

26

calories contributed by each class of fatty acids. The
constant -1.68 and the coefficient for AM of 0.05 did
not contribute significantly to the equation which was
modified to

ACholesterol (mg/100 m1) = 2.74 AS - 1.31 AP
indicating that the relationship of saturated and poly-
unsaturated fatty acids to serum cholesterol could be
described as a function of ZS -P.

Hegsted et a1. (1965) conducted a series of studies
that were similar in design to those of Keys et a1.

Groups of schi20phrenic patients were fed a low-fat,
natural foods diet to which various test oils were added
at levels varying from 22 to 38% of total calories from
fat for four week diet periods. The following equation
was derived to relate the changes in serum cholesterol
to changes in dietary fat and cholesterol

ACholesterol (mg/100 m1) = 2.16 AS - 1.65 AP +
6.66 AC - 0.53 where S and P are the changes in the percent
of dietary calories derived from saturated and polyunsatu-
rated fatty acids, respectively; C is the change in
cholesterol intake in 100 mg/day.

Both the equations by Hegsted et a1. (1965) and Keys
et a1. (1965d) suggest that saturated fatty acids have, per
weight basis, approximately twice the effect on the change
in serum cholesterol as do the polyunsaturated fats, which

act in the opposite direction.

27

Based on the small coefficient for monosaturated fatty
acids Keys et a1. concluded that monosaturated fatty acids
have no effect on serum cholesterol. Although the data of
Hegsted et al. also yielded an equation with a small co-
efficient for monosaturated fatty acids, they were reluctant
to conclude that monosaturated fatty acids were neutral
and could be isocalorically exchanged for starch in the
diet without affecting serum cholesterol. This conclusion
was based on the fact that significant regression coeffi-
cients for monosaturated fatty acids were found when the
data for each of the different trial diets were analyzed
separately, as opposed to analyzing all the data from the
different trial diets together.

Chain length of fatty acids. There is considerable
evidence that saturated fatty acids may vary substantially
in their hypercholesterolemic effects (McGandy and Hegsted,
1975). Evidence that.shortchain fatty acids have much
less influence on serum cholesterol than the longer chain
fatty acids has been reported in the literature. Keys
et a1. (l965d)demonstrated that saturated fatty acids
containing fewer than 12 carbon atoms had little or no
effect on serum cholesterol in man. They attributed this
lack of effect to the fact that fatty acids with fewer than
12 carbon atoms in the chain are more polar and less
hydrophobic than the other fatty acids and appear to be

metabolized differently, being absorbed via the intestinal

28

capillaries and the hepatic portal system rather than via
the lymphatics.

The relative effects of the different fatty acids have
been studied by various investigators. Based on the data
available from their multiple regression equations Hegsted
et a1. (1965) concluded that, aside from linoleic acid,
the only other fatty acids clearly influencing the level
of serum cholesterol were myristic ($14) and palmitic
(516) acids; lauric ($12) and stearic (518) acids, satu-
rated fatty acids with 10 carbon atoms or fewer, and
monosaturated fatty acids had little effect on serum
cholesterol concentration. Keys et al. (1955d) in con-
trast, found that the cholesterol-promoting effect of
saturated fatty acids was due to lauric, myristic, and
palmitic acids. They concluded that since palmitic acid
was much more abundant than lauric and myristic acids in
most diets, it was primarily responsible for the contri-
bution of dietary fat to the serum cholesterol level in
man. They similarly reported a lack of effect on serum
cholesterol level by stearic acid. This confirmed the
findings of Connor et a1. (1964) that when diets containing
substantial amounts of cocoa butter, which has a high
content of stearic acid, were fed an unexpectedly low serum
cholesterol level was observed.

Several mechanisms have been proposed to explain the

cholesterol lowering effect of polyunsaturated fats. The

29

literature contains evidence both for and against each of
the proposed mechanisms. To date no single mechanism has
been shown to apply in all cases. Grundy and Ahrens (1970)
provide a good review of the effects of polyunsaturated

fats on absorption, excretion, synthesis, and the distri-
bution of cholesterol in man. The mechanisms are summarized
briefly below:

Fecal excretion of neutral steroids and/or bile
acids. Polyunsaturated fat has been reported to cause both
an increase (Lewis, 1958) and a decrease (Ali et al., 1966)
in bile acid excretion; Grundy and Ahrens (1970) reported
no change. An increase in bile acid excretion may be the
result of decreased reabsorption of bile acids or increased
conversion of cholesterol into bile acids.

Cholesterol absorption. Several recent studies
(Nestel et al., 1976; Grundy and Ahrens, 1970) have shown
no difference in the absorption of dietary cholesterol
when high polyunsaturated fat diets were consumed. This
finding conflicts with an earlier report by Wood et a1.
(1966) that showed a reduction in the absorption of choles-
terol following a diet high in polyunsaturated fat.

Cholesterol synthesis. It has been proposed that
a decrease in the rate of cholesterol synthesis in the
liver may contribute to the lowering of plasma cholesterol
that follows the feeding of diets high in polyunsaturated

fat. There have been no direct measurements of cholesterol

30

synthesis in vivo in man. Indirect measurements, however,
in normolipemic (Nestel et al., 1976) and in hypercholes-
terolemic subjects (Grundy and Ahrens, 1970) have shown
no significant changes in cholesterol synthesis following
ingestion of diets high in polyunsaturated fat.
Distribution of cholesterol between plasma and tissue.
Grundy and Ahrens (1970) proposed that polyunsaturated fat
may lower serum cholesterol by causing its redistribution
from plasma to one or more tissue compartments. Direct
evidence for the transfer of cholesterol to tissues during

polyunsaturated fat feeding in man is lacking.

Summary

Coronary heart disease is a major health problem in
the United States as well as in many other countries.
The results of epidemiologic, experimental, and clinical
investigations have implicated elevated serum cholesterol
levels as an important risk factor in the development of
atherosclerosis. Although no causal relationship has been
proven between serum cholesterol concentration and CH0,
there is abundant evidence indicating that the risk of
developing CHD is positively correlated with the level of
cholesterol in the blood.

A need exists for well-controlled research to study
the effects on serum cholesterol of reducing cholesterol

intake and modifying the amount and type of fat that is

31

normally consumed in the American diet. In order for the
diet modifications to be meaningful they must be economi-
cally feasible and easily incorporated into daily food
preparation. The modified diet must be palatable and easily

adapted to the individuaPs lifestyle.

METHODS AND PROCEDURES

Subject Recruitment

Male subjects were recruited to participate in a 10-

week feeding study by advertising in the campus newspaper.

A total

of 32 subjects was needed to participate in the

two-part study: 16 in the Winter term and 16 in the Spring

term.

The same protocol was used in both terms. The

criteria for eligibility in the study which were established

in the protocol stated that the subjects should

1')

ii)

iii)

like eggs, habitually consume eggs, and be willing
to eat two eggs/day

have serum cholesterol levels greater than 200 mg/
100 ml

agree to eat only the foods provided by the study

at the arranged facilities

be within i 20% of their desirable body weight and
be willing to maintain this weight throughout the
study

be willing to maintain a relatively constant pattern

of physical activity.

32

33

Screening‘Procedures

A total of 107 men were screened for total serum cho-
lesterol following a 14-hour overnight fast. Forty-seven
men were found to have serum cholesterol values 2 200 mg/
100 ml. Ultimately several people with screening serum
cholesterol values in the 190 mg/100 ml range were invited
to participate in the study because some of the men with
higher serum cholesterol levels were unwilling to make a
complete commitment to the study.

The subjects who were selected received a physical
examination and routine laboratory tests that included
blood glucose, electrolytes, SGPT, SGOT, hematologic tests,
and urinalysis. All screening laboratory tests were per-
formed by a commercial medical laboratory that serves many
of the local hospitals, the Student Health Center, and
many of the research projects conducted at Michigan State
University.

The subjects were interviewed to obtain information
about normal eating patterns, food allergies, food dislikes,
coffee and tea consumption, smoking habits, and family
medical history. The men were asked to keep a diet record
on three consecutive days for the purpose of estimating
energy requirements. They were given specific verbal and
written directions in a cover letter accompanying the food
record form instructing them to list all foods consumed,

portion sizes, food preparation method and time of

34

consumption. An example of a complete diet record was
provided. Despite the specific directions which were given,
the diet records were returned in such an incomplete state
that they were of limited value in predicting energy
requirements. They did prove useful, however, in providing
information, albeit sketchy, on the frequency of consumption

of eggs, butter, milk and meat.

Subject Characteristics

Age, Race and Marital Status. With the exception of
one subject who was 69 years old, subjects ranged in age
from 19 to 42 years, with a mean age of 24.8 years (Table 1).
Thirty subjects were Caucasian; two were Black. There were
two sets of brothers participating in the study (subjects 3
and 4; 9 and 10); two of the brothers were fraternal twins.
Three of the subjects were married and two had children.
All but three subjects were students at Michigan State Uni-
versity. The three non-student subjects worked adjacent to
the university campus.

Body Weight and Anthropometric Measurements. The body
weights of the subjects measured at the time of their
physical examinations ranged from 84 to 134% of the average
weight/height for men of medium frame according to the 1960
Height-Weight chart of the Metropolitan Life Insurance Com-
pany.

Lean body mass (LBM) and body fat (BF) were estimated

using measurements of flexed biceps circumference, height,

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37

and weight. All measurements were taken by one technician
before breakfast during the second or third weeks of the
study. Two consecutive measurements of biceps circumference
were taken at the fullest point of the fully flexed dominant
arm using a plastic coated tape measure and the mean value
was reported. Height was measured with the subjects in
stocking feet, eyes straight ahead, with heels and shoulders
touching a vertical surface to which a Lufkin steel tape was
attached. Weight was measured on a beam scale by the sub-
jects and later recorded by the technician. The following
prediction equation (Wright and Wilmore, 1974) was used to
calculate LBM:

LBM (kg) = -74.58 + 0.5949 x height (cm) + 0.0286 x

[biceps circumference (cm)]2
Percent body fat was calculated from the difference between
LBM and total body weight.

The 32 subjects who were selected were free from
detectable disease. The results of the laboratory tests
performed at the time of the physical examination were
within the normal range except for serum triglyceride
values of 311, 225, and 175 mg/100 ml is subjects 9, 20
and 30, respectively. Total serum cholesterol was found to
be greater than 250 mg/100 ml in six subjects, thus exceed-
ing the laboratory's range for normal values (Table 1).

The 16 subjects from each term were assigned to one of
four groups so that the average age, body weight, and

screening serum cholesterol levels were approximately the

38

same for all groups.

Orientation

An orientation meeting was held prior to the start of
the study to introduce subjects to the staff and their
fellow volunteers, to familiarize them with the surroundings,
to review the procedures, and to answer any questions that
may have arisen. The subjects were reminded to avoid
taking any medication (including aspirin) or vitamin sup-
plements unless prescribed by the consultant physician for
the study. They were instructed to notify us and to call
the physician in the event of illness.

We stressed the point that each subject would serve
as his own control and hence it was important that he main-
tain a relatively steady pattern of activity and that his
body weight remain stable. They were asked to be sure to
eat two eggs/day if they were not already doing so, so that
the 10 day control period would approximate as closely as

possible a diet to which they were accustomed.

Informed Consent

The project was reviewed and approved by the University
Committee on Research Involving Human Subjects. The purpose
and the nature of the study and the possible risks were
explained to the prospective subjects and a consent form
for the screening blood test was signed. The consent form

explained that the subject could withdraw from the study at

39

any time without penalty and that results would be treated
in strict confidence (Appendix A-l). A second consent form
for participation in the study was signed at the orientation
session by those subjects selected to be in the study

(Appendix A-2).

Experimental Diet Plan

During the first ten days of the study all subjects
consumed a control diet. The control diet was designed to
represent a "typical" American diet with two eggs/day, 42 to
45% of total calories as fat, and a polyunsaturated: satura-
ted fat (P/S) ratio of 0.3 to 0.5. Other foods in the diet
were controlled to provide an average of 350 mg of choles-
terol. The control period allowed for estimation of energy
needs to maintain weight, adjustment of subjects to experi-
mental procedures, and collection of baseline data. During
the next eight weeks, half of the subjects (8 subjects/
Winter and Spring terms) received each of the following .
diets for four weeks in a crossover design (Table 2):
1) Diet C-E: the control or typical American diet described
above; 2) Diet C-ES: the same diet as the control diet
except that two eggs were replaced by a cholesterol-free egg
substitute. The remaining half of the subjects received
each of the following diets in a similar crossover design:
1) Diet MF-E: a modified fat diet similar to the one recom-
mended by the Inter-Society Commission for Heart Disease

Resources (1972) except that it included two eggs/day,

4O

 

 

 

Table 2. Experimental diet plana
Weeks
Group
1-2 3-6 7-10
1 C-E C-E C-ES
2 C-E C-ES C-E
3 C-E MF-E MF-ES
4 C-E MF-ES MF-E

 

aDiet designations are as follows:

C-E:

C-ES:
MF-E:

MF-ES:

Control or typical American diet with 2 eggs daily
and 42 to 45% of total calories from fat, P/S
ratio m 0.3 - 0.5.

Control diet using cholesterol-free egg substitute
to replace eggs.

Modified fat diet providing 32 to 35% of total
calories from fat, P/S ratio 2 1.0. Diet included
2 eggs/day.

Same as diet MF-E except that egg substitute was
used to replace the eggs.

41

approximately 35% of the total calories as fat with a P/S
ratio 2 1.0. 2) Diet MF-ES: this diet was the same as the
modified fat diet except that the egg substitute was used

instead of eggs.

Menus

A two-week cycle of basal menus with 2800 kcal/day was
planned for each of the control and modified fat diets (see
Appendix B for sample menu). The amounts of carbohydrate,
protein, and calories in the diets were calculated using
the 1975 USDA Handbook No. 456 (Adams, 1975) for all foods
except for dairy products, the values for which were taken
from USDA Handbook 8-I (Posati and Orr, 1976). Values for
total lipids, saturated fatty acids, polyunsaturated fatty
acids and monosaturated fatty acids were taken from a
series of tables prepared by the USDA Agricultural Research
Service (Anderson, 1976; Anderson et al., 1975, 1977;
Brignoli et al., 1976; Exler and Weihrauch, 1976; Fristrom
and Weihrauch, 1976; Posati et al., 1975; Weihrauch et al.,
1976). Values for cholesterol were taken from the table
prepared by Feeley et a1. (1972). Nutrient composition
values supplied by the food manufacturers were used in

menu calculations when applicable.

The basic menus were planned so that as many of the

foods as possible could be used in all four diets.

Calorie adjustments and allowances for food preferences

42

for individual subjects were accomplished by adding or
subtracting food portions to obtain the desired calorie
level while maintaining the specified percent of the
calories from fat and the appropriate P/S ratio. The
subjects weighed themselves daily. Adjustments in caloric
intake were made at regular intervals if subjects were
gaining or losing weight. Adjustments for the change from
the control diet to the modified fat diet were accomplished
primarily by substituting margarine for butter and skim
milk for whole milk. The reduction in calories resulting
from the decrease in fat in the modified fat diet was
compensated for by increasing fruit, vegetable, and bread
servings. The items allowed ad libitum in all four diets
were coffee, tea, and spices. Subjects were given the
choice between whole wheat, cracked wheat, and white bread.
The amount of commercial cholesterol-free egg substi-
tute used was comparable in nutrient composition to the
equivalent of two large whole eggs. The egg substitute
was made from egg whites (82%), liquid corn oil (10%),
nonfat dry milk (7%), emulsifiers, preservatives, coloring

and flavor additives (Table 3).

Meals
Meals were served in the dining room of the Student
Health Center. Three meals were served each day except on

Sunday when only brunch and dinner were served. The meal

.43

Table 3. Comparison of the nutrient composition of two
large whole eggs and an equivalent serving
portion of the cholesterol-free egg substitute
used in this study

 

Egg Substitute1 Whole Eggs

 

Serving size, portion 1/2 cup two large
. g 120 105
Calories, 180 160
Protein, 9 14 14
Carbohydrate, g 6 2
Fat, total 9 12 12
polyunsaturated g 6 2
saturated g 2 2
Cholesterol, mg 0 480
Sodium, mg 260 140

 

1Egg Beaters, Standard Brands, Inc., Stamford, Conn.

44

hours were: breakfast 7:00 - 9:00 a.m.; lunch 11:00 -

1:00; and dinner 4:30 - 6:30. Trays bearing name cards

were set up in advance in refrigerated carts so that when

the subjects came in for meals the cook only had to add

the foods from the hot cart and double-check the food on

the tray with the diet sheets. For those subjects who

expressed the desire for an early morning snack on Sundays,

a box of cereal, a carton of milk, and a piece of fruit

were provided to take home Saturday evening. Occasionally

meal hours were extended to accommodate problems arising

from time conflicts. In some instances field trips or

lunch hour meetings necessitated packing a meal to go.

For one subject a bag lunch was provided on Mondays

because of a lunchtime schedule conflict. All meals that

were packed and taken from the Health Center were recorded.
Occasionally friends and family members purchased food

from the Health Center cafeteria line and joined the sub-

jects for meals. It was not unusual on a Saturday night

to see several guests sitting at the subjects' table next

to their dates.

Food Preparation

The majority of foods served in the study were pur-
chased through the University Food Stores. The meat, fish
and poultry were purchased in large quantities at the

beginning of each term and weighed into approximate portion

45

sizes by the Food Stores and frozen to ensure uniformity
of source. Eggs, milk and bread were purchased as needed
from the same suppliers throughout the study. Bread and
angel cake were the only two baked goods not prepared in
our kitchen. Foods containing appreciable amounts of fat
(e.g. salad dressings, peanut butter, cheese, nuts, and
meats) were weighed on a Toledo scale to the nearest gram,
whereas foods containing little or no fat (e.g. cooked and
salad vegetables, juices, and canned fruit) were served

by measurement or by commercially available preportioned
servings (e.g. ketchup, jelly, raisins, sugar and prepared
cereal). Preportioned pats of butter and margarine and
cartons of milk were used. They were weighed randomly and
found to be of consistent weight. Similar procedures were
used to weigh or measure ingredients for recipes prepared
during the study. Many of the recipes were prepared indi-
vidually, particularly when appreciable amounts of fat and
cholesterol were involved, rather than in large quantities
to be divided into individual portions. For example, a
vegetarian chili recipe was prepared in a large kettle and
divided into the appropriate number of servings. Then to
each serving of chili was added a carefully weighed portion
of cooked ground beef. Whenever possible recipes were
portioned and cooked in individual pans. At breakfast

the eggs and egg substitute were prepared in separate

frying pans.

46

As many of the entrees as possible were prepared and
frozen before the beginning of the Winter and Spring terms
(e.g. macaroni and cheese, chili, chicken cashew, potato
kugel) to reduce the amount of daily preparation and facili-
tate serving. Weighed or measured portions of several kinds
of cookies, cakes and muffins as well as cherry tarts,
pumpkin pies, pizza, waffles, and french toast also were
prepared in advance and frozen. Nearly all of the recipes
for these products were made with egg substitute, margarine,
and corn oil, so that they could be used on both the control
and modified fat diets. A11 ingredients in these recipes

were weighed except for spices and flavorings.

Food Intake Records

Careful food intake records were kept for each subject.
All trays were checked after meals for food that may have
been left. The few food mistakes that occurred in serving
food were recorded and the daily nutrient calculations were
corrected when the mistakes involved any appreciable amounts
of fat. Occasionally extra servings of certain food items
(e.g. raw or cooked vegetables) were given if requested;
records of these foods were also kept.

Subjects who were accustomed to consuming alcoholic
beverages on a regular basis were given the option of having
12 ounces of beer (to be supplied by the subject) included

in their daily diet plan. Six subjects chose to do so.

47

The rest of the subjects were told that they could substi-
tute one 12 ounce beer (or two light beers) for two slices
of bread on any day except immediately prior to the blood
tests. Alcohol substitutions were recorded.

The daily intake of calories, carbohydrate, protein,
total fat, saturated fatty acids, polyunsaturated fatty
acids, monosaturated fatty acids and cholesterol were
recorded for each subject. From this the percent of total
calories from fat, the percent of calories from protein,

and the P/S ratio were calculated.

Blood Tests

Blood samples for serum cholesterol determinations
were drawn before breakfast following an overnight fast on
the first day of the study, on Friday of each week, and on
Tuesday at the end of each experimental diet period (weeks
2, 6, and 8) (Table 4). The blood samples were centrifuged,
and the serum divided into two portions and frozen for
subsequent serum cholesterol analysis by our laboratory
and by the commercial medical laboratory.

Total serum cholesterol was determined in our labora-
tory by a colorimetric method using ferrous sulfate (Searcy
and Berquist, 1960) and a chloroform-methanol extraction
(Leveille et al., 1962). Samples were analyzed in tripli-
cate and the mean value was used. See Appendix C for

laboratory method.

48

Table 4. Schedule of blood tests

 

 

 

 

 

 

 

 

 

 

 

Weeks Tuesday Friday
. 1 Chola Chol
Control Per1od
2 Chol Chol
3 ChOI
. . 4 ChOI
Experimental Per1od 1
5 Chol
5 Chol Chol
7 ChOT
8 ChOI
Exper1mental Period 2 9 Chol
10 Chol COO]

 

aChol=total serum cholesterol

49

At the end of Winter and Spring terms an aliquot of
the frozen serum samples was sent to the commercial labora-
tory for determination of serum cholesterol using an auto-
mated Hycel Super SeventeenTM method, which is basically
the Liebermann-Burchard reaction]. The automated procedure

made it possible to analyze all samples at one time.

Statistical Design

The statistical design of the study was that of a
split-plot design with crossover treatments for the sub-
jects. The values for total serum cholesterol were averaged
within experimental diet periods and the differences between
the average cholesterol values for each of the two experi-
mental diet periods and the average control period value
were calculated for each of the parameters. Averaging was
done in two ways: (1) averaging all the values within each
diet period and (2) averaging only the last two values from
each diet period, that is, those values from blood samples

drawn on Tuesday and Friday at the end of each diet period.

1Hycel, Inc., Houston, TX.

RESULTS

Body Weights

 

Weekly body weight values were calculated by averaging
daily weights from Friday through Thursday so that the
weekly averages corresponded to the weekly serum choles-
terol values for bloods drawn each Friday. See Appendix
D for weekly body weight values for individual subjects.

The average change in body weight that occurred within
each of the 4-week experimental diet periods and the control
periods was calculated for the two groups of subjects
participating in the Winter and Spring Terms (Table 5).

The greatest weight change within a given diet period
was a 3.6 kg decrease in one subject (#4) during the first
experimental diet period of the Winter term while consuming
the control diet with eggs, followed by a 2.3 kg decrease
in the second experimental diet period while consuming the
same diet with egg substitute. This subject, as well as
other subjects from the Winter term, claimed to have gained
weight during the preceding Christmas holiday season. This
weight loss may have, in part, reflected a reversion to his
normal body weight. A second subject (#15) experienced a

2.7 kg decrease during the first experimental diet period

50

51

Table 5. Average change in body weight within the control
period (weeks 1—2) and each ofathe experimental
diet periods (weeks 4-6, 7-10)

 

 

Winter term Spring term
kg kg
Control Period -0.54 -0.09
Experimental Period 1 -l.00 -0.27
Experimental Period 2 -1.23 -0.77

 

aThe average body weight change for the control period was
found by calculating the changes in body weight between
weeks 1 and 2 for each subject and computing the mean
value. The average body weight change for the first
experimental diet period was calculated by comparing the
weekly weights from week 6 (the end of the first experi-
mental diet period) with the weekly weights from week 2
(the end of the control period). The average body weight
change for the second experimental diet period was deter-
mined by comparing the weekly weights from week 10 (the
end of the second experimental diet period) with the
weekly weights from week 6.

52

while consuming the control diet with egg substitute,
followed by a 2.3 kg decrease during the second diet period
when eggs replaced the egg substitute. Prevention of this
weight loss was a perplexing problem because the caloric
intake of this individual was increased by increments of
100 to 300 calories each week, yet he continued to lose
weight even while consuming 4000 calories/day.

During the last four weeks of the Spring term one
subject (#27) experienced a 2.3 kg decrease in average body
weight while consuming the modified fat diet with eggs;
another subject (#30) experienced a 2.7 kg decrease while
consuming the modified fat diet with egg substitute.
Whereas the body weights of the subjects in the Spring term
were relatively stable during the first five weeks, there
tended to be a gradual decrease in body weight that began
during the sixth week of the study. This decrease may have
reflected the arrival of spring and the shredding of layers
of heavy winter clothing. The subjects were instructed to
weigh themselves in their normal indoor clothing after
removing their shoes/boots. During the winter months
normal indoor clothing included long underwear, sweaters,

and extra heavy socks not worn during the warmer months.

Nutrient Intake
The daily intake of calories, carbohydrate, protein,

total fat, polyunsaturated/saturated fat (P/S), and

53

cholesterol were tabulated for each subject and the average
intakes for diet periods were calculated. See Appendix E
for individual data. Table 6 is a compilation of the
ranges and averages of protein, fat, and cholesterol intake
for subjects by diet.

The subjects consumed between 2600 and 4000 kcal/day.
The average protein intake, percent of total calories from
protein, intake of total fat, and percent of total calories
from fat were nearly the same for each of the control diet
groups (subjects on the control diet with eggs or egg
substitute) and for each of the modified fat diet groups
(subjects on the modified fat diet with eggs or egg sub-
stitute). Because the egg substitute had a higher content
of polyunsaturated fat, replacement of eggs with the egg
substitute resulted in a slightly higher P/S ratio. The
average P/S ratios of the control diet with eggs or egg
substitute were 0.45 and 0.57, respectively; the average
P/S ratios of the modified fat diets with eggs or egg
substitute were 1.10 and 1.34, respectively.

The difference between the average cholesterol intakes
on the control and modified fat diets reflect the decrease
in dietary cholesterol that would naturally accompany the
reduction in dietary fat. The average decrease in dietary
cholesterol seen in the control diet when eggs were replaced
by the egg substitute is approximately equal to the

reduction in dietary cholesterol when eggs were replaced

54

 

 

 

Table 6. Dietary intake of protein, fat, and cholesterol
Diets
c-Ea c-Esa MF-Eb MF-Esb

Protein, 9

Range 89-167 90-166 89-163 98-205

Average 120.1 119.2 124.8 126.0
Protein-% kcal

Range 12-22 12-23 12-21 12-21

Average 16.0 16.1 16.5 16.5
Fat, 9

Range 121-185 122-175 96-153 94-160

Average 145.0 145.1 116.9 119.7
Fat-% kcal

Range 39-47 33-47 32-37 31-47

Average 43.8 44.0 34.6 35.0
P/S

Range 0.18-0.66 0.26-0.89 0.77-1.68 0.98-1.74

Average 0.45 0.57 1.10 1.34
Cholesterol, mg

Range 751-1033 207-491 553-952 97-394

Average 894 339 728 186

 

aValues represent intake of subjects fed diets C-E and

b

C-ES.

Eight subjects consumed diet C-E during the first

experimental diet period (weeks 3-6) and diet C-ES during
the second experimental period (weeks 7-10).
ing eight subjects consumed the diets in the reverse order.

The remain-

Values represent intake of subjects fed diets MF-E and

MF-ES following diet C-E in the control period.

Eight sub-

jects consumed diet MF-E during the first experimental
period and diet MF-ES during the second experimental

period.

in the reverse order.

The remaining eight subjects consumed the diets

55

by egg substitute on the modified fat diet. These average
decreases in dietary cholesterol are approximately equal
to the cholesterol content of two large whole eggs

(500 mg).

The wide range in caloric intake among subjects
explains in part the large group ranges seen here in the
amount of protein, fat, and cholesterol consumed. The day-
to-day variation for individual subjects tended to be less,
particularly for those subjects whose caloric requirements
remained relatively constant throughout the study. It is
nearly impossible when using a diet of conventional foods
to increase calorie intake without increasing cholesterol
if the same percent fat and P/S ratio are maintained.

It is important to note that although nutrient intake
did vary somewhat from day-to-day within the two-week
cycle of menus, unless calorie requirements changed, each
subject received the same menu every two weeks, and hence

the same nutrient intake.

Total Serum Cholesterol

 

Serum cholesterol values from our laboratory tended
to be lower than the values reported by the commercial
laboratory because of differences in methodologY; however,
the trends in the response of serum cholesterol to dietary
changes and the magnitude of response tended to be similar

for both laboratories. The commercial laboratory had the

56

advantage of being able to run all the serum cholesterol
determinations at one time by an automated method. Only

the values for serum cholesterol from the commercial labora-
tory will be reported here.

The average cholesterol values for individual subjects
during each diet period are presented in Tables 7 to 10.
These values represent the average of the two cholesterol
values for blood samples drawn on Tuesday and Friday of the
last week of each diet period. (See Appendix F for the
complete listing of all values).

Six of the eight subjects who continued on the control
diet with two eggs/day for four weeks following the 10-day
control period experienced decreases in serum cholesterol
ranging from 3 to 30 mg. One subject demonstrated a 1 mg
increase and the eighth subject showed an average increase
of 8 mg. The average change in serum cholesterol for this
group during this period was a 10.5 mg decrease. The per-
cent change ranged from an 8.3% decrease to a 3% increase,
with an average change of a 4.3% decrease from the average
control period value.

When eggs were removed from this diet six of the eight
subjects demonstrated an additional decrease in serum cho-
lesterol ranging from 6 to 52 mg. One subject experienced
a 7 mg increase and one a 15 mg increase. The average
change in serum cholesterol between the first and second

diet periods when eggs were removed from the diet was a

57

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61

16.6 mg decrease. This represents a 6.6% decrease from the
average values from the first experimental diet period.

The serum cholesterol values during the egg-free period was
on the average 27.1 mg below the control period values,
with decreases ranging from 17 to 44 mg in six of the sub-
jects. One subject had a final serum cholesterol value

1 mg above his control period value, while the eighth
subject's cholesterol level was 4 mg above his control
period level.

When subjects were placed on the egg-free control diet
for four weeks following the 10-day control diet with two
eggs/day, all eight subjects experienced a decrease in
serum cholesterol ranging from 6 to 32 mg, with an average
decrease of 23.8 mg for the group (Table 8). This repre-
sents an average decrease of 10.8% below the average
control period value.

When eggs were added back to this diet six of the
eight subjects demonstrated an increase in serum choles-
terol levels ranging from 16 to 58 mg. One subject showed
an additional 10 mg decrease in serum cholesterol when eggs
were added back to the diet, and one subject showed no
change. The average serum cholesterol value for this
group during the last week of the four week diet period
was 25.1 mg/100 ml higher than the value for the egg-free
diet period of the previous four weeks. This change repre-

sents an average increase of 13.3% above the average values

62

from the egg-free diet period. Five of the eight subjects
had higher serum cholesterol values when eggs were added
back to the diet than they had when consuming the same diet
eight weeks previously.

When placed on the modified fat diet with two eggs/day
following the 10-day control diet with two eggs/day, seven
of the eight subjects experienced a decrease in serum cho-
lesterol ranging from 15 to 48 mg (Table 9). The eighth
subject demonstrated a 4 mg increase. The average change
for the group was a 28.1 mg or 12.5% decrease below the
control period value.

When eggs were replaced by an egg substitute during
the last four weeks of the modified fat diet, five of the
eight subjects experienced an additional decrease in serum
cholesterol ranging from 2 to 33 mg. Two subjects showed
no change, and one subject demonstrated a 25 mg increase
when eggs were removed from the diet. The average change
in serum cholesterol when eggs were removed from the modi-
fied fat diet was an 8.9 mg or 3.5% decrease. The average
serum cholesterol level during the last week of the final
experiment diet period on the egg-free modified fat diet
was 35.8 mg below the average serum cholesterol value during
the lO-day control period.

When subjects were placed on the egg-free modified fat
diet for four weeks following the lO-day control diet with

two eggs/day, all eight subjects experienced a decrease in

63

serum cholesterol ranging from 25 to 65 mg, with an average
decrease of 41.1 mg or 19.0% (Table 10). When eggs were
added back to this modified fat diet for four weeks all
eight subjects experienced an increase in serum cholesterol
ranging from 8 to 31 mg, with an average increase of 18.9
mg, or 11.2%. The average group serum cholesterol level
during the last week of this final experimental period was
21.5 mg below the average value during the 10-day control
period when subjects were consuming a diet with more total
fat, saturated fat and cholesterol.

Figure 1 summarizes the changes in serum cholesterol
concentration relative to the control period values for the
four diet groups. Replacement of eggs with egg substitute
resulted in a decrease in serum cholesterol in both the
control and modified fat diets groups. The addition of
eggs back to the egg-free control and modified fat diets
produced an increase in serum cholesterol concentration in
both groups. The decrease in serum cholesterol concentra-
tion that occurred when subjects were placed on the modified
fat diet was greater than the decrease in serum cholesterol
that occured when eggs were replaced by egg substitute in
the control diet.

It is not known why the 8 subjects who continued on the
same control diet with 2 eggs/day during the first experi-
mental diet period demonstrated a mean reduction in serum

cholesterol concentration of 10.5 mg, or 4%. Four of the

Figure 1.

64

The changes in total serum cholesterol con-
centration during the two 4-week experimental
diet periods relative to the control period
were found for each of the four diet groups
(8 subjects/group) by subtracting the average
values of the last two serum cholesterol
values for the experimental diet periods

from the mean value of the last two serum
cholesterol values of the lO-day control
period. The symbols used are as follows:

C-E =the control diet with 2 eggs/day

C-ES =control diet with egg substitute
replacing eggs

MF-E =modified fat diet with 2 eggs/
day

MF-ES=modified fat diet with egg sub-
stitute replacing eggs

65

 

   

Control Diets _
C-E
' +1.4
-23.8
-27-1
Modified Fat Diets
C-E MF-E MF-ES C-E MF-ES MF-E

 

-35.8

 

66

subjects in this group experienced a decrease in serum
cholesterol concentration, with the greatest decrease equal
to 30 mg. One subject experienced an 8 mg increase in
serum cholesterol and 3 subjects showed essentially no
change. This group demonstrated the greatest variability
in response to dietary changes. The reason for this is
unclear.

Analysis of variance of the serum cholesterol values
obtained by the commercial laboratory revealed significant
diet and treatment effects at the 5% level, indicating that
the changes in serum cholesterol seen in this study were
related to the type of diet (control vs modified fat diet)
and to treatment with either eggs or egg substitute (Table
11). No significant group or seasonal effect was found,
thus allowing us to combine the data for subjects from both
the Winter and Spring terms. No significant period effect
was seen, indicating that the changes in serum cholesterol
seen in this study were not related to the sequencing of the
experimental diets (i.e. eggSOegg substitute vs egg substi-
tuteOeggs). The same level of significance was found when
the data were analyzed by averaging all the serum choles-
terol values within a diet period or when averaging only
the last two values of each diet period.

Correlation between average weekly body weights and
weekly serum cholesterol levels was computed for the entire

group of 32 subjects. No correlation was found at the 5%

67

Table 11. Analysis of variance of the data for total serum
cholesterola

 

 

Sources of Variation DF F1 F2 CV
Seasons 1 0.09 0.09

Diets (Control vs MF) 1 13.77* 12.23* 4.20
SXD 1 3.25 0.08

Error (Subject/SD) 28

Periods 1.78 2.48
Treatments 12.88* 28.19*

SXT 2.89 0.02 4.21
DXT 0.47 1.24

SXDXT 1 0.21 0.01

Error2 (Residual) 27

63

 

*Significant at p<0.05.

aSymbols used are as follows:
DF=degrees of freedom
CV=critical value
S=Seasons (Winter vs Spring terms)
D=Diets (Control vs Modified Fat)
T=Treatment (Eggs vs Egg Substitute)
F1=f ratio for the average of all values
F2=f ratio for the average of the last two values

significance level

68

(r=0.66).

DISCUSSION

The purpose of this study was to compare the effects
on total serum cholesterol of replacing 2 eggs/day in the
diet with a cholesterol—free egg substitute and changing
from a typical American diet to a modified fat diet that
is lower in total and saturated fat and higher in polyun-
saturated fat. The modified fat diet utilized was similar
to the diet recommended in the Report of the Inter-Society
Commission for Heart Disease Resources (1972). The menu
was made up entirely of natural foods that are customarily
consumed by the United States population. The amount and
type of dietary fat and cholesterol fed in the study were
within the normal range of consumption in the United States
(Keys et al., 1974). Unlike other studies reported in the
literature the subjects were not adapted to a cholesterol-
free diet, or to a diet to which they were not accustomed.
Attempts were made to individualize the menu for each
subject in order to accommodate food dislikes and preferences,
to maintain weight, and to provide a diet that approximated

as closely as possible the customary diets of the subjects.

69

70

The Design of the Study

 

The split-plot design of this study allowed for
analysis of the effects of various factors (e.g., diet,
treatment) as well as of their combined effects (e.g.,
diet x treatment). The crossover design in which each
subject served as his own control eliminated the possi-
bility of factors other than those related to diet influen-
cing the results.

The study was designed to correspond to the lO-week
academic term. The study was planned to terminate before
the week of final exams in order to avoid the stress that
often accompanies studying for exams. The influence of
stress on serum cholesterol levels has been documented by
Dreyfus and Czaczkes (1959), who have shown an increase in
serum cholesterol concentration in medical students at
exam time.

Since one of the criteria for eligibility in the
study stated that subjects must like and habitually consume
eggs, the control period was assumed to be an extension of
the subjects' customary diets. A lO-day control period was
believed to be adequate for the estimation of caloric
needs, collection of baseline data, and adjustment of
subjects to experimental procedures. Keys et a1. (1974)
pointed out that all, or nearly all, of the dietary fat-
serum cholesterol response of man is exhibited in two or

at the most three weeks. In view of this fact, four weeks

71

was determined to be an adequate period of time for the

experimental diet periods.

Subject Selection

 

Young male subjects with moderately elevated serum
cholesterol levels were selected to participate in the
study because they are considered to be at high risk for
CHD and a segment of the population who would benefit from
preventive dietary measures. Enos et a1. (1955) provided
pathological data on young American soldiers killed in the
Korean war that demonstrated that atherosclerosis was
already present in young men in their 20's. It has been
assumed that elimination of the risk factors for athero-
sclerotic heart disease (of which serum cholesterol is one)
at an early age would be more effective than after athero-
sclerosis is in an advanced state. In the Oslo diet-heart
study, Leren (1970) showed that the reduction in the recur-
rence of heart attacks after dietary modification was
evident only in the younger men in the study.

Because of the relationship of serum cholesterol
levels to the menstrual cycle, female subjects were not
included in the study. Serum cholesterol levels have been
shown to decrease in women at the time of ovulation (Oliver

and Boyd, 1953).

72

Subject Compliance

 

All 32 subjects completed the lO-week study. There
were very 'few food rejections or complaints. The excep-
tionally high subject compliance that was seen in this
study was probably related to their genuine concerns, in
view of their family histories, about the risk of CHD.
Also, many of the subjects had participated in or were
involved in their own research and they were aware of the
importance of reliable information. It would seem, too,
that the gourmet menu contributed, at least in part, to
the adherence to the diet. At the end of the ten weeks,
even after having seen the same menu five times, many of

the subjects requested copies of the recipes.

Body,Weight

 

Subjects were weighed daily and adjustments in caloric
intake were made regularly because it has been shown
that rapid weight gain or loss was paralleled by elevations
or depressions, respectively, in serum cholesterol levels
(Anderson et al., 1957). Brunner et a1. (1979) reported
similar findings. Although several subjects in the present
study experienced some fluctuation in body weight, the
changes were not rapid or extreme, and no correlation was
found at the 5% significance level between weekly body
weight values and weekly serum cholesterol concentrations

0.66).

for the total group of subjects (r

73

Alcohol Consumption

 

One 12 ounce can of beer was included in the daily
diet plan of six subjects. Other subjects occasionally
substituted 12 ounces of beer for two slices of bread, which
are approximately comparable in calorie content. There was
no apparent difference between the subjects who consumed
beer on a daily basis and the subjects who consumed no
alcohol during the 10-week study in either the direction
or the magnitude of response of serum cholesterol to diet
modifications. Similarly, there was no apparent difference
in serum cholesterol response to diet modifications in the
subjects who, on occasion, substituted beer for bread.
These subjects were asked to refrain from substituting
beers on the days preceeding blood tests. It was believed
that the beer would be fully metabolized before bloods
were drawn and that such small quantities of alcohol con-
sumed on an occasional basis would have little or no
effect on total serum cholesterol levels.

Although the short term effect of alcohol on serum
lipid levels has been studied experimentally in man the
methodology is often questionable. Many of the partici-
pants in such research were alcoholics who were temporarily
deprived of alcohol before the experiments. Also the
number of subjects in each study generally was small.
Friedman et al. (1965) demonstrated no short term effect

of alcohol on serum cholesterol levels in daily drinkers

74

who continued their usual pattern of imbibing. Carey et a1.
(1971) showed that "moderate" alcohol intake, 20% of total
calories as alcohol,ingested during the evening for 12
consecutive days, had no noticeable effect on cholesterol.
Ostrander et a1. (1974), in contrast, found that frequency
of alcohol ingestion was significantly related to serum
cholesterol concentration among the 202 male participants

in the Tecumseh epidemiological study. These findings were
based on data obtained from questionnaires on the drinking
habits of each subject, followed by a blood test for serum
lipid determinations. Again, the methodology to provide
this type of data is questionable because of the possibility
of the subjects' reports of alcohol intake being influenced

by their concept of society's view on alcohol consumption.

Changes in Serum Cholesterol

 

The results reported here indicate that dietary cho-
lesterol may influence serum cholesterol concentration in
healthy males. Addition or deletion of 2 eggs/day from the
diet generally produced parallel changes in total serum
cholesterol concentration. Replacement of eggs with an
equivalent serving of cholesterol-free egg substitute
resulted in average reductions in serum cholesterol of
approximately 17 and 24 mg/100 ml, or 7 and 11%, respec-
tively, in each of the two diet groups consuming the high

saturated fat diets. The addition of eggs to the diet

75

of the subjects who consumed the egg substitute in the
first experimental diet period resulted in a 25 mg/100 ml
or 13% increase in serum cholesterol.

These findings conflict with those of Slater et a1.
(1976), Porter et a1. (1977) and Flynn et a1. (1979), who
found a lack of association between egg consumption and
serum cholesterol concentration in healthy subjects. The
intake of foods other than eggs, however, was not controlled
in these studies. It is conceivable that the variation in
cholesterol intake from other foods and the variation in
the amount and type of fat may have been sufficient to
mask the effect of adding or deleting two eggs from the
diet.

During the control period with 2 eggs/day subjects
were consuming approximately 270 mg of cholesterol/1000
kcal compared to approximately 105 mg cholesterol/1000 kcal
on the control diet with egg substitute. According to the
following prediction equation proposed by Keys et al.
(1965b)

ACholesterol (mg/100 m1) = 1.5 ((Adietary

cholesterol mg/lOOO kcal)1/2)
one would expect a 19 mg decrease in total serum cholesterol
concentration with the removal of 2 eggs/day from the

control diet. The results of this study are in agreement

with the predicted value from the Keys equation.

76

Based on the results of a comprehensive study on
schizophrenic patients under metabolic conditions, Hegsted
et a1. (1965) found that for each 100 mg change in dietary
cholesterol one could predidt a change in total serum
cholesterol of approximately 5 mg/100 ml. Using this
equation one would expect a 27 mg decrease in serum cho-
lesterol concentration following the removal of 2 eggs/day
from the control diet. The observed value was somewhat
lower than the predicted value.

A prediction equation was developed by Mattson et a1.
(1972) which related changes in dietary cholesterol to
serum cholesterol levels. This equation predicted a 12 mg/
100 m1 change for each 100 mg change in dietary cholesterol/
1000 kcal, or a 20 mg decrease in serum cholesterol concen-
tration for subjects on the control diet following the
removal of eggs from the diet.

Based on the evaluation of the combined results of 19
sets of experiments by several different investigators,
Keys et a1. (1974) predicted that the serum cholesterol
response to the addition of two eggs/day to the diet would
be an increase of 10 or 12 mg. In the present study mean
increases in serum cholesterol concentration of 25.1 and
19.1 mg were observed when eggs were added back to the
control and modified fat diets, respectively, following
the 4-week diet period in which eggs were replaced by egg

substitute. These increases in serum cholesterol

77

concentration are approximately twice as large as the
values predicted by Keys et a1.

When the percent of total calories from fat was re-
duced from 45 to 35 and the P/S ratio was increased from
0.45 to 1.10 on the modified fat diet the subjects demon-
strated a statistically significant decrease (p<.05) in
total serum cholesterol. When eggs were replaced by egg
substitute in the modified fat diet an additional decrease
in serum cholesterol was seen.

The decrease in serum cholesterol concentration that
occurred when subjects were placed on the modified fat
diet was greater than the decrease in serum cholesterol
that occurred when eggs were replaced by egg substitute.
These findings are in agreement with those of Keys et a1.
(1965) and McGandy and Hegsted (1975), who found that the
potential for altering serum cholesterol was greater by
the modification of dietary fats than by restriction of
dietary cholesterol. The results of the present study
disagree with the conclusion by Mattson et a1. (1972) that
a change in the intake of cholesterol may be more important
than a change in dietary fat in reducing serum cholesterol
levels.

The degree of responsiveness of serum cholesterol
levels to removal of eggs from the diet did not appear to
be related to the subjects' initial serum cholesterol

levels or to the order in which dietary changes were

78

initiated.. For example, subject 19, whose control period
cholesterol level was 224 mg/100 ml experienced a 44 mg
decrease (19.6%) in cholesterol when eggs were removed
from the diet (Table 7). Subject 4, on the other hand,
whose cholesterol level during the control period was

262 mg/100 ml, experienced only a 6 mg or 2.6% decrease
when eggs were removed from the diet. For both of these
subjects eggs were replaced by egg substitute during the
last four weeks of the study. The same lack of correlation
was seen between initial serum cholesterol levels and
magnitude of response to removal of eggs from the diet
when the eggs were replaced by egg substitute immediately
following the control period. Subject 31 whose control
period serum cholesterol level was 224 mg/100 ml experi-
enced a 23 mg or 10.3% decrease in serum cholesterol when
eggs were replaced by egg substitute (Table 8). Subject 7
whose control period serum cholesterol level was 262 mg/
100 m1 demonstrated a 28 mg decrease (10.6%) when placed
on the egg-free diet.

Similarly, the magnitude of response to cholesterol
intake on the modified fat diets did not appear to be
related to the subjects' initial serum cholesterol levels
or to the order in which eggs were added or deleted from
the diet. Subject 22 whose control period serum cholesterol
level was 236 mg/100 ml showed a 48 mg or 20.3% decrease

in total serum cholesterol after consuming the modified fat

79

diet with two eggs/day for four weeks (Table 9). In
contrast, subject 27 from the same diet group experienced
a 30 mg or 11.3% decrease from his control period value of
264 mg/100 ml. When eggs were removed from their diet
during the last four weeks of the study, subject 22
experienced no additional decrease in serum cholesterol
whereas subject 27 experienced a 17 mg or 7.3% decrease.
The degree of responsiveness of serum cholesterol concen-
trations to changes in the diet did not appear to be
related to family history of heart disease, hypertension,

diabetes, or to tendency to overweight.

CONCLUSIONS

The results of this study indicate that mean reduc-
tions in serum cholesterol of approximately 5 to 10% can
be achieved in healthy young men when two eggs/day are
replaced by a cholesterol-free egg substitute in a typical
American diet. A 10 to 12% reduction in serum cholesterol
was achieved when subjects consumed diets lower in total
and saturated fat, and higher in polyunsaturated fat;
when eggs were replaced by the egg substitute in this
modified fat diet a 15 to 19% mean reduction in serum
cholesterol was achieved. These findings are in agreement
with those reported by Christakis et a1. (1966), the
National Diet-Heart Study Research Group (1968), Turpeinen
et a1. (1968) and Leren (1970), who demonstrated mean
reductions in blood cholesterol of 10 to 15% with diet
modifications in men participating in longitudinal studies.
These studies have shown that with continued diet modifi-
cation these reductions in serum cholesterol can be
maintained for years.

Although a causal relationship between serum choles-
terol concentration and coronary heart disease has yet to

be proven, strong evidence exists that suggests that

BO

81

elevated serum cholesterol is an important risk factor in
the development of atherosclerosis. The results of this
study have shown that it is possible to decrease serum
cholesterol level by diet modification. Whether diet
modification will lead to a reduction in the prevalence
of CHD remains to be seen. It is only logical, however,
that an attempt should be made to reduce or remove the

risk factors over which man has control.

SUGGESTIONS FOR FURTHER RESEARCH

The results of the present study indicate that addi-
tion or deletion of two eggs/day and modification of the
amount and type of fat in the diet do influence the concen-
tration of cholesterol in the serum of healthy, young men
with moderately elevated levels of serum cholesterol. A
similar study conducted on larger population samples would
enable one to more accurately quantitate the effects of
these two types of diet modifications on serum cholesterol.
It would seem highly desirable to increase the length of
the experimental diet periods to determine whether further
changes or adaptation of serum cholesterol levels would

occur with time.

82

APPENDICES

APPENDIX A

83

APPENDIX A-1

Consent form for screening serum cholesterol determination

Michigan State University Human Cholesterol Study
Department of Food Science and Human Nutrition 1977

CONSENT FORM

I, , agree to participate in a screening

 

test for a study of the effect of the dietary intake of eggs and an
egg substitute on serum cholesterol. The purpose and the nature of

the study have been explained to me by ,

 

and I have been given an opportunity to ask questions. Criteria for
selection of participants in the study have been described and I agree
to the preliminary blood tests and physical examination, realizing that
they will be used to determine my eligibility for further participation.
I understand that my participation in these preliminary tests does not
guarantee that I will be selected as a subject for this study. I
understand that I can withdraw from the study at any time without

penalty and that results will be treated in strict confidence.

Signed:

 

Date:

 

84

APPENDIX A-2

Consent form for participation in the cholesterol study

DEPARTMENT OF FOOD SCIENCE AND HUMAN NUTRITION
MICHIGAN STATE UNIVERSITY
EAST LANSING, MICHIGAN

I, , agree to participate in a
study of the effect of the dietary intake of eggs and an egg substitute
on serum cholesterol. The purpose and nature of the study have been
explained to me by , and I have been
given an opportunity to ask questions. Criteria for selection of
participants in the study have been described and I agree to the pre-
liminary blood tests and physical examination, realizing that they will
be used to determine my eligibility for further participation. If I
am selected for the study I am aware of the potential risks which may
be involved as well as any possible benefits. However, I understand
that my participation in the study does not guarantee any beneficial
results to me as an individual. My participation in this experiment
can be terminated by me at any time without penalty. I understand
that the results of the study will be treated in strict confidence
and that I will remain anonymous. A summary of the results of this

experiment will be provided to me at my request.

Signed:

 

Date:

 

APPENDIX B

85

APPENDIX B

Two week sample menu for subjects on the control diet

with 2 eggs/daya

 

 

Week 1
BREAKFAST LUNCH DINNER
Pineapple juice Macaroni & cheese Baked pork loin
Eggs Tossed salad (170 g)/cr. mush—

English muffin

Kraft cr. Russian

room soup

 

 

 

MON Butter, jelly drsg Stovetop dressing
Milk Canned peaches Brussel sprouts
Peanut butter cookies SPICY applesauce
Cherry bavarian
Bread;butter
Apricot juice Hamburger (180 g)/ Baked fish (170 g)/
Eggs bun dill and wine sauce
Toast Potato chips Baked potato/sour
Butter, jelly Raw spinach salad/ cream
Milk creamy Russian Cut green beans
TUES dressing Tossed salad/blue
Sherbet cheese drsg
Angel cake/frozen
strawberries
Bread, butter
Milk
Orange juice Beef noodle Cup-A- Beef kabobs (170 g)/
Cold cereal Soup green pepper, onion,
Banana Egg salad/tomato & mushrooms
Toast cucumber slices Rice
WED Butter, jelly Rye bread Carrot sticks
Milk Oatmeal cookies Baked custard/grape-
Milk nuts
Bread, butter
Milk
Grapefruit juice Sliced ham (80 g)/ Chicken cashew - 1
Eggs Syrian bread serving
Toast Potato salad , Chow mein noodles
Butter, jelly Dill pickle spear Broccoli
THURS Milk Baked apple/dates, Coconut mandarin

cinnamon & brown
sugar
Milk

salad
Lemon bisque
Bread, butter
Milk

86

 

 

BREAKFAST LUNCH DINNER
Canned peaches Roast beef sandwich Omelet/mushrooms
Cold cereal (120 9) French fries
Raisins Celery sticks Sausage links

FRI Date nut muffin Banana Tossed salad/French

Butter, jelly Sugar cookies dressing
Milk Butter Chocolate cake
Sugar Milk Bread, butter

Milk

 

Apple juice

Turkey (113 g)/

Swiss steak (170 g)

 

French toast/sirup
Butter
Milk

Fried eggs sandwich Parsley potato
Toast lettuce, tomato, Pea pods
SAT Butter, jelly mayonnaise Cottage cheese/pine-
Milk Coleslaw apple ring
Jello/peach & pear Spiced apple cake
halves Bread, butter
Milk Milk
Snack Orange juice Baked pork loin (170
Milk Eggs g)/barbecue sauce
Cereal Canadian bacon Oven fried potato
SUN Fruit (85 g) Peas

Waldorf salad

Ice cream/chocolate
sauce, pecans

Banana

Bread, butter

Milk

aReplacement of eggs with egg substitute was the only change made in
the diet when subjects were placed on their respective egg-free diets.

The portion sizes of many food items (e.g. juice, bread, vegetables,
fruit, baked goods, butter/margarine, salad dressings, and milk)
varied from subject to subject according to type of diet (Control vs
Modified Fat), energy requirements, and food preferences.

Coffee and tea were available at each meal and were allowed ad libitum.

87

 

 

 

 

 

 

Week 2 - Control
BREAKFAST LUNCH DINNER
Grapefruit half Chili con carne Baked veal cutlet
Eggs Crackers (142 g)
Toast Tossed salad/Italian Rice
Butter, jelly dressing Cut green beans
MON Milk Oatmeal cookies Cucumber & onion/
Butter mock sour cream
Milk Yellow cake/praline
topping
Bread, butter
Milk
Pineapple juice Fish fillets (135 Broiled steak (170 9)
Eggs g)/bun Baked potato
Bran muffins Tartar sauce Zucchini vegetable
Butter, jelly Carrot & celery medley
TUES Milk sticks Tomato juice
Peanut butter Sherbet
cookies Bread, butter
Apple Milk
Milk
Banana Hamburger (180 g)/ Quiche/cheese, BacO-
Orange juice bun bits
Cold cereal Carrot sticks Broccoli
WED Toast Pineapple upside Tossed salad/French
Butter, jelly down cake dressing
Milk Milk Fresh mixed fruit cup
Bread, butter
Milk
Cranapple juice Chicken noodle Cup- Meatloaf - 1 serving
Eggs A-Soup Potato kuegel
Date nut muffin Peanut butter/jelly Peas/mushrooms
THURS Butter, jelly sandwich Pear/shredded cheese
Milk Potato chips on endive
Orange Ice cream
Sugar cookies Bread, butter
Milk Milk
Apricot juice Hot dog (91 g)/bun Oven baked chicken
Eggs catsup, mustard, (120 g)
Toast pickle relish Acorn squash/brown
FRI Butter, jelly French fries sugar

Milk

Celery sticks
Brownie
Milk

3 bean salad
Cherry cobbler
Bread, butter
Milk

 

88

 

 

 

BREAKFAST LUNCH DINNER
Grapefruit juice Ham (113 g) sandwich/ Pizza/sausage-l ser-
Eggs lettuce, mustard ving
Toast Potato salad Tossed salad/Italian
SAT Butter, jelly Apple crisp dressing
Milk Milk Canned peaches
Sugar cookies
Milk
Snack Orange juice Roast beef (150 g ck)
Milk Eggs Mashed potatoes/gravy
Cereal Sausage (41 9) Green beans
SUN Banana Waffles/sirup & butter Tossed salad/French
Milk dressing

Pumpkin pie
Bread, butter
Milk

89

Two week sample menu for subjects on the modified fat diet

Week 1

with 2 eggs/daya

 

BREAKFAST

LUNCH

DINNER

 

Pineapple juice

Eggs
English muffins

Macaroni & cheese (skim

milk)
Tossed salad/creamy

Baked pork (142 91/
cr mushroom soup
Stovetop dressing

 

 

 

MON Jelly, margarine Russian dressing Brussel s routs
Skim milk Canned peaches SPICY app esauce
Peanut butter cookie Cherry bavarian
Skim milk Bread, margarine
Skim milk
Apricot juice Hamburger (120 g)/bun Baked fish (170 g)/
Eggs Potato chips dill & wine
Toast Raw spinach salad/ Baked potato/margarine
Margarine, jelly creamy Russian dres- CUt green beans
TUE Skim milk . sing Tossed salad/blue
Sherbet cheese dressing
Margarine Angel cake/frozen
Skim milk strawberries
Bread, margarine
Skim milk
Orange juice Beef noodle Cup-A-Soup Beef kabobs (142 g)/
Cold cereal Egg salad/ green pepper, onion,
Banana tomato & cucumber mushrooms
Toast slices Rice
WED Margarine, jelly Rye bread Carrot sticks
Skim milk Oatmeal cookies Baked custard (skim)/
Sugar Skim milk grapenuts
English walnuts Bread, margarine
Skim milk
Grapefruit juice Pineapple juice Chicken cashew - 1
Eggs Ham (80 gl/Syrian serving
Toast bread Chow mein noodles
Margarine, jelly Potato salad Broccoli
Skim milk Dill pickle spear Coconut mandarin
THURS Baked apple/dates, salad

brown sugar, Eng
walnuts, cinnamon
Skim milk

Lemon bisque (skim)
Bread, margarine
Skim milk

 

9O

 

BREAKFAST

LUNCH

DINNER

 

Canned peaches
Cold cereal
Bran muffin

Roast beef (120 g)
sandwich
Celery sticks

Omelet (skim, marga-
rine)/mushrooms
Sausage links

 

 

Margarine, jelly Banana French fried potatoes
FRI Skim milk Sugar cookies Tossed salad/French
Skim milk dressing
Chocolate cake
Bread, margarine
Skim milk
Apple juice Turkey (113 g) sand- Swiss steak (170 g)
Fried eggs wich/lettuce, Parsley potato
Toast tomato, mayonnaise Pea pods
SAT Margarine, jelly Coleslaw Cotta e cheese (low
Skim milk Jello/peach & pear fat /pineapp1e ring
halves Spiced apple cake
Skim milk Bread, margarine
Skim milk
HS snack Orange juice Baked pork loin (114
Cold cereal Eggs g)/barbecue
Skim milk Morningstar breakfast Oven fried potatoes
Apple slices Peas
French toast/sirup Tossed salad/French
SUN Margarine dressing, 5 hard
Skim milk cooked egg
Apricots Sherbet/chocolate
sauce/pecans
Banana

Bread, margarine
Skim milk

Week 2 - Modified

91

 

 

 

 

 

 

BREAKFAST LUNCH DINNER
Grapefruit half Apple juice Baked veal cutlet (142 9)
Eggs Chili con carne Rice
Toast Crackers Cut green beans
Margarine, jelly Tossed salad/Italian Cucumber & onion/mock
MON Skim milk dressing sour cream
Oatmeal cookies Yellow cake/praline
Rosano cheese (chol- topping
free) Bread, margarine
Skim milk Skim milk
Pineapple juice Fish fillets (100 91/ Broiled steak (142 9)
Eggs bun Baked potato
Bran muffins Tartar sauce Zucchini vegetable medley
TUE Margarine, jelly Tossed salad/French Tomato juice
Skim milk dressing Sherbet/chocolate sauce
Peanut butter cookie Margarine
Apple Skim milk Snack: fruit
Skim milk
Banana Hamburger (90 gl/bun Quiche (skim)/Bac0bits
Orange juice catsup, mustard Broccoli
Cold cereal Carrot sticks Tossed salad/French
WED Toast Pineapple upside down dressing
Margarine, jelly cake Fresh mixed fruit cup
Skim milk Skim milk Bread, margarine
Skim milk
Cranapple juice Chicken noodle Cup- Meatloaf-l serving
Eggs A-Soup Potato kuegel
Date nut muffin Peanut butter/jelly Peas/mushrooms
THURS Margarine, jelly sandwich Pear/shredded cheddar
Skim milk Orange cheese on endive
Sugar cookies Ice cream
Skim milk Bread, margarine
Skim milk
Apricot juice Hot dog (91 g)/bun Oven baked chicken (1209)
Eggs catsup, mustard, Acorn squash/brown sugar
Toast pickle relish 3 bean salad
FRI Margarine, jelly Celery sticks Cherry cobbler
Skim milk Brownie Bread, margarine

Skim milk

Skim milk
Snack
apple

 

92

 

 

 

BREAKFAST LUNCH DINNER
Grapefruit juice Ham (113 g) sandwich/ Pizza/Morningstar
Eggs lettuce, mustard sausage
SAT Toast Potato salad Tossed salad/Italian
Margarine, jelly Apple crisp dressing
Skim milk Skim milk Canned peaches
Sugar cookies
Skim milk
HS snack Orange juice Roast beef (150 g
Skim milk Eggs ck)
Cold cereal Sausage (41 g) Mashed potatoes/
SUN Banana Waffles/sirup & gravy

Sugar packet

butter
Skim milk

Green beans
Celery sticks
Pumpkin pie
Bread, margarine
Skim milk

 

APPENDIX C

93

APPENDIX C-l

Serum Cholesterol Determination by the Laboratory at

Reagents

1.

Procedure

1.

Michigan State University

FeSo :CH COOH

4 3

Place about 60 g ferrous sulfate (FeSO4:7H20)

in 1 liter glacial acetic acid. Allow to stand
for at least 3 hours. Filter and store in glass
bottle at room temperature. Solution remains
stable for several months (00 not use if solution
has any color).

Stock Cholesterol Standard

5 mg cholesterol/m1 chloroform

Store in refrigerator

A series of working standards were prepared daily
using the stock cholesterol standard and chloro-

form methanol.

Stock Cholesterol

 

 

mg/100 ml Standard ONCE;
100 0.2 0.8
200 0.4 0.6
300 0.6 0.4
500 1.0 0.0

\OCDNOS

10.

11.
12.

94

With micropipeter, transfer an aliquot of serum or
standard (0.1 ml)to small test tube. For blank,
use chloroform.

Add 2.5 m1 chloroform-methanol solution (2:1).
Mix.

Cover test tubes with marbles and place in hot
water bath (50-60) for 15 min.

Cool.

Mix.

Centrifuge at 3000 rpm for 15 min.

With automatic dilutor, remove 1.0 ml of the super-
natant and add to it 6.0 m1 FeSO4-CH3COOH in large
test tube.

Add 2.0 ml H2504 (conc.) and immediately mix
thoroughly.

Cool.

Read Optical Density at 490 mp.

95

Principles of the Procedures for Quantitative Determination

of Cholesterol in Serum by the Hycel Super Seventeen Method

1. Extraction: Cholesterol and cholesterol esters
are soluble in organic solvents but almost insoluble in
aqueous solvents.

2. Color Reaction: Cholesterol is dehydrated in an
acidic medium to yield 3,5-cholestadiene. There is some
question about the reactions that follow which produce the
green color measured at 625 nm. It has been thought that
the cholestadiene is dimerized to bis-3,5-cholestadiene
which in turn reacts with sulfuric acid to form cholestadi-
ene-3-monosulfonic acid. Recently, however, evidence has
been presented which indicates that the cholestadiene is
oxidized by sulfuric acid to yield a pentaenylic cation

which is the colored species.

APPENDIX D

 

 

 

 

 

 

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omp omp mew mOF map map may mep map ma— mp
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mm: mm: mm: NNN NNN NNN NNN NNN 2: 3: N—
nep map may omp omp Pmp Nm— «mp mm_ mm_ @—
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sup mmp wmp wmp omp Pmp Pwp pr Pap pr OF
moF mo— mop mop mop mop mop cup Fur mmp m_
mop amp cop cop mop mop mm— mop mop cup NF
rm? omr Pmp omp om” omp vmp mmp vm— mmp FF
pr Pmp owp Fmp omp mnp cm, on— mnp mnp op
may wep map mep Pmp om— Nmp mmp amp «mp m
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eep evp me— mew nap NON we— mep wv— om, m
mON OON «ON vow mom NoN mom mow mON opN o
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amp cap oep pep Nap Nap ¢¢F vep meF mep m
cop mop mop mop sop mop mop mop any Pup N
mmp emp mmp mmp mmp mmp mmp “mp mm? wmp p
op m m N m m a m N P pumnnzm
Oxmmz

Omuomwnam POOOO>NOON cow Amnpv mugmwmz Anon xpxmmz mmmcm><

o x~ozmmm<

97

.xmmz :umm mo Omumcasp cmzocgp NOONLO co Omcammme Ousmwmz
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mmp Omp Omp mmp mm— mm? mmp mmp mm? mm_ mN
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map sup omp ONO Ow? mnp Fm? pr mwF Nmp mN
amp omp omp pr pr Pwp omp pr Pwp ONO NN
«mp mmp nmp Nmp Nmp wmp wm— mmp mmp 1 PN
Op O m N O O O m N P pumnazm
mxmmz

.N.O.OOOOO O NOOZOOOO

APPENDIX E

98

 

 

 

 

 

 

 

 

 

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99

 

 

 

 

 

 

 

 

 

 

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mum x~ozmmm<

101

 

 

 

 

 

 

 

 

 

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APPENDIX F

102

 

 

 

 

 

 

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103

 

 

 

 

 

 

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104

 

 

 

 

 

 

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105

 

 

 

 

 

 

 

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ON OF NO ON ON O O N O O O m N N

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NOONOOOO N van .uowgma umwu Foucmswgmaxm pmgwm mzu chgau Ammumzv mpzaNamazm OOm OON:
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0-0 xHoszO<

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