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THE BLOOD SERUM CHOLESTEROL OF OVEKUEIGHT COLlEGE
WOMEN ON A CONTROLLED WEIGHT REDUCTION DIET

BY

Hazel Chang Amen

A.THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and.Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Foods and NUtrition

1951

THESIS

 

 

 

C

11*" /5‘/

a .

43(5)

ACKNOWLE DGL‘EI‘H‘S

The writer wishes to express her sincere
appreciation to Dr. Wilma D. Brewer for her interest,
patience, and helpful assistance throughout this study;
to Dr. Margaret A. Ohlson for her helpful suggestions
and advice; to Miss Doris Smith who assisted in recruit—
ing control subjects; to the Department of Foods and
Nutrition of Michigan State College for use of the data
on the overweight subjects; and to the twenty-one women
students whose cooperation made this study possible.

The writer wishes also to acknowledge with deep
appreciation the scholarship provided by Michigan State
College for the past two years which made it possible for

her to complete this study.

TABLE OF CONTENTS

MODUCT I OIJ C O O O O O O O O O O 0 O O 0
REVIEW OF LITERATURE . . . . . . . . . . .
Chemical Structure of Cholesterol . .

Concentration of Cholesterol in Blood

Factors Influencing the Blood Cholesterol . . . .

sex. . . . . . . . . . . . . . . .
age. . . . . . . . . . . . . . . .
menstruation and pregnancy . . . .
fasting and starvation . . . . . .
diet . . . . . . . . . . . . . . .

Ob881ty. O O O O O O O O O O O O 0

Methods on the Determination of Cholesterol . . .

historical develOpment . . . . . .

colorimetric determination, extraction . . . .

colorimetric determination, development of

color. . . . . . . . . . . . . .

other Methods of the Determination of
MEREZEII‘ITAL PROCEDURE . . . . . . . . . .
Subjects. . . . . . . . . . . . . . .
Sampling Techniques . . . . . . . . .

chemical Procedure. . . . . . . . . .

Cholesterol

Page

12

14

16
17
20
20
21

22

RESULTS AND DISCUSSION .

L

Serum Cholesterol during Weight Reduction .

Comparison of Serum Cholesterol Values for Over-

weight Women and Women of.Average Weight. .

Comparison of Serum Cholesterol of Capillary and

Venous Blood.

momma 0 1mm .

. ZI'D'ARY AND CC-I‘ICLUS IONS .

25

29

4'7

50

53

 

1.... .Jfl. N

 

TABLES
Page

TABLE 1. Concentration of Cholesterol in.Human Blood

as Reported by Various Investigators . . . . . 3
TABLE 2. The Age, Height, and Body Weight of ten Over-

weight Women on a Weight Reduction Diet. . . . 26
TABLE 3. Caloric Intake of Overweight Subjects for each

two Week Period on a Weight Reduction Diet . . 28
TABLE 4. Total Cholesterol of Blood Serum of Overweight

Women on A Self-Selected Diet, A Weight

Reduction Diet, and a Diet Planned for Main-

tenance of Desired Weight. . . . . . . . . . . 30
TABLE 5. Analysis of'Variance of Total Cholesterol of

Seven Subjects on a Weight Reduction Diet

for Ten Weeks. . . . . . . . . . . . . . . . . 35
TABLE 6. Free Cholesterol of Blood Serum of Overweight

Women on a Self~Selected Diet, a Weight

Reduction Diet, and a Diet Planned for

Maintenance of Desired Weight. . . . . . . . . 36
TABLE 7. Analysis of'Variance of Free Cholesterol of

seven Subjects on a Weight Reduction Diet for

Ten Weeks. . . . . . . . . . . . . . . . . . . 37
ABLE 8. Ratio of Free to Total Cholesterol of Blood

Serum of Overweight Women on a Self-Selected

Diet, A Weight Reduction Diet, and a Diet

Planned for Maintenance of Desired Height. . . 39

TABLE 9.

TABLE 10.

TABLE 11.

TABLE 12.

TABLE 13.

Cholesterol Ester of Blood Serum of Over-
weight Women on a Self-Selected Diet, A

Weight Reduction Diet and a Diet Planned for

maintenance of Desire Weight .

Analysis of'Variance of Cholesterol Ester of

Seven Subjects on a Weight Reduction Diet

for Ten WEEKS o o o o o o o o 0

Ratio of Combined to Total Cholesterol of
Blood Serum of Overweight Women on a Self—
Selected Diet, a Weight Reduction Diet, and

a Diet Planned for Maintenance of Desired

1:59 i ght O O O O O O O O O O O O O

The Age, Height, Weight, and Serum Choles-

terol of Eleven Healthy Women of.Average

Body Weight . . . . . . . . . .

Comparison of Serum Cholesterol Values for

Capillary and Venous Blood. . .

43

45

 

 

FIGURES
Page
FIGURE I. Graph Showing the Average Changes in Body
Weight, in Total, Free, and Cholesterol
Ester of Blood Serum of Overweight Women . . 31
FIGURE II. Graph Showing the Changes in Body Weight,
in Total, Combined and Free Cholesterol
of Blood Serum of Overweight Women on a

Weight Reduction Diet. . . . . . . . . . . . 33

INTRODUCT IO.’L\T

Cholesterol has been known since the eighteenth century as the
chief constituent of human gall stones. It is the characteristic
sterol of higher animals and is present in all cells of the animal
organism, in the brain and nerve tissue, the suprarenal glands and in
egg yolk.

A possible physiological function for cholesterol in blood was
suggested by Kelsey (1941) who believed that blood cholesterol was asso-
ciated with the transport of unsaturated fatty acids. Studies with deu-
terium as a trace element have demonstrated that cholesterol is converted
by the body to other biologically active substances, including both bile
salts and hormones. (Schoenheimer, Rittenberg, and Graff 1955).

There is general agreement among several authors that the concen-
tration of cholesterol in the blood or any person remains constant and
is not easily changed by diverse ph"siological conditions (Kirk, Page,
Lewis, Thompson, Van Slyke 193 , Sperry 1956, Offenkrantz 1938), Any
change in the concentration of blood cholesterol, therefore, would be
expected to result only from a disturbance of some metabolic function.

The relationship of blood cholesterol to diseased conditions has
been the subject of both clinical and experimental investij:tions. Since
cholesterol is present in high quantities in atherosclerotic lesions,
several investigations have been directed toward studies of factors
influencing the concentration of blcod cholesterol in atherosclerosis.

1

. . ‘. ‘ ..
:1 SIMIL) 01):.68 111

‘
| —
.

Cb

Since the incidence of atherosclerosis is relativ'ly hig
vicuals, and since the metabolisn of cholesterol appears to be allied

closely with the lipid metabolism, studies of the blood cholesterol of

overweieht individuals may be heluful in understandins the hrsiolou‘
D J U :95 VJ

of obesit". The following study mas conducted to inv;stigate the

possible chin: s in the blood serum cholesterol of ov::we ght

women on a controlled weight reduction diet a

(

ship between excess body weight and serum cholesterol Values.

REVIEW OF LITERATURE

T‘T‘IT'TW'J "‘ T m- ._
C: J—nIA.

<.
.u‘gl gadoi

Chemical Strugtgre f Chole; ero’

 

Cholesterol is one of the primer: constituents of body cells. It is

A

a monatonic alcohol contiining one d uole bond and possesses the formula

CD
.3
'1

H)

O

H

‘r—J

9

a

(D

co

er

Ca HQCOH. The structure 1
~ J

 

The four rings A, 3, C, and D form the cyCIOpentraOperhydrophenanthrene

nucleus, which is characteristic not only of cholesterol but also of other

plant and animal sterols and a wide v riety of n turallroccurring compounds.

Some of these compounds hrve importen physiological significance. These

g

comgoun-s include the bile salts, the steroid hormones, the sterol
vitamins, the eglyccne p rtion of the ceréiec glycosides, the ssponins

serived from the plant seyonin, and the c rcinorenic hydrocarb;ns of the
:hensnthrene type (Feiser, Teiser 1939). The possible reletionchip of

the varied com ounds wnose chemical str icture ere fu.dementally similar

has attrrcteu conside able nttentio

*Jo

I"! .2
n ~’-LOOKL.

 

as "flr 3‘ p ‘1‘ . 1
_Qonceno1ution oi Choiester i

 

Values reported by verious investifetors for whole blood cholesterol
for aiiorentl" hezltl‘y ad lts are given in Table l. The tote l blood
cholesterol rsnjed from 107.5 to 332 millifiram.per 130 cubic centimeters.
Values for free cholesterol ranked from 2@.3 to 110 millirrem per 100
cubic centimeters.

Cr oles terol cor tnins en slco .01 group and therefore is copeble of
forming esters; 'n tle bl 00d, e lhrge proportion of cholesterol is combined
wit‘ fatty acids. Sgerry (1936) fornd that the ratio of free to total
cholesterol in the blood was rems Enbly constant for humans. The amount
of free in the total cholesterol varied within narrow limits of 2%.? to
30.1 per cent with an average of 26.9 per cent. In an eerlier re crt,
K'rk and co-worhers (1935) had stated that the ratios of free to total
cholesterol varied widely between ins-Vi.uzl Values t.ere reported which
ranged fro. 22 to 72 per cent. The reasons for the differences between
the nor: of Kirk and hat of Sherry were not evide fit el houfh both inves-
ti stors yresentei rerres for the co ncentraticn of total choles terol alliCh
were &D;-Cde{t€1‘ the same. Subsequent studies from other laboratories

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- 5 -

the ratio of free to total blood cholesterol in a variety of condi-
tions. (Offenkrantz and Kurshan l 36, Offenkrantz 1938, Smith and
Marble 1957, Gardner, Gainsborough, and Hurray 1938, Peters and nan
1943).

The remarkable constancy of the ratio between free and combined
cholesterol may indicate the presence of an enzyme system.uhich
regulates the proportions of the two substances in the blood. Sperry
(1956) found that esterification of free cholesterol occurred in the
serum. Thus the ratio may be maintained through continuous and
simultaneous esterification of free cholesterol.

Bugnard (1930) observed that the venous plasma of dogs contained
more cholesterol than arterial plasma, whereas the concentration in
whole venous blood did not differ from that of arterial blood. He
concluded that there was an exchange of cholesterol between the
corpuscles and the plasma which was dependent upon the pH of the
circulating blood. According to this theory, when carbon dioxide is
given up by the blood in the lungs, and the blood becomes relatively
alkaline, cholesterol migrates from the plasma to the corpuscles. The
reverse shift takes place as the blood becomes saturated with carbon
dioxide. Bugnard found alSO.EEJKlE£2 experiments that the saturation
of whole arterial blood with oxygen resulted in lower plasma
cholesterol values than saturation of the blood with carbon dioxide.
Levine and Soskin (1939) also found an inverse relationship between
the total serum lipids and the carbon dioxide combining power of the

blood.

Shope (1928) reported that the concentration of cholesterol esters
in heparinized plasma did not differ from that in the serum. When
sodium citrate or potassium oxalate were used as anti-coagulants, the
plasma contained less bound cholesterol than the serum. Sperry and
Schoenheimer (1935, 1937) found that oxalated plasma contained
significantly smaller amounts of total and free cholesterol than
serum or heparinized plasma. These workers considered that the lower
values resulted from the effect of the oxalate in altering the serum
and plasma volumes. Gardner, Gainsborough, and Murray (1938) stated
that the use of potassium oxalate resulted in plasma choleSLerol values

which were about four per cent lower than heparinized plasma.

Factors Influencing Elood Cholesterol.

 

The wide range of values for blood cholesterol which are given
in Table 1 raises the question of whether the variations represent
differences between individuals or variations in a single individual
over a period of time. Sperry (1937) and Turner and Steiner (1939)
agreed that the concentration of cholesterol in the blood remained
remarkably constant for erch person. ban and Gildea (1937) and
Schube (1936) reported that variations occurred in blood cholesterol
concentration of individuals although the values which were reported
vete within fifteen per cent of the average concentration. .A review
of the f ctors which may influence the cholesterol values of blood
follOWs:

S359 Peters and Len (1943) found no distinction between serum

lipids of normal male and female adults but Lornerup (1950) observed

that there was a tendency toward higher serum lipid values in females
than in males.

.§§E' Sperry (1936) found that the concentration of blood
cholesterol was low at birth and that a rapid increase in cholesterol
content occurred during the first four days of life. After the fourth
day, the values for blood cholesterol appeared to remain constant during
infancy. The rvnge of plasma cholesterol values for infants four to
25 days old ranged from 71 to 192 milligram per 100 cubic centimeters
of plasma. The ratio of cholesterol ester to total cholesterol varied
from 51 to 72 per cent for infants. Thus, the constancy of the ratio
of free to total cholesterol which was observed for adults was not
found for infants. Hulbock and Kaufmann (1938) obtained values that
were in agreement with those of Sperrv.

A gradual but slight increase of cholesterol with age throughout
childhood was reported by Baylac and Sendrail (1928) and hard (1931);
however, Offenkrantz and Karshan (1936) reported values for blood
cholesterol for children which were within the same range as for
adults. Kirk and his co-worhers (1935) studied a group of men who
ranged from 21 to 91 years of age. These workers found that age did
not have a significant effect either on the concentration of blood
cholesterol or on the ratio of total to free cholesterol.

Keys and co-workers (1950) found that the values of serum
cholesterol for men and women were not significantly different in the
age group of 17 to 30 years. However, above this age, there was an
average increase per year of 2.2 milligrams of total cholesterol per

100 cubic centimeters of serum. For the age range 17 to 78 years in men,

Keys and co-workers found a pronounced curvilinear relationship
I
between age and serum cholesterol concentration with a maximum in

the sixth decade.

‘genstruation and Erggnangy. Ckey and Boyden (1927) reported

 

that exceptions to the usual constancy of blood cholesterol occurred
during menstruation. The study included me surenents of sixteen
healthy women over a period of 26 menstrual cycles. The usual
fluctuations in the concentration of blood cholesterol during the
menstrual cycle involved a slight preucnstrual rise, followed by a
distinct fall in ediately before or during menstruation, then a sudden
rise during or slightly after the bleedin: phase followed by a gradual
decline to the average value. Offenkrantz (1938) observed that the
fluctuations during menstruation were within the limits of variations
of adult men. Both Okey and Offenkrantz found that the cholesterol
concentration in blood of normal women during the intermenstrual
period was constant.

The cholesterol content of blood apparently increases during
pregnancy. Grigaut (1913) and Hermann and heumann (1912) found no
change in the first trimester; however a gradual rise in all lipid
constituents occurred in the fourth month. The concentration remained
high during the rest of pregnancy, and through puerperium, then
declined gradually to pre-pregnancy values. Boyd (1934) showed that
this change occurred only in the plasma, and that the erythrOCyte

lipids remained constant.

Fasting and Starvation. The eff ct of fasting on blood chol-

 

esterol w s first studied by Gardner and Landen (1913) who observed

considerable increases in free and esterified cholesterol during
periods of hunger. ShOpe (1927) observed that hypercholesteremia
occurred during periods of fasting up to six days. The blood
cholesterol decreased within a few hours after feeding. This subse-
quent lowering of blood cholesterol occurred regardless of the type
of food eaten, whether fat, czrbohydrate, protein or a mixed diet.
Bose (1946) found that low hholesterol values occurred in starvation.
In cases of starvation, nan and Gildes (1936) found that the
cholesterol concentration in the blood seemed to vary directly with
the state of nutrition.

2333, The effect of diet on the concentrations of blood chol-
esterol over extended periods of time was first reported by Luden
(1917) who from self observation found that a prolonged vegetable
diet resulted in a decreased value for cholesterol and that an
exclusive meat diet increused the concentration. Gardner and Gains-
borough (1927) reported that blood cholesterol varied with the amount
of cholesterol in the diet. On the other hand, Turner and Steiner
(1939) concluded that no relationship existed bethen the type of
diet and cholesterol concentrations in the blood. Neither maintenance
on a high or low fat diet nor addition of cholesterol to the food
influenced the blood cholesterol in their study.

okay and Stewart (1933) determined cholesterol in the whole blood
of four normal women who were kept on diet which contained varying
amounts of cholesterol for approximately one month. Values reported
for blood cholesterol were slightly higher when the diet contained

3.1 grams of cholesterol per day than when a control diet was used

- 10 _

which contained 0.77 grams. Values were higher when egg yolks were
fed as a source of cholesterol than when cholesterol was mixed with
butter, but these differences were slight and were not considered

to be significant. Corwin (1938 reported that long term feeding on
a high fat diet produced only slight hypercholesteremin.in dogs but
when the high fat diet was supplemented with lecithin there was a
marked elevation of free cholesterol and combined cholesterol. Cho-
1esterol administered in the solid state had no effect on the blood
levels.

The specific effect of lecithin on the concentration of blood
cholesterol was investigated by Steiner and Domanski (1941). Eight
subjects were given 14 grams of lecithin and eight grams of cholesterol
for five weeks. The average increase in the cholesterol concentration
in the serum was about 101 milligrams per 100 cubic centimeters of
blood at the end of this period.

These studies indicate that ingested cholestcrol when given alone
or when mixed with fat, apparently has little or no influence on the
concentration of cholesterol in the blood, but when cholesterol is
mixed with lecithin, an appreciable rise in the concentration of
cholesterol in the blood may occur, probably through finer emulsifi-
cation and more efficient absorption.

Keys, hickelsen, Miller and Chapman (1950) studied the relation-
ship between the cholesterol content of the diet and the concentration
of cholesterol in the blood. Blood cholesterol was found to be
independent of the intake over a wide range; however, when the diet

was free of cholesterol, blood cholesterol was lowered at a rate

-11...

related to the previous concentration of cholesterol in the blood.
A severe reduction of cholesterol in the diet was not easily achieved
since cholesterol occurs only in foods of animal origin and is
relatively stable. Hovever, {eys found that a cholesterol free diet
readily lowered the blood cholesterol in normal men and in men with
extreme hypercholesteremia.

obesity. The influence of the amount and the character of the
blood lipids on the development of atherosclerosis has been studied
extensively; however, the results which have been reported have been
highly controversial. Diagnostic symptons of atherosclerosis do not
appear until the advanced stages of the disease; therefore, the deter-
mination of its presence and extent in humans is difficult. Considerable
evidence which has been obtained in experimental studies with rabbits
has indicated that hypercholesteremia may be a factor in the develop-
ment of atherosclerosis (Page, 1941). Investigations have been
directed toward the possibility that this also may be true of the
development of atherosclerosis in humans (Ueinhouse 1943, Mann 1951).
Further, the possibility that obesity is a prediSposing f ctor toward
atherosclerosis and that hypercholest remia may be a factor in this
relationship has been studied. Rony and Levy (1929) measured the
blood cholesterol of 15 normal and 21 obese adults preceding the
following a test meal of 500 milliliters of 20 per cent cream. There
was no apparent change in the concentration of blood cholesterol.
Conversely, Blotner in 1935 administered a test meal which was high

in fat to 21 obese individuals and found that there was a marked

- 12 -

increase in blood cholesterol; this increase in blood cholesterol

was not observed when the same test meal was given to 13 normal
adults. The nature of the test meal wts not described in the report.
The results of Rony and Levy were confirmed by Oppenheimer and Bruger
(1943) who repeated the experimental conditions of the previous study
with seven normal and 13 obese individuals and found no inorease in
blood cholesterol following the test meal. These studies have
indicated recognition of the problem relating to cholesterol metabo-
lism in obesity. There is need for further investig tion of the

problem.

fiethods on the Determination of Cholesterol.

w-‘ .-v

 

Eistorica1_peve10pment. Chemical methods for the determination

 

of cholesterol date back to the early part of this century. Windaus

in 1909 published a gravimetric procedure and Grigaut in 1910 published
a gravimetric method for cholesterol determinations. hindaus found
that cholesterol formed stoichiometric coordination compounds with
digitonin and other sap nins. On the basis of this observation windaus
precipitrted cholesterol by digitonin in alcohol and determined it
gravimetricelly. Grigaut applied the Liebermann-Burchard reaction
(1885) to an ether extract of cholesterol and measured the intensity
of color produced by the reaction. In the Liebermann-Burchard reaction,
acetic anhydvide and concentrated sulfuric acid were added to a dilute
solution of cholesterol in chloroform. The color MhiCh resulted was
first blue, tlen green, and finally after long statding, a yellow

brown.

- 13 -

For twenty years Windaus' gravimetric method was considered
the standard procedure for the estimation of cholesterol. The method
is considered excellent but it is laborious and time consuming.

The method was not applicable to routine analyses of small
quantities of blood and tissue available for study as it was not
possible to weigh accurately one or two milligram quantities of
cholesterol digitonide with ordinary analytical balances.

Grigaut's method required five to ten cubic centimeters of blood
and required three to five hours to extract the cholesterol. anten-
rieth and Funk (1913) modified Grigaut's method and reduced the
quantity of blood needed to two cubic centimeters and introduced the
use of strong alkali to extract the cholesterol. The extraction
time was thus reduced to 25 to 35 minutes. The use of strong alkali
introduced a new problem. .A brown tint developed which was believed
to interfere with the colorimeter reading.

From 1916 to 1928, Bloor published a series of articles which
presented modifications and improvements of the colorimetric proce-
dure. He found that the cholesterol in blood behaved differently
from pure standard cholesterol. Blood cholesterol reacted more
readily and faded f ster than pure cholesterol. Bloor questioned
whether the blood cholesterol was wholly or in part different from
the pure cholesterol, or whether the difference resulted from changes
in the rate of the reaction caused by impurities in the blood extracts.
Later (1922), Bloor reported that pure cholesterol was not affected
by alkaline saponification, and it was probable that some substance

other than cholesterol was present in the blood which was sensitive

- 14 -

to alkali and gave a brown tint. In 1928, Bloor published another
paper in which he reported the determination of total fatty acids
and cholesterol in an alcohol-ether extract obtained by saponifica-
tion and extraction; in 1929, Bloor published complete details for
the oxidative determination of phOSpholipids, fatty acids, and
cholesterol from the same extract.

Bloor had undoubtedly carried out the most extensive ground work
for the colorimetric determination of cholesterol. In the same
period of time, other laboratories also were concerned with improve-
ments of met ods for cholesterol determinations. Various modifications
of the colorimetric procedure involved two processes, 1. e., the
isolation of cholesterol by extraction and the duvelOpment of color
in the extract.

Colorimetric determination of Cholesterol: Extraction. The

 

extraction process may follow one of two steps: (1) the wet method
which Bloor used extensively, and (2) the dry method of extraction.
In wet extraction, blood is treated with alcohol-ether and filtered
to remove the precipitated protein. The solvent is evaporated and
the lipid material is taken up by chloroform. This method is
preferred when more than one phase of the various lipid fractions
of blood are studied. hith the dry method, only cholesterol can be
analyzed in the extract. The blood is dried on some substance as
filter paper and then extracted with chloroform in a continuous
extraction process. A comparison of the methods for wet extraction
and dry extraction of cholesterol for blood which have been reported

in the literature indicated that equa ly reliable results have been

_ 15 -

obtained by the two methods. Only the total cholesterol content of
the blood can be determined when the procedure of the dry extraction
is used. The wet extraction method has the advantage that both total
and free cholesterol may be determined from the same extract and that
special continuous extraction apparatus is not needed.

The wet extraction method was used by Autenrieth and Funk (1913)
and Grigaut (1910). Both investigators used ether for extraction.
When used alone, ether re uires a long extraction period; however,
when ether is used with alcohol or when alcohol and acetone are used
for extraction, only a few minutes are required to obtain complete
extraction. Bloor, in his experiments (1916, 1917, 1928) used ether
and alcohol in the ratio of three to one. Alcohol precipitates the
protein and mixes with the water in the blood, and ether extracts the
lipoid substances more completely. schoenheimer and Sperry (1934)
used a mixture of hot alcohol-acetone (1:1) for extraction of
cholesterol. Sobel and Mayer (1945) found that cold extraction with
alcohol and acetone (1:1) was as effective as hot extraction.

A method for dry extraction of cholesterol has first introduced
by myers and Wardell (1918). Blood was adsorbed on plaster of Paris
and dried before extraction with chloroform. .After adsorption, blood
was in a finely divided state on the plaster of Paris. This condition
facilitated extraction. The calcium salt also retained substances
which added to the color deve10pment by Bloor's procedure. Results
of this method agreed favorably with the method of Windaus (1909).

Lieboff (1924) used paper discs to adsorb blood and reported that

drying rt high temperatures impaired the test slightly and gave lower

-16-

results. Lieboff reported that drying the blood on filter paper was
not necessary. Ling (1928) reported the use of fat—free filter paper
for the adsorption of blood and Abrahamson (1938) substituted
absorbent cotton for filter paper and obtained similar results.

Cglorimetric Determination of Cholesterol: Development of Color.
Most methods using chloroform as the initial solvent used the
Liebermann-Burchard color reaction. The blue-green color produced
is me sured colorimetrically or photometrically. This reaction in
spite of its apparent simplicity, has proved difficult to control.

The intensity and the shade of the color have been shown to be
influenced by small differences in the concentrations of the reagents,
by the presence of traces of water or other impurities, time and
temperature (Hoffman 1941).

Schoenheimer and Sperry (1934) precipitated cholesterol with
digitonin before application of the Liebermann-Burchard reaction to
the digitonide dissolved in acetic acid. It was believed that isola-
tion of cholesterol as the digitonide avoided errors which were
introduced into other colorimetric methods by interfering chromo-
genic substances present in fatty extracts. Digitonin has an
absorption curve similar to that of cholesterol. However, Schoen-
heimer and Sperry found that digitonin did not have an absorption
peak in the range of 610 to 620 millimicrons where the color develop-
ment by cholesterol was at its maximum. .After precipitation of
cholesterol digitonide, Schoenheimer and Sperry dissolved the precipi—
tate with glacial acetic acid and then added separately acetic anhydride

and sulfuric acid. The mixture was allowed to stand for fifteen

_ 17 -

minutes at 25° C. and the intensity of the color was measured
spectrophotometrically at a wave length of 610-620 millimicrons.
Schoenheimer and Sperry were able to achieve by this method a degree
of accuracy formerly obtained in the sacrogravimetric method of
Windaus; more-over, this method required only 0.2 cubic centimeters
of blood where as hindaus employed 15 to 20 cubic centimeters of
blood.

Sperry and Brand (1943) preposed a number of improvements of the
earlier method of Schoenheimer aid Sperry. Sperry and brand suggested
mixing the acetic anhydride and sulfuric acid beforehand so that the
evolution of heat from the reaction did not affect the rate of color
development. The color reaches a maximum in about fifteen minutes and
then fades, so the temper ture and time of reading must be selected
for maximrm color intensity.

The most recent modific tion of the method of Schoenheimer and
Sperry was published by Foldes and gilson (1950). The reagents were
the same '3 in the earlier method with the exception that a solution
of 0.5 per cent alcoholic digitonin was used instead of a solution
of 0.4 per cent aqueous digitonin. The authors claimed that the use
of the alcoholic digitonin resulted in a fine, evenly dispersed
precipitate which was e sier to handle than the coarse, sticky .

precipitate obtained when aqueous digitonia was used.

Ether Nethods for_the Determination of Cholesterol

 

 

 

Most of the methods reported in the liter ture for the determin-
atio of cholesterol have applied the Liebermann-Burchard r action

for color formation; however, ether reactions also have been employed

- 13 _

although the methods have not been as widely read.

Okey (1930) developed a micro method for the estimation of
cholesterol in which the digitonide WTS oxidized with silver
chromata-sulfuric acid and the excess dichromate was titrated with
sodium thiosulfate. This method has given excellent results; however
it is more time consuming than the colorimetric proced re of
Schoenheimer and Sperry.

Bernoulli (1932) employed acetic chloride and anhydrous zinc
chloride for color development in the determination of cholesterol.
This reaction, called the Chugaen test, was considered to result in
the formation of a compound which had a more stable color than that
formed by the Liebermann-Purchard reaction. Bernoulli claimed that
one part of cholesterol in 80,000 parts of solution could be detected
by this procedu 8. This method has the disadvantage that fatty acid
esters of cholesterol and ergosterol and phytosterol will give the same
reaction as cholesterol with the test.

A color reaction which employed salicylaldehyde in a solution
of chlorofom, sulfuric acid and water was applied by Ohysama (1938)
for the determination of cholesterol. This reaction resulted in a

violet-colored substance which formed in the chloroform layer.

EXPEJEIEJI'AL PROCEDURE

BEER BETTAL PROCEDURE

A series of determinations were made of the total and free
cholesterol in blood serum of ten overweight college women who were
subjects on a weight-control metabolism study conducted by the Foods
and Nutrition Department at Michigan State College. Records of body

weights and food intakes were available for inspection.

Subjects.

The subjects were overweigit college women who ranged from 18
to 23 years of age. In September 1950, a preliminary study on weight
reduction was begun. Subjects A.L., I.P., and V.S. participated in
the study from Se_tember to December. After the Christmas vacation
these women returned to the diet table and were continued on a weight
reduction study with seven additional subjects wno began the experi-
mental study in January. The caloric intake for subjects A.L., I.P.,
and V.S. on their usual self-selected diet was determined initially
in September. In January, these subjects were placed immediately on
the weight reduction diet. However, the other subjects were permitted
to eat without restriction from a miXed diet of foods typical of this
area. All servings of food were weighed to determined the customary
food intake of the subjects. Following this, all subjects were given
the weight reduction diet. The calories of the diet were supplied
chiefly by protein and fat. Subjects V.S. and I.P. reached expected

weight before the period of observation was concluded. At this time,

_ 21 -

the diets of these subjects were adjusted to maintain the desired
weight. Since subjects 8.8. and A.L. did not show appreciable
weight loss on the reduction diet, their food intakes were reduced
by ten and twenty per cent of the basic reduction diet, respectively.

Determinations of serum cholesterol were made at intervals of
approximately two weeks from.3anuary, 1951 through march 1951. Thus
values for serum cholesterol were obtained for seven subjects (A.w.,
M.J., D.VA., E.H., A.S., 8.8., and F.G.) before weight reduction and
at two week intervals during the period of weight reduction. Serum
cholesterol values were not obtained for subjects A.L., V.S., and I.P.
before weight reduction. analysis of serum cholesterol were made
during weight maintenance for subjects V.S. and I.P.

A group of eleven women students of average body weight volun-
teered to become control subjects. A series of three blood samples
were taken at about one week intervals and serum cholesterol was
determined. k record of a typical day's food intake was obtained from
each subject and the caloric intake w«s calculated by the method of

Donelson and Leichsenring (1945).

Sampling Techniques.

 

Samples of blood were taken from the subjects in the f sting
state at approximately two weeks intervals. The blood was drawn from
the fingertip from a small incision made by a lancet or a Bard-Parker
blade. Approximately 1.0 cubic centimeter of blood was taken for each
sample. After the blood clotted, the samples were centrifuged to .
separate the blood cells from the serum. Serum was used for choles-

terol determinations in all cases.

During the period of study, cholesterol Values were determined
also on the serum of venous blood samples taken frem the subjects.
The samples were taken at least two hours after a me 1. The blood
was treated in the same manner as the fingertip sample and the serum
was held in frozen storage until analysis. A comparison was made of
the cholesterol of the venous blood serum and the cholesterol content
of the serum of fingertip (capillary) blood which was analyzed within

a week of the tire that the venous samples were taken.

Chemical Procedure.

 

The concentration of total and free cholesterol in blood serum
was determined by the method of Schoenheimer and Sperry (1954) with
modifications according to Sperry and Brand (1943), Sobel and Mayer
(1945) and Foldes and Wilson (1950). Three cubic centimeters of
alcohol-acetone (1:1) solution were added to 0.2 cubic centimeters of
serum in a five cubic centimeter volumetric flask. The solution was
brought to boiling and held at boiling temperature for 50 seconds. The
Solution was cooled and made to volume. The protein precipitate was
separated from the eXtract by filtration through fat-free filter paper.

An aliquot of one cubic centimeter of extract was used for the
determination of total cholesterol. One drop of ten per cent potassium
hydroxide was added to the extract to hydrolize the cholesterol esters.
The solution was allowed to stand for 30 minutes and then titrated with
ten per cent acetic acid in alcohol, using phenolphthalein as an
indicator.

An aliquot of two cubic centimeters of the extract was taken for

the determination of free cholesterol. Hith the exception of the

hydrolysis of cholesterol eSters, the determinations of total and
free ch lesterrl were car ied out in the same manner. Both total
and free cholesterol were precipit ted with a 50 per cent alcoholic
digitonin solution and allo ed to stand overnight. 'fhe precipitate
was separated by centrifuging 'nd then washed Lit: an acetone-ether
solution and with anhydrous ether. The ether w‘s removed by
evagorfition.

The digitoni: precigit te was dissolved in glacial acetic acid
and a solution of acetic rnhydride-sulfuric acid (20:1) was ad ed.
The solution wws allowed to stard for 27 minutes (no lcrger than 50
minutes) and the absorption of lifht was measured in the Beckman
spectrOphotometer at a revelength of 620 millimicrons. Determinations
of serum cholestercl were M‘de in duplicate; however in some instances
sufficient blood was not obtained to permit dupliccte analyses.

A series of solutions of 0.01 to 0.10 milligram of Lure
cholesterol per one cubic centimet-r of acetic acid rere prepared
and treated according to the above procedure. The absorption of light
was me aired and the values were plotted on semi-log papar against
concentration of cholesterol. The graph was used as a reference curve
for ,he calculation of the anornts of total and free cholesterol in
blood serum.

In a prelimina-y study of this method, reCOVeries of pure

cholesterol added to blood serum ranged from 94 to 98 3er cent.

I

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RESULTS AND DISCUSSION

The subjects were overweight women who were apparently healthy
in other respects. Routine physical examinations made by the student
health service were negative. However, subject F.G. had received one
grain of thyroid preparation since childhood and continued thyroid
therapy during the study. The desired weights of the subjects were
predicted from height and body stature on the basis of anthropometric
measurements.1 The subjects were moderately active college women as
judged by their ability to carry a full time college program
successfully. The age, height and predicted weights of the subjects
are given in Table 2.

The age of the subjects ranged from 18 to 25 years; the subjects
were from 8.0 to 51.2 per cent overweight at the beginning of the
study. Initial weights are given for subjects V.S., AsL., and I.P.
both at the beginning of the preliminary study and at the beginning of
the period of observation. Estimations of the average loss per week
for A.L., V.S., and I.P. were based on the period of preliminary
study and the experimental period; thus the total reduction period
was 24 weeks for subject A.L. and 19 weeks for subjects V.S. and I.P.
The average loss of weight for all subjects was 0.67 kilogram per
week. The rate of weight loss was satisfactory for all of the sub-

jects except for A.L. who averaged only 0.20 kilogram per week. Two

 

1 The anthropometric measurements and predictions of desired
Weights were made by Dr. Hargaret A. Ohlson.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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-27-

subjects, V.S. and I.P., achieved the desired weight loss which was
12.4 kilogram for V.S. and 8.9 kilogram for I.P. These subjects

were then given diets planned to maintain body veight. At the end
of the study, subjects A.S. and 8.8. also had lost the desired amount
of weight. The other subjects were from.14.4 to 35.9 per cent over-
weight at the end of the observational period.

The caloric intakes of the subjects on the unrestricted diet,
the weight reduction diet, and the weight maintenance diet was given
in Table 3. Since the women were apparently healthy individuals, it
was assumed that the excess weight resulted from ingestion of calories
greater than the output of energy. The caloric intake of the subjects
during the period in which they ate without restriction averaged 1983
and ranged from 1789 to 2280 calories per day. It is probable that
these intakes may be slightly below their previous customary intakes
since weight losses from one to four pounds occurred during the two
weeks on the self-selected diet. The caloric intakes for this period
were below the recommendations of the Food and Nutrition Board of
the National Research Council. This suggests that the recommended
intake for calories for women may be high.

After a period of two weeks on the self-selected diet, the food
intake of each subject was reduced to 1403 calories per day; these
calories were supplied chiefly by protein and fat. The basic
reduction diet supplied from 61.6 to 78.5 per cent of the caloric
intakes of the subjects during the self-selected period. The basic
reduction diet proved satisfactory for weight reduction for all of

the subjects except for A.L. and S.S. for whom it was desirable to

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- 29 -

increase the rate of weight reduction. After six weeks on the
reduction diet, A.L. and 8.8. were given diets which supplied twenty
and ten per cent less respectively of the foods of the basic reduction
diet. Only two subjects, V.S. and I.P. attained adequate loss of
weight so that they could be observed on diets planned to maintain
body weight. Since I.P. was of small body build, the caloric intake
was increased only to 1513 calories per day after weight reduction.
This increase apparently was not adequate since she continued to lose
weight. Subject V.S. was given 1709 calories per day for weight
maintenance. This intake appeared to maintain body weight for the

subject during the period of observation.

gggpmrpholesterol during weight Reduction.

 

The total cholesterol concentration in the blood serum of
individual overweight subjects at two week intervals is shown in
Table 4. The total serum cholesterol during the period on self-
selected diets ranged from 141.9 to 227.5 milligram per 100 cubic
1 centimeters and averaged 175.4 milligram per 100 cubic centimeters
serum. Since V.S., A.L., and I.P. were on weight reduction diets at
the beginning of the study, no values for the total serum cholesterol
on the self-selected diets were available for these subjects.

The average values for total serum cholesterol were 167.9, 172.4,
172.3, 188.2 and 186.9 milligrams per 100 cubic centimeters for each
two week interval, respectively. A graph showing the average changes
in the total serum cholesterol and body weight for the subjects for

each two week period is given in Figure 1. Changes in body weight and

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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in serum cholesterol for the indiVidual subjects are given in

Figure II. There pas an initial decrease of total serum cholesterol
for four of the subjects in the two we‘k period following the intro-
duction of the basic reductinn diet. Following this, there was a
tendency torard an increase in total serum cholesterol. This pattern
was char cteristic of four subjects, n. ., E.E-, S.S., F.G.; an
increase in total s-rtn cholesterol during the efperimental period
also occurred for subjects M.J., D.Va., A.S. and I.P. however, the
influence of time on total serum cholesterol values was determined
statistically by analysis of variance (Table 5), and it was found that
the changes in cholesterol for the successive periods vere not statis-
tically significant. Differences between individuals were highly
si*nificart.

The concentration of free cholesterol in the blood serum of the

3

experimental subjects is given in Table 6. These alues also are

shown graphically in Figure II and tie average of values for free
cholesterol in the serum for the entire grOUp is shown in Figure I.

The concentration of free cholesterol averaged 41.2 milligram per

100 cubic centimeters of serum during the period of the self-selected
diet and the values for the entire group ranged from 29.4 to 55.0
milligrams per 100 cubic centimeters for the group. Variations in
concentration of free cholesterol in the serum followed the same
pattern as for total cholesterol in hat a decrease in free cholesterol
values occurred in the two week period after the subjects wer given
the reduction diet. An analysis of variance showed that changes in the
concentretion of free cholesterol during the period on weight reduction

vere not statistically significant (Table 7).

()

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Total Cholesterol
Cholesterol Ester

Free Cholesterol
Weight

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O 2 ll E 8 1.0
Subject F, .G.

Figure II (continued)

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2 Cholesterol Ester
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Table 5

.A"AIYSIS CF V.1EI3"C3 CF ECIAL CHCLZgil: on L: o;.h-.
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Source of Variance r Ealues
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The ratios of free to total cholesterol for the individual

ile 8. The ratio of free to total cholesterol

”a

subjects are given in in
varied from 13.6 to V1.5 per cent dU‘ing the s lf—selected diet period
with en everoge of 23.9 per cent. The average ratios of free to total
cholesterol for each two week interval during w-ight reduction were

.1."

cent. The averages lor the

o
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greeter during the sucues ive geriods. The ratio of fr.e to total
cholestervl eried from 2.9 to 23.3 per cent duri 3 weight reduction
for subject H.J. and from 16.1 to 27.6 per cent for subject 3.8.

Values for the concentration of cholesterol ester in the blood
serum were obtained from the difference of the totsl and free choles-
terol velu s for e'ch period and are shown in Table 9. The cholesterol
ester of blood serum during the self-selected diet period ranged from
39.4 to 186.9 milliérs 8 per 100 cubic centimeters and averaged 134.1
milli rams per 1J0 cubic centimeters. The rverage cholesterol ester
concentrations for each tvo week interval during Weight reduction were
131.9, 132.6, 128.9, 145.6 and 143.3 milligr'ns per 100 cubic centi-
meters of serum respectively. The relative constancy of the values
for totul cholesterol for the individual subjects during weight
red ction me, be seen in Fig re II. Analysis of variance also showed

that these changes were not significant (Table 10).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Table 10

 

 

.KLLYSIS OF VZF.I FCE 'F mLZSTERCL ESTER CF SEEQJ
STBJFCTS O‘T A JEIGE RED"CTIC" DIET FOR TEE WEEK
De~rees Predicted F Values
Source of Vsrience F Vilues !
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- 42 -

The ratio of cholesterol ester to total cholesterol in the serum
rag calculated for each subject and the values are given in‘Table 11.
From 65.9 to 87.1 per cent of the total cholesterol was present in
the blood serum Us the c olest rel ester fJECthH. average values
for the entire grorp during the successive geriods were remarkably
constant. Variations for the individual subjecxs during weight
reduction mere from 5.4 per cent for subj ct E.h. to 11.5 per cent
for subject A.S.

The values pros nted above indicate that for this grou; of over-
weirht women there was lit le ch nae in the total serum cholesterol
or in the fractions of serum cholest r'l during weight reduction. The
decrease in total cholesterol and free cholesterol which occurred for
some of the subjects in the two week period after the subjects were
given a restricted diet suggested the possibility of a relationship
between the c loric intake and the concentration of serum.cholesterol.
The gradual increase which occurred following the initial decrease
may have represented an adjustment t- the restricted caloric intake
or the influence of a diet which supplied a relatively high ratio of
the calories from animal fats. Since the changes in serum cholesterol
for the entire group were not found to be statistically significant,
it would seem that the grrdual increase in serum cholesterol which Was
observed for four of the subjects may have represented an adjustment
to the restricted caloric intake. The values for serum cholesterol
obtained in this study tould seem to indicate that the excess body
weight for these subjects at the beginning of the study did not influ-

ence the cholesterol metabolism.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Serum cholesterol values also were ohtained for subjects V.S.
and I.P. on diets which were planned to maintain the desired weights
of these subjects. This portion of the study was limited to six
weeks for subject V.S. and four weeks for subject I.P. For both
subjects, the serum cholesterol values on maintenance diets were

within the ranges of values for these subjects during weight reduction.

Comparison of Serum Cholesterol Values for Overweight h men and “omen

of Average height.

 

Eleven healthy women of average body weight were selected to act
as controls for a comparison of serum cholesterol values of overweight
women and women of average weight. The age, height, weight, caloric
intake and cholesterol concentrations of free, total, and cholesterol
ester of the control subjects are given in Table 12. Ages of the
women in the control group varied from 19 to 28 years; this was
slightly above the range of ages for the subjects on the reduction
diet.

The caloric intakes of the women of average weight varied from
1499 to 2498 calories per day. The average intake whs 1859 calories
per day or about 140 calories less than the average intake of the
experimental subjects on self-selected diets.

The total serum cholesterol values for the control subjects range
from 115.0 to 192.5 milligrams per 100 cubic centimeters; the average
was 153.6 milligrams. Both the range of values and the average total
cholesterol concentration of the serum of the women of average weight

were below the values for the experimehtal subjects. However the

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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- 45 -

individual differences within both groups were great enough that
there was not a significant difference between the total serum
cholesterol values of the overweight women and the women of average
body weight (t-test, Fisher, 1938).

The combined cholesterol we the fraction of the totcl choles-
terol which was lower for the women of average weight than the

experimental subjects. The range of values for cholesterol ester

 

i'

for the control subjects was from 81.9 to 142.1 milligrams per 100

.‘tia- lg-

cubic centimeters of serum as compared with a range of 99.4 to 186.9
miiligrams of cholesterol ester per 100 cubic centimeters of serum. E
for the experimental subjects; the average concentration of cholesterol
ester was 110.2 milligrams per 100 cubic centimeters for the control
subjects and 134.1 milligrams per 100 cubic centimeters for the over-
weirht women. Again, the difference was not statistically significant
according to the "t" test (Probability = 0.10).
The values for free cholesterol for the eleven women of average
weight was similar to the values for the experimental subjects. The
range of values was 33.1 to 56.0 milligrams per 100 Cubic centimeters
with an aver ge of 43.5 milligrams for the control group and 29.1 to
55.0 milligrams with an average of 41.2 milligrams for the experimental
group. The mean ratio of free to total cholesterol concentrat on of
the women of average weight was 28.3 per cent; this was five per cent
less than the average ratio for the experiment 1 subjects.
The women of the two groups were similar both in the range of
ages and of heights. However the similarities were not close enough

to justify pairing the subjects on the basis of desired weight, height

Jan“... ...... nm—anwflallhthuv?‘ gr

- 47 _

and age. The tendency toward higher serum cholesterol values for the
overweight women when compared with women of average body weight
sugvests the desirability of extending this study to include a larger

number of subjects and by pairing subjects on the basis of body build.

gangggnison of Serum Cholesterol of Capillary and Venous Blood.

A comparis n of serum cholesterol for capillary and venous blood
is presented in Table 13. The average values for total cholesterol
and cholesterol ester concentration in venous blood serum we'e within
one milligram of the average values for capillary blood serum, but
he average of free cholester l in venous blood serum was three milli-
grams less than that of capillary blood. The average ratio of
cholesterol ester to tot 1 cholesterol in the venous blood was within
one yer cent of that for capillary blood.

On the basis of this study, it would seem that the cholesterol
content of capillary blood serum does not differ from that of the

serum of venous blood.

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S'LEE”..1-ZY AD CONCLUSIOIS

SULLm‘Y all) CUITCLUSIUI‘TS

The total and free cholesterol concentrations of blood serum
were set rzin d for ten overweight colle;e women at two week interVals
for a period of twelve re ks. The sutjects were given controlled
weiyht redrction diets after the'r customary caloric intakes were
determined.

The serum cholcsterol during the self-selected diet g-riod
~ed 175.4 milLigraxs per 140 cubic centimeters. There was an
initial decrease of total cholusterol for four of the subj'CZS after
the introduction of the basic red ct en diet; this was followed by a
trend toward an incresce in total seizn cholester l. The other
subjects also sho:ed 3 gr dual increase in tetal serum Cholesterol.
Korever in serum cholester“l during the successive Lcrfods were not
statistically significant.

The free cholvsterfil concentration of blood serum averaged 41.2
milligram per 100 cubic centimeters of se rm during the :eriod or the
se f-Felected diet. Variations in the cancentration of frre cholesterol
in the serum followed the same patte n as that for total cholesterol.
nn analysis of variance showed that changes in the concentrations of
free cholesterol during the period on weight reduction were not
statistically significant. hatios of free to total cholesterol
averaged 21.8 per -ent durirg the Meight reduction period.

The concentration of cholrsterol ester t 5 obtained fro; the
diffs ence of total and free cholesterol v:lues for each period. The

cholesterol ester averaged 154.1 milligram per 100 cubic centimeters.

Juli-Ll.
. 40-...

The average ratios of chol sterol ester to total cholesterol during
successive perio s were re» rhably co>stant. Individral variations
ranfed from 5.4 fer cent to 11.5 per cent.
rhe values attained indie to that for this group of overweight
women, there was little change in the total serum cholesterol and in
fractions of ser“m cholesterol durir; reight reduction.

The subjects lost an average of 0.67 kilograi of body weight
per reek. The rate of weight reduction was satisfactO'y in all of
the subjects but one. Two subjects achieved the desired weight loss
and were then given diets planned to naintain body Leights. The
values for blood serum cholesterol d ring the maintenance diet period
for these subjects were witrin the range of the values for thesc
subjects durin? weight reduction.

A comparison of serum chol sterol values for overweight women
and vonen vith average teigbt indic ted that the values for the average
total cholesterol concentration of ear m of the women of ave age body
weight were below the values of the experiment 1 subjects. Houever,
the difference was not statistically significant. Valves of free
cholesterol for the contr 1 group and the experimental group mere
similar. The mean ratio of free to total cholesterol concentration
was five per cent less for the experimental subjects than the control
group.

A comparison of ser m cholesterol for capillary and venous blood

shored that the cholesterol content of the blood serum of capillary

blood does not differ from that of the serrm of venous blood.

lEl‘EI‘LENCEflS C ITED

REFERENCES CITED

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1938 Extraction. Science 87: 420

Autentrith, W., A. Funk. Neber Koloremtrische Bestimmungs nethoden,
1913 Die Bestinmung de Gesantcholesterins in Blut und in
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Baylac, J., M; Sendrail. Cholesterinemia in Infants. J..A. h..A.
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Bernoulli, A. L. New method for the Colorimetric Determination of
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Bloor, W. R. The Determination of Cholesterol in Blood. J. Biol.
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Bloor, W. R. The Determination of Small Amounts of Lipids in blood
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Bloor, H. R. The oxidative Determination of :hospho-lipids (Lecithin
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Bloor, h. R., K. F. Balkan, D. M. Allen. Determination of ratty acid
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Blotner, H. Blood Fat Tolerance Tests in Malnutrition and Obesity.
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Bose, J. R., U. E. De, P. Mukerjee. A Preliminary Study of the
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Boyd, E. H. The Lipemia of Eregnancy. J. Clin. Invest. 13: 347
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Boyd, E. M. Diurnal Variations in Plasma Lipids. J. Biol. Chem.
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Bugnard, M. Bull. Soc. Chim. Biol. 12: 97 (Arch. Path. 35: 438, 1943}
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Corwin, W. C. Experimental Rypercholesteremia in DOgs. arch. fath.
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Donel on, E. G., J. K. Leichsenring. Food Composition Table for Short
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Feiser, L. F., M. F. Feiser. Natural iroducts Related to hhenanthrene,
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Fisher, g._1, st~tisticali I fit ods for Tesearch workers, Oliver and
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Gardner, J. R., P. B. anden. Liv. on the Cholesterol Coritent of the
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1915)
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Groen, J., C. E. Kaxainga, J. H. Reise:1, A. F. Willebrands. Het
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Hermann, R., J. Neumann. Biochem. Ztschr. 43: 47 (Arch. fath. 35: 438,
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Hoffman, h. S., Photoelectric Study of LiebP1mann-Burchard Reaction of
1940 its Sig ific cance in Determinations of Cholesteiol. Proc.
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Hoffman, W. S., rhotelometric Clinical Chenistr h_111a1 Morrovv and
l 94:]. C O O ’ IV 81-1? Yorl‘: .

Kelsey, F. E., H. E. Longenecker. Distribution and Characterization
1941 of Elasma Fatty Acids. J. Biol. Chem. 139: 727

Keys, A., 0. Hickelsen, E. Miller, C. Chapman. The Relation in Man
1950 Between Cholest rol Levels in the Diet and in the Blood.
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Keys, A., 0. Mickelsen, E. Miller, B. Hayes, H. Todd. The Concentration
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Kingsely, G. R., R. R. Schaffert. Determination of Free and Total
1949 Cholesterol by Direct Chloroform Extraction. J. Biol.
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Kirk, E., I. H. Page, R. H. Lewis, N. R. Thompson, D. D. Van Slyke.
1935 Plasma Lipids of Normal Men at Different Ages. J. Biol.
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Kornerup, V. Serum Cholesterol, Total Lipids and Phosyholipids in
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Kornerup,'V. Concentrations of Cholesterol, Total Fat and PhOSFholipid
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Leiboff, S. L. A Simplified Method for Cholesterol Determinations in
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Levine, R., S. Soskin. Acid-Base Balance and the Distribution of Eat in
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Ling, S. 1h The Determination of Cholesterol in Small Amounts of Blood.
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Luden, G. Studies on Cholesterol. IV. Experiments Concerning the
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Man, E. B., E. F. Gildea. Serum Lipoids in Malnutrition. J. Clin.
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Man, E. B., E. F. Gildea. variations in Lipemia of Normal Subjects.
1937 J. Biol. Chem. 119: 769

Man, E. B., J. P. Peters. Gravimetric Determinations of Serum
1933 Cholesterol adapted to the Man and Gildea latty Acid Method,
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Mann, G. V. Research in Cholsterol Metabolism.Re1ated to Cardiovascular
1951 Disease. J. Am. Diet. hssn., 27: 209

Mata, M. Simplified Determination of Blood Lipids, Rev. Farm Cuba
1948 26: 29 (Chen. Abst. 43: 3872a, 1949)

Muhlbock, 0., C. Kaufmann. Ztschr. f.d. ges. Exper. Med. 102: 461
1938 (Arch. Path. 35: 438, 1943)

Myers, V. G., E. L. Wardell. The Colorimetric Estimation of Cholesterol
1918 in Blood vith a Note on the Estimation of Caprosterol in
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‘—

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