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A study of methods of cholesterol analysis

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A study of methods of cholesterol analysis

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Content A STUDY OF METHODS
OF CHOLESTEROL ANALYSIS
by
KATHERINE WONG CHENG
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In. Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Nutrition)
January 1959
U N IV E R SITY O F S O U T H E R N C A L IF O R N IA
G R A D U A T E S C H O O L .
U N IV E R S IT Y PA R K
L O S A N G E L E S 7
Bio 'St c 5>S
This thesis, written by
Katherine Wong Cheng
under the guidance of h-$X...Faculty Committee,
and approved by all its members, has been pre
sented to and accepted by the Faculty of the
Graduate School, in partial fulfillment of the
requirements for the degree of
.iJsr..
Dean
Date..
Faculty Committee
Chairman
ACKNO WLE DGEM EN T S
I would like to express my sincere gratitude to
Dr. John W. Me hi for his invaluable guidance, counsel, and
sponsorship throughout the course of this study.
I would also like to express my appreciation to the
other m em bers of my committee, Dr s. Roselyn B. Alfin-Slater
and Walter Marx, for their invaluable criticism .
My special thanks go to Dr. Hallie F. Bundy for her
generous aid in the preparation of this thesis.
It is to my under standing husband, Luke, that this
thesis is dedicated.
TABLE. OF CONTENTS
CHAPTER
PAGE.
I. h is t o r ic a l in t r o d u c t io n . . . . . . . 1
II. STATEMENT OF THE PROBLEM AND
PLAN OF ATTACK . . . . . . . . 7
III. MATERIALS AND METHODS . . . . . 8
IV. EXPERIMENTAL RESULTS
Prelim inary Results . . . . . «
0 0
Experiments on the F erric Chloride and
Pearson .Methods of Cholesterol
D e te rm in a tio n ..........................................
. . 19
The Recovery of Cholesterol and
Cholesteryl Palmitate from the
Silicic Acid. Column . . . . . . 29
The Recovery of Cholesterol from
-an Acetone:95% Ethanol Extract
of Serum after { 1 ) Precipitation
with Digitonin and ( 2 ) Silicic
Acid Column Chromatography . . 35
V. THE FORMULAE FOR CALCULATION . . 3?
V
CHAPTER. PAGE
VI. DISCUSSION . . ................................................ . . . 40
VII. . SUMMARY . . . . . . . . . . . . . . 52
B IB L IO G R A P H Y .................................................................. 5 4
LIST OF TABLES
TABLE
PAGE
I. Total Cholesterol of a 1:1 Acetone:9 5%
Ethanol Extract of Pooled Serum
before and. after Digitonin Precipitation. . 2 2
II. The Effect of Time on the Color
Development in the F erric Chloride
Method for Cholesterol Determination
. 2 6
III. The Recovery of Added Cholesterol from
Serum by Extraction, with Acetone:95%
Ethanol 27
IV.
The Color Recovery from Recrystallized
Cholesterol after ( 1 ) Precipitation
with Digitonin and ( 2 ) Hydrolysis
with Potassium Hydroxide followed by
Precipitation with Digitonin 30
Va. Data for the Standard Curve using the
F erric Chloride Method 32
Vb. Data for the Standard Curve using the
Pearson Method 33
VI. The Recovery of Cholesterol and Cholester'yl
Palmitate from the Silicic Acid Column .
. 34
V l l
TA BLE PAG E
VII. Thee Recovery of Cholesterol from an
Acetone: 95% Ethanol Extract of
Serum after ( 1) Precipitation with
Digitonin and ( 2 ) Silicic Acid
Column Chromatography .................................................36
VIII. A Comparison of the Recovery of
Cholesterol and Cholesteryl
Palmitate from ..Silicic Acid I
and .Silicic Acid II (Statistical
Analysis of Data from Table VI ) . . . . 42
IX. A Comparison of the Recovery of
Cholesterol from .Serum .Extract '
A- Silicic Acid. I and Serum
.Extract B- Silicic Acid II
(Statistical Analysis of Data from
Table VII ) . ........................................................... 45
X. A Comparison of the Recovery of Total
Cholesterol after Digitonin Precipitation
and after Silicic Acid Column Chromatography
of an Acetone:95% Ethanol Extract of
Serum (Statistical Analysis of Data
_____________from Table VII ) 47
v iii
TA B L E PAGE
XI. A Summary of Results, after Silicic
Acid Column Chromatography, from
some Other Publications and The
Present S t u d y .............................................................................4 9
■ r -
LIST O F FIGURES
FIGURE PAGE
1. The silicic acid column ......................................... . 17
2. The standard curve, for the F erric
Chloride method .......................................... 2 0
3.t The standard curve for the Pearson
' m e t h o d .......................................... ....... 21
4. The standard curve for the F erric
Chloride method using recrystallized
cholesterol ......................................... 23
5. The standard curve for the Pearson
method, using re crystallized
cholesterol ..................................................................................24
6 . A comparison of the color development
of cholesterol and of cholesteryl
palmitate using the F erric Chloride
and Pearson methods ......................................................31
C H A PTER I
HISTORICAL INTRODUCTION
Methods of cholesterol analysis have been extensively
reviewed ( 25, 26, 27, 31 ), and from the vast amount of
information available on this subject only those aspects direct
ly pertinent to the present study will be discussed. These
are:
Extraction of free and esterified cholesterol from the
o r i gina.1 sampl e .
Separation and/or isolation of free and esterified
cholesterol.
Analysis of free and esterified cholesterol.
Extraction
In the wet extraction of free and esterified
cholesterol, the tissue is extracted with a solvent, usually
a lower aliphatic alcohol, which is generally combined, with
another solvent. The use of a 1:1 acetone:ethanol solvent
system for extraction ,wa.s reported in 1943 by . Schoenheimer
o
and Sperry ( 1 ). These authors obtained the extract by
using 5 ml. of 1:1 acetone: ethanol for 0.2 ml. of serum and
2
reported that good results were obtained with extracts made
up in 10 ml. of 1:1 acetone:ethanol from 0.5 ml. of serum,
and that the cholesterol added to 1:1 acetone:ethanol solution
used for extracting sera was recovered within lim its of
accuracy of the method ( 97.2 to 99.3% ). In 19 50 Sperry'
and Webb ( 2 ) published a revision of the Schoenheimer —
Sperry method for the determination of cholesterol but the
extraction procedure was maintained without modification.
In 1955. Sperry and Brand ( 32 ) published a procedure Using a
2:1 chloroform:methanol extraction for the direct determination
of the unmodified lipids of the blood serum, and the recovery
of cholesterol in the 1:1 acetone:ethanol extraction was used
as a control. The results from both extractions were in
good agreement.
Separation and/or Isolation
In I9 O 9 Windaus ( 3 ) showed that cholesterol and
digitonin form an insoluble precipitate and that the reaction
is specific for 3B~-hydroxy sterols. In 1910 the same author
( 4 ) reported on the cholesterol content of kidney tissue
using digitonin precipitation and also showed tha.t the
cholesterol digitonide is a 1:1 addition compound. About
3
a year later Grigant ( 5 ) published ah adaptation of Windaus'
method for the determination of serum cholesterol. The
validity of Windaus' work was confirmed by Thaysen ( 6 )
in 1914. During the year 1934 Schoenheimer and Sperry ( 1 )
published a micromethod for the determination of free and.
esterified cholesterol using digitonin precipitation. This,
micromethod was studied by Sobel and Webb ( 7 ) who
suggested the use of an alcoholic solution of digitonin, in
place of the aqueous solution, to eliminate the technical
difficulties encountered in the washing of the digitonide
precipitate. This micromethod was revised by Sperry and
Webb ( 2 ) in 1950 adopting the above mentioned modification
of Sobel and Mayer. The Sperry and Webb procedure was
used in the present study.
The use of a silicic acid column for the fraction
ation of lipids was introduced by Trappe ( 8 ) in 1940.
With such a column Borgstrom { 9 ) was able to separate
free cholesterol from esterified cholesterol as well as other
lipids by elution with, various solvents. In 1953 Fillerup
and Mead ( 10) published their findings on the chromatographic
separation of plasma lipids with a silicic acid column using
ether :petr oleum ether solvent systems. In 1955 Malmberg (11.)
4
investigated the effect of the particle size of the silicic acid
upon fractionation, and found that the performance of silicic
acid could be improved by decreasing the particle size.
The special preparation of silicic acid before use has been
indicated in all of the recent papers. Wycoff and Parsons
( 12 ) used the silicic acid column with chloroform:petroleum
ether and ethyl acetateipetroleum ether solvent systems for
the m icro-assay of esterified cholesterol and free cholesterol
respectively from serum or plasma. Recently Barron and
Hanahan ( 13) published their observations on the silicic acid
chromatography of neutral lipids from rat liver, beef liver
and yeast using ether:h.exance solvent systems; and Hirsch
and Ahrens ( 14 ) published their results on the separation of
complex lipid mixtures with silicic acid.
In silicic acid chromatography the sample to be
fractionated is placed on a column of absorbent and the
fractionation is carried out by eluting with solvents of
increasing polarity.
Analysis
The classical Liebermann and Bur chard reaction
( 15, 16 ) involves the treatment of a solution of cholesterol
5
in acetic anhydride with concentrated sulfuric acid, which
results in a color display which goes from red to violet to
green. Grigant ( 17 ) introduced this procedure for the
quantitative estimation of serum cholesterol in 1 9 1 0 using
a ehloroform:acetic anhydride: sulfuric acid system. Reinhold
( 18 ) in 1935 reported a difference in the rate of formation
of color by free cholesterol and esterified cholesterol;
Sperry and Brand ( 19 ) in 1943 studied the effects of light,
time and temperature upon the color development. As a
result of these, and other studies defining conditions for
reproducible color development, this reaction has been accept
ed as the standard in many laboratories. In 1953 Pearson
et al. ( 2 0 ) ' introduced a rapid method involving the addition
of p-toluenesulfonic acid, acetic anhydride and sulfuric acid
to serum. This direct addition of reagents to serum for
color development was found by Best et al. ( 23) to give
higher results than the Schoenhei.mer and Sperry method.
In 1901 Hammarsten and Mandel ( 21 ) stated that
cholesterol, with the addition of two or three volumes of
concentrated sulfuric or hydrochloric acid and one volume of
a "medium solution" of ferric chloride would, after careful
evaporation, first yield a reddish violet residue and then a
6
bluish violet residue. This reaction was subsequently employee
for the quantitative determination of cholesterol, but had not
been employed in recent years until it was used in 1953 by
Zlatkis et al. ( 22 ) for a rapid method for the direct deter
mination of cholesterol in blood serum and was also found by
Best et al. ( 23 ) to give higher results than the Schoenheimer
and Sperry method. The presence of interfering substances in
serum was also recognized by Zak et al. { 24 ), who recom
mended the application of the method to extracts of serum in
the same year.
Cholesterol reacts with a variety of strong acids to
yield colored products. The mechanism of formation of these
colored products is complex and not well defined, though it
has been the subject of some speculation which is discussed
in recent books by Kriche vsky ( 25 ) and by Cook ( 26 ).
Despite the fact that the reactions are only imperfectly under
stood, and that they are not specific for cholesterol, it has
been possible to establish conditions which make some of them
suitable for quantitative determinations. The Pearson m odifi
cation of the Liebermann-Bur chard reaction and the Zak
modification of the F erric Chloride reaction are only two such
examples, with which we will be .concerned in the present
study.___________________________________________________________________________
C H APTER II
STATEMENT OF THE PROBLEM AND PLAN OF ATTACK
The general purpose of this project has been to
study some: possible improvements in the methods of
cholesterol analysis, and particularly the separation of
free and esterified cholesterol from small samples of blood
serum by silicic acid column chromatography.
Before attacking the problem of silicic acid column
chromatography, a method for the color development of
cholesterol and a procedure for checking the recovery of
I
free and esterified cholesterol from the silicic acid column
had to be established.
CH APTER III
MATERIALS AND METHODS
Cholesterol
C. P. cholesterol was obtained from Mallinckrodt
Chemical W.orks and recrystallized from hot methanol.
Three recrystallizations were necessary to obtain a sharp
melting point.
The cholesterol standard for the F erric Chloride
method was prepared by dissolving 10 mg. of cholesterol
in 1 0 0 ml. of acetic acid.
The cholesterol standard for the Pearson method
was prepared by dissolving 50 mg. of cholesterol in 100 ml.
of acetic acid.
Acetic Acid
Baker and Adamson's Special Reagent Grade acetic
acid was used.
F erric Chloride Color Reagent
The stock solution was prepared by dissolving 1 g.
9
of C. P. ferric chloride lump in 1G ml. of acetic acid.
* i " '
The working solution was prepared by diluting 1 ml.
of stock solution in 1 0 0 ml. of concentrated sulfuric acid.
p - Toluene sulfonic Acid Reagent for the Pearson. Method.
p-Toluene sulfonic acid monohydrate was obtained from
Eastman Organic Chemical, Rochester, New York.
The reagent was prepared by dissolving 12 g. of
p-toluenesulfonic acid monohydrate in 1 0 0 ml. of acetic acid.
Potassium Hydroxide Solution
The solution was prepared by dissolving 10 g. of
reagent grade potassium hydroxide in 2 0 ml. of water.
Digitonin Solution
Digitonin was obtained from H. M. Chemical Co. Ltd. ,
Santa. Monica, California.
The solution was prepared by dissolving 500 mg. of
digitonin in 55m l. of 9 5% ethanol and 45 ml. of water.
Ethyl Ether
Reagent grade ethyl ether was used and stored with •
10
the addition. of a piece of metallic sodium.
n-Heptane
Pure grade with 99 mol percent minimum of
n-heptane was obtained from Phillips Petroleum Co.,
Bartlesville, Oklahoma.
It was further purified by distilling twice and
running once through a column of silicic acid.
Silicic Acid
Silicic acid, obtained from Mallinckrodt Chemical
Works, was prepared for use by:
I.) Grinding in. a .mortar by hand to a fine powder.
2.) Washing twice with twice its volume of
methanol.
M
Washing twice with, twice its volume of ethyl
ether.
4.)
Drying in air under the hood.
5.) Washing twice with twice its volume of
n-heptane.
The silicic acid thus prepared was stored in
n- heptane.
11
Cholesteryl Palmitate
A sample of cholesteryl palmitate was obtained from
Dr. Jam es Mead. This sample was found to be free of
cholesterol by digitonin precipitation.
-The Extraction of Serum
1.) An 0.5 ml. volume of serum was introduced
into an .Erlenmeyer flask containing 10 ml. of 1:1 acetone:
95% ethanol.
2.) The solution was heated to boiling in a water
bath.
3.) The solution was then cooled and filtered
through Whatman No. 1 filter paper.
4.) The precipitate was washed on the filter paper
with 1 :1 acetone:9 5% ethanol and. the washings were used to
bring the final volume of the filtrate to 50 ml. This filtrate
will be referred to as the serum extract.
The Precipitation of Total and Free Cholesterol by Digitonin
For the determination of cholesterol by the F erric
Chloride methods 1 m l. of serum extract, 2 drops of
potassium hydroxide solution for saponification, and . 1 ml. of
12
digitonin solution, were used.
For the determination of cholesterol by the Pearson
method, 5 m l. of serum extract concentrated to a volume of
1 ml. under a stream of nitrogen, 4 drops of potassium
hydroxide solution, and 2 ml. of digitonin solution were used.
Precipitation of total cholesterol.
1.) The potassium hydroxide solution was introduced
into centrifuge tubes containing the serum extract and the
tubes were incubated for 30 minutes at 60°.
2.) The resulting solution was neutralized with 10%
acetic acid to a phenolphthalein end point and an additional
drop was added to each tube.
3.) The digitonin solution was then added, and the
contents of each tube were mixed well by shaking. The
tubes were allowed to stand at room temperature oyer-night.
4.) The following day the tubes were again mixed,
then centrifuged for 15 minutes at 2800, RPM, and the
supernatants discarded.
5.) The precipitates were washed with. 2 ml. of
1:2 acetone:ether and then with 2 ml. of ether for 5 minutes,
centrifuging each time at 2800 RPM.
6 .) The tubes containing the cholesterol digitonide
13
were evaporated to dryness under a stream of nitrogen and
were then ready for color development.
Precipitation of free cholesterol. Steps 1.) and
2.) above were omitted. The digitonin solution was added
directly to the serum extract and steps 3.) through 6 .)
were carried out.
Silicic Acid Column Chromatography
Preparation of the column.
1.) A small amount of glass wool and filter paper
pulp were placed at the bottom of the column to prevent the
silicic acid from coming through.
2.) The silicic acid was poured into the column as
a slurry in n-heptane and a. small amount of nitrogen
pressure was used to insure the firm ness and uniformity of
the column. The final height of the column was about 8 cm.
( about 2. 5 g. of silicic acid ).
3.) A small amount of filter paper pulp was placed
on the top of the column of silicic acid to prevent it from
being disturbed.
4.) The column was then washed with about 5 ml.
of n-heptane and it was , ready for use.
Operation of the column. The column was operated
. ; , -------------------------------------
14
under a very small amount of nitrogen pressure and it was
never permitted to run dry. The flow rate was about 10
drops per minute. F resh silicic acid was used for each run.
1.) The sample, in n-heptane,' ( about 0,2 ml. in
volume ) was introduced onto the column and washed down
into the column with about twice the sample volume of
n-heptane.
2.) 14 ml. of n-heptane was allowed to run through
the column, the eluent was collected. This fraction contained
esterified cholesterol.
3.) 15 ml. of 30% ethyl ether in n-heptane was
allowed to run through the column, the eluent was collected.
This fraction contained free cholesterol.
The s ilic ic acid, colu m n is shown in F ig u r e 1.
F erric Chloride Method for the Determination of Cholesterol
The stan d ard and blank w e r e run e a ch tim e and all
e v a p o ra tio n s w e r e c a r r ie d to d r y n e s s under a s tr e a m of
n itro g en .
1.) 3.2 ml. of acetic acid were added to 1m l. of
the cholesterol standard solution. For the serum extract
samples, the cholesterol precipitated by digitonin, and the
15
eluents from the silicic acid column, 4 .2 ml, of acetic acid
were added to the residues from evaporation.
2.) To each tube 2. 8 ml. of the ferric chloride
color reagent were added, by layering, and the tubes were
mixed well by shaking.
3.) After 25 to 30 minutes the absorbance at 560
mji was read in the Beckman Model B Spectrophotometer.
Pearson Method for the Determination of Cholesterol
The standard and blank were run each time, and all
evaporations were carried to dryness under a stream of
nitrogen.
1.) For the standards, 1 ml. of the cholesterol
standard solution was added to each tube.
For the serum extract samples, 1 ml. of
acetic acid was added to the residue from the evaporation, of
5 ml. of serum extract.
For the cholesterol precipitated by digitonin,
lm l. of acetic acid was added to the residue from step 6 .),
page 1 1 .
For the eluents from the silicic acid column,
1 ml. of acetic acid was added to the residue from the
16
evaporation of each fraction.
2.) This was followed by the addition of 1 .2 m l. of
p-toluene sulfonic acid to each: tube.
3.) The tubes were placed in a .20° w ater bath,
and 4 ml. of acetic anhydride were added.
4.) After 15 minutes at 20° 0.5 ml. of concen
trated sulfuric acid were added and the contents of each
tube were mixed well by shaking.
5.) After an additional 15 minutes at. 2 0 ° the
absorbance at 615 znp was read in the Beckman Model B
Spe c t r op h o t ome te r .
P ressu re head
for reservoir
17
P ressure head
for column
Column
m r
Figure 1 The silicic
Reservoir
acid column
CHAPTER IV
EXPERIMENTAL RESULTS
Prelim inary Results
A prelim inary study of the F erric Chloride and
Pearson methods for the determination of cholesterol was made
in Dr. J. W. Mehl's laboratory by Takao Ushiyama before the
present work wa,s undertaken. Some of the pertinent findings
of Dr. Ushiyama were:
1.) The use of A. C. S. Special Reagent Grade
acetic acid for the F erric Chloride method, in place of
Reagent Grade acetic acid, improved the linearity of the
standard curve.
2.) Maintaining a tem perature of .20° during the
color development in the Pearson method improved the stabili
ty of the color developed.,
*, 3.) The concentration of p-toluenesulfonic acid re a
gent in the Pearson method was found not to be critical over
a. range of 2 to 1 2 %.
After undertaking the present study certain, additional
information, was obtained:
19
1.) The standard curve for the F erric Chloride
method using the C. P. cholesterol, shown in Figure 2, was
not linear.
2.) The standard curve for the Pearson method
using the C. P. cholesterol, shown in Figure 3, was
satisfactory.
3.) The total cholesterol of 1:1 acetone:95% ethanol
extract of pooled serum before and after digitonin precipitation
determined ' by the F erric Chloride method is in good agree
ment with the results obtained using the P earson .method, as
shown in Table I.
Experiments on the F erric Chloride and Pearson Methods of
Cholesterol Determenation
Standard curves. Cholesterol was recrystallized as
indicated in the Section on m aterials. The wavelength of
choice was confirmed by absorption spectra to be 560 mji for
the F erric Chloride method and 615 mp for the Pearson
method. The standard curves for the F erric Chloride and
Pearson methods using the recrystallized cholesterol are
shown in Figures 4 and 5.
2.0
1 . 00
0 . 80
o
'- D
IT)
0 . 60
0. 40
0 . 20
0. 14 0 . 06 0 . 10 0 . 0,2
mg. of cholesterol
F ig u re 2 The standard curve for the
F erric Chloride method
21
ID
*H
sO
-M
« 5
< a
u
fl
o j
X
u
o
CD
<
0 . 80
0 . 60
0 . 40
0 . 20
0 . 6 0 . 8 0. 4 0 . 2
mg. of cholesterol
F ig u re 3 The standard curve for the Pearson
method
TABLE I
TOTAL. CHOLESTEROL OF A 1:1 ACE,TONE:9 5%. ETHANOL EXTRACT OF
POOLED SERUM BEFORE AND AFTER DIGITONIN PRECIPITATION
Method Total Cholesterol, mg'. %
Serum Extract. After Digitonin Precipitation
Average Range Average Range
Ferric Chloride 218 217 - 228 197 183 - 217
Pearson. 218 .215 - 226 198 196 - 202
tv)
tv)
23
1 . 00
0 . 80
0 . 60
0 . 40
0 . 20
0 . 04 0 . 08 0 . 16 0 . 12
mg. of cholesterol
Figure 4 The standard curve for the F erric
Chloride method using
recrystallized cholesterol
Absorbance at 615 mp.
1. 60
1. 20
0 . 80
0. 40
0.25 0. 75 1. 25
mg. of cholesterol
F ig u re 5 The standard curve for the Pearson
method using recrystallized
cholesterol
25
The effect of time on the color development in the
F erric Chloride method for cholesterol determination. The
intensity of the color developed in the F erric Chloride method
was observed to decrease with time. A study of the rate of
color development was made and the results are shown in
Table II. From the data in Table II, the best time for
reading the color intensity is 25 to 30 minutes after the addi
tion of the ferric chloride color reagent.
The recovery of added cholesterol from serum by •
extraction with 1:1 acetone:95% ethanol. Three extracts of
serum were prepared. Extract 1 contained 1 ml. of serum.
Extract 2 contained 1 m l. of serum plus 1 mg. of cholesterol,
and Extract 3 contained 1 ml. of serum plus 2 mg. of choles
terol. If the extraction of added cholesterol is complete, the
difference between Extract 2 and Extract 1 should be half the
difference between Extract 3 and Extract 1. The results are
shown in Table III and it is apparent that the recovery of
added cholesterol from serum by extraction with 1:1 acetone:
95% ethanol is quantitative.
26
TA BLE II
THE EFFECT OF TIME ON THE COLOR DEVELOPMENT
IN THE FERRIC CHLORIDE METHOD FOR
CHOLESTEROL DETERMINATION
Time —^ . Absorbance Absorbance Absorbance
in - with 0. G 1 mg. (with 0. 05 mg. • ■ with 0.10 mg.
minutes cholesterol cholesterol' cholesterol
8 0. 072 0. 338 0. 677
10 0 . 0 6 8 ,0. 338 0. 674
15 0 . 0 6 8 - 0.333 0. 667
2 0 0 . 0 6 6 0. 328 0. 664
25 0 . 064 0. 322 0. 655
30 0. 064 ,0. 318 0. 653
35 ; 0 . 0 64 0. 318 0. 648
40 0 . 0 6 0 0. 318 0. 648
45 0. 058 0. 316 0 . 646
50 0. 056 0. 311 0 . 640
55 .0. 055 0. 303 0 . 629
60 0. 055 0. 301 0. 625
1/ 0 time : The addition of ferric chloride color reagent
into the tube
TABLE III
THE RECOVERY OF ADDED CHOLESTEROL FROM SERUM
BY EXTRACTION WITH ACETONE:95% ETHANOL.
Pearson method
Sample Choles- Choles--- Recovery
terol terol of added
added, found, choles-
mg. mg. terol,
mg.
Ferric Chloride method
Sample Choles- Choles- Recovery
terol ■ terol of added
added, found, choleS-
mg. • mg. terol,
mg.
5 ml. of . 0. 13 —
Extract 1—
5 ml. of 0.10 0.23 0. 10
Extract 2 —
5 ml. of 0.20 0. 33 0.20'
Extract 3 —
1 ml. of --------- 0.030
Extract 1
1ml. of .0.020 0.050 0.020
Extract 2 .
1ml. of 0.040 0.071 0.04,1
Extract 3
1 / 1 ml. of serum in. 50 ml. of 1:1 acetone:95% ethanol
2./ 1 ml. of serum plus 1 mg. of cholesterol in 50 ml. of 1:1 acetone:95%. ethanol
3/ 1 ml. of serum plus 2 mg. of cholesterol in 50 ml. of 1:1 acetone:95% ethanol
-4
28
The color recovery from recrystallized cholesterol
after ( 1 ) precipitation, with digitonin and { 2 ) hydrolysis
with potassium hydroxide followed by precipitation with
digitonin. The results are shown in Table IV. They indi
cate that there is no loss of cholesterol during precipitation
with .digitonin or during hydrolysis with potassium hydroxide.
A comparison of the color development of cholesterol
and cholesteryl palmitate. These results are shown in
Figure 6 . They indicate that there is no difference in the
*
color development for cholesterol and cholesteryl palmitate
using the F erric Chloride method; however, there is a
slight difference using the Pearson, method.
The p r e c is io n and S e n sitiv ity of the two m e th o d s.
The data from the two standard curves, shown in Table V a
and V b were used for the calculation. The precision is
expressed as the standard deviation of duplicates. The sensi
tivity is expressed as slope of the standard curve divided by
the standard deviation. The formulae are given in Chapter V .
For the Pearson method:
-6 i/
P r e c is io n .............................................. 13.8 x 10 A —
-6
S en sitiv ity .............................................. 0. 0456 x .10 A
29
For the F erric Chloride method:
-6
P r e c is io n ................................................ 17.2 x 10 A
-6
S en sitiv ity ................................................ 0.382. x 10 A
JL/ A = Absorbance units
The Recovery of_ Cholesterol and Cholesteryl Palmitate from
the Sillicic Acid Column
Two b a tch es of s ilic ic a c id w e r e p r e p a r ed s e p a r a te ly
according to the procedure on page 10. The first batch was
designated as Silicic Acid I and the second batch as Silicic
.Acid IX . A total of 0'. 1 mg. of cholesterol in .xx-heptane, was
applied to each of two columns and 0. 15 mg. of cholesteryl
palmitate in n-heptane was applied to each of two columns.
The r e c o v e r y of c h o le s te r o l and c h o le s t e r y l p a lm ita te fr o m
Silicic Acid I and Silicic Acid II is shown in Table VI.
Cholesterol and cholesteryl palmitate were determined by the
F erric Chloride method. To calculate the per cent recovery,
«
the absorbance, after color development, of each eluent was
divided by the absorbance after color development of the
corresponding un- chromatographed sample.
TA BLE IV
THE COLOR. RECOVERY FROM RECRYSTALLIZED CHOLESTEROL AFTER ( 1 )
PRECIPITATION WITH DIGITONIN AND ( 2 ) HYDROLYSIS WITH POTASSIUM
HYDROXIDE FOLLOWED BY PRECIPITATION WITH DIGITONIN
Pear son, method
• < %
Amount of Absorbance
cholesterol at 615 mu
Average
k Ferric
Amount of
cholesterol
Chloride method
I
Absorbance Average
at 560 mu
0 . 5 mg . 0. 588 0. 574 0 . 1 mg. 0 . 646 0 . 65.1
0.560 0. 655
0. 5 mg. 0.586 0 . 579 0 . 1 mg. .0.652. 0. 638
digitonin 0. 572 digitonin 0. 623
precipitation pr ecipitation
0. 5 mg. 0. 572 0. 578 0 . 1 mg. 0.623 0. 637
hydrolysis .0.584 hydrolysis 0. 650
and digitonin. and digitonin
precipitation precipitation
O J
o
31
-Ghol-e-s-terol
1. 00 -i
Cholesteryl
palmitate
F erric Chloride
method
0 . 80 -
‘Pearson method
0 . 60 -
0 . 40 -
0 . 20 -
mg. of cholesterol
Figure 6 A . comparison of the color development
of cholesterol and cholesteryl palmitate
using the F erric Chloride ( 560 mji )
and Pearson ( 615 mp. ) methods
TABLE Va
DATA FOR THE STANDARD
THE FERRIC CHLORIDE
32
CURVE USING
METHOD
mg. of Absorbance Average
cholesterol at 560 m/i .Absorbance
Blank 0 . 0 0 0
, 0 . 004
0 . 0 0 2
0 . 01 0. 056
0. 058
0 . 057
0 . 0 2 0 . 109
0 . I l l
0 . 1 1 0
0. 03 0 . 180
0 . 170
0. 17 5
0 . 04 0. 236
0. 238
0. 237
0. 05 0. 304
0. 312
0. 308
0 . 0 6 0. 364
0. 367
,0. 366
, 0.08 0. 500 0. 501
0. 502
.0 . 1 0 0.628
0. 633
0. 635
0 . 12. 0. 771
,0. 773
0. 772
0 . 15 0.995
0.980
0. 990
TABLE Vb
DATA FOR THE STANDARD CURVE
USING THE PEARSON METHOD
33
mg. of Absorbance Average
cholesterol at 615 mp Absorbance
Blank ,0 . 0 0 0 0 . 0 0 2
0. 004
0.25 0 . 288 0.289
0 . 2 9 0
0 . 40 0. 476 0. 476
0.-475
0. 50 0. 596 0 . 597
0. 597
0 . 80 0 . 9 6 8 0. 967
0. 965
1. 0 0 1. 213 1. 204
1. 195
1.25 1. 522 1. 523
1. 524
TABLE VI
THE RECOVERY * OF CHOLESTEROL AND
FROM THE SILICIC ACID
CHOLESTERYL PALMITATE
COLUMN
Silicic Acid I — ^
Silicic Acid II ''
% recovery of % . recovery of % recovery of % recovery of
chol e sterol chol e ste ryl cholesterol cholesteryl
palmitate palmitate
10 3.4 ' 93.4.’ - 101. 3 99.2
10 3.2. 94.7 101. 5 .99. 1
104.7 95. 5
10 3.9 99.2
102.9 95.8
10 3.8 95.0
94.4
95. 2
x l / = 103. 5 x =95. 4 x =101.4 x = 9 9 . 2
S.D. ^ = r Q . 66 S.D.sai. 69 S. D. = 0.20 S. D. =3 0. 14
\! Silicic Acid I and II refer to two .batches of silicic acid prepared as indicated on page 10
2/ Mean O J
3 / Standard deviation
35
The Recovery of Cholesterol from a 1:1 Acetone:9 5% Ethanol
Extract of Serum after ( 1 ) Precipitation with Digitonin
and ( 2 ) Silicic Acid Column Chromatography
Two separate batches of an acetone :9 5% ethanol
extract of pooled serum were prepared according to the
procedure on page 11. They were designated as Serum
Extract A and Serum Extract B. Serum Extract A was
studied with Silicic Acid I and Serum Extract B was studied
with. Silicic Acid II. Silicic Acid I and II were the same
preparations as used for the recovery study of cholesterol
and cholesteryl palmitate described on page 29. The size of
each sample in this experiment was about 0. 08 ml. of serum
or 0. 15 mg. of total cholesterol. The recovery of cholester
ol is shown in Table VII. The absorbance of the untreated
serum extract was taken as- 100%, so that the results from
different batches of silicic acid and the different serum
extracts can be compared, and also the results from silicic
acid column chromatography can be compared with results
from the digitonin precipitation.
TABLE VII
THE RECOVERY OF CHOLESTEROL FROM 1:1 ACE.TONE:95% ETHANOL EXTRACT
OF SERUM AFTER ( 1 ) PRECIPITATION WITH DIGITONIN AND
( 2 ) SILICIC ACID COLUMN CHROMATOGRAPHY
Digitonin prec:ipita,tion after
hydrolysis by potassium
hydroxide
V
Silicic acid column chromatography
Recovery, % of the total Recovery, % of the total Recovery, % of the free
cholesterol in the untreated. cholesterol in the untr ea,ted jcholesterol in the untreated
serum extracts serum, extracts serum extracts
Serum A SeJum B Serum A- Serum B-
Silicic Silicic
Acid I Acid II
Serum A- Se rum B -
Silicic Silicic
Acid I Acid II
97. 1 95.0 93. 7 93.6 109. 3 102, 8
99. 5 96.2 92. 3 92.2 107. 5 102, 5
96.4 95. 4 9 0 . 1 92. 7 110. 4 102. 5
97. 5 91. 8 93.6 110.2
97.9 92. 1 92.7 112.3
x U = 97.7 xJ /= 9 6 .4 x = 9-2.0 x = 93.0 x = 109.9 x - 102,6
S. D. Z-I - 1.59 S. D.— / - 1,2.7 S.D. = 1. 30 S.D. = 0. 60 S.D. = 1.73 S. D. = 0.17
1/ Mean 2/ Standard deviation
w
CHAPTER V
THE FORMULAE FOR CALCULATION
The formulae used in the statistical analysis are given
below:
Standard Deviation of the Duplicates (28)
S. D. (dup. ) zzz ~
d2
2n
= summation.
:d = difference between duplicate observations
n zssz number of degree of freedom
.S e n s it iv it y (27)
dx.
Sen. = slope =
'S. D. S. D. (dup. )
dx i=s change in quantity measured (absorbance)
dy = change in quantity determined (cholesterol)
38
Mean: (30)
x
value of individual observation
Standard Deviation (30)
S. D.
39
Formulae for the t Test for a Significant Difference between
Two Means (30)
Standard deviation of the two .samples.
Xi
The t test.
S. D. (t)
L = t • S. D. (m) confidence limits at the
chosen level of significance
- standard, deviation
of the difference in
the means of the
two samples
tg sss Value in. th e T a b le o f t at th a t le v e l o f
s ig n ific a n c e a n d w ith th a t n u m b er o f
d e g r e e s o f fr e e d o m
At 5% level of significance, there is a chance of
being correct in the prediction .that the true value of the
difference in the means of the two samples lies between the
confidence limits (i L).
S. D. (m) sss S. D. (t)
^ n r + n 2
n i n :
l n2
C H A PTER VI
DISCUSSION
The prelim inary results indicate that the F erric
Chloride and Pearson methods for the color development of
cholesterol are in good agreement. It was also found that
the total cholesterol concentration, of 1:1 acetone:9 5% ethanol
extracts of blood serum after digitonin precipitation is about
10% lower than in the . original untreated serum extracts.
Most of this difference was found to be the result of losses
sustained during the precipitation step. When conical screw
capped centrifuge tubes w ere used, in place of the round
bottom tubes, a tightly packed precipitate was obtained and
the difference was reduced to about 3%. Our results after
digitonin precipitation of recrystallized cholesterol indicate
that there is no loss of cholesterol during precipitation with
digitonin or during hydrolysis with potassium hydroxide, th ere
fore the 3% difference is thought to be due to the presence
of chromogens other than cholesterol in the original serum
extracts.
41
The recovery of added cholesterol from serum by
extraction with 1:1 acetone:95% ethanol was also studied.
The results indicate that the recovery is quantitative and are
in agreement with those obtained by Schoenheimer and
Sperry ( 1).
The fractionation of free and esterified cholesterol
from a .1:1 acetone :9.5% ethanol extract of serum by silicic
acid column chromatography was found to be rather unsatis
factory when the present study was initially undertaken. The
column was redesigned to achieve a m ore simple operational
technique. The solvent system for eluting the esterified and
free cholesterol was investigated with recrystallized cholester
ol and with cholesteryl palmitate obtained from Dr. J. Mead.
It was found that pure n-heptane for the elution of choles-
teryl palmitate and 30% ethyl ether in n-heptane for the
elution of cholesterol were satisfactory. Two separate
batches of silicic acid were prepared and the average recove
ry of cholesteryl palmitate and cholesterol using Silicic I
and Silicic Acid II was significantly different at the 5% level
of significance. The results are shown in Table VI and
their statistical analyses in Table VIII. This difference may
TABLE VIII
A COMPARISON OF THE RECOVERY OF CHOLESTEROL AND CHOLESTERYL
PALMITATE FROM SILICIC ACID I AND SILICIC ACID II
(STATISTICAL ANALYSIS OF DATA FROM TABLE VI)
Standard
deviation
of the two
samples
Calculated Confidence t value
value of t limits from
Table t
At 5% level
O f
significance
Comparison of %
recovery of
cholesterol from
Silicic Acid I and
Silicic Acid II
0.61 4. 21 ±1.22 2.45 Significantly
different
Comparison of %
recovery of
•cholesteryl palmitate
from.Silicic'Acid I
and Silicic Acid II
1. 58 3.01 ±2.99 2.31 Significantly
different
ro
43
be explained by the fact that both batches of silicic acid
were ground by hand, a process which is difficult to control.
It . is suggested that with, improvement in the method of
preparation of silicic acid before use, better results can be
achieved.
For the fractionation of free and esterified cholester
ol from a 1:1 acetone:95% ethanol extract of serum on the
silicic acid column, it was thought best to compare the r e
covery from the column with the well studied and accepted
digitonin precipitation. However, free and esterified choles
terol are obtained from silicic acid column chromatography,
but free and total cholesterol are obtained by digitonin
precipitation. Since the recovery of cholesterol and choles
teryl palmitate is different with, different batches of silicic
acid, some problems are presented in making the comparison.
In. order to compare the recovery of total cholesterol by the
two methods and also the results with different batches of
silicic acid, the total cholesterol in the original serum
extract was taken as 100, and recoveries expressed in per
cent. In regard to the recovery of free cholesterol from
serum extracts after silicic acid column chromatography, the
4 4
free cholesterol after the digitonin precipitation, was taken to
be 100. The recovery of cholesterol from 1:1 acetone:95%
ethanol extracts of serum after precipitation with digitonin
and after silicic acid column chromatography is shown in
Table VII. A statistical comparison of the recovery of
c h o l e s t e r o l from Serum Extract A with Silicic Acid I and
Serum Extract B with Silicic Acid II is shown in Table IX,
these statistical analyses indicate that:
1.) The per cent recovery of total cholesterol from
untreated Serum Extract A and B by digitonin precipitation
after potassium hydroxide hydrolysis is not significantly
different at the 5% level of significance. This is interpreted,
to mean that the two serum extracts are sim ilar in their
per cent composition of digitonin precipitable m aterial.
,2.) The per cent recovery of total cholesterol from
an untreated serum extract after silicic acid column chrom a
tography using Serum Extract A with Silicic Acid I and
Serum Extract B . with ..Silicic Acid .II is not significantly
different at the 5% level of Significance. This lack, of
difference can be explained by. the effect of compensation
between the recoveries of cholesterol and esterified
cholesterol ( see Table VI and VIII ).
/TABLE IX
A . COMPARISON OF THE RECOVERY OF CHOLESTEROL FROM SERUM EXTRACT A-
SILICIC ACID I AND SERUM EXTRACT B-SILICIC ACID II
(STATISTICAL ANALYSIS OF DATA FROM TABLE VII)
Standard
deviation
of the two
samples
Calculated
value of t
I
Confidence. t value
limits from
, 'Table i
At 5% level
of
significance
. 1 l
Comparison of % recovery of
total cholesterol from untreated
Serum Extract A and B by
digitonin. precipitation after
KOH hydrolysis
1. 38
0 0
o
r * 4
±2.. 84 2. 45 Not
significantly
different
Comparison of % . recovery of
total cholesterol from untreated
serum extract after silicic acid
column chromatography using
Serum Extract A-Silicic Acid I
and Serum Extract B-Silicic
Acid II
1.01 1. 52. ±1.49 2.31 Not
significantly
different
Comparison of % recovery of
free cholesterol from
digitonin. precipitation of
untreated serum extract after
silicic acid chromatography
using Serum Extract A-Silicic
Acid I and Serum Extract B a
silic ic Acid II
1. 44.
6.97
±2.. 58 2 . 45 Significantly
different
> n
46
3.) The per cent recovery of free cholesterol from
the digitonin precipitation of an untreated serum extract after
silicic acid column chromatography using Serum Extract A .
with Silicic Acid I and Serum Extract B with Silicic Acid II
is significantly different at the 5% level of significance.
This significant difference can be attributed to the significant
difference between the average recovery of cholesterol from
Silicic Acid I and Silicic Acid II ( see Table VI and VIII ).
Another statistical comparison of the recovery of total choles
terol after digitonin precipitation and after silicic acid column
chromatography of an 1:1 acetone:95% ethanol extract of serum
is shown in Table X. These statistical analyses indicate
that the per cent recovery of total cholesterol from an un
treated serum extract after precipitation with digitonin and
after silicic acid column chromatography is significantly
different at the 5% level of significance for both the Serum
Extract A with Silicic Acid I and Serum Extract B with
Silicic Acid II, these significant differences are attributed to
the limitations of silicic acid column chromatography and to
the difference in the chemical principles of the two methods.
TABLE X
A COMPARISON o f t h e. r e c o v e r y o f t o t a l c h o l e s t e r o l a f t e r d ig ito n in
PRECIPITATION AND AFTER SILICIC ACID COLUMN CHROMATOGRAPHY OF.
AN ACE TONE: 9 5%. ETHANOL .EXTRACT OF SERUM
(STATISTICAL ANALYSIS OF DATA FROM TABLE VII)
Standard
deviation,
of the two
samples
Calculated'
value of t
Confidence
limits
t value
from -
Table t
At 5% level
of
significance
% dif
ference
Comparison of % recovery
of total cholesterol from
untr eated Serum Extract A
after (1) precipitation with
digitonin and (2 ) Silicic Acid
I column chromatography
L 4.0 5. 55 ±2.98 2. 45 significant
ly
different .
5. 68
Comparison of % recovery
of total cholesterol from
untr eated Ser um E xtract B
after (1) precipitation with
digitonin and (2.) Silicic Acid
II column chromatography
0.98 5. 56
±1.60 2. 31 significant
ly
different
3.45
-j
48
It is rather difficult to compare the results of the
present study with other publication# mainly due to the use
of different solvent systems. An attempt is made in Table
XI to summarize both, the results of some other publications
and of the present study.
TABLE XI
A SUMMARY OF RESULTS AFTER SILICIC ACID COLUMN CHROMATOGRAPHY
FROM -SOME OTHER PUBLICATIONS AND THE PRESENT STUDY
Source Column size Sample size, solvent system, and % recovery
Method of color
development of
cholesterol
Filler up
and Mead
(10)
Silicic acid:
13 g.,
column height:
7 cm.
50 mg. of cholesteryl oleate were eluted „by
280 ml. of 1% . ethyl ether in petroleum ether
with 98. 4 to 100 % recovery
50 mg. of cholesterol were eluted by 210 ml. of
10% ethyl ether in petroleum ether with
99 to 100 % recovery
Liebermann
and Burchard
Wycoff
and
.Parsons
(12)
Column height:
5. cm .,
diameter of
outside column:
8 m m .,
one hour was
required ..for the
operation
Stearyl cholesterol was eluted by 4 ml. of 1:1
chloroform:petroleum ether.
For 2., 0 mg. sample, the recovery was 103, 5%.
For 0. 2 mg. sample, the recovery was 100.5%.
For 0.02 mg. sample , recovery was 99. 5%.
Cholesterol was eluted by 4 ml. of I; 1 methyl
or ethyl acetate :petroleum ether.
Ferric
Chloride
sO
Source Column size Sample size, solvent system, and % recovery
Method of color
development of
cholesterol
For 2. 0 mg. sample, the recovery was 103.5%.
For O '. 2 mg. sample, the recovery was 103. 5;% .
For 0. 02 mg. s am pi a the. recovery was 100. 5%.
Serum sample used.: 0.02 ml.
Barron
and
Hanahan
(13)
Silicic acid:
60 g .,
column height:
15. 5 cm .,
flow rate: 2 .to
2. 5 ml. per
minute.
Total lipid, sample: about 1. 1 g. . Liebermann
Recovery was based on per cent of total lipid weight, and Bur chard
For beef liver, the recovery was 101%.
For’ rat liver, the recovery was 98%.
For yeast, the recovery was 104%.
Cholesteryl esters were eluted by 500 ml. of
15% benzene in hexane.
Cholesterol was eluted by 1 liter of 15 to 20 %
ethyl ether in hexane.
o
Source Column size Sample size, solvent system, and % recovery
Method of color
development of
cholesterol
Hirsch Silicic a.cid:
and 18 g .,
Ahrens outside diameter
(14) of column: 18 mm.
flow rate: 0. 3 to
0.6 ml. per
minute.
The Silicic acid:
present 2.5 g .,
study outside diameter
of column: 11 mm.
flow rate: about
0. 5 ml. per
minute. ,
two hours were
required for
the operation.
31. 7 mg. of cholesteryl palmitate was eluted by
225 ml. of 1% ethyl ether in petroleum ether
and 60 ml, of 4% ethyl ether in petroleum ether
with 9 6 . 5 to 98. I % recovery.
17. 2 mg. of cholesterol wa.s eluted by 450 ml.
of 8% ethyl ether in petroleum ether and 50 ml.
of 25% ethyl ether in petroleum ether with 94. 7
to 96.5 % recovery.
0. 15 mg. of cholesteryl palmitate wa,s eluted by
15 ml. of n-heptane with 95. 4 to 99. 2 % recovery.
0. 1 mg. of cholesterol was eluted by 14 ml. of
30% ethyl ether in n-heptane with .101. 4 to
103,5 % recovery.
Serum sample used: 0.08 ml.
L iieberm ann
and B u rch ard
Ferric Chloride
CHAPTER VII
SUMMARY
A comparison of the F erric Chloride and Pearson
methods for cholesterol determination was made. The F erric
Chloride method was found to be approximately eight times
more, sensitive than the Pearson method.
A silicic acid column .was designed with, which the
free and esterified cholesterol from about 0. 08 ml. of blood
serum can be separated by a stepwise elution in less than
two hours.
The average recovery of re crystallized cholesterol
after silicic acid column chromatography was 103. 5% for one
batch of silicic acid and 101.4% for another batch.
The average recovery of cholesteryl palmitate was
95.4% for one batch of silicic a,cid and 99.2% for another
batch.
. The average recovery of total cholesterol of serum
extracts after silicic acid, column chromatography is 5%
lower than the recovery after hydrolysis with potassium
53
hydroxide followed by precipitation with digitonin which in
turn is 3% lower than that in. the .original serum extract.
The average recovery of the free cholesterol of
serum, extracts after silicic acid column chromatography was
1 0 9 . 9 % for one batch of silicic acid and 102.6% for another.
*
batch based on the digitonin precipitable cholesterol of the
original extract.
BIBLIOGRAPHY
BIBLIOGRAPHY
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Asset Metadata

Creator Cheng, Katherine Wong (author)

Core Title A study of methods of cholesterol analysis

School Graduate School

Degree Master of Science

Degree Program Biochemistry and Nutrition

Degree Conferral Date 1959-01

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, analytical,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Mehl, John W. (committee chair), Alfin-Slater, Roselyn B. (committee member), Marx, Walter (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-778381

Unique identifier UC11348041

Identifier EP41337.pdf (filename),usctheses-c17-778381 (legacy record id)

Legacy Identifier EP41337.pdf

Dmrecord 778381

Document Type Thesis

Rights Cheng, Katherine Wong

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA