A CCMPARATE‘VE STUDY OF
METHODS FOR THE DETERMINATKCN
CF CHOLESTEROL 1N BLOOD

Tl‘esis In the Degree of 3.4.5.
MECHEGAN STATE. COLLEGE '
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A oonpmnvm STUDY or umnons roa m
Dnmmanou or cacwcrmoz. IN BLOOD.

by
Annabel Studebaker Larzelere

A THESIS

submitted to the Graduate school of Michigan

State College of Agriculture and Applied
Science in partial fulfilment of the
requirements for the degree of

MASTER 0? 801330!

Department of Chemistry
1940

AOKNOWLEDGHEIT

The author wishes to acknowledge the gen-
erous assistance given by Dr. 0. A. Hoppert
in the planning of the experiment and in

the preparation of the manuscript.

V...“

Historical Review -
IIperimental Procedure
Data ==—- — —-
Discussion of Results ‘
Summary —— "—-—-~—-‘
Bibliography -—— -

A

‘

CONTENTS

1.
HISTORICAL REVIEW

The chemical literature contains many methods, with
various modifications, for the quantitative determination
of cholesterol in blood. One of the earliest procedures
was suggested by Windaus(1)in 1910. This was based on the
precipitation of cholesterol by digitonin in alcohol solution.
The cholesterol digitonide was then determined gravimetri-
cally. Since 15 to 20 cc. of blood was necessary, this
method was not well suited to clinical work.

In 1910 also, Grigaut$2)reported a method which deter-
mined the cholesterol by the Liebermann-Burchard reaction.
This reaction($)was developed in 1885, but it was not
applied to quantitative work previous to 1910. The majorb
ity of methods devised since that time have utilized this I
reaction. Grigaut's precedure required 5 to 10 cc. serum
and ether extraction in a Soxhlet extractor for 3 to 5
hours. This was later adapted to clinical use by Auton-
rieth and.Funk(f)who used only 2 cc. serum.and reduced
extraction time to two hours.

Bloor(§)in 1915, further simplified this method so
that it could be applied in his system of analysis of
blood lipoids. These colorimetric methods involved sap-
onification or esters, including the cholesterol esters.

‘In 1916, Bloor‘6)reported that this saponification was
unnecessary. Although in 1922, Bloor,_Pelkan, and.Allen‘7)

2.
published a revised method again using saponification, in

1928, Bloor's latest directions(8)omit this step.

Bloor has undoubtedly done the most extensive work
in the determination of cholesterol, but many of his con-
temporaries have submitted other methods which have been
widely accepted.

,All of these methods have certain features in common.
They involve: first, the extraction of cholesterol by some
fat solvent, and second, the treatment of the extract ob-
tained for the quantitative estimation of cholesterol.

The methods differ in the manner in which the chole-
sterol is extracted. Some methods employ dry extraction.
In the procedures of Myers and Wardell‘?) the blood is
absorbed on several grams of plaster of Paris and dried
before extraction. Leiboff(1°)and Ling(11)use fat-free
filter discs to absorb the blood. They claim this pre-
vents caking often experienced with plaster of Paris. It
also eliminates any loss due to changing the plaster of
Paris from the crucible to the extraction thimble and
further loss when the extract is transferred to a volu-
metric flask. Abrahamson‘12)recommends absorbent cotton
to replace the filter paper.‘ This method sometimes shows
incomplete extraction probably due to caking of the blood
during drying. Other workers have used permutit and washed
sea sand.

In this study cheesecloth was used to absorb the

3.
blood: Besides being much less expensive than most of the
above absorbents, it is easy and convenient to handle.
MicroscOpic examination shows that the blood is absorbed
in the cotton fibers and is dispersed so the solvent can
readily extract lipoid substances.

In nearly all cases, the absorbed blood is dried
before extracting. Ling says this step is unnecessary
in his method but it can be done. Chloroform is the sol-
vent usually employed because the Liebermann-Burchard
color can be develOped in the chloroform extract.

In the wet extraction methods, preliminary drying
is unnecessary., The blood is run into the solvent, and
the mixture is heated and filtered to remove the pre-
cipitated proteins. Bloor(5’6’7'8)uses a mixture of
absolute alcohol and ether (3:1). The alcohol preci-
pitates the proteins and mixes with the water in the
blood. The ether extracts the lipoid substances more
completely. Schoenheimer and Sperry(13)in their digi-
tonin precipitation method use a mixture of equal parts
of acetone and absolute alcohol for extraction. Anten-
rieth and Funk‘4), like grigaut(2), use only ether which
requires longer extraction time. When alcohol and ether
or alcohol and acetone are used, it is necessary to heat

on a water bath at the boiling point for only a few minutes

 

eThe use of cheesecloth was originated in this
laboratory by E.C. Tabor in an attempt to find an
inexpensive , convenient substitute for the filter
discs for use in Biochemistry classes.

4.
before filtering to obtain complete extraction.

The various methods also differ widely in the treat-
ment of the extract to determine the cholesterol. Most
methods employing chloroform as the initial solvent utilise
the Liebermann-Burchard color reaction. The green color
produced can be compared with standards in the colorhmeter.
It can also be measured with photometer or photelometer
using a suitable filter.

0hyama‘14)has devised a new color reaction. The
chloroform solution is treated with salicylaldehyde,
water, and sulfuric acid. After shaking for two hours,
the red-violet color of the chloroform layer is determined
colorimetrically. I

A red color is produced in a glacial acetic acid
solutibn of cholesterol when heated with acetyl chloride
and anhydrous zinc chloride. .Although the color is given
by several other sterols, Bernoulli‘15)claims it is
applicable to the determination of cholesterol in blood.
The advantage of this reaction is that the color is more
stable than that given by the Liebermann-Burchard.reagents.
However,_these two color reactions have not as yet been very
widely accepted.

The alcohol-ether extract in Bloor's method may or
may not be subject to saponification with sodium ethylate
or sodium hydroxide and later acidified. In either case,
it is evaporated to dryness, extracted with chloroform, and

the Liebermann—Burchard reaction carried out. The initial

5.
extraction with alcohol and ether eliminates the necessity
for drying and extracts the lipoids more completely.

The alcohol-acetone extract of Schoenheimer and
Sperry is saponified by potassium. hydroxide and then
treated with a solution of digitonin. The digitonide,
after washing and drying, is dissolved in 1 cc. acetic
acid, which is substituted for the 5 cc. chloroform or-
dinarily used in the Liebermann-Burchard reaction. The
color in this solvenfrequires about twice as long to
develop.

Szent-Gyorgyi(16)has modified the Windaus method
so that the digitonide precipitate can be determined on
'a micro scale. He also reports a titrimetric method in
which the digitonide is oxidized by potassium chromate.
The excess chromate is determined by adding potassium
iodide and titrating with a standard thiosulfate solution.
Ruth Okey(17), working in Bloor's laboratory has simplified
certain parts of this procedure. The latter methods are
receiving increasing attention in the literature and many
modifications are reported.

The very fact that there are so many and varied
methods indicates that probably no one is entirely satis-
factory. The literature contains contradictory claims

as to which gives the highest or most consistent results.

6.
EXPERIMENTAL PROCEDURE

Although gravimetric and titrimetric methods have re-
ceived some attention, the colorimetric methods seem to be
used most frequently because they are less time consuming and
Just as accurate.

For this study only colorimetric methods Were used.
Leiboff'aémethod, as given in the Journal of Biological Chem-
istry, g1, 17?, Was chosen as a representative of the dry ex-
traction method. Three—inch squares of cheesecloth were sub-
stituted for the fat-free filter paper discs.

Bloor'sjmethod'Was used as an example of Wet extraction.
It is one most frequently found in text books for the deter-
mination of cholesterol. The method Was used Without saponi-
fication as given in the Journal of Biological Chemistry, 11,
53. The extract was made up to 50 cc. after 2.5 cc. blood
had been added and the mixture heated. Aliquots of 5 cc. of
this extract,after filtration,were taken so that the quantity
of blood in each Was 0.25 cc. This is the same quantity em-
ployed in the Leibcff method and allowed the use of the same
standard. Aliquots of this extract were saponified with con-
roentrated sodium hydroxide according to the directions by
‘Bloor, Pelkan, and Allengin the Journal of Biological Chemis-
try, .53., 191.

The digitonin method was also chosen since it is being
Irather Widely accepted. )The methcdf?olched as outlined by

ESchoenheimer and Sperry in Journal of Biological Chemistry,

7.
ige, 745. A 3 cc. fraction of the original extract was
taken in place of 1 cc. to give a deeper color and thus re-
main within the more sensitive range of the photolometer.
subsequent reagents except the acetic anhydride and sulfuric'
acid were also doubled. An alcohol solution of digitonin was
used instead of the water solution. This is recommended by
Wandfimwm) in a later publication. The
potassium chloride which is formed when the saponification
mixture is neutralized does not dissolve when this alcohol
solution of digitonin is added. A few drape of distilled
water must be added till the solution is clear. ‘This does
not interfere with the precipitation of the cholesterol
digitonide.

Rat's blood was used in all the work. Since samples
vary slightly, only results obtained on the same samples can
be compared. The extractions were made as soon as possible
after the samples were obtained.

In the first work the calorimeter was used to compare
the color obtained from the blood with that given by standard
solutions. This did not prove very satisfactory because of
the yellow tints deveIOped in the unknown making comparison
difficult. This coloration was especially marked in runs by
the Leiboff and saponified Bloor methods and was worse in
some tubes than others. In nearly all cases the unsaponified
Bloor extracts gave colors which could easily be matched with

that Of the standard.

a.

The color produced by the digitonin method did not have
a yellow tinge but rather a gray appearance which made com-
parison by the calorimeter almost impossible.

In this case much better results were obtained with the
phctelometer using a red filter. The center cell of the
photelometer was filled with a blank solution containing the
solvent, acetic anhydride, and sulfuric acid in the same pra-
portions as in the samples. The instrument was set at 100
when this cell was in place. Then this blank was replaced
by the cell containing the sample and the reading was record?
ed. A reference curve was prepared by making up several stane
dards of varying cholesterol content. The photolometer read-
ings were plotted against the cholesterol content on semi-
logarithmic paper and resulted in a straight line. The cho-
lesterol content was recalculated to give milligrams per 100
cc. blood or plasma directly from the graph. 1‘his was done
by using the following formula:

m cholesterol in standard x 100 = mg cholesterol
W per 100 cc.
The dilution of the standard was the same as that of the un-
known made later. A different graph was required for the
chloroform solution than for the acetic acid solution. The
line given by the chloroform standard had a greater slaps.
The results obtained.by these methods using whole

blood are contained in Table I. Table II contains the results

given by plasma treated in the same ways.

DATA

Table I
Results using whole blood

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11.
DISCUSSION OF RESULTS

With whole blood the Leiboff method gave the highest
results and the digitonin precipitation method the lowest in
most cases. Duplicate samples determined by the Leiboff
method usually agreed better than those by the other methods.
This is probably due to the fact that the entire process is
carried out in the same container.

_Variations between the different methods may be explained
as follows: Although the first directions given by Bloor are
for plasma, serum, or whole blood, he states in 1988(8)that
the cholesterol is not entirely extracted from whole blood by
alcohol and ether.. This undoubtedly accounts for the lower
results obtained by this method. “The values obtained corrob-
crate Bloor's statement that saponification is unnecessary
since both gave practically the same results. Moreover the
color was.not improved and so this step seems a waste of time
and materials.

- In the first tests saponification caused very low re-
sults. According to the directions, the sodium sulfate ob-
tained when the saponification mixture is acidified is porous
and allows complete extraction. The directions also state,
'If the drying and distribution of the salt have been care-
fully carried out, very little of the salt will break loose
from the bottom of the i'lask.‘I An attempt was made to find a
cause for the lower results. After extraction according to

directions, the salt was broken up with a stirring rod and

13.

further extracted. This extract gave considerable green
color when treated with acetic anhydride and sulfuric acid,
showing that extraction had been incomplete. Therefore, in;
subsequent tests the salt was stirred and broken during ex-
traction. The salt was allowed to settle and the chloroform
extract decanted through a small filter into the graduated
cylinder. This avoided the use of large amounts of chloroform
which must first be evaporated down in a larger container and
then transferred to the graduated cylinder with further danger
of loss. Determinations made using this modification agreed
quite closely with values obtained without saponification.

Careful neutralization and drying are important in
Bloor's procedure. Complete neutralization liberates free
fatty acids which are soluble in the chloroform. These acids
may be a contributing factor to the yellow color later devel-
Oped. Failure to neutralize may result in the decomposition
of the cholesterol. Heating on the steam bath after the alco-
hol has disappeared is apt to cause oxidation of some of the
more unstable compounds. There is equal danger at this point
in the unsaponified extract. On the other hand, if the drying
is incomplete, small amounts of moisture may be extracted by
the chloroform. Unless the extract and reagents are completely
anhydrous, the color will not develop completely.

The digitonin method is also described for whole blood,

as well as for serum and plasma. Probably the same difficulty

is involved here as in Eloor's method in securing complete ex—

133
traction from the whole blood. The directions of Schoen-
heimer and Sperry call for heating the acetone-absolute al-
cohol mixture to the boiling point before adding the blood.
This causes formation of large clots which are difficult to
break up so that complete extraction can be made. It was
found more satisfactory to run the blood into the cold mixture
and heat to boiling later. This causes a flooculent precipi-
tate which does not form large clots on heating if the tube is
kept in motion. This motion also prevents superheating.

Schoenheimer and Sperry claim, “The isolation of choles-
terol as digitonide entirely avoids the errors which are intro-
duced into other colorimetric methods by other chromogenic
substances present in fatty extracts.’l This absence of 'other
chromogenic substances' may account for the slightly lower re-
sults obtained. The term 'dntirely avoids the errors“ is not
quite correct since later in the same report they state that
“digitonin precipitates practically all natural sterols, not
all of which give color reactions.“ 1'hose sterols which give
the Liebermann-Burchard reaction are likely to interfere in
all methods.

As stated in the previous section, the yellow color
often develcped in the samples makes comparison in the color-
imeter very difficult. This difficulty with the colorimetric
determinations has been recognized since Grigaut first devised
his method. The cause of the yellow color has been explained

in several ways all of which may be partially correct. Bloor‘ng)

14.
showed that temperature control is important. A temperature
of 33° 0. is most satisfactory. At higher temperatures the
color assumes a yellowish tinge. ‘At lower temperatures the
samples, which contain some cholesterol esters, develop faster
than the standard which contains only pure cholesterol. This
results in high values.

The yellow cast may be due to other color-producing sub-

stancss in the blood. Luden‘zo)

states that bile derivatives
may be a contributing factor. hueller‘31)suggests that oxy-
cholesterol may interfere, but too little is known of it to
make a positive statement.

Prolonged heating of the residue after the alcohol and
ether are evaporated from the Bloor extract may cause oxidav
tion and decomposition of some of the less stable fatty acids.
The oxidation products, if extracted by chloroform, may give
that extract a yellow color even before the Liebermann-Bur-
chard reagents are added.

Several attempts have been made to improve the color in
the unknown. It was thought that saponificaticn would remove
some of the interfering substances, but the color after such
treatment still had a yellow tinge. Mueller suggested washing
the chloroform extract with water and dehydrating with anhydrous
sodium sulfate. This process requires a great deal of time,
and it is difficult to know when this extra step is necessary

‘beoause the extract seldom shows a yellow color before it is

treated with acetic anhydride and sulfuric acid. The use of

15.
a red glass filter with the colorimeter has been suggested by
Bloor. This is a simple expedient if such a filter is avail-
able. The red glass filter in the photelometer accomplishes
the same results by allowing only certain rays to be trans-
mitted. The red filter is also used in the photelometer with
the digitonide color. Schoenheimer and Sperry studied the
absorption of the color produced by cholesterol digitonide
and found it was the same as that given by pure cholesterol
at all wave lengths.

The determinations were made using plasma because
Bloor admits his method is not entirely satisfactory for
whole blood. These results are shown in Table II. Here
the Leiboff method gave the lowest results in contrast with
the highest for whole blood. Since this method was devised
for whole blood, the literature contains no information on
values given by it for plasma. It is possible that the more
complete extraction of cholesterol from plasma by the alcohol
and ether causes that method to give higher results in this
case. Using plasma, the Bloor method gave values slightly
higher than those obtained with digitonin. This was also the
case when whole blood was used. It is probable then that wet
extraction is more satisfactory with plasma while dry extrac-
tion gives getter results for whole blood.
In evaluating the methods, there are advantages and

disadvantages to each. In the class laboratory when only
one or two determinations are to be made, the Leiboff method
would probably give the most satisfactory results. There is

16.
little technique involved beyond that of accurate pipetting
and proper use of the calorimeter or photelometer. The en-
tire procedure is carried out in one container and takes a
relatively short time. The main disadvantage is the yellow
color which makes it difficult to compare the unknown with
the standard in a calorimeter. The photelometer overcomes
this difficulty, but it is not worth while to standardise
it unless a series of determinations is to be made.

Bloor's method involves at least three containers, two
extractions, and two filtrations. Moreover, the evaporation
of the alcohol and ether requires great care, and makes it
necessary for the operator to have considerable experience
before the results are likely to be dependable. More blood
is required than for either the Leiboff or digitonin meth-
ods. However, after the preliminary steps are carried out,
the color is better.

In the digitonin method the centrifuge tube into which
the extract is pipetted is used throughout the remainder of
the determination. fThis is an advantage over Blcor's methr
od. However, the necessary washings and decantations re-
quire careful manipulation to avoid loss. Duplicate deter-
minations require at least three hours more time than dup-
licates by the Lsiboff method. However, the determination
is more specific for cholesterol. In this method, as in
Bloor's, the extract is filtered before being used. With

such volatile solvents as alcohol, ether, and acetone it is

17.
rimpossible to avoid some evaporation which may affect the
concentration of the filtrate.

Regardless of the procedure used preliminary to
developing the color, the final step requires great care.

The reaction is a delicate one. The color develops to a
maximum and then soon starts to fade. Therefore, it is
necessary to read the color within a specific time after the
.sulfuric acid is added. This is 15 to 35 minutes for chlor-
oform solutions and 37 to 35 minutes for acetic acid solution.
The speed with which the color develops depends on the temp-
erature. As previously stated 330 O. has been found most
satisfactory. The amount of sulfuric acid is a very import-
ant factor, a 0.1 cc.pipette being most satisfactory for add-
ing the acid.

In conclusion, it is impossible to say which method
gives the most accurate results. It is not necessarily the
method giving the highest results since the color is not en-
tirely specific for cholesterol. Per cent recovery of added
cholesterol does not give much information as to accuracy,
since there are claims of high per cent recovery by all the
methods. It seems probable that added cholesterol would be
extracted more readily than cholesterol and its esters which
are mixed with other blood lipoids. consequently the methods
can only be compared and the differences recognized when they
are used. Any value for cholesterol content should be accompn

anied by specific directions as to the method used.

18.
BUMMARI

1. In the estimation of cholesterol in whole blood, the
Leiboff method gave the highest results and the digitonin
method of Sahoenheimer and Sperry the lowest results. Val-
ues by Bloor's method were intermediate, being only slightly
higher than those by the digitonin method. .

2. Using plasma in place of whole blood, Bloor's method
gave the highest values. Results by Schoenheimer and Sperry's
method were slightly lower while the Leiboff method gave much

lower values.

3. The color produced by extraction according to Leiboff's
method was frequently distinctly different from that of the
standard. Colors by Bloor's method, especially when saponi-
fication was omitted, were easier to match with a standard.
The digitonin precipitation method gave a very light green

color which appeared.gray when compared with the standard.

4. The Leiboff procedure was found to be much simpler than*
the other methods used in this study. 'chever, it is doubt-

ful whether absolute values are obtained by any of the meth-
ods now employed.

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1.

3.

3.

4.

5.

19.
BIBLIOGRAPHY

Windaus, A. L.
Uber die qmantitative Bestimmung des cholesterins
und der cholestarinester in einigen normalen:und
pathologischen iertn.

Z. physiol. Ohem., 1910, §§, 110..# ;

Grigaut ’ ‘0

Dosage colorimetrique de la cholesterins dans 1'
organisms.

Oompt. rend. 860s 3101s, 1910, fig, 83?.

Lieberman, 0.
fisher das Oxychinoterpen
Ber. deut. chem. 668., 1885, lg, 1803.*i7

Autenrieth,‘l., and Funk, A.

Haber kclorimetrische Bestimmungs methoden; Die Best-
immung de gesant-cholesterins in Blut und in Organen.

Munch. med. loch., 1913, eg, 1243.
3100:, W. R.

Studies on blood fat. II Fat absorption and blood
lipoids.

J. 8101. Chemo, 1915, 3-3., 317.

Bloor, l. R.
The determination of cholesterol in blood.

J. 5101. Chem., 1916, $53, 227.

7.

8.

9.

10.

11.

13.

13.

20.
Bloor, W. 3., Pelhan, K. F., and Allen, D. H.

Determination of fatty acids (and cholesterol) in
small amounts of blood plasma.

Jo 8101. OhCMe, 1923,‘§§J 191s

Bloor, I. R.

The determination of small amounts of lipoid in
blood plasma.

J. 9101. 0hem., 1929, 21, 53.

Myers, V. Oe, and Wardell, Es L0

The colorimetric estimation of cholesterol in blood,
with a note on the estimation of caprosterol in fices.

J. Biol. chem., 1919, is, 147.

LeibOff, 8. Re

A simplified method for cholesterol determination
in blood.

J. Biol. 0hem., 1924, eg, 177.

Ling, 80 M.

The determination of cholesterol in small amounts
of blood.

J. Biol. chem" 1929, 1g, 361.

Abrahamson, E. u.

Use of cotton to absorb blood for cholesterol ex—
traction.

Science, 1938,Ԥz, 480.

Schoenheimer, R., and Sperry, W. n.

A micromethod for the determination of free and com-
bined cholesterol.

J. Biol. Chem., 1934, 106, 745.

14.

15.

16.

17.

18.

19.

31.

Ohyama , 8.

Microdetermination of cholesterol based on a new
color reaction.

Chemical Abstracts, 1938, 3;, 8466.

Bernoulli, Ac Lo

New method for the colorimetric determination of
cholesterol.

35/3
Chemical Abstracts, 1932, 2Q, 3732;

Szent - Gyorgyi, A.

Die gravimetrische mikrocholesterin bestimmung.

Biochem. 3., 1923, 136, 107.

Die titrimetrische mikrocholesterin bestimmung.

Biochem. 2., 1923, 136, 112.

Okey, Re

A micromethod for the estimation of cholesterol
by oxidation of the digitonide.

J. 9161. Chem., 1930, §§, 367.

,Sturges, 8., and Knudson, A.

Application of the Schoenheimer - Sperry method to
the determination of cholesterol and cholesterol
esters in tissues.

J. 9161. Chem., 1939, 126, 543.

Bloor, I. R.

The determination of cholesterol in blood.

J. Biol. Chem., 1917, 29, 437.

22.
20. Luden, G.

*Studies on cholesterol. III. The influence of bile
derivatives in Blcor's cholesterol determination.

Jo 3101. Chem., 1917, 3-9., 4630

21. nueller, H. G.

A comparison of the results obtained by the colorimet-
rho and gravimetric determinations of cholesterol.

JO B101. Chem., 1916, fig, 549.

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