AMERICAN JOURNAL OF CLINICAL PATHOLOOY
Vol. 31, No. 4, April, 1959, pp. 310-313
Printed in U.S.A.
ESTIMATION OF CHOLESTEROL I N SERUM
HARRISON H. LEFFLER, M.D.
The Professional Laboratories, Washington, D. C.
Zlatkis and his associates,3 in 1953,
described an iron sulfuric acid reagent for
the direct estimation of cholesterol in
serum. The next year, Zak and his associates2
adapted this reagent to the estimation of
both free and total cholesterol. The reagent
of Zlatkis has one undesirable feature; it
is unstable. This past year Rosenthal and
co-workers1 dissolved ferric chloride in 87
per cent phosphoric acid and developed a
color reagent of excellent stability. A
simple technic, using Rosenthal's color
reagent, is described in this paper.
PRINCIPLE
The serum proteins are precipitated and
total cholesterol simultaneously extracted
by isopropyl alcohol. The addition of the
iron color reagent to an aliquot of the clear
extract produces a stable color with a density
proportional to the amount of cholesterol
present. Free cholesterol is precipitated
from a second aliquot by means of adding a
solution of digitonin, and the cholesterol
is then determined after adding color reagent
to the washed and dried precipitate.
METHOD
ReageJits
1. Isopropyl alcohol. Reagent grade 99
per cent is used throughout the procedure.
2. Iron stock solution. Ferric chloride
(FeCl 3 -6H 2 0), 2.5 Gm., is dissolved in 100
ml. of phosphoric acid (H3PO4, 87 per cent).*
It is stable for at least 9 months.
3. Color reagent. Iron stock solution, 8
ml., are diluted to 100 ml. with concentrated
sulfuric acid (H 2 S0 4 , sp. gr. 1.84). This
Received, December 12, 1958; accepted for
publication December 19.
Dr. Leffler is Director. The Professional
Laboratories are located at 1515 U street, N.W.,
Washington 9, D. C.
* Inasmuch as phosphoric acid is sold only under
the label "S5 per cent," it is important to request
the 87 per cent strength when ordering this
reagent.
solution is stable for at least 6 weeks when
kept in the dark at room temperature. I t
should be discarded when a precipitate
forms.
4. Solution ojdigitonin. Digitonin (Merck),
1 Gm., is dissolved in 50 ml. of ethyl alcohol
(reagent grade, 95 per cent) and diluted to
100 ml. with distilled water.
5. Cholesterol stock standard. Cholesterol
(Pfanstiel) is dissolved to the state of
saturation in hot absolute ethyl alcohol
(approximately 11 Gm. per 100 ml.) and
the solution cooled in the refrigerator until
recrystallization is complete. The supernatant fluid is discarded and the cholesterol
recrystallized 2 more times in the same
manner. The purified crystals are dried in a
vacuum desiccator to constant weight.
Purified cholesterol, 100 mg., is dissolved
in isopropyl alcohol to a volume of 100 ml.
and kept in a ground glass-stoppered bottle.
6. Cholesterol working standard. Stock
standard, 2 ml., is pipeted into a chemically
clean glass-stoppered bottle. Isopropyl alcohol, 23 ml., is added and the tightly stoppered bottle stored at room temperature.
One milliliter of this standard is equivalent
to 200 mg. of cholesterol per 100 ml. of
serum.
Apparatus
1. Matched cuvets with vinylite lined
screw caps; size of cuvets to suit the colorimeter used. Kimble culture tubes No. 45080
are suitable for this purpose.
2. Special aeration apparatus (Fig. 1).
Procedure
Into a screw-capped tube, 9.6 ml. of
isopropyl alcohol and 0.4 ml. of serum are
pipeted. The tube is capped, shaken for 5
sec, and permitted to stand for 5 to 10 min.
I t is again shaken and centrifuged at 3000
r.p.m. for 5 min. The supernatant extract is
treated as follows:
Free cholesterol. Into a matched cuvet
(labeled FREE), 4 ml. of the clear extract are
310
April 1959
CHOLESTEROL IN SERUM
311
labeled, matched cuvets as indicated in
Table 1.
The glacial acetic acid is added from a
buret and the contents mixed. From a
second buret, the color reagent is allowed to
flow down the side of the slanted tube and
underlie the contents of the tube without
mixing. With the screw caps adjusted
tightly, each of the tubes is inverted 4
times and allowed to stand for 10 min. The
per cent transmittance is read at X 540 m/x
against the blank set at T = 100.
TABLE 1
PROTOCOL FOR DETERMINING TOTAL CHOLESTEROL
200-mg.
Blank Stand- Total
ard
Isopropyl alcohol
Working standard
Clear extract
Glacial acetic acid
Color reagent
ml.
1
0
0
2
2
ml.
0
1
0
2
2
ml.
0
0
1
2
2
Free
ml.
1
0
0
2
2
.FIG. 1. Diagrammatic sketch of the apparatus
for the estimation of cholesterol in serum: (1)
cuvets with extract; (2) Vacutainer stoppers;
(3) air-inlet needles; (4) plastic needle-adapter
from transfusion set; (5) needle-adapters with
needles attached; (6) plugged needle-butt attached in order to prevent air entering when not
in use; (7) plastic tubing; (8) multiple aspirating
glass tube; (9) connection to suction pump; and
(10) level of water in water-bath.
pipeted. The tube, connected to the aeration
apparatus (Fig. 1), is placed in a water-bath
at 60 to 80 C , and air drawn through the
apparatus. The extract is evaporated to
approximately a 1-ml. volume. Solution of
digitonin, 1 ml., is added, mixed, and let
stand for 10 min. The tube is centrifuged at
3000 r.p.m. for 5 min. and the supernatant
fluid poured off, being careful not to disturb
the precipitate. Acetone, 5 ml., is blown into
the tube to disperse the precipitate, the
contents mixed, and the tube centrifuged
for 5 min. The acetone is poured off and,
with the use of the aeration apparatus, the
precipitate is dried by air current. The tube
is then treated as given below for the total
cholesterol.
Total cholesterol. Reagents are added to
100 ng
200 mg
300 mg
Frc 2. Curve for estimating cholesterol in serum
312
Vol. 31
LEFFLER
On semilogarithmic graph paper, the
per cent transmittance of the standard is
plotted against its value in mg. of cholesterol
per 100 ml. of serum. Connecting this point
to the point of 100 per cent transmittance
yields a straight line that, when projected
downward, forms a graph on which values
for cholesterol from 0 to 300 mg. may be
found (Fig. 2).
Example: (1) Per cent transmittance, X
540 mM; (2) blank, 100; (3) 200-mg. standard, 30; (4) total, 25; and (5) free, 32. From
the graph (Fig. 2), the following results are
determined:
25 % T = 230 mg. of total cholesterol
per 100 ml. of serum.
32 % T = 190 mg. of cholesterol in 4 ml.
of extract
190 H- 4 = 48 mg. of free cholesterol per
100 ml. of serum.
The total minus the "free" indicates the
amount of cholesterol esters.
DISCUSSION
The 99 per cent isopropyl alcohol is an
excellent reagent for extracting cholesterol.
I t precipitates serum proteins in a finely
divided state and, without the use of heat,
extracts total cholesterol in a minimal
amount of time. It yields a low blank with
glacial acetic acid and color reagent and thus
makes it unnecessary to evaporate the
extract to dryness before adding these
reagents. I t is also an ideal solvent for the
preparation of cholesterol standards.
Evaporating the extract to a small volume
and adding the solution of digitonin results
in rapid precipitation of free cholesterol.
The washed and dried precipitate is not
dissolved in the isopropyl alcohol, but is
partly soluble in the acetic acid. On adding
the color reagent and mixing, the heat of
reaction dissolves the rest of the precipitate
and yields a color that reaches its maximal
intensity in approximately 10 min. The
color is then stable for several hours.
Nonhemolyzed serum should be used, but
the presence of even large amounts of
bilirubin does not interfere.
The procedure may be used as a micromethod for the estimation of total cholesterol. Serum, 0.1 ml., is added to 2.4 ml. of
isopropyl alcohol, the contents mixed,
centrifuged, and 1 ml. of the clear extract
treated as for total cholesterol. No modification is required.
Matched cuvets are easily prepared from
the screw-capped culture tubes. They are
cleaned in hot potassium dichromate solution, washed in tap water, rinsed several
times in distilled water, and dried in the
hot air oven. The per cent transmittance
at X 540 mn of a freshly prepared and
filtered solution of 30 per cent copper sulfate
is recorded for each tube. The tubes yielding
identical readings are marked and used as
matched cuvets.
Sources of Error
The manner of mixing with the color
reagent is most important. The methods of
rotation, shaking, bumping, twirling, and
so on, all yield different degrees of mixing
and cause variations in the colorimeter
readings. When the cuvets are gently
inverted the same number of times for each
estimation, the results obtained by different
workers are in close agreement.
Inasmuch as the steps in the procedure
have been reduced to a minimum, many of
the previous sources of error have been
largely eliminated.
SUMMARY
A simplified procedure is described for
the estimation of total and free cholesterol
in serum, and a minimum of time and
reagents are required for the analysis. The
procedure is based on the use of isopropyl
alcohol for the simultaneous precipitation
of serum proteins and the extraction of total
cholesterol. Addition of a solution of digitonin to a second aliquot results in the
precipitation of free cholesterol. In both
analyses, an iron reagent is used for the
development of a stable colored complex,
the density of which is proportional to the
amount of cholesterol.
Duplicate determinations, in the normal
range, check within 5 mg. per 100 ml. of
serum. The estimation of both free and total
cholesterol requires less than 50 min., and
when the special technic in mixing the extract and iron reagent is used, analyses of the
April 1959
CHOLESTEROL IN SERUM
same sample of serum by different laboratories give results that are in close agreement.
SUMMARIO IN INTERLINGUA
Es describite un simplificate methodo pro
le estimation del cholesterol total e del cholesterol libere in le sero. Le amonta de
tempore e le quantitate de reagentes requirite es minimal. Le methodo se basa
super le uso de alcohol isopropylic pro le
precipitation simultanee de proteinas serai
e le extraction de cholesterol total. Le addition de un solution de digitonina a un secunde portion del mesme specimen resulta in
le precipitation del cholesterol libere. In ambe
analyses, un reagente a ferro es usate' pro le
313
disveloppamento de un stabile complexo
colorate. Le densitate de isto es proportional
al quantitate de cholesterol presente.
Determinationes duplicate, intra le limites
normal, es congruente intra 5 mg. per 100
ml. de sero.
REFERENCES
1. ROSENTHAL, H. L., PFLUKE, M. L., AND BUS-
CAGLIA, S.: A stable iron reagent for determination of cholesterol. J. Lab. & Clin.
Med., 50:318-322, 1957.
2. ZAK, B., DICKERMAN, R. C , WHITE, E. G.,
BURNETT, H., AND CHERVEY, P. J.: Rapid
estimation of free and total cholesterol. Am.
J. Clin. Path., 24: 1307-1315, 1954.
3. ZLATKIS, A., ZAK, B., AND BOYLE, A. J.: A
new method for the direct determination of
cholesterol. J. Lab. & Clin. Med., 41:
486-492, 1953.