A RAPID METHOD FOR T H E PREPARATION OF SEROLOGICALLY
ACTIVE PHOSPHOLIPIN AND PURIFIED LECITHIN*
T. V. LETONOFF, M.S.f
From the Venereal Disease Research Laboratory, U. S. Public Health Service, Stapleton,
Staten Island, New York
Before the use of laboratory tests based on immunologic factors, the reports of
studies of the phospholipids appeared in the publications of the biochemists.
Erlandsen 2 in 1907 presented a comprehensive review of the literature and at the
same time reported an intensive research on the phospholipids of beef heart
muscle. I t is interesting to note that the method used at the present time for
the isolation and purification of lecithin is almost as described in that early
paper.
The role of phospholipids as antigens in the serologic tests for syphilis was
reviewed and summarized by Eagle1 in 1937. Up to that time no worker had
claimed success in isolating a single chemical compound possessing the necessary
antigenic property.
Within the last decade Pangbom 5 has prepared a phospholipid from beef heart
muscle which, in combination with lecithin and cholesterol, has been successfully
employed in serologic tests for syphilis. This so-called, "cardiolipin" is apparently much nearer a chemical entity than the former lipoidal extracts used as
antigens, but the intricate and time-consuming methods described for its extraction and purification have not made it practical to produce the substance in
sufficient quantities for wide-spread study and subsequent adoption in routine
serologic procedures.
The methods which follow have given satisfactory yields of lecithin and
serologically active phospholipin, have not presented any problem in reproducibility of products and can be carried out in a period of approximately seven days.
EXTRACTION OF PHOSPHOLIPIN AND LECITHIN
Five beef hearts, weighing 4 to 5 pounds each, are used for the preparation of
phospholipin and lecithin. The organs are removed immediately after the
animals have been slaughtered. They are opened, drained rapidly and frozen
with crushed carbon dioxide. (No more than thirty minutes should elapse
between the death of the animals and the freezing of the tissue.) The hearts
are kept in the presence of solid carbon dioxide, while the fat, connective tissue,
blood and blood vessels are removed as completely as possible. The remaining
heart tissue is cut into pieces while cooled with solid carbon dioxide. The cooled
tissue is finely ground by repeated passages (8 times) through a meat grinder
(one-eighth inch openings).
The wet, minced beef heart tissue, about 4 kilograms, is mixed with 3 liters of
* Received for publication, April 9, 1948.
t Present address: Department of Biochemistry, Clinical Laboratory, Veterans Administration Hospital, Coatesville, Pennsylvania.
625
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LETONOFF
acetone per kilogram of tissue in a six-gallon crock. The mixture is stirred
continually with an electric stirrer (1500 r.p.m. or more) for about twenty hours',
then filtered by suction. The tissue is washed once on the filter paper with
acetone; this extraction with acetone is repeated once. The acetone-extracted
tissue, after being well drained, is then transferred to a special Eppenbach Q V
6-2 Colloidal Mill containing 3.5 liters of absolute methyl alcohol per kilogram of
original wet tissue. Larger quantities of tissue cannot be satisfactorily extracted
in a mill of this size. The mixture is ground and extracted for about four hours
and then filtered by suction. The colloidal mill and the tissue on filter paper are
washed several times with absolute methyl alcohol; then, the washed tissue is
discarded.
To the clear, freshly filtered methyl alcohol extract is added a 20 per cent
aqueous solution of barium chloride until no further precipitate is formed. The
mixtures are allowed to stand overnight in the refrigerator. The supernatant
extract is separated from the insoluble barium phospholipin by decantation and
centrifugation and retained for the preparation of lecithin as described below.
PURIFICATION OF PHOSPHOLIPIN
The crude barium phospholipin precipitate is collected in 250 cc. centrifuge
cups (40 cc. per cup) and washed once with 100-150 cc. of absolute methyl
alcohol. After centrifuging, 80 cc. of ether, 20 cc. of absolute ethyl alcohol and
100 cc. of half-saturated sodium chloride solution are added and mixed thoroughly
with a glass or porcelain spatula to dissolve the precipitate. The material is
then transferred to a separatory funnel; the centrifuge cups are rinsed twice with
a mixture of 40 cc. portions of ether, 10 cc. of ethyl alcohol and 50 cc. of halfsaturated sodium chloride solution, and the washings are added to the material in
the funnel. The two layers are separated without mixing, and the aqueous layer
is discarded. The ethereal solution is treated with 20 cc. of ethyl alcohol for each
80 cc. of ether, followed by the addition of half-saturated sodium chloride solution equal in volume to that of ether-alcohol mixture. The solution is shaken
vigorously for about five minutes. If an emulsion is formed, it is broken by the
addition of small portions of ethyl alcohol and ether. The layers are separated,
and .the aqueous layer is discarded. The treatment with ethyl alcohol and halfsaturated sodium chloride solution is repeated three times. The ethereal solution is then washed once more with half-saturated sodium chloride solution. The
ethereal solution of sodium phospholipin is dehydrated overnight on about 100
grams of anhydrous sodium sulfate (Na^SCM, then filtered. The filtrate is concentrated by vacuum distillation to about 200 cc. or just to the point where the
solid starts to separate from the ether. (In this and subsequent vacuum distillations the air is displaced by carbon dioxide, and the temperature of the water bath
is kept at 37 C.) The cloudy solution is poured into about 1200 cc. of methyl
alcohol with rapid mixing. The flocculent precipitate is separated by nitration,
washed twice with 100 cc. of methyl alcohol and discarded. The solution is
concentrated in vacuum to remove the ether.
To the methyl alcohol solution of partially purified sodium phospholipin is
P H O S P H O L I P I N AND
LECITHIN
027
added 20 per cent aqueous barium chloride solution until no further precipitate is
formed. The end point is readily detected by chilling the mixture in ice. After
standing overnight in the refrigerator, the precipitate is collected by centrifugation and washed once with methyl alcohol and once with acetone. To the
precipitate is added 80 cc. of ether (anhydrous); then the latter is mixed thoroughly so that all the precipitate is transformed into a stiff gel. The gel is
precipitated by the gradual addition of 80 cc. of acetone, and the ether-acetone
purification is repeated until the supernatant is colorless. Four to six etheracetone purifications are sufficient.
The barium phospholipin salt, purified by ether-acetone precipitation, is
mixed with 100 cc. of ether followed by 20 cc. ethyl alcohol and 100 cc. of halfsaturated sodium chloride solution; the whole is thoroughly mixed to dissolve the
barium phospholipin precipitate and then transferred into a separately funnel.
The centrifuge cup is washed twice with a mixture of 100 cc. portions of ether,
20 cc. of ethyl alcohol and 100 cc. of half-saturated NaCl solution, and the washings are added to the material in the funnel. The mixture is shaken vigorously
for about five minutes; the layers are separated, and the aqueous layer is discarded.
The treatment with sodium chloride solution is repeated three times, each time
with the addition of alcohol in proportion of 20 cc. for each 100 cc. of ether remaining in the funnel and 300 cc. of half-saturated sodium chloride solution.
The ethereal solution is then washed once more with the sodium chloride solution.
The colorless ethereal solution of the sodium phospholipin is dried overnight on
about 30 grams of anhydrous sodium sulfate salt, then filtered and concentrated
by vacuum distillation to about 100 cc. With rapid mixing it is then transferred
by means of pipet to 1200 cc. of absolute ethyl alcohol. (The flask is washed
with small portions of ether which are added to the alcohol.) The slighly cloudy
solution is concentrated in vacuum to about one liter. The small amount of
flocculent precipitate which separates during distillation is removed by filtration.
If the alcoholic solution of sodium phospholipin is not entirely free from color, add
an equal portion of absolute mzthyl alcohol, precipitate the phospholipin with 20
per cent barium chloride solution and repeat purification with ether-acetone precipitation described above.
The concentration of sodium phospholipin in alcoholic solution is calculated
from the phosphorous content of this solution. The phosphorous conversion
factor is 4.18 per cent. The sodium salt of phospholipin is soluble in absolute
alcohol to 3.0 mg. per cc. at 1-3 C. and to 10 nig. per cc. at room temperature.
It contains no nitrogen. The iodine number is 128-130.
PREPARATION OF LECITHIN
After removal of the crude barium phospholipin precipitate, 50 per cent
aqueous solution of cadmium chloride is added to and mixed with the clear
methyl alcohol extract until no further precipitate is formed. This mixture is
allowed to stand in the cold overnight, and the precipitate is then collected and
washed twice with absolute methyl alcohol and once with absolute ethyl alcohol.
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LETONOFF
PURIFICATION OF LECITHIN
To the 40 cc. of cadmium lecithin precipitate in centrifuge cup is added a
mixture of equal parts of petroleum ether and 80 per cent ethyl alcohol. The
solution is mixed thoroughly with a glass or porcelain spatula to dissolve the
precipitate and transferred to a separatory funnel. The centrifuge cups are
rinsed with a mixture of petroleum ether and 80 per cent ethyl alcohol. The
total volume of petroleum ether and 80 per cent ethyl alcohol is about 500 cc.
After a preliminary shaking by hand to release the air, the funnel is placed in
a variable speed electric shaker for forty-five minutes and shaken with a motion
simulating hand shaking. The alcoholic extract is separated from the petroleum
ether so that no petroleum ether is added to the alcoholic extract. The above
process is repeated four times using 500 cc. portions of 80 per cent ethyl alcohol
and adding petroleum ether to keep the volume fairly constant.
The alcoholic extracts are combined and concentrated in vacuum to about
500 cc. (This process may be started during the extraction period.) A heavy
precipitate of the lecithin cadmium salt separates as the petroleum ether is
removed. The concentrated solution is allowed to stand in the cold overnight
or longer. The precipitate is collected by centrifugation and washed once with
absolute ethyl alcohol.
The purified cadmium lecithin salt is distributed in volume of approximately
25 cc. into 250 cc. centrifuge cups where it is washed once with 100-150 cc. of
ethyl alcohol per cup. After centrifuging, 100 cc. of ether, 20 cc. of ethyl
alcohol and 100 cc. of half-saturated sodium chloride solution are added, and the
mixture is thoroughly stirred with a glass or porcelain spatula to dissolve the
precipitate. I t is then transferred to a separatory funnel, the centrifuge cups
are rinsed twice with a mixture of 40 cc. of ether, 10 cc. of ethyl alcohol and 50 cc.
of half-saturated sodium chloride solution, and the washings are added to the
material in the funnel. The two layers are separated without mixing, and the
aqueous layer is discarded. The ethereal solution is treated with 20 cc. of
ethyl alcohol for each 80 cc. of ether, then with half-saturated sodium chloride
solution equal in volume to that of the ether-alcohol mixture.
The solution is shaken vigorously for five minutes. If an emulsion is formed,
it is broken by the addition of small portions of ethyl alcohol and ether. The
layers are separated, and the aqueous layer is discarded. The treatment with
ethyl alcohol and half-saturated sodium chloride solution is repeated three times.
The ethereal solution of sodium lecithin is dried overnight on about 100 grams
of anhydrous sodium sulfate, filtered and concentrated by vacuum distillation to
approximately 100 cc. or just to the point where the solid starts to separate from
the ether; then, about one liter of absolute ethyl alcohol is added, and the mixture
is distilled in vacuum to remove the ether. The alcoholic solution of lecithin
containing a small trace of color is filtered and purified once more. The entire
process of purification (beginning with the cadmium chloride precipitation) is
repeated to obtain a colorless alcoholic solution of sodium lecithin.
The concentration of sodium lecithin in alcoholic solution is calculated from
PHOSPHOLIPIN AND LECITHIN
629
the phosphorous content of the solution. The phosphorous conversion factor
is 3.95 per cent.
The sodium salt of lecithin is soluble in absolute ethyl alcohol up to 20 mg.
per cc. at 1-3 C. and up to 100 mg. per cc. at room temperature. Nitrogen to
phosphorous ratio is 1:1. The iodine number is 78-80.
REAGENTS'
Barium chloride, 20 per cent, dissolved in water and filtered
Cadmium chloride, 50 per cent, dissolved in water and filtered
Sodium chloride, saturated solution. Half-saturated sodium chloride solution prepared
from preceding solution and filtered
Sodium sulfate, anhydrous, C.P.
Acetone, C.P.
Methyl alcohol, anhydrous, C.P.
Ethyl alcohol, absolute, U.S.P.
Ethyl alcohol, 95 per cent, U.S.P.
Ethyl alcohol, 80 per cent
Ether, anhydrous, C.P.
Ether, petroleum, B.P. 30-65 C.
DISCUSSION
Early workers, as cited by Erlandsen, recognized the necessity for preventing
enzymic activity and oxidation or other chemical changes resulting from exposure
to air by keeping the tissues in solvents as much as possible or by operating at
reduced temperatures. Solid carbon dioxide was chosen as the preservative
agent since it prevents or greatly retards all enzymic or bacterial activity and
chemical processes. Throughout the preparation these deleterious influences
were minimized by exposing the material to atmospheric conditions as little as
possible.
Care should be taken to carry out complete acetone extractions because
residual acetone-soluble substances interfere with the precipitation of the
phospholipin by barium chloride. This becomes evident from the formation of a
sticky precipitate instead of the easily dispersed flocculent precipitate of the
barium phospholipin salt.
As a means of preventing the undesirable changes in tissues, the Eppenbach Q
V 6-2 colloidal mill was used. As originally designed, the apparatus stalled
from clogging of the grinding mechanism by connective tissue. This resulted in
overheating the material and slowing down the process, thereby causing significant losses of the sought-for phospholipin. There is now available a similar
model of the colloidal mill* which incorporates a rotor and stator designed for
micro-shearing action with a special extended shaft of fluted contour for the
purpose of agitating suspended material in order to eliminate the necessity of
using an auxiliary stirrer. The later model carries other mechanical improvements to minimize the hazards inherent in such equipment.
* This mill is manufactured and sold by Eppenbach, Inc., 45-10 Vernon Boulevard, Long
Island City, New York.
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LETONOFF
Experience showed that the use of large volumes of ether in the ether-acetone
purification process results in a pure product. An increase in the number of
times that the ether-acetone purification was repeated with small volumes of
solvents did not result in a product of like purity, probably because the solubility
of the undesirable adventitious substances was decreased in the presence of
relatively large amounts of the barium salt of serologically active phospholipin.
The formation of a precipitate during the solution of the sodium salt in
absolute ethyl alcohol is due to one of two causes. If present in traces only, it is
probably attributable to residual unconverted barium salt; large amounts of
precipitate indicate that an insufficient amount of alcohol has been used. In this
case the sodium phospholipin may be recovered by dissolving the precipitate in
ether and adding it to a proportional volume of absolute alcohol.
For the chemical assay of the phospholipin and purified lecithin, any reliable
quantitative analytic method for phosphorous, nitrogen and iodine number
determination is applicable. The factors 4.18 and 3.95 for converting phosphorous content to phospholipin and lecithin are those of Pangborn.
The yields of serologically active phospholipin vary from 1 to 1.5 grams per
kilogram of wet tissue; for lecithin, 4 to 5 grams per kilogram of wet tissue.
The phospholipin prepared by this method differs from the cardiolipin of
Pangborn in that it is less soluble in absolute ether and absolute alcohol, and it is
not precipitable as the cadmium salt. Weil and Ritzenthaler 6 also found that
the active antigen factor is not precipitable by cadmium chloride.
Assay of the six lots each of the phospholipin and purified lecithin prepared as
described above in 1945 and 1946 by the author at the Venereal Disease Research
Laboratory, U. S. Marine Hospital (USPHS), Staten Island, New York, has
shown satisfactory and reproducible serologic reactivity in the complementfixation procedures employed at that time in the same laboratory. Harris and
his associates have reported the satisfactory use of these lipoid antigens in
microflocculation tests. 3 ' 4
The sodium salts of the phospholipin and purified lecithin in absolute ethyl
alcohol solution preserved in brown bottles have remained stable for more than
two years.
Phospholipin and purified lecithin, which from preliminary tests appear to be
chemically and serologically identical with those obtained from beef heart, have
been isolated from other animal tissue (liver, kidney, brain) and from some
plants.
The term cardiolipin, therefore, appears to be too restrictive; at the present
time, the substance prepared by this method is best referred to by the general
name of phospholipin.
SUMMARY
A rapid method for the preparation of serologically active phospholipin and
purified lecithin is described. The method gives satisfactory yields of purified
lecithin and serologically active phospholipin. The lipoid serologic antigens
PHOSPHOLIPIN AND LECITHIN
631
prepared by this method are chemically pure and readily reproducible; the
process can be carried out in a period of approximately seven days.
REFERENCES
1. E A G L E , H A R R Y : T h e Laboratory Diagnosis of Syphilis. St. Louis: C. V. Mosby Co.,
1937,440 p p .
2. ERLANDSEN, A.: Untcrsuchungcn uber die lecithinartigen Substanzen des Myocardiums
undderquergcstreiftenMuskeln. Ztschr. f. physiol. Chem., 51:71-155,1907.
3. H A R R I S , A., ANDMAHONEY, J . F . : Cardiolipin and purified lecithin as reagents in syphilis
serology. Am. J. P u b . Health, 37:997-1001,1947.
4. H A R R I S , A., R O S E N B E R G , A. A., AND R E I D E L , L. M . : A microfloccualion t e s t for syphilis
using cardiolipin antigen; preliminary report. J. Ven. D i s . Inform., 27:169-174,1946.
5. PANCBORN, M . C : A simplified preparation of cardiolipin, with a note on purification
of lecithin for serologic use. J. Biol. Chem., 161:71-S2,1945.
6. W E I L , A. J . , AND RITZENTHALER, B . : Zur K e n n t i s der sogenannten Lepoidantigene.
Ueber das antigen wirksame Prinzip in alkoholischen Organextrakten. Zentralbl. f.
B a k t . (Abt. 1), 127:194-198,1932.