THE SO CALLED ETHER-INSOLUBLE PHOSPHOLIPIDS IN BLOOD AND TISSUES*

BY R. G. SINCLAIR AND MARGERY DOLAN

(From the Department of Biochemistry, Queen’s University, Kingston, Canada)

(Received for publication, September 19, 1941)

Ever since it was introduced by Altmann in 1889 (l), acetone has been widely and successfully used for isolating and purifying the phospholipids. However, acetone alone is unable to bring about a complete precipitation of the phospholipids, especially from mixtures with fat and cholesterol. In 1910 Nerking (2) pointed out that the addition of a little magnesium chloride to the acetone made the precipitation of the phospholipids practically complete. Numerous workers since that time have confirmed the fact that acetone and magnesium chloride precipitate all phos- phorus compounds soluble in alcohol and ether, even though they may not all be lipids.

While developing his oxidative micromethod, Bloor (3) dis- covered that phospholipid, prepared from tissues, was insoluble in dry ether after it had been precipitated by acetone and magnesium chloride. If, however, the ether was saturated with water, the phospholipid slowly but completely dissolved. Later Kirk, Page, and Van Slyke (4) observed that, even though they adhered strictly to the directions prescribed by Bloor, some of the phospholipid, from both tissues and blood, did not redissolve in moist ether. They analyzed some of the ether-insoluble material; “The N:P ratio found was 0.875 (instead of the usual ratio for cephalin and lecithin of 0.438), showing this fraction to be a diaminomono- phosphatide.”

On the basis of this observation, Kirk (5) later developed a method for the determination of the ether-insoluble phospholipids in blood and tissues. And, although careful to point out that the

* Supported by a grant from the National Research Council of Canada. 659

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660 Ether-Insoluble Phospholipids

ether-insoluble phospholipids did not consist entirely of sphingo- myelins, he felt sure that the latter, if present, were included in this fraction. The lecithins and cephalins were believed to be extracted by the moist ether. Using the Roman method, Kirk determined the lecithins by their choline content and estimated the cephalins by difference. Kirk (6) has applied his method in an extensive study of the comparative amount of the three types of phospholipids, lecithins, cephalins, and ether-insoluble phospho- lipids, in normal and pathological conditions. Dziemian (7) has used the Kirk procedure for the study of the lipids in the red cells of various animals.

Meanwhile Folch and Van Slyke (8) and Christensen (9) have demonstrated that a considerable part of the nitrogen in the pe- troleum ether-soluble fraction of the alcohol-ether extract of blood is non-lipid in nature, much of it being urea. It is obvious there- fore that the N:P ratio in such extracts is of no value as an index of the nature of the lipids present.

About 2 years ago the Kirk procedure1 was applied to a study of the changes in the lecithin, cephalin, and sphingomyelin contents of blood in conditions in which pronounced changes of the total phospholipid had been observed. With fairly pure brain sphingo- myelin, we confirmed the finding that sphingomyelin was com- pletely insoluble in moist ether after precipitation by acetone and magnesium chloride. When the method was applied to extracts of blood plasma, there was excellent agreement between duplicates and the relative proportions of the lecithins, cephalins, and sphin- gomyelins in blood plasma were more uniform from one individual to another than had been found by Kirk (6).

In due time, the solution of magnesium chloride had to be replenished. For several successive analyses the concentration of the solution used increased from one analysis to another. In these analyses, higher and much less consistent values for the ether- insoluble phospholipids in blood plasma were obtained instead of the low and uniform values found previously. We were led to suspect, therefore, a relationship between the percentage of ether- insoluble phospholipids and the amount of magnesium chloride added to the acetone to bring about precipitation. Accordingly

l With Mr. E. J. Hanna an attempt was made to apply the method of Thannhauser and Sete (10) without success.

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R. G. Sinclair and M. Dolan 661

we undertook a detailed study of this point, the results of which are described in this paper.

Kirk (5) copied Bloor (3) in prescribing that the phospho- lipids should be precipitated from petroleum ether solution with 7 cc. of acetone and 3 drops of a saturated solution of magnesium chloride in 95 per cent alcohol. However Boyd (11) for his lipid analyses on blood uses 7 cc. of acetone and 0.1 cc. of 90 per cent magnesium chloride in 95 per cent alcohol. Accordingly both con- centrations of magnesium chloride were employed.

In brief, this study has made it clear that the amount and con- centration of the magnesium chloride solution used have a pro- found effect on the accuracy and the significance of the results obtained by methods that involve precipitation of the phospho- lipids by acetone. The percentage of ether-insoluble phospho- lipids is a function, more or less linear, of the amount of magnesium chloride added. However, the source of the phospholipid is also of great importance. All other conditions being constant, the per- centage of ether-insoluble phospholipid may reach about 90 to 100 per cent of the total phospholipid of blood plasma and yet it rarely exceeds 20 per cent of the phospholipid of various tissues. The N:P ratios of the total phospholipid and of the ether-insoluble fraction indicate that the latter does not consist primarily of sphingomyelins but rather is merely a portion of the mixture of phospholipids present.

EXPERIMENTAL

Phosphorus was determined by the method of Fiske and Sub- barow (12), perchloric acid (13) being used for digestion. A Klett- Summerson photoelectric calorimeter was used. Nitrogen was determined by the micro-Kjeldahl method. Choline was deter- mined by the method of Brante (14), slightly modified.

All solvents were redistilled. Acetone was dried over drierite or anhydrous calcium chloride.

Experiments with Saturated Magnesium Chloride2

On Plasma Extracts-A suitable volume of heparinized plasma from a normal man and from a dog was extracted with about 20

2 The saturated solution of magnesium chloride was prepared as follows: 10 cc. of alcohol were pipetted into a weighed glass-stoppered flask and

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Ether-Insoluble Phospholipids

volumes of 3: 1 alcohol-ether and filtered. The proteins were washed several times with small amounts of alcohol-ether and the filtrate made up to a suitable volume. An aliquot was taken for phosphorus determination. The remainder was evaporated to dryness at 40” under reduced pressure and in a continuous small stream of nitrogen. The residue was repeatedly extracted with petroleum ether and, in the case of human plasma, by chloroform as well. The centrifuged extract was made to volume. An aliquot was taken for phosphorus determination and then suitable aliquots were pipetted into a series of 15 cc. centrifuge tubes. The petroleum ether was evaporated to 1 cc. in a stream of nitrogen. If chloroform was also present, all solvent was evaporated and the residue was dissolved in 1 cc. of petroleum ether. To each tube 7 cc. of acetone were added and then 0, 1, 2, etc., drops of the saturated magnesium chloride solution. After being vigorously stirred, the tubes were stoppered and set away in the refrigerator for at least 2 hours and generally overnight.

The tubes were centrifuged. The acetone was poured off, and the tube rinsed with 3 cc. of cold acetone. Now, 5 cc. of freshly distilled ether, saturated with water, were added and the contents of the tube were thoroughly stirred with the ether. The tubes stood for at least 10 minutes, generally longer, and then were centrifuged. The clear ether solution was carefully aspirated into a 25 X 200 mm. test-tube for phosphorus determination. Another 3 cc. of moist ether were added to each centrifuge tube, and the stirring, standing, and centrifuging were repeated. The ether was added to the first lot.

The contents of the centrifuge tube, consisting of a clear drop of

weighed. Enough MgC12.6HzO was added to approximate but not make a saturated solution, and the flask again weighed. When the salt had com- pletely dissolved, successive small amounts of MgC12+6H20 were added, until a small excess remained undissolved. Thus, the weight of MgC12. 6H20, within less than 1 per cent, required to saturate 10 cc. of alcohol was determined. This method avoided the uncertainty of determining the concentration by evaporating and drying a portion of the saturated solu- tion. At the prevailing room temperature, 10 cc. of alcohol dissolved 9.40 gm. of MgC12.6H20. From the total weight and the specific gravity, it, was found that 1 cc. of the saturated solution contained 596 mg. of MgC12.6Hz0. Since the pipette used delivered 56 drops per cc., each drop carried 10.7 mg. of MgC12.6HzO.

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R. G. Sinclair and M. Dolan 663

water with a whitish layer on top, were transferred with several volumes of hot alcohol to a 25 X 200 mm. test-tube for combustion.

The results are shown in Figs. 1 and 2. The total acetone- insoluble phospholipid (A. I. P.) is the sum of the phosphorus in the ether-soluble and ether-insoluble fractions (E. I. P). Both the A. I. P. and the E. I. P. are expressed as a percentage of the total lipoid P, as determined on an aliquot of the petroleum ether extract. Thus values above 100 per cent are due to the summation of experimental errors.

HUMAN PLASMA

.I .2 .3 .4 Impi

A .l 4.3 6 9

ace.

FIG. 1. Effect of amount and concentration of magnesium chloride solu- tion on solubility of phospholipids in acetone and in moist ether. Experi- ment with human plasma. Each aliquot contained 0.0629 mg. of phos- phorus. A. I. P., acetone-insoluble phospholipid; E. I. P., ether-insoluble phospholipid. Dash lines, saturated magnesium chloride; solid lines, 30 per cent magnesium chloride solution.

On Phospholipids Isolated from Ox Serum and from Rat Tissues- Rat organs were ground with crushed glass and extracted by re- fluxing twice with 95 per cent alcohol and once with ethyl ether. The combined extracts were evaporated to dryness below 50” under reduced pressure. The residue was thoroughly extracted with ether. After removal of the suspended ether-insoluble ma- terial by centrifuging, the ether solution was concentrated in a stream of nitrogen and several volumes of acetone were added. No magnesium chloride was added. The acetone-insoluble frac- tion was redissolved in ether and reprecipitated with acetone.

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664 Ether-Insoluble Phospholipids

This crude mixed phospholipid was then dissolved in ether and aliquots taken for weighing and analysis and for the precipitation experiments. The ethyl ether was evaporated and the residue taken up in 1 cc. of petroleum ether.

The nitrogen and phosphorus analyses are given in the legends to Figs. 3,4, and 5 which show the yields of A. I. P. and of E. I. P. with increase in the amount of saturated magnesium chloride solu- tion. It is quite obvious that the yields of E. I. P. from rat tissue phospholipids were much smaller than with human and dog plasmas. However, they were all alike in that the percentage of

DOG PLASMA

I I I I I I I I I

.I .2 3

.3 .4 6

.s .6 .7 9 12dw

-8cc

FIG. 2. Experiment with dog plasma. Each aliquot contained 0.0663 mg. of phosphorus. A. I. P., acetone-insoluble phospholipid; E. I. P., ether-insoluble phospholipid. Dash lines, saturated magnesium chloride; solid lines, 30 per cent magnesium chloride solution.

E. I. P. increased with increase in the amount of magnesium chloride.

It is interesting that the total acetone-insoluble lipid of brain

was the only tissue preparation to give a measurable amount of E. I. P. in the absence of any magnesium chloride. And yet, with magnesium chloride, rat liver phospholipids gave practically the same yield of E. I. P. as did the acetone-insoluble lipids of the brain. Removal of the “protagon” that settled out of the brain lipids on standing in ether solution in the cold materially decreased the percentage of E. I. P. (cf. Figs. 4, B and 5).

Experiments were next carried out with phospholipid isolated

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R. G. Sinclair and M. Dolan 665

RAT /KIDNEY IPL. I I

FIG. 3, A. Experiment with phospholipid isolated from rat kidneys. Analysis, N 3.11, P 3.38; N:P 2.04. Each aliquot contained 0.0639 mg. of phosphorus.

FIG. 3, B. Experiment with phospholipid isolated from rat hearts. Analysis, N 2.25, P 3.27; N:P 1.52. Each aliquot contained 0.0508 mg. of phosphorus. A. I. P., acetone-insoluble phospholipid; E. I. P., ether- insoluble phospholipid. Dash lines, saturated magnesium chloride; solid lines, 30 per cent magnesium chloride solution.

RAT LIVER PL. RAT BRAIN -ACETONE INSOLUBLE

FIG. 4, A. Experiment with phospholipid isolated from rat livers. An-

alysis, N 2.45, P 3.08; N:P 1.76. Each aliquot contained 0.0500 mg. of phosphorus.

FIG. 4, B. Experiment with total acetone-insoluble lipids isolated from rat brains. Analysis, P 2.62. Each aliquot contained 0.0434 mg. of phos- phorus. A. I. P., acetone-insoluble phospholipid; E. I. P., ether-insoluble

phospholipid. Dash lines, saturated magnesium chloride; solid lines, 30 per cent magnesium chloride solution.

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666 Ether-Insoluble Phospholipids

from ox serum, and thus still more comparable with that from tissues. Except for the extraction of the serum with 3: 1 alcohol- ether, the method of isolation of serum phospholipids was the same as that used with tissues. Unpublished data show that the pre- cipitation of serum phospholipids by acetone without magnesium chloride was less complete than in the case of tissue phospholipids.

The results of the experiments with ox serum phospholipid are shown in Figs. 6 and 7. It is quite clear that the behavior of the isolated phospholipid is essentially the same as in plasma extracts.

.I 3

.2 .3 .4 .5 .6 .7 .8cc 6 9 124rops

FIG. 5. Experiment with the ether-soluble fraction of the acetone- insoluble lipids isolated from rat brains. Analysis, P 3.28. Each aliquot contained 0.0527 mg. of phosphorus. A. I. P., acetone-insoluble phospho- lipid; E. I. P., ether-insoluble phospholipid. Dash lines, saturated mag- nesium chloride; solid lines, 30 per cent magnesium chloride solution.

Experiments with 30 Per Cent Magnesium Chloride Solution3

These experiments were carried out concurrently with those already described. The yields of A. I. P. and of E. I. P. are shown in Figs. 1 to 7.

These experiments with 30 per cent magnesium chloride show that the alcohol which is used as a vehicle tends to inhibit the precipitation of phospholipids.

In the two instances in which it was tried (Fig. 3), 0.05 cc. of 30 per cent magnesium chloride in 7 cc. of acetone produced maximum

8 The 30 per cent solution was prepared by dissolving 15 gm. of MgClz. 6H10 in 95 per cent alcohol and bringing to 50 cc.

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R. G. Sinclair and M. Dolan 667

precipitation, although in one case it was only 90 per cent com- plete. In general it may be said that with volumes up to 0.2 CC.

OX SERUM PHOSPHOLIPID

.I .2 3 .4 .5 .6 .l .a< c. 3 6 9 12 drops

FIG. 6. Experiment with phospholipid isolated from ox serum. Analysis, N 2.80, P 1.90; N:P 3.27. Each aliquot contained0.0501 mg. of phosphorus. A. I. P., acetone-insoluble phospholipid; E. I. P., ether-insoluble phospho- lipid. Dash lines, saturated magnesium chloride; solid lines, 30 per cent magnesium chloride solution.

OX SERUM PHOSPHOLIPID

100 .___.. -.c -w.... . . . . -4I.P

I----L

1

.I .2 3 6

.3 .4 .5 .6 .l Jc L. 9 IZdraps

FIG. 7. Experiment with phospholipid isolated from ox serum. Analysis, N 2.57, P 2.25; N:P 2.53. Each aliquot contained 0.0366 mg. of phosphorus. A. I. P., acetone-insoluble phospholipid; E. I. P., ether-insoluble phospho- lipid. Dash lines, saturated magnesium chloride; solid lines, 30 per cent magnesium chloride solution.

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668 Ether-Insoluble Phospholipids

the effect of the magnesium chloride is dominant; above 0.2 cc. the inhibitory effect of the alcohol becomes dominant. At the same time, the percentage of E. I. P., in that precipitated, steadily increases with the increase in amount of magnesium chloride added, even though the actual amount increases and then declines. Con- sequently, with about 0.6 cc. all of the phospholipid precipitated is insoluble in moist ether.

In the case of the tissue phospholipids, the yield of E. I. P. depends only on the amount of magnesium chloride added; the concentration, at least as between 30 per cent and saturated, has no effect. With plasma phospholipids, on the other hand, the differences in the percentage of E. I. P. with the same amount of magnesium chloride are quite considerable. There does not seem to be any explanation for these differences or their irregularity.

N : P Ratios of Plasma Extracts and of Ether-Insoluble Phospholipids

These experiments were carried out in order to determine whether or not the separation of phospholipids into the ether- soluble and ether-insoluble fractions actually produced a chemical fractionation.

As stated earlier, the determination of the N : P ratio on ordinary blood extracts is useless. However, Folch and Van Slyke (15) have introduced the method of precipitating the proteins and lipids with colloidal iron and magnesium sulfate, washing with water to remove non-lipid nitrogenous substances, and then ex- tracting the lipids with alcohol and ether. The Folch-Van Slyke method was used on one sample each of dog and ox plasma. The alcohol-ether extract was evaporated to dryness at 40’ in a stream of nitrogen and the residue was extracted with petroleum ether. Aliquots of this extract were taken for nitrogen, phosphorus, and choline analyses. Another aliquot, containing 0.675 mg. of lipoid P in the experiment on ox serum and 1.428 mg. of lipoid P in the experiment on dog plasma, was evaporated to 4 cc. and then 28 cc. of acetone and 48 drops of saturated magnesium chloride were added. The same procedure as that described above was followed, all volumes being multiplied by 4. The E. I. P. was also analyzed for nitrogen and phosphorus.

The results are given in Table I. The N : P ratios of the purified lipids show that, at the most, 47 per cent of the phospholipids of

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R. G. Sinclair and M. Dolan 669

the ox serum and 35 per cent of the phospholipids of the dog plasma consisted of sphingomyelins. And yet, on precipitation with acetone and magnesium chloride, 100 and 89 per cent, respec- tively, of the phospholipids were insoluble in moist ether. Clearly, the E. I. P. did not consist exclusively or mainly of sphingomyelins. Indeed, the N:P ratio of the E. I. P. was actually lower in both instances than the N : P ratio of the total phospholipids.

It should perhaps be pointed out that extraction by the Folch- Van Slyke method followed by solution in petroleum ether resulted

TABLE I N:P Ratios of Plasma Extracts and of Ether-Insoluble Phospholipids

Method of extraction

mg. WJ. m@. Pm cent

Ordinary alco- hol-ether. Ox serum 3.74 3.48

Folch-Van Slyke “ ‘I 3.22 2.81 8.8 1.47 0.80 101 1.37 Ordinary alco-

hol-ether.. D og plasmr 14.01 13.18 45.2 Folch-Van Slyke “ “

I I

3.98 0.88’ 33 5.89 11.91 37.2 1.35 0.80” 89 1.24

- * It is likely that one or other of these ratios is in error. The decrease

from 0.88 to 0.80 would mean an absolute as well as relative increase in the cephalin content.

Animal

Petroleum ether-soluble ether-

T

Cho- line: P N:P N:P

ratio ratio

Ether- insoluble fraction

in a significant decrease from the amount of phosphorus extracted in the usual way by alcohol-ether and then by petroleumether.

SUMMARY

1. Under the standard conditions employed (0.3 to 2 mg. of phospholipid in 1 cc: of petroleum ether and 7 cc. of acetone), acetone alone precipitated about 40 to 70 per cent of the phos- pholipid of plasma and tissues. Addition of 1 drop of satu- rated or 0.1 cc. of 30 per cent MgClt .6HzO in 95 per cent alcohol brought about complete precipitation. With more than 0.2 cc. of 30 per cent MgC1,.6HzO, precipitation again became incomplete.

2. In the absence of MgCl, .6HzO, all of the tissue phospholipid

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670 Ether-Insoluble Phospholipids

and most of the plasma phospholipid that was precipitated were soluble in moist ether. With the addition of MgC12.6Hz0, the percentage of ether-insoluble phospholipid increased in proportion to the amount of salt used. A maximum of about 20 per cent of the tissue phospholipid and about 90 per cent of the plasma phospholipid became insoluble in moist ether.

3. N : P ratios of plasma phospholipids and of the ether-insoluble fractions show that the latter do not consist solely or even mainly of sphingomyelins. The data indicate that the ether-insoluble fraction is simply a portion of the whole mixture of phospholipids.

4. These findings have an important bearing on the accuracy and significance of the results obtained with methods that involve precipitation of the phospholipids.

BIBLIOGRAPHY

1. Altmann, R., Arch. Anat. u. Physiol., Physiol. AU., 524 (1889). 2. Nerking, J., Biochem. Z., 23, 262 (1910). 3. Bloor, W. R., J. Biol. Chem., 82, 273 (1929). 4. Kirk, E., Page, I. H., and Van Slyke, D. D., J. Biol. Chem., 106, 203

(1934). 5. Kirk, E., J. Biol. Chem., 123, 623 (1938). 6. Kirk, E., J. Biol. Chem., 123, 637 (1938). 7. Dziemian, A. J., J. Cell. and Comp. Physiol., 14, 103 (1939). 8. Folch, J., and Van Slyke, D. D., J. BioZ. Chem., 129, 539 (1939). 9. Christensen, H. N., 1. BioZ. Chem., 129, 531 (1939).

10. Thannhauser, S. J., and Setz, P., J. BioZ. Chem., 116, 533 (1936). 11. Boyd, E. M., Am. J. Clin. Path., 8, 77 (1938). 12. Fiske, C. H., and Subbarow, Y., J. BioZ. Chem., 66, 375 (1925). 13. King, E. J., Biochem. J., 26, 292 (1932). 14. Brante, G., Biochem. Z., 306, 136 (1940). 15. Folch, J., and Van Slyke, D. D., Proc. Xoc. Exp. BioZ. and Med., 41,

614 (1939).

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R. G. Sinclair and Margery DolanTISSUES

PHOSPHOLIPIDS IN BLOOD AND THE SO CALLED ETHER-INSOLUBLE

1942, 142:659-670.J. Biol. Chem. 

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