THE SEPARATION OF SPHINGOLIPIDES BY ADSORPTION CHROMATOGRAPHY*

BY BENJAMIN WEISS

(From the Departments of Biochemistry, New York Stats Psychiatric Institute, and the College of Physicians and Surgeons, Columbia University,

New York, New York)

(Received for publication, April 20, 1956)

It, has been shown that the sphingolipide fraction is labeled during per- fusion of monkey brains with either acetate-l-U4 or octanoate-l-C14 (1, 2). To ascertain the site, or sites, of labeling, it, is necessary to separate and identify the components of the sphingolipide mixture. The early classical procedures for the isolation of individual sphingolipides from tissues of the central nervous system have depended essentially on differences in solubil- ity. For example, glycosphingosides’ (cerebrosides) have been separated from phosphingosides’ (sphingomyelins) by means of cold and hot pyridine, respectively (4, 5), or by use of a series of chloroform-methanol solutions of graded concentrations (6). Separation of the closely related glyco- sphingosides, kerasin and phrenosin, has been achieved by fractional pre- cipitation from acetone (4). Such methods are inadequate for our pur- poses; they involve lengthy procedures, require large amounts of material, do not give quantitative recovery of the individual sphingolipides, and do not insure the complete removal of other lipides, owing to the dissolving effect, of one lipide on another.

In recent, years, other techniques have been applied to the problem of lipide separation. Phosphosphingosides have been freed from glycosphin- gosides by adsorption of the latter on alumina (7), and from glycerol-con- taining lipides either by transesterification with sodium ethylate in ethanol (8) or by mild saponification with dilute aqueous alkali (9). Glycosphingo- sides have been freed from phospholipides either by precipitation from chloroform-methanol solution with an aqueous solution of trichloro- acetic acid (10) or by passage through a column of magnesium silicate (11). Taurog et al. (12) separated choline-containing from non-choline-containing phospholipides of liver by adsorption on magnesium oxide. In the present, study the last, mentioned approach was tried first without success; about 35 per cent, of the phosphorus and 43 per cent, of the choline were adsorbed

* This investigation was supported in part by research grant No. B-344(C5) from the Institute of Neurological Diseases and Blindness of the National Institutes of Health, Public Health Service.

1 The nomenclature recommended by Folch and Sperry (3) is used throughout this paper.

523

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524 SEPARATION OF SPHINGOLIPIDES

by magnesium oxide from sphingolipide mixtures dissolved in methanol- petroleum ether.

Silicic acid has been used as the adsorbent in a variety of lipide fraction- ations. Borgstriim (13) achieved the quantitative separation of phospho- lipides from mixed lipides by adsorption on silicic acid from chloroform solution and elution with methanol, and he fractionated mono-, di-, and tri- glycerides on similar columns by the use of chloroform, benzene-chloroform, and benzene solutions, respectively (14). Silicic acid columns have also been used to fractionate the plasma lipides (15), to separate fatty acids from phospholipides (16)) to isolate acetal phospholipides (17)) and to sepa- rate phosphatidyl ethanolamine, lecithin, and lysolecithin from a phospho- lipide mixture (18). The present communication adds to this list a method for the quantitative separation of small amounts of sphingolipides by chro- matography on silicic acid columns.

EXPERIMENTAL

Materials-Merck’s silicic acid and reagent grade acetone, chloroform, and methanol were used without further purification.

Preparation of Sphingolipides-A modification of the procedure of Carter et al. (19) was used in isolating sphingolipides. One-half of a fresh monkey brain weighing about 25 gm. was homogenized with 250 ml. of acetone in a Waring blendor, and the suspension centrifuged. The residue was treated successively with three 200 ml. portions of acetone and three 200 ml. por- tions of petroleum ether, the suspension being centrifuged after each addi- tion of solvent. In those experiments in which the brain had been perfused with a P-labeled compound, the homogenate in acetone was first dried at low temperature in 2racuo so that the acetone- and petroleum ether-soluble lipides could be obtained in the absence of water for other studies. The residue, dried in air, from the foregoing extractions was treated with three 200 ml. portions of boiling 95 per cent ethanol, and the suspension filtered with suction after each treatment. The precipitate which formed when the combined ethanol extracts were chilled to 5” was filtered and washed with acetone. The white powder contained 16.5 to 17.6 per cent hexose (20) and 0.81 to 1.00 per cent phosphorus (21). For purposes of comparison, sphingolipides were also isolated by the same extraction pro- cedure from 10 pounds of lipides of beef spinal cord,* and the resulting light tan powder weighed 940 gm. and contained 16.3 per cent hexose and 1.04 per cent phosphorus.

All sphingolipide preparations were examined for the presence of impuri- ties (i.e. compounds not containing sphingosine) by determination of ester

* The spinal cord lipides were obtained from Armour and Company, Chicago. The author is indebted to Dr. Norman S. Radin for suggesting this source.

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B. WEISS 525

groups (22), fatty aldehyde,” free cholesterol (23), and free hexose (Bene- dict’s solution). The qualitative ninhydrin and orcinol reactions were also applied, and in some cases the sphingolipides were subjected to purification by the method of Folch et al. (24).

Preparation of Column-A suspension prepared by thoroughly stirring 50 gm. of silicic acid in 100 ml. of chloroform-methanol (2: 1, v/v) was slowly poured into a 20 X 400 mm. column fitted with a fritted disk of coarse porosity, above which was a bit of glass wool. Entrainment of air was avoided. As solvent passed through the column, with the concomitant settling of the silicic acid, more chloroform-methanol was added until a total of 250 ml. had been used. When the level of solvent approached the surface of the adsorbent, chloroform was added in several portions totaling 200 ml. If the column had been properly prepared, after most of the chloroform had passed through, the silicic acid was uniformly translu- cent. If any areas of opacity remained, probably because of inadequate mixing of the silicic acid and chloroform-methanol, the column was rejected. A fresh column was prepared for each determination.

Attempts were made to accelerate the preparation of the column and to increase its flow rate either by mixing Hyflo Super-Cel with the silicic acid (14) or by adding the silicic acid, previously washed by decantation with chloroform-methanol, directly to the column from a chloroform suspension. Both measures diminished the resolving power of the column.

Significant amounts of silicic acid were carried into the effluent during the preparation of the column, washing with chloroform being continued until the effluent was free from residue. If silicic acid was present in the lipide fractions, as occasionally happened despite this precaution, it was removed by treating the residue obtained on evaporation with chloroform- methanol and filtering it through sintered glass.

Operation oj Column-The last chloroform wash was allowed to flow until the surface of the adsorbent was barely exposed. At this instant, the sphin- golipide sample, dissolved in 10 ml. of chloroform-methanol, was added with care to avoid turbulence in the adsorbent. The solution was allowed to enter the column until the silicic acid surface was just exposed when 10 ml. of chloroform were added. After this had drained to the adsorbent surface, 50 ml. of chloroform were added. The column was clamped into position on a constant volume fraction collector and connected to a gradient elution apparatus similar to the one described by Busch et al. (25). Meth- anol was transferred by the pressure of dried air from a reservoir containing 500 ml, to a flask which contained 400 ml. of chloroform, and in which the

a Aldehydes were determined by a method developed by Dr. J. Wittenberg and Dr. S. R. Korey, to whom the author is indebted for details of the procedure prior to publication.

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526 SEPARATION OF SPHINCiOLIPIDES

solvents were mixed by magnetic stirring. The solvent was carried to the column by a glass tube through a rubber stopper which sealed the chro- matographic tube. A 21 gauge needle to which was attached a short piece of rubber tubing with a pinch clamp, and which was inserted through the stopper, provided a manual pressure release valve. The eluting solvents came in contact only with glass, and the pressure was regulated to maintain a flow rate of 40 to 45 ml. per hour. The eluates were collected in 5 ml. fractions.

Analytical Methods-The eluates were combined in groups of five and analyzed for hexose, with galactose as standard (20), and phosphorus (21). The fractions which lay within a given band, as established by these deter- minations, were combined, concentrated under a stream of nitrogen in a water bath at 50”, transferred quantitatively to tared 10 ml. volumetric flasks, and dried to constant weight. Determinations of the following were then carried out: fatty aldehyde, free amino groups, ester groups (22), hexose, phosphorus, iodine number (26), neuraminic acid by the orcinol reaction (27), choline, and nitrogen. The mercuric acetate catalyst gave erratic results and was omitted in the determination of the iodine num- ber (28). The method used for choline determination is based on the iso- lation of the reineckate from butanol solution (29) and application of the Cazeneuve reaction (30) after oxidation to chromate. Nitrogen was deter- mined by digestion with concentrated H$O, in sealed tubes at 370” for 18 hours and subsequent nesslerization. Radioactive samples were counted to a standard error of 5 per cent; all the values were corrected to in&rite thickness.

Iso,?athm of Fatty A&Is--Portions of the corresponding fractions from several chromatographic separations were obtained, evaporated under nitrogen, and hydrolyzed with 25 ml. of 1.8 N methanolic H&JO, for 5 hours (31). The fatty acids and their methyl esters were extracted with several portions of petroleum ether (b.p., 60-70”) totaling 40 ml., the solvent was removed by a stream of nitrogen, and the residue was saponi- fied by refluxing it for 30 minutes with 20 ml. of N NaOH in 85 per cent ethanol. The solution was acidified and the fatty acids were extracted with petroleum ether. The extract was dried over NafiO,, and, after removal of the solvent, the fatty acids obtained from each band were subjected to fractional precipitation from absolute, 80 per cent, 67 per cent, and 50 per cent ethanol and recrystallized three or more times from the solvent of or- igin.

Results

The analyses of the sphingolipides before chromatography indicated the absence of fatty aldehydes, free cholesterol, and free hexose, whereas the

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B. WEISS 527

ester group, ninhydrin, and orcinol reactions were positive. Application of the purification procedure of Folch et al. (24) had no detectable effect on the analyses.

In a typical chromatographic separation of a sphingolipide preparation from monkey brain, the presence of at least seven components was demon- strated (Fig. 1). Of these, four emerged from the column in well defined fractions, the smallest of which was eluted in band I by chloroform con- taining small amounts of methanol in the second 50 ml. of effluent. Band II appeared after 175 ml. of solvent had passed through the column.

PXIO - HEXOSE -

200

VOLUME OF40EO;UATE (ii$ 800

0

,

FIQ. 1. Chromatographic separation of 94.7 mg. of a sphingolipide preparation from monkey brain. A silicic acid column and gradient elution with chloroform and methanol were used. Band I was eluted with chloroform containing small amounts of methanol. The concentration of methanol was approaching 100 per cent during the elution of band V. Each sample contained 25 ml. of eluate. The phosphorus values were arbitrarily multiplied by 10 for convenience in plotting.

Although this band comprised the major fraction (Table I), its emergence was sharp and spread over a relatively small volume of effluent. Band III, the second largest fraction, followed immediately after band II and was equally sharp. There next appeared three peaks, A, B, and C, which were less well defined and could not be further resolved. They were grouped under the designation of band IV and analyzed together. Band V was most strongly adsorbed, as seen by its position and broad, plateau- like shape. It has been shown that the strength with which lipides are held by an adsorbent varies as a function of their polarity (32). During each determination, a yellow band, adsorbed at the top of the column, re- mained immobile during the development of the chromatogram. Accord- ing to weight, the five fractions accounted for an average of 78 per cent of

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528 SEPAFtATION OF SPHINGOLIPIDES

the material placed on the column, but average recoveries of 94.4 and 93.4 per cent, respectively, of hexose and phosphorus were obtained. Some of the loss represented by these values may be explained by retention of mate- rial on the column, particularly in the yellow band. Also, in pooling the samples, some eluates at the extremities of the bands were omitted. It is evident that the material which was lost in these ways contained much smaller concentrations of hexose and phosphorus than the fractions which were analyzed. A sphingolipide preparation from beef spinal cord gave results (Fig. 2) which were similar to those yielded by the preparations of monkey brain. The same number of bands was obtained in the same rela-

C””

VOLUME O??LUAT E <iti”Lq 0””

FIQ. 2. Chromatographic separation of 98.0 mg. of a sphingolipide preparation from beef spinal cord. For the procedure and meaning of the characters used in plot- ting, see the legend to Fig. 1.

tive positions; but there were differences. Band I was much larger, band IV much smaller, and hexose Peak C was absent. Bands I to V con- tained 21.0, 37.6, 14.3, 7.3, and 19.8 per cent, respectively, of the total weight recovered.

In some experiments, aliquot portions of the sphingolipide preparations were chromatographed with and without prior purification by the method of Folch et al. (24). The results, which were unaffected by the purification, render unlikely the possibility that water-soluble substances such as hexose phosphates were present in any of the fractions.

Occasionally, the solution of sphingolipides in chloroform-methanol was turbid. Such solutions were usually filtered, but the results were the same when they were applied to the column without treatment.

Most of the data reported here were obtained on sphingolipide samples

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B. WEISS 529

weighing from 60 to 100 mg. These amounts are well within the resolving power of the column. In several experiments, 200 mg. samples (4 mg. per gm. of silicic acid) were cleanly separated, but a pronounced overlapping of bands occurred when 400 mg. samples were used.

The analytical data (Table I) show that hexose was present in all bands except band V and phosphorus only in bands IV and V, and choline was absent from all bands except band V. The percentage values and molar ratios for bands I and II agree closely with the theoretical composition of

TABLE I Composition of Bands Obtained from Chromatography of Monkey Brain

Sphingolipide Mizture on Silicic Acid

Component Original mixture

Choline, %. ................. . 2.46 Hexose, y. ................... 16.04 Nitrogen, %. ................ 2.17 Phosphorus, %. ............. . 1.05 Choline-N, molar ratio ....... 0.13 Choline-P, “ “ ....... . 0.61 Hexose-N, “ “ ....... . 0.57 Hexose-P, “ “ ....... . 2.71 Phosphorus-N, molar ratio. .. . 0.21 Ester group-P, “ “ ... Fatty acid, “C. (m.p.). ......

Weight, %. ................ . ...1100 .o -

I II --

0 0 20.16 21.57

1.66 1.67 0 0 0 0 0 0 1.01 1.02

III --

0 21.44 3.24 0 0 0 0.55

0 0 0

84 101-102 84-85

4.4 38.1 24.5

Band No.

0 0.4 (a) 3.23 (b) 2.22 (c) 0 0 0.32 (d) 0.84 (e) 0.39 (f) 0.31 (8)

9.3

V

13.84 0 3.27 3.45 0.51 0.99 0 0 0.50 0

73-74,t 52-53t 13.7

* These quantities represent averages of values ranging from 1.89 to 21.5, 2.40 to 4.15, 1.95 to 2.52,0.05 to 0.50,0.16 to 1.66,0.24 to 0.68, and 0 to 0.66 for (a), (b), (c), G-0, (e), (f), and 004, respectively.

t Two fatty acids, melting at the points indicated, were obtained from this band.

conventional glycosphingosides. The data for band III indicate that it consisted of a glycosphingoside which contained 2 atoms of nitrogen per mole of hexose, and those for band V are in close agreement with the theo- retical composition of phosphosphingosides. The widely variable data from band IV are in accord with the chromatographic findings (Figs. 1 to 3) which show that a mixture of substances was present in this fraction. The substances which reacted with orcinol and ninhydrin in the original prepa- ration were present in band IV, these tests being negative in the other bands. The presence of neuraminic acid, indicated by the orcinol reaction, was also suggested by the observations that a brown precipitate formed when aliquots of the material in band IV were heated with 16 per

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530 SEPARATION OF SPHINGOLIPIDES

cent H&O, and that the solution remained colorless with the formation of a fine, white precipitate in the presence of 0.2 N NaOH (33). In most ex- periments, ester groups were found in band IV in significant amounts (Ta- ble I). The other bands showed at the most a very slight ester group re- action. The average ester group-phosphorus molar ratio in band IV from beef spinal cord preparations was much smaller (0.06) than that from monkey brain sphingolipides. The average ratios of total band V to total band IV phosphorus were 1.78 and 3.03 for monkey brain and beef spinal cord preparations, respectively. In agreement with the analyses of the total sphingolipides,‘no fatty aldehydes were detected in any of the bands.

FIQ. 3. Chromatographic separation of 76.2 mg. of a CP-labeled sphingolipide preparation from monkey brain. Radioactivity in total counts per minute is shown by X, and plotted according to the scale on the right vertical axis. For the pro- cedure and meaning of the other characters used in plotting, see the legend to Fig. 1.

The average iodine number of the sphingolipide preparations was 31.8. The iodine numbers of the five bands, which were all fairly close to this value, gave no indication of the separation of a fraction with a consistently high or low degree of unsaturation. The theoretical iodine numbers of the glycosphingoside, kerasin, and the phosphosphingoside, lignocerylsphingo- sylphosphorylcholine, are 31.3 and 30.5, respectively.

In an attempt to identify further the compounds present in the various fractions, the fatty acids were isolated from all of the bands except band IV. Bands I and III each yielded a fatty acid which crystallized from 67 per cent ethanol. Band II gave a fatty acid which crystallized from absolute ethanol. Two fatty acids were derived from band V, one crystallizing from 80 per cent and the other from 50 per cent ethanol. The limited amounts of these fatty acids precluded any examination of them beyond the deter-

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B. WEISS 531

mination of their melting points (Table I). No conclusions can be drawn from such data, but the results are in accord with the assumption that the fatty acids had the following identities: Bands I and III, lignoceric; band II, cerebronic; and band V, arachidic (higher melting of the two present). The reported melting points of these fatty acids are 83-84”, IOO-lOl”, and 74-76”, respectively. No identity is suggested for the acid from band V with a melting point of 5233”. Its lead salt melted at 99-101”.

Labeled sphingolipide preparations from monkey brains which had been perfused with acetate-l-Cl4 or octanoate-l-Cl4 were purified by the pro- cedure of Folch et al. (24) and chromatographed. In a typical experiment with sphingolipides from an acetate-perfused brain, one peak of radioactiv- ity coincided with phosphorus peak (A) of band IV, another larger peak coincided approximately with the hexose peak (C) of band IV but over- lapped band V, and a third small peak was over band V (Fig. 3). Similar results were obtained with labeled sphingolipides from brains perfused with octanoate, except that a single peak of radioactivity was eluted; it coincided with the hexose peak (C) of band IV. The possibility that the radioactiv- ity of the sphingolipides was the result of contamination by the perfused labeled fatty acid was ruled out by an experiment in which labeled octanoic acid was added to unlabeled sphingolipides. When this preparation was chromatographed, all the radioactivity emerged with the glycosphingoside of band I.

DISCUSSION

The data obtained in this investigation give conclusive evidence that three glycosphingosides were separated in bands I, II, and III, and that two species of phosphosphingoside were present in band V; they give strong, though not conclusive, support to the assumption that the compounds in bands I and II are kerasin and phrenosin, respectively. The presence of 2 atoms of nitrogen per mole of hexose in band III indicates that it consists of a hitherto unrecognized glycosphingoside. Since this compound gave no ninhydrin reaction, it is reasonable to assume that the 2nd nitrogen atom may be present as an acetylated amino group attached to the carbohy- drate portion of the molecule.

The data for band IV from monkey brain indicate that it contains at least one phosphorus-containing and two carbohydrate-containing com- pounds. The positive orcinol reaction suggests that the carbohydrate was present in gangliosides, which are reported to contain no free amino group and to yield on hydrolysis 5 moles of hexose, 2 moles each of fatty acid, sphingosine, and neuraminic acid, and 1 mole of acetylated hexosa- mine (33). The presence in band IV of a free amino group, shown by the ninhydrin reaction, and the absence of choline remain to be explained. It

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532 SEPARATION OF SPHINGOLIPIDES

would be possible to account for both findings by the assumption that the phosphorus-containing compound was a cephalin which had not been re- moved in the preparation of the sphingolipides. This explanation is ren- dered unlikely by the ester group-phosphorus molar ratios, which were zero in some experiments in which the usual amounts of phosphorus were pres- ent, and which appeared to vary to some extent with the concentration of hexose-containing material. The findings could be explained by the pres- ence of a sphingolipide containing phosphorus, a free amino group, and no choline. These requirements would be satisfied by sphingosyl phosphate or analogues of sphingomyelin containing ethanolamine or serine such as the cephalin B of Brante (34), or possibly the akali-stable, choline-free frac- tion described by Dawson (35). Compounds such as sphingosyl phosphate, psychosine, and ceramide, which may be intermediates in the synthesis of sphingolipides, have not been found in the brain, and no direct evidence for their presence was obtained in this investigation. Thannhauser and Boncoddo (36) found dipalmityl lecithin to be a contaminant of phospho- sphingoside preparations. No evidence for its presence was obtained in this study, since no fraction contained both choline and significant amounts of ester groups. If it was present in the sphingolipide preparations, it was probably retained on the column.

Chemical changes have been shown to occur on various adsorbents used in chromatography (37,38), including the isomerization of p-monoglyceride to the LY form on silicic acid (14). No indication of structural alterations was seen in the present studies, although it is possible that they occurred, particularly in the compounds of band IV. It was previously reported (2) that choline, fatty acids, and sphingosine, isolated from labeled sphingo- lipides, contained no radioactivity. This finding and the results of the present study suggest that the Cl4 activity of sphingolipides resides in the carbohydrate moiety. This interpretation leaves unexplained, however, the presence of activity in the phosphosphingosides of band V.

SUMMARY

A chromatographic method is described for the separation of the sphingo- lipides of the central nervous system by gradient elution with chloroform and methanol from a silicic acid column.

Sphingolipides from monkey brain and beef spinal cord gave similar chro- matographic profiles, but there were considerable quantitative differences in the size of the fractions, and a carbohydrate-containing fraction, present in monkey brain preparations, was not detected in sphingolipides from beef spinal cord.

Three glycosphingosides were separated (in bands I, II, and III) in rela- tively pure form. Two of these (in bands I and II, respectively) appeared

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B. WEISS 533

to be kerasin and phrenosin; the third contained 2 atoms of nitrogen per mole of hexose and a fatty acid which was probably lignoceric acid.

Two species of phosphosphingoside were eluted in band V. The fatty acid in one of these was probably arachidic acid.

One of the fractions (band IV) from monkey brain sphingolipides in- cluded at least one phosphorus-containing and two carbohydrate-containing components, probably gangliosides. Possible explanations of the presence of a free amino group in, and the absence of choline from, this fraction are discussed.

Most of the radioactivity of sphingolipides, labeled by perfusion of mon- key brains with acetate-l-C4 or octanoate-1-CY4, was found in band IV. Octanoate-1-CY4, added to unlabeled sphingolipides, emerged with the gly- cosphingoside of band I.

BIBLIOGRAPHY

1. Meltzer, H. L., Federation Proc., 14, 100 (1955). 2. Weiss, B., Federation Proc., 14, 482 (1955). 3. Folch, J., and Sperry, W. M., Ann. Rev. Biochem., 17, 147 (1948). 4. Rosenheim, O., Biochem. J., 8, 110 (1914). 5. Levene, P. A., J. Biol. Chem., 18,453 (1914). 6. Loening, H., and Thierfelder, H., 2. physiol. Chem., 68,464 (1910). 7. Thannhauser, S. J., and Setz, P., J. Biol. Chem., 116,527 (1936). 8. Klenk, E., and Rennkamp, F., Z. physiol. Chem., 267, 145 (1940). 9. Thannhauser, S. J., Benotti, J., and Boncoddo, N. F., J. Biol. Chem., 166, 677

(1946). 10. Uzman, L. L., Arch. Biochem. and Biophys., 46, 149 (1953). 11. Radin, N. S., and Brown, J. R., Federation Proc., 14,266 (1955). 12. Taurog, A., Entenman, C., Fries, B. A., and Chaikoff, I. L., J. Biol. Chem., 166,

19 (1944). 13. Borgstriim, B., Acta physiol. Stand., 26,101 (1962). 14. Borgstrom, B., Acta physiol. Stand., 26, 111 (1952). 16. Fillerup, D. L., and Mead, J. F., Proc. Sot. Exp. BioZ. and Med., 83,674 (1953). 16. Reiser, R., and Dieckert, J. W., Proc. Sot. Ezp. BioZ. and Med., 87, 622 (1954). 17. Rapport, M. M., Lerner, B., and Alonzo, N., New York State Dept. HeaZth, Ann.

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28. Lips, H. J., J. Am. Oil Chem. Sot., 30,399 (1953). 29. Sperry, W. M., and Brand, F. C., Federation Proc., 4,104 (1945). 30. Caeeneuve, A., Analyst, 26, 331 (1900). 31. Carter, H. E., Norris, W. P., Glick, F. J., Phillips, G. E., and Harris, R., J. Biol.

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Benjamin WeissCHROMATOGRAPHY

SPHINGOLIPIDES BY ADSORPTION THE SEPARATION OF

1956, 223:523-534.J. Biol. Chem. 

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