A new procedure for the extraction, purification and fractionation of brain gangliosides

Biochimica ‘i‘l Elsevier BBA

A

et Biophysicn Actn, Scientific Publishing

296 (I 973) 160-170 Company, Amsterdam

- Printed

in The Netherlands

56175

h’EWT PROCEDURE

FRACTIONATIOK

FOR

OF BRAIS

THE

EXTRACTION,

PLTRIl;ICATION

=\KD

GX~GI,IOSIDISS

I. A procedure for the extraction, separation and purification of brain gangliosides is described, which involves: exhaustive extraction of brain with buffered tetrahydrofuran; partition of the extract first with ethyl ether, then with distilled water; dialysis of the obtained aqueous phase, and chromatography of the dialyzed solution on silica gel column. The residue after exhaustive extraction contains all brain glycoproteins. 2. This procedure,

compared

with the conventional

chloroform-methanol

pro-

cedure, was proved to achieve a more complete extraction of ganghides with no concurrent solubilization of glJq”oteins and to guarantee the absence of any significant degradation. Thus, the present procedure is able to provide a final preparation of gangliosides free of glycoproteins, and a preparation of gl!.coproteins free of gangliosides

The procedure for the extraction and purification of gangliosides developed b, l;olch-Pi ct ul.1 and perfected by Suzuki’ has been conveniently adapted by Brunngraber ct ~1.:~for separating brain gangliosides from glycoproteins and glycosaminoglycans. Using this procedure gangliosides (especially the most polar ones) may not be completely extracted2,3, whereas some sialic acid containing glycoproteins are solubilized by chloroform and methanol 4 ‘. Furthermore, after partitioning, the less polar gangliosides (Gh13, GMz, X-acetylneuraminic acid (SANA%)-galactose (Gal)-ceramide) partly remain in the organic plia~e~,~. Efforts were made in our laboratoq~ to devise a procedure suitable for the complete extraction of gangliosides from brain tissue and the clear cut separation of brain gangliosides from sialoglycoproteins. The most promising approach9 was obtained with the method of lipid fractionation of Trams and Lautcr”, reliable for the isolation Alblxe\-iations:

N.AS.2,

S-acctylnent-;LminIc

acid, sialic acid;

Cd,

galactose

FRACTIONATION OF GANGLIOSIDES of gangliosides, modifications

but

yielding

161

low recovery

had to be introduced

of the multisialogangliosidesll.

into the original

procedure,

which,

Several in its final

form, provided a quantitative yield and was mild enough to prevent degradation of the products1Z-14. The present work, carried out on the brain of several animals (trout, chicken, rat, rabbit, pig, ox, human), describes the adapted procedure and compares it with the conventional method of Folch-Pi et al.’ and Suzuki”. EXPERIMENTAL Chromatographic

materials

Silica gel for column chromatography (0.02-0.5 mm, 70-325 mesh ASTM), Silica Gel G for thin-layer chromatography and precoated silica gel thin-layer plates were provided by Merck A.G.; precoated polycarbonate sheets (K 511 V) were purchased from Eastman Kodak. Dowex 2-X8 resin (200-400 mesh) (Dow Chem. Co.) was prepared in acetate form according to Svennerholm15. Sol7lents, chemicals ad other jwoducts The solvents used for the extraction

and partition

of lipids from tissue were of

reagent grade quality. All solvents for column and thin-layer chromatography were redistilled before use. All other chemicals were the best commercially available. Glucosamine, galactosamine and other reference materials, except for gangliosides, were purchased from Sigma Chem. Co. ; standard amino acid from the Spinco Beckman Co. Standard gangliosides [GiM3, GM2, GMI, GDra, GDIb, GTIb, GQI”, Gga, Ggb, G7a’::‘j were prepared, as the potassium salts, from beef brain according to Tettamanti and Zambottil”. The purity of the final preparation of each ganglioside was in general higher than 93-95%, the contaminants being silica gel and other gangliosides. They were stored in a vacuum dessiccator at -IO “C. Dialysis tubing (0.25 inch width) was purchased from A. Thomas on a glass apparatus.

Co. The water routinely

used was freshly

redistilled

Adult animals were used. Pig and ox brains were removed at the slaughterhouse within 20 min after sacrifice and processed (if not frozen) in the next 30 min. The brain from smaller animals (rat, rabbit, chicken, trout) was removed immediately after sacrifice and processed. Human brain (frontal lobe), provided by the Department of Eorensic Medicine of the Medical School of Milan, was removed from cadavers, stored at 2 “C, 2 days after death. Analytical methods Total bound NANA was determined measuring free NANA liberated after hydrolysis at 80 “C in 0.05 M H,SO, for 2 h; protein-bound NANA was determined under identical conditions with I h hydrolysis. The obtained values were multiplied by the correcting factor 1.17 (total bound NANA) and 1.20 (protein-bound XANA) * The gangliosides of known structure are named according to Svennerholm16. ** The gangliosides of unknown structure (G3, Gga, Ggb, Gib) are provisionally cording to Tettamanti et ~2.“.

named

ac-

162

G. TETTAMASTI

ft d.

established with experiments reported elsewhere (S. Marchesini, I;. Bonali and (;. Tettamanti, unpublished). Fret ?;ANA, if necessary purified on Dowex z-S8 column according to SvenrlerlloIm~S, u-as determined by the method of \Varrenl”. Ganglioside bound SANA was determined according to Svcnnerholml”, after exhaustive dial!.& of the sample against distilled water (at least three days, 3 cllanges a day). Total phosphorus and lipid bound phosphorus was determined according to Zilversmit and Davis (ref. 20) ; nitrogen by the metllod of I(jieldahl-Sessler”l. Glucosamine and galactosamine, liberated from the sample 1)~ Itydrolysis in 1.5 M HU at IIO Y‘ for 5 II, \vere _ separated and quantitated (as free amino group, ninhydrin reaction) with the aid of tllc Beckman amino acid analyzer, Model Iynicllrom (column : hq cm Y o.cj cm; pH of the eluting buffer: 5.~8 ; temperature: 55 “C ; resin type : Beckman JJ 72). l;ucose, liberated from the sample by hydrolysis in 0.75 11 H&O, at 1x0 ‘C for z 11, was cllromatographically identified according to Rloczar ct uZ.~~,and cluantitated by tlrc mctllod of Dische and Shettles2”. The amino acid analysis was performed with the Beckman amino acid analyzer, Model Unichrom, using the standard manifacturer’s conditions. Prior to analbrsis tile sample was hydrol\zcd in 6 M HCl at IIO 'C for zo h in vials scaled under S,. Thin-layer ciu-omatography of gangliosides was performed under the following conditions: (a) precoated silica gel plates or freshly prepared silica gel plates (la>,er thickness, 250 pm, llcatcd 2: 11 at 120 "C before use) stored, when not in use, in a dessiccator over (‘aC1,; (b) solvent systems : I, n-propanol-32(j!;, ammonia-water (6:2: I, yh/v) (2 11); IT, cliloroforln-metllanol-2.5 M KH,OH (60:35:S, v/\-/v) (1.5 11); II I, cliloroforni~metlianoLwatcr (70 : 30 : 5, v/v/v) (I+rh II) ; (c) run temperature : between IS and 22 "C; (d) detection of the spots: anisaldellyde spray rcagent”a, or exposure

to iodine vapours.

The brain, washed from blood and freed from meninges, was homogeneousl! smashed in a mortar and stored in plastic bags at -20 “C if not immediately processcd.

In the case of big auimals dissected grossly grcy matter was used. Extractio7l of gungliosid~5 The extraction of gangliosides, generally from small amounts of brain (I g), was accomplished by the I;olch-Pi chloroform-methanol method (with the double, reversed-ratio, estraction introduced by Suzuki”) according to Brunngraber ct nl.:~and by the tetrallydrofuran method set up in our laboratoryl”. The tetrahydrofuran mcthod is, briefly, as follows: I g tissue was llomogeniaed at top speed in a steel tube (adapted to an NISI: blendor) I min with I ml 0.01 R1 potassium phosphate buffer (pII 6.8) and one further min after addition of 8 ml tetrahydrofuran. The misture XIS centrifuged at IZOOO xg for IO min at 15 ‘C. The supernatant was poured into a graduated centrifuge tube. The residue was homogenized again (2 min) with I ml above buffer and 4 ml tctrahydrofuran, and centrifuged. The pellet was extracted with the buffer-tetrahydrofuran mixture twice again. The pooled supernatants, after addition of 0.3 \.oI. eth\.l ctllcr, were vigorously shaken for 2 min, tllen centrifuged at 600 XR for 20 min at‘15 -‘C. The aqueous phase was accurately transferred into a \Tacuum flask. The organic phase, after addition of 0.1 vol. bidistilled water, was shaken and centrifuged as before. The newl!,-formed aqueous plla~ was added to the former. The

FRACTIONATION OF GANGLIOSIDES

pooled aqueous perature

163

phases were concentrated

not exceeding

35 “C) anddialyzed

under vacuum

to about

2 ml (bath

for 3 days at 2-4 “C against

tem-

I 1 bidistilled

water, changed three times a day. The dialyzed solution, which is the “crude ganglioside mixture” was lyophilized and stored, if necessary, at -IO “C. Purification,

fvactionatiolz and quantitation of gangliosides

Dry silica gel was poured into a column,

0.7 cm in diameter,

and uniformly

packed with an electric vibrator till reaching 25 cm in height. The lyophilized crude ganglioside mixture (corresponding to I g fresh tissue) :’ was dissolved with few drops of the mixture

1z-propanol-isopropanol-3z”/0

ammonia

(35 : 35 : 30, v/v/v) and poured

with a thin Pasteur pipet at the top of the dry silica gel column. After 2-3 rinsings with 3-4 drops of the same solvent, the elution was accomplished under the following conditions: solvent, the same as above; flow rate, 8-10 ml/h; temperature not exceeding IS “C ; effluent automatically collected in volumes of I ml. An aliquot from every third tube was assayed chromatographically on freshly prepared silica gel G thinlayer plates, developed with solvent system I. After thin-layer chromatographic monitoring, the tubes showing ganglioside spots were pooled, their content collected and concentrated under vacuum to 3-4 ml. The residue was dialyzed at 2-4 “C for 3 days against I 1 bidistilled water, changed three times a day. The dialyzed solution, which constitutes the “purified ganglioside preparation”, was lvophilized _ and stored, if necessary, at -10 “C. Fvactionatiofa &After silica gel column chromatography (see above) the eluates showing the same thin-layer chromatographic pattern were pooled, 5 different fractions being usually obtained (an additional fraction, containing very slow moving gangliosides,

Fraction

I

II

III

IV

w

Fig, I. Fractionation of pig brain gangliosides on silica gel column chromatography. 1:luting solvent: n-propanol-isopropanol-3z”/0 ammonia (35 : 33 : 30, v/v/v). Thin-layer chromatography of the eluted fractions was performed on freshly prepared silica gel plates with solvent system I; the spots were located by an anisaldehyde spray reagent. A and B, contaminants. * The present procedure was used for purification of the crude ganglioside either with the chloroform-methanol and the tetrahydrofuran method.

preparation

obtained

164

G.

could be obtained

from chicken,

frog and fish brain.)

as indicated

TETTAMAKTI

c’t d.

in I’ig. I. =\ known

aliquot of each fraction was submitted to thin-layer chromatography under the folowing conditions : Fraction I : freshl?. prepared thin-layer plates; first run, solvent system I followed, after complete drying, by a second run with solvent system Fractions II, III, I\: and V: precoated silica gel plates; solvent system III. In each plate known amounts

of the correspondent

pure gangliosides

11;

were spot-

ted and run as reference standards. Then the spots of both the samples and the correspondent standard gangliosides were located with iodine, scraped off and assayed for sialic acid by Svennerholm’s’5 method.

The general features

of the chloroform-methanol

and the tetrahydrofuran

me-

thods were first assessed on pig brain, then verified on the brain of other animals. E?!aluatioz of the chloroform-~ltcthallol a?zd the tctrahydrofuran w&hods & big bvnilz The data exposed in Table I show that the total content of bound SASA in the defatted residue, dialyzed aqueous phase and organic phase obtained by tllc two methods was practically identical (1242 pg and 1246 ,~g/g fresh tissue). However the

Lhalpzcd aqueous phase I’inal organic phase Ikfattetl residue Total l
content of bound NANA in the aqueous phase cantly higher (7-8’:/,) with the tetrahydrofuran fresh tissue), while that in the defatted residue with the cllloroform-metllanol method (345 il;g organic phase obtained with the tetrahydrofuran

(“gangliosidic NASA”) was signifimethod (931 yg against 855 yg/g (“glJ,coproteic NANA”) was higher against 312 kg/g fresh tissue). The method was SASA free while that

FRACTIONATION TXRLX

THE

165

II

EXTR.ICTIOS BY

OF GANGLIOSIDES

OF

THE

CHLOROFORM~METHASOL

TETRAHYDROFURAN

DEFATTED

RESIDUE

(OBTAISED

FROM

PIG

BRAIN)

PROCEDURE

Distribution of bound NAN.A in the newly formed fractions (insoluble residue, aqueous phase, errors are reorganic phase). The mean values of IO experiments with the correspondin, - standard . ported Round NAXLY was determined as indicated in the Experimental section. _____ lz?ozfnrlx.4 A“4 .Ilati,rLnl f/q I)EY g startilrgfvrsh tlsslw) (:hlorofornl~n~ethanol defatted residue Same chloroform-methanol dcfattcd residue processed by the tetrahydrofuran procedure ~nsolublc residue aqueous phase organic phase -~

___~~

____-

345 *

4.9

302 L 3.7-l 38 -L 1.4.5 0

-___

~~~~

._______

W-lb

-

_

,..,

standard giangfiosides

.,

__ AP 95

Elution

115

135

time (min)

Fig. 2. Evidence for the presence of gangliosidcs in the chloroform-methanol dcfatted residue obtained from pig brain. The chloroform~mcthanol defatted rcsiduc was extracted by the tctrahydrofuran method and the obtained aclueous phase (AI’), after clialysis, chromatographed on prccoatcd silica gel plate. Solvent: chloroform-methanol-water (60: 3.5 : 8, v/\r/v) ; run at 18 “C for I.+ h; location of the spots by an anisaldehyde spray rcagcnt. Fig. 3. Galactosamine and glucosamine present in the crude ganglioside mixture (dialyzed final aqueous phase) obtained from pig brain by the tetrahydrofuran and the chloroforn-methanol methods. The hcxosamines were scparatcd and assayed (as free amino-group) with the aid of the Beckman automatic amino acid analyzer (for the conditions used see Experimental). r, tctrahydrofuran crude gangliosidc mixture @us I @I standard glucosamine; a, tctrahydrofuran crude ganglioside mixture; 3, chloroform+methanol crude ganglioside mixture. The reproduced graphs correspond to experiments in which the crude gangliosidc mixture obtained from 1.5 g fresh tissue was processed.

166

G. TETTAMAXTI

et d.

provided by the chloroforrr-methanol method contained low but measurable amounts of NANA (46 ,~g/g fresh tissue). The amount of bound phosphorus in the dialyzed aqueous phase was significantly higher with the tetrahydrofuran than with the chloroform-methanol method (o.zo,~g against 0.05 ,~g/g fresh tissue). The content of nitrogen in the parallel fractions obtained with the two methods was practically identical. The final recoveq of all the examined parameters was excellent with both methods (over oj”;,). The defatted residue obtained from brain with the chloroform-methanol mcthodwas processed by the tetrahydrofuran metliod. The distribution of bound NIZNA in the newly obtained insoluble residue, aqueous phase, and organic phase is reported in Table II. About II “/ (38 ,~g over 345 p,g/g fresh tissue) of the NANA present in the chloroform~methano1 defatted residue was extracted with buffered tetrahvdrofuran and recovered in the novel aqueous phase. This NAKA, as shown by- the thin-layer chromatogram reproduced in I’&. 2, is linked to gangliosides, namely, GTrb and GDra (in higher amounts), GDrb and GMr (in lower amounts). \I’hen the dcfatted residue obtained with the tetrahydrofuran method was treated wit11 the chloroform-methanol procedure, no NAKA could be extracted. A number of contaminants is present in the crude ganglioside preparations. The content of phospholipicls was 4 times higher (see Table I) in the crude ganglioside preparation obtained method. The content

-~ ~_~ * Significant. ** Highlv significant.

with the tetrahydrofuran than with the chloroform-methanol of peptides, indicated by detection of amino acids (after acid

-

--

FRACTIONATION

hydrolysis),

OF GANGLIOSIDES

was practically

(0.5-0.7,~mole

167

the same in the preparations

obtained

amino acid/g fresh tissue). The presence of glycoproteins

by both methods was ascertained

by determining glucosamine and fucose. Both the chloroform-methanol and the tetrahydrofuran dialyzed aqueous phase (see Fig. 3) contained galactosamine (0.9 and 1.0 pmole/g fresh tissue, respectively) related to the presence of gangliosides. But thechloroform-methanol preparation showed a considerable amount of glucosamine (0.043 pm&/g fresh tissue) while the tetrahydrofuran preparation only traces (0.005 pmole/g fresh tissue). Fucose was never detected in either the chloroform-methanol and tlie tetrahydrofuran preparation. The purification step on silica gel column removed all contaminants. Namely phospholipids disappeared from the tetrahydrofuran crude ganglioside preparation; the glucosamine containing material from the chloroform-methanol one ; and amino acids were reduced to trace amounts. On the other hand the recovery of gangliosides after purification on silica gel column was excellent: 922 pg over 931 p-g NAXA/g fresh tissue (c)9.20/,) for the tetrahydrofuran preparation; 821 ,cg over 855 j-g (c$“;) for the chloroform-methanol preparation. Gmgliosidc $atter?a The ganglioside pattern, on thin-layer plate, of the crude ganglioside preparation obtained by the chloroform-methanol method strictly paralleled that of the tetrahydrofuran preparation. After fractionation on silica gel column five fractions were parallely obtained, each of them having the same ganglioside composition: Gkl3, GM2 and G3 in Fraction I; GNI and GDra in Fraction II, Gga and Ggb in Fraction III; GDrb and GTzb in Fraction IV; GQI and G7a in Fraction V. As shown in Table IV practically identical values for the same ganglioside were obtained, regardless of the different preparation procedure used. Ganglioside pattern in the brain of different animals The application of the chloroform-methanol

QUAXTITATIVE

PATTERN

TETRAHYDROFURAS

(.I)

OF GAKGLIOSIDES AND

OBTrlINEL)

CHLOROFORM--METHANOL

FROM

(B)

and the tetrahydrofuran

THE

RRAIS

OF DIFFERENT

methods

ANIMALS

HY

PROCEDURES

The quantitation of each ganglioside (expressed as “,A individual ganglioside XhN A on total gangliosidc X.4N.A) was performed under the conditions described in the Experimental section. The ganglioside nomenclature followed is that of Svennerholm’“; gangliosides G3, Gga, G5b and G7b, of unknown structure, are provisionally named according to Tettamanti et d.“.

__~ Gang-

Piy

.-__

liosidc

A

GM.3

1 ’

Gil12

I

0% B

A_ 0.3

0,s

0.9

0.6

Rat A

B 0.3

\

0.j

I

0.9

12.1

19.9 3.9

19.1 3.1

I.j

I.4

‘Traces Traces

HllWlfZ~

A

n

Traces

Traces

J

.4

B _~_____

0.4

0.1

0.7

0.0

0.6

0.6

20.0

IO.2

1O.j

IL.0

I2.1

IT.1

38.6 0.6

34.i 0.3

34.9 0.3

Il.0

11.6

2j

0.6

0.6

II.0

1X.-j

IX.1

25.1

28.j

28.2

0.9

IO.7

Rabbit u

3.0 0.0

7.4

0.0

7.0 0.0

)Traccs

II.4 28.6 4.0 0.0

Traces II.7

27.x 3.6 0.0

i

Traces

17.3 24.z 4.2 0.0 -

17.5

26.2 Traccb 27.4 13.7 3.6 0.0

168

CC.TETTARIAKTI

Pt d.

to the brain of trout, chicken, rat, rabbit, ox, and human gave the following results (see Tables III and IV) : (a) the crude ganglioside preparation obtained by the tetrahydrofuran method had, in all the examined animals, a higher content of bound SANA than the chloroform-methanol one (from 7-15 ‘:/odepending on the animal). Conversely the chloroform-methanol defatted residue had higher values of bound SANA than the tetrahydrofuran one; (1~)approximately 10% of the NAN,4 contained in the chloroform-methanol defatted residue was extracted by buffered tetrahydrofuran and recovered in the newly formed aqueous phase. This NASA had in all cases ganglioside nature; (c) the recovery of gangliosides (as bound NANA) after purification on silica gel column was over 97o/Ofor the tetrahydrofuran preparations, and over oq@,, for the chloroform-methanol preparations; (d) the qualitative and quantitative pattern of brain gangliosides for the same animal, obtained by the two methods was practically identical, slightly higher values of GDrb, GTrb and GQr being observed in the prep;lration obtained by the tetrahydrofuran method.

The procedure commonly used for the extraction and separation of gangliosides and sialoglycoproteins from brain is that devised by Folch-Pi et a1.l and perfected b\, Suzuki”, which uses a mixture of chloroform and methanol in different ratios as the extracting solvent (chloroform-methanol method). Generally the procedure works well. However an incomplete extraction, an incomplete revovery in the aqueous phase of gangliosides, and a partial solubilization of glycoproteins are reported2m-6 to occur with this method. These phenomena pose, especially when studying the subcellular location and physiological involvement of both gangliosides and sialoglycoproteins, not negligible problems. An alternative procedure, set up in our laboratory, employs buffered tetrahydrofuranUJ as the extracting solvent (tetrahydrofuran method). The efficiency of the new procedure in extracting and separating brain gangliosides from sialoglycoproteins was compared with that offered b!. the conventional clllorofornl~~rrlrtllanol method. The amount aqueous phase was the tetrahydrofuran NANA was surely sulted linked only

of bound NANA extracted from brain and recovered in the final systematically higher (from 8 to 15 o/O depending on the animal) with than the chloroform~methanol procedure. The nature of this gangliosidic: in fact after purification on silica gel column it rcto recognized ganglioside material and quantitatively rcco\,vred.

In addition about ro”/b of the NANA present in chloroform-methanol defattcd residue could be extracted by buffered tctrahydrofuran and recovered in the novel aqueous phase. This NANA, as shown by thin-layer chromatographic analvsis, is also linked to gangliosides, especially CDIa, GTIb and GQI. This clearly indicates that buffered tetrahydrofuran is more effective than chloroform-methanol mixtures in extracting brain gangliosides. A further advantage of the tetrahydrofuran method concerns the preparation of sialoglycoproteins. Since brain gangliosides do not contain glucosaminc while gl~-coproteins do, glucosamine can be considered a good marker for glycoproteins. \I’ell, the crude ganglioside preparation obtained from pig brain by the chloroform~mttl~anol method contained 0.043 yiM glucosamine/g fresh tissue, the galactosamine content

FRACTIONATION

r6q

OF GANGLIOSIDES

being ZI times higher. When the chloroform-methanolcrude preparation was purified on silica gel column, the content of glucosamine in the purified preparation fell down, the galactosamine/glucosamine ratio rising from ZI to about 150. A possible explanation of this behaviour, suggested also by previous reports4p8, is that the crude ganglioside preparation obtained by the chloroform-methanol method contained small amounts of glycoproteins, which were removed from gangliosides by silica gel column chromatography. traces

Conversely the tetrahydrofuran crude ganglioside preparation of glucosamine. It seems so clear that by the tetrahydrofuran

contained only method glyco

proteins remain entirely in the defatted residue. On the other hand the crude ganglioside preparation obtained by the tetrahydrofuran method contains more contaminant phospholipids than the chlorofornimethanol preparation. However these contaminants, as well as other minor substances present in both the chloroform-methanol and the tetrahydrofuran crude ganglioside preparations (peptides), are completely removed by silica gel column chromatography, purification step yielding quantitative recovery of gangliosides. Finally, the qualitative and quantitative pattern of brain gangliosides prepared from the same animal by either the chloroform-methanol and the tetrahydrofuran procedure appeared practically identical. Somewhat higher amounts of gangliosides GDra, GTrb and GQI were recorded in the tetrahydrofuran preparation; the difference, if meaningful, may be explained by the observation that the residual gangliosides present gangliosides.

in the chloroform-methanol

In conclusion,

the procedure

defatted

residue

for the preparation

are primarily

of gangliosides

multisialo-

presented

here

(tetrahydrofuran method) achieves the same excellent performances as the procedure of l~olch-Pietal.1andSuzuki2 (chloroforrnmetlianol method) with regard to the final yield of gangliosidesand the absence of appreciable degradation phenomena. In addition the tetrahydrofuran method offers some relevant advantages : complete extraction of gangliosides from brain, absence of concurrent solubilization of glycoproteins, quickier execution and lower cost. Thus the new procedure can be considered alternative and possibly better than the conventional one for studies which require the most accurate separationofgangliosides from glycoproteins. Conversely the described procedure is inferior to the conventional one if a simultaneous lipid analysis is wanted, because larger amounts of phospholipids are extracted into the crude ganglioside fraction. The suitability of the new procedure for the preparation of gangliosides from extraneural organs and pathological tissues is actually under investigation. ACKNOWLEDGEMEiSTS

This work was supported the (C.N.R.), Italy.

by a grant from the Consiglio Kazionale

delle Riccr-

IiEFEKENCES I L 3 4 5

J. Ii. E. L. D.

Volch-Pi, 11. Lees and G. H. Sloane-Stanley, ,[. f3iol. Chew., 126 (1957) Suzuki, /, Newochrvn., 12 (1965) 629-638 G. Brunngraber, B. D. Brown and H. Hof, Clin. Chim. Acta, 12 (1971) Svcnncrh~lm, Acta Chew. &mad., IO (1956) 694-696 ‘1. Booth, ,I. Neuvochrm., 9 (1962) 26j-276

497-509 r59-17o

170

G. TETTAMAKTI

ct

al.