- Email: [email protected]
A procedure for the quantitative isolation of brain gangliosides
97
Biochimica et Biophysics 0 Elsevier/North-Holland
Acta, 617 (1980) Biomedical Press
97-109
BBA 57489
A PROCEDURE FOR THE QUANTITATIVE GANGLIOSIDES
LARS
SVENNERHOLM
Key
May 18th,
words:
OF BRAIN
and PAM FREDMAN
Department of Neurochemistry, Psychiatric St. Jiirgen Hospital, S-422 03 Hisings Backa (Received
ISOLATION
Research (Sweden)
Centre,
University
of Giiteborg,
1979)
Ganglioside
isolation;
Ageing;
(Human
forebrain)
Summary In a systematic study of the optimal conditions for the quantitative isolation of gangliosides from brain tissue and their further purification the yield of gangliosides obtained by extraction of the tissue twice with twenty volumes of chloroform/methanol/water (4 : 8 : 3, v/v) was larger than that obtained with all other solvents tested, including tetrahydrofuran/phosphate buffer. The gangliosides were separated from other lipids by phase partition, water was added to the total lipid extract to give a final chloroform/methanol/water volume ratio of 4 : 8 : 5.6. Isolation of gangliosides from the total lipid extract with the aid of anion-exchange resins was not practical as a routine procedure on a large scale. The crude gangliosides extract was freed from low molecular weight contaminants by dialysis against water. This method was superior to the purification on gel filtration media or on anion-exchange resins, which required large columns with selective losses of gangliosides as a result. The present method has been applied to human brain, and the concentration and distribution of gangliosides in the human forebrain in infancy and old age are given.
Introduction Brain gangliosides are a large family of acidic glycosphingolipids common basic neutral tetrasaccharide moiety gangliotetraose to which from 1 to 5 sialic acids [ 11. During the last decade a large number of gangliosides with different carbohydrate moieties have been isolated structure determined. These include gangliosides with an extra
Abbreviation:
NeuAc. N-acetyheuraminic
acid.
with a are bound new brain and their N-acetyl-
98
galactosamine [ 2 1, with N-acetylglucosamine [ 3-51, several fucose-containing gangliosides [ 6,7] and oligosialogangliosides with 3-5 sialic acid residues [8,9]. In some species, particularly in the pig N-glycolylneuraminic acid has been demonstrated besides N-acetylneuraminic acid [6] and in gangliosides with a disialosyl linkage the terminal sialic acid also exists in vivo in the lacton form
[lOI. Thin-layer chromatography has proved to be invaluable in the quantitative study of the major gangliosides of brain tissue, but because of the complexity of the gangliosides, identification of the gangliosides by TLC alone can never be accepted: it requires also isolation of the proper gangliosides and determination of their structures. This is important in the study of the biological function of the gangliosides, particularly as receptors, since the receptor ganglioside can constitute only a small percentage of a ganglioside band at TLC [ 111. Determination of the ganglioside composition of tissues, cells or subcellular particles requires complete extraction of the gangliosides. The optimal conditions for the isolation of gangliosides from tissues have recently been extensively investigated by Tettamanti and coworkers [ 123, and the solvent system suggested by them, tetrahydrofuran/phosphate buffer, has already proved the solvent of choice for the extraction of complex neutral glycosphingolipids from various tissues [ 131. We have used the same principle as they, a large proportion of water in the extraction solvent, and succeeded in improving the extraction of the gangliosides. With the new procedure we have obtained larger yields of gangliosides than with any existing method. It is particularly useful for the quantitative isolation of higher gangliosides, and has been applied by us for inter alia the preparation of tetra- and pentasialogangliosides of human brain (Fredman, P., Nilsson, 0. and Svennerholm, L., unpublished results). Materials Chemicals. Sephadex G-25 fine and G-50 fine, DEAE-Sepharose CL-GB, DEAE-Sephadex A-25, QAE-Sephadex A-25 and DEAE-Dextran were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. The gels were swollen overnight in distilled water and then transferred to Biichner funnels and rinsed with distilled water until the eluate was free from carbohydrates, assayed with the orcinol reaction [ 141. At least 10 1 of water was required for 400 g of Sephadex. The anion exchanges were converted to the acetate form in a column by passing 2 M sodium acetate in water through the anion exchanger until the effluent was negative for chloride. The gels were then rinsed with distilled water and dried by letting ethanol flow slowly through the column until the volume of the gel remained constant. They were then dried by suction on a Biichner funnel. Spherosil DEA and SpherosiI XOB 015, a porous silica gel with a mean pore diameter of 125 nm, particle size 100-200 m, surface area 25 m2/g and pore volume 1 mg/g, were obtained from Rhone Poulence Industries, Division Chimie Fine, Paris, Spherosil-DEAE-Dextran was prepared as previously described [15]. 1 kg of Spherosil was poured into 2.3 1 of a 7.5% aqueous solution of DEAE-Dextran at pH 11.5 and then dried 15 h at 80°C. The DEAEDextran was cross-linked with 1,4-butanediol/diglycidyl ether (Aldrich
99
Chemical Company, Milwaukee, WI). It was used in acetate form. Precoated thin-layer plates, silica gel 60, 20 X 20 cm, were purchased from Merck AG, Darmstadt, F.R.G. Dialysis tubing (20 mm diameter) was from Union Carbide, Chicago, IL 60638. Four N-acetylneuraminic acid (NeuAc) preparations were used for the standardization of the methods: two were obtained from commercial sources, Sigma Chem. Co., St. Louis, MI 63178 and Merck AG, one was a gift from Dr. G. Strecker, Lille, France, and the fourth was prepared in our own laboratory. The molar absorbance differed less than 1% between the four samples when tested with the resorcinol method [16,17]. Tritiated GMl-ganglioside (100 000 cpm/nmol) was prepared as previously described [ 181. Organic solvents and other chemicals were of analytical quality. Tissue material. Human brains from neonatal to old age subjects were obtained by courtesy of Dr. Kerstin Bostriim, Head, Department of Forensic Medicine. Written permission to examine these brains was given by the Royal Swedish Medical Board. Brains from l&month-old cattle were from Scan Vast, Varberg, Sweden. Assay methods. The yield of gangliosides from each isolation was measured with the assay of lipid-bound NeuAc using the resorcinol method. Portions of the lipid extract, containing lo-30 nmol of NeuAc, were evaporated in a small conical tube (10 X 100 mm). Water (0.5 ml) was added and the gangliosides were solubilised by carefully mixing them on a cyclomixer or on a water bath ultrasonicator. To the unknown samples and standards of 15 and 30 nmol NeuAclO.5 ml water was added 0.5 ml of resorcinol reagent [16]. The mixture was heated for 15 min in a boiling water bath and the chromogen was extracted with 1 ml of butylacetate/butanol(85 : 15, v/v) [17]. The solvent mixture was then centrifuged for 5 min at 400 X g and the absorbance of the organic phase was read at 620 nm. The coefficient of variation was 1.0%. The ganglioside pattern was quantified by scanning the TLC plates sprayed with the resorcinol reagent diluted with an equal volume of water. The plates were heated for 5 min at 140°C and the absorbance of each ganglioside fraction was then recorded with a Zeiss KM3 chromatogram scanner at 620 nm (unpublished data). ‘The following solvents and chromatographic conditions were used: chloroform/methanol/0.25% KC1 in water (60 : 35 : 8, v/v) for 2 h, lpropanol/0.25% KC1 in water (3 : 1 and 7 : 3, v/v) for 4 h, and chloroform/ methanol/2.5 M ammonia in water (60 : 32 : 7 and 60 : 40 : 9, v/v) for 2 h. All runnings were performed at 21 * 1°C. Method Extraction of gangliosides from brain tissue. Cerebral tissue (0.5 kg) was homogenized with 1.5 1 of distilled water in a Waring blendor at a maximum speed for 2 min at +4”C. The brain homogenate was poured into 5.4 1 of methanol at room temperature under constant stirring and afterwards 2.7 1 of chloroform was added. The mixture was stirred for 30 min at room temperature and was then centrifuged at 2000 X g for 30 min. The supernatant was cleared by filtration through Celite 535. The brain residue was reextracted once by homogenization in 1.0 1 of water in the Waring blendor and then poured into 4.0 1 of chloroform/methanol (1 : 2, v/v). Centrifugation and filtration was done as before.
100
Isolation of crude gangliosides by solvent partition. The two extracts were combined in a large funnel, and 2.6 1 of water was added to give a final chloroform/methanol/water (+tissue) ratio of 1.0 : 2.0 : 1.4. The solvents were carefully mixed by turning the funnel up and down several times but shaking was omitted to prevent emulsification. When the two phases were distinctly separated (generally within 6 h), the lower phase was removed and the upper phase set aside. To the lower phase (approx. 3 1) was added 1.5 1 of methanol. After thorough shaking of the funnel 1.0 1 of 0.01 M KC1 in water was then added slowly, and carefully mixed with the extract. The funnel was left overnight. The two upper phases were combined and evaporated to dryness after addition of isobutanol to prevent foaming. Purification of the crude ganglioside fraction. The residue of the upper phases was dissolved in 500 ml of chloroform/methanol/water (60 : 30 : 4.5, v/v), and left for 24 h at room temperature. The precipitate was removed by centrifugation. The ganglioside extract was evaporated and the residue dissolved in 200 ml of water. It was dialysed against running tap water for 48 h, and then against two changes of distilled water for 24 h each. The dialysed gangliosides were evaporated and then redissolved in 200 ml of chloroform/methanol/water (60 : 30 : 4.5, v/v). Results
Extraction
a f gangliosides
From our previous studies [ 191 and a large series of preliminary experiments we found the lowest recovery of gangliosides to be that from brain white matter. Another prerequisite for a tissue to be suitable for the elaboration of a new isolation procedure is that it has a relatively high concentration of gangliosides. Therefore, we felt that the most suitable material for the isolation of gangliosides would be one containing both grey and white matter. In the experiments described below we used adult human and bovine forebrains. The homogenized brain tissue was extracted with different solvents, alcohols, chloroform/alcohol mixtures and tetrahydrofuran. We found that the yield of gangliosides was lower when extracted with alcohols alone than with alcohol/chloroform mixtures, and that chloroform/methanol mixtures extracted more efficiently than chloroform/ethanol or chloroform/propanol mixtures. Therefore, in the continued experiments we used only chloroform/ methanol mixtures and tetrahydrofuran. The chloroform/methanol ratio was altered from 2 : 1 to 1 : 4, the water content of the final extract from 5 to 25% (when possible) and the ratio brain tissue to solvent from 1 : 6 to 1 : 40. The recovery of gangliosides was highest when the ratio chloroform/methanol was between 1 : 2 and 1 : 3, and it was 2-5% lower when the chloroform/ methanol ratio was 1 : 1 or 1 : 4 (Table I). In our standard procedure for the isolation of gangliosides we selected the chloroform/methanol ratio of 1 : 2, since the content of contaminants was reduced with the higher chloroform proportion. When the chloroform/methanol ratio was 1 : 2, the optimal water content was 20%. The highest ganglioside recovery was obtained with the largest ratio between tissue and solvent (1 : 40) (Table I), but two extractions with 20 solvent vols./
101 TABLE YIELD
I OF
EXTRACTED RATIOS All
OF
extractions
Solvent
GANGLIOSIDES, WITH SOLVENT were
DETERMINED
DIFFERENT AND
triplicate
(v/v)
AS
MIXTURES
WATER
OF
IN RELATION
TOTAL
NeuAc.
FROM
HUMAN
CHLOROFORM/METHANOL TO
AND
BRAIN VARYING
TISSUE
or quadruplicate. Ratio
tissue/total
volume
water
content
(%)
pm01
Chloroform/methanol
1
2
1
20
4
2.73
Chloroform/methanol
1
4
1
20
4
2.66
Chloroform/methanol
1
2
1
20
9
2.88
Chloroform/methanol
1
4
1
20
9
2.75
Chloroform/methanol
1
2
1
20
14
2.97
Chloroform/methanol
1
4
1
20
14
2.81
Chloroform/methanol
1
2
1
20
20
2.98
Chloroform/methanol
1
2
1
20
20
3.12
Chloroform/methanol
1
2
1
40
10
2.94
Chloroform/methanol
1
3
1
40
10
2.94
Chloroform/methanol
1
4
1
40
10
2.86
Chloroform/methanol
1
2
1
40
20
3.05
Chloroform/methanol
1
3
1
40
20
3.05
Chloroform/methanol
1
4
1
40
20
2.98
Chloroform/methanol
1
2
1
40
25
3.03
twice
NeuAc/g
tissue
tissue volume increased the recovery further by 2-3%. Since it is convenient, when possible, to reduce the solvent volumes in preparative work we decided to use a 1 : 20 ratio of tissue to final extract volume and to make reextractions with half the volume of solvent but unchanged chloroform/methanol/water ratio. In repeated experiments with brain from individuals of varying age, the recovery of gangliosides was 92-96s in the first extract, 4-7s in the second extract and less than 1% in the third + fourth extracts. Assay of the extracts with thin-layer chromatography showed no significant difference in the ganglioside patterns of the extracts. On the other hand, the proportions of non-lipid substances increased with each subsequent extraction. We therefore decided on only one reextraction. In the recommended procedure the recovery of gangliosides from adult normal mammalian brain was estimated to be 99 f 1%. We found some lacton formation of the terminal sialic acid of the gangliosides with a disialosyl linkage and tried to reduce it by homogenization in a neutral or weakly basic buffer. Most of the studies were performed with a phosphate buffer of pH 7.8 with a final buffer concentration up to 0.03 M. The addition of buffer did not reduce the percentage of lactons but rendered the further processing of the ganglioside fraction more difficult, since the dialysis time and the volume of dialysate had to be increased. Homogenization in phosphate buffer instead of water had no effect on the yield of gangliosides or the ganglioside pattern. The buffer thus had no advantage. An organic buffer, Tris/maleate, had similar disadvantages as phosphate buffer. Exaction with tetrahydrofuran was performed as described by Tettamanti et al. [12]. When the brain tissue was extracted with either tetrahydrofuran or chloroform/methanol (1 : 2, v/v) with 5--8% of water [19], chloroform/ methanol extracted slightly more from grey matter, but tetrahydrofuran gave a 10% higher recovery from white matter. With the extraction method devel-
102
TABLE
II
COMPARISON AND All
OF
THE
YIELD
OF
GANGLIOSIDES
EXTRACTED
WITH
TETRAHYDROFURAN
CHLOROFORM/METHANOL determinations
were
triplicate. firno1 NeuAc/g
Tissue
tissue
Tetrahydrofuran/O.Ol (PH Cerebral
grev
Cerebral
white
matter
matter
6.8,
4
: 1, v/v)
M phoshate
buffer
Chloroform/methanol/water (4
: 8 : 3,
3.42
3.53
1.37
1.57
v/v/v)
oped the recovery of gangliosides from mammalian grey matter was 3-10% and that from white matter was lo-15% higher than with the tetrahydrofuran method, which is illustrated by one experiment in Table II. Other advantages of the chloroform/methanol method is a shorter and simpler extraction. Tetrahydrofuran is unstable, and the risk of peroxidation of the solvent and the oxidation of especially phosphoacylglycerol fatty acids, requires rapid handling of the extracts. Isolation of a crude ganglioside extract by solvent partition In preliminary experiments the gangliosides were separated from the other lipids by chromatography on DEAE gels. The procedure described by Ledeen et al. [20] was followed but the load of lipids on the gels was increased. The capacity of the resins tested, DEAE-Sepharose, DEAE-Sephadex, QAESephadex and Spherosil-DEAE-Dextran, was too low to be of interest for large scale isolation of gangliosides from brain tissue. Therefore, all lipid extracts were partitioned, and a crude ganglioside fraction was isolated. When the new extraction procedure with a large proportion of water was adopted there was no need to add extra salt at the first partition. In a previous study [19] we showed that a solvent ratio chloroform/methanol/water of 1.0 : 1.0 : O.~(V/V) gave a higher recovery of gangliocides, particularly from white matter, than the classical Folch-Pi ratio 2.0 : 1.0 : 0.75(v/v). Since we found that the optimal ratio between chloroform/methanol was 1 : 2(v/v) for extraction of gangliosides from brain tissue, we tried to keep the same ratio between chloroform/methanol during the partition. A varying volume of water was added to give ratios between chloroform/methanol/water from 1.0 : 2.0 : 1.0 to 1.0 : 2.0 : 1.4(v/v). When the proportion of water was increased further, irreversible emulsification occurred. The highest ganglioside recovery was obtained with the largest proportion of water, but the differences were small, and chloroform/methanol/water (1.0 : 2.0 : 1.0, v/v) gave 95% of the maximum value (Table III). The yield of gangliosides at the second partition of the lower chloroform phase with methanol/water (1.0 : 0.7, v/v) was not reduced by the salt content of the water, but the salt gave a more rapid and distinct separation of the two phases. Approx. 5% of the total NeuAc was recovered in the second upper phase, but more important was the different ganglioside pattern compared with
103 TABLE
III
GANGLIOSIDE SOLVENT Solvent
CONTENT
FOUND
IN THE
UPPER
PHASE
AFTER
PARTITION
USING
DIFFERENT
MIXTURES ratio
(v/v/v)
l~mol
NeuAc/g
tissue
Chloroform/methanol/water 1.0 1.0 1.0
: 2.0 : 1.0 : 2.0 : 1.2 : 2.0 : 1.4
2.19 2.26 2.31
the first partition (Table IV). Isolation of the remaining gangliosides in the lower phase by anionexchange chromatography is often more rational than to make a third solvent partition. At the second partition the solvent ratio of chloroform/methanol/water was approx. 2.0 : 1.0 : 0.7 (v/v). In small-scale experiments we used larger volumes of methanol/water to secure a solvent ratio chloroform/methanol/water of 1.0 : 2.0 : 1.4 (v/v) but the yield of gangliosides was only negligibly higher and did not warrant the large increase of upper phase volume. When normal human brains were assayed only minor amounts (less than 1%) of gangliosides remained in the lower phase, mainly GM3 and sialosylgalactosylceramide. When pathological brain tissue, containing large proportions of monosialogangliosides, was extracted a third partition should be done (Fredman, P., Nilsson, 0. and Svennerholm, L., unpublished results). Purification
of the crude ganglioside
fraction
In order to exclude evaporation of the upper phase and at the same time remove other lipids, proteins and low molecular weight impurities, serious efforts were made to attach the gangliosides to DEAE gels. Columns with 150 ml bed volumes were loaded with the upper phase of adult mammalian brain extracts, and the leakage of gangliosides was assayed in the effluent. The concentration of gangliosides in the upper phase varied in different experiments between 200 and 300 pm01 of NeuAc/l, and the same upper phase was used for
TABLE
IV
COMPARISON
OF
Ganglioside
Total
GM2 GM1
THE
GANGLIOSIDE
Molar
NeuAc:
percentage
PATTERN
AT
FIRST
partition
Second
58.4
pm01
3.4 8
26
71
4
2
GDla
23
9
GDlb
24
9
GTlb
21 2
2 _
GD3
GQ1
SECOND
of gangliosides
First
1
AND
/ml01
partition
PARTITION
104
the different gels. Spherosil-DEAE-Dextran and QAE-Sephadex had the lowest capacity, and a significant amount of gangliosides appeared in the effluent already after 1 1 of the upper phase. The concentration of gangliosides in the effluent was rather constant, 5-10% of NeuAc in the upper phase, when the columns were loaded further with 3 1 of the upper phase. Only monosialogangliosides appeared in the effluent. DEAE-Sephadex had the largest capacity of the tested gels, but GM1 appeared in the effluent already after 11. The concentration of this ganglioside was, however, very low, only 1% of the gangliosideNeuAc in the upper phase placed on the column, and it remained so low during the load of the column with a further 3 1 (approx. 750 pmol NeuAc) of the upper phase. This means that 1 mmol of ganglioside-NeuAc could be loaded on 150 ml (25 g) of DEAE-Sephadex with a loss of less than 1% of NeuAc. DEAESepharose had the same capacity as DEAE-Sephadex to retain the gangliosides (only loss of 1% ganglioside NeuAc in form of GMl) but after 600 pmol of ganglioside-NeuAc, the concentration of monosialoganglioside rapidly increased in the effluent to 20% of the ganglioside-NeuAc concentration in the upper phase. In view of these results it was evident that the crude ganglioside fraction had to be freed from salts and other non-lipid contaminants before it should be applied to an anionexchange resin. The upper phase was evaporated to dryness in a rotating evaporator after the addition of isobutanol to prevent foaming. The major portions of proteins (peptides) and a portion of low molecular weight substances were removed by redissolving the extract in chloroform/ methanol/water (60 : 30 : 4.5, v/v). The recovery of gangliosides was better than 99% in this step. The gangliosides that remained in the precipitate had the same composition as the dissolved gangliosides. Remaining peptidic material was removed by passing the ganglioside fraction dissolved in chloroform/ methanol/water (4 : 8 : 3, v/v) through a small silica gel column. All gangliosides were eluted with 10 column ~01s. of the solvent. When the evaporated gangliosides were dissolved in chloroform/methanol mixtures with ratios varying from 2 : 1 to 1 : 2 (v/v) but without water, a large portion of the higher oligosialogangliosides, GTl and G&l was not dissolved. Chloroform/methanol/ water (30 : 60 : 5, v/v) gave a lower recovery of gangliosides than the more chloroform-rich mixture used, chloroform/methanol/water (60 : 30 : 4.5, v/v). Two different procedures were used for the removal of salts and other low molecular weight substances in the crude ganglioside extract, chromatography on molecular sieves or dialysis against water. When molecular sieves were used the gangliosides were either dissolved in organic solvent (chloroform/methanol/ water, 60 : 30 : 4.5 (v/v) and the method elaborated by Wells and Dittmer [21] was applied, or they were dissolved in water and separated on Sephadex G-100. With Wells and Dittmer’s method a large portion of trisialogangliosides, and practically a.lI tetra- and pentasiologangliosides were retained on the Sephadex column. When 1 l~mol of crude gangliosides from human brain was chromatographed on 1 g of Sephadex G-25 the recovery of NeuAc was 8590%. Nor separation on Sephadex in water was successful since the crude ganglioside fraction was difficult to dissolve in a small volume of water and the dimensions of the Sephadex G-100 column had to be large but even then the separation was incomplete. These results ruled out the molecular sieve separa-
105 TABLE
V
CONCENTRATION
OF
All
of four
values
Subject’s
are means age
GANGLIOSIDES
IN HUMAN
FOREBRAIN
determinations.
N-Acetylneuraminic (wet)
weight)
Total
lipid
acid
(mmol/kg
NeuAc
fresh
recovery
in upper
phase
(%)
extract
Upper
phase
lipids
2 months
2.83
2.60
92
3 months
2.96
2.78
94
4 months
2.81
2.61
93
61
years
2.31
2.13
92
72
years
2.53
2.39
95
tion method for large-scale isolation of brain gangliosides. Dialysis against water for 72 h resulted in a ganglioside extract with only a negligible salt content and relatively small losses of gangliosides. When 100 nmol/ml water of labelled ganglioside GM1 was dialysed the losses to the dialysate was 5% when the ganglioside was dissolved in 0.5 M KC1 in water or in methanol/water (1 : 1, v/v) but only 3% when dissolved in distilled water. When crude gangliosides of the upper phase in a concentration of 10 pm01 NeuAc/ml were dialysed against distilled water for 72 h, less than 0.5% of total NeuAc was found in the dialysate. The ganglioside pattern of the dialysate was very similar to that of the original crude ganglioside fraction. In the dialysate free NeuAc was approx. 10% of the total sialic acid. Determination
of the concentration
of gangliosides
in human
brains
One half of a forebrain was homogenized and extracted as in the standard procedure. NeuAc and the ganglioside pattern were determined in the total lipid extract and in the upper phase lipids. The recovery of NeuAc, determined from the absorbance in the resorcinol method, was in the upper phase 9295% of that in the total lipid extract. Since the ganglioside pattern was exactly TABLE
VI
DISTRIBUTION All
values
mo., Gang-
OF
are given
months;
yr.,
GANGLIOSIDES
in molar
IN HUMAN
FOREBRAIN
percentage.
years.
% N-Acetylneuraminic
acid
% Ganglioside
lioside 2 mo.
3 mo.
4 mo.
61
yr.
72
Yr.
2 mo.
3 mo.
4 mo.
61
yr.
72
GM3
1
1
1
3
4
2
2
2
6
6
GD3
4
6
5
4
3
4
5
4
4
3
GM2
4
3
4
1
1
7
6
7
2
2
GM1
13
13
15
15
16
24
24
27
28
30
GDla
49
46
45
42
39
18
17
GDlh
8
GTlb
16
GQlb
4
44
20
18
9
9
22
23
17
19
30
31
5
3
5
5
7 10 2
9 10 2
8 11 1
21
21
19
19
2
2
YI.
106
the same in the two extracts the difference in NeuAc absorbance presumably depends on unspecific colour given by substances in the lipid extract. The resorcinol reaction has been criticized because it gives a false colour, mainly for a large number of sugars other than sialic acids, but when the absorbance is read at 620 nm, as in the present study, this contamination is negligible, which is evident from the fact that the same recovery of NeuAc was obtained from the adult brains, which contain several-fold higher concentrations of cerebrosides than the infant brains. The diminution of sialic acid colour occurred during the partition and not during the dialysis, which suggests that the lower phase lipid contained chromogens that reacted with the resorcinol reagent. The ganglioside pattern shows that the proportion of GMl-ganglioside in the human brain is very constant, while GDla and GM2 are higher in infancy than later in life. The portion of GDlb and GTlb increases with age so that in old people most sialic acid resides in ganglioside GTlb. Discussion During the last decade important progress has been made in the development of new procedures for the separation of gangliosides [22-251. These procedures have proved very useful in the discovery and isolation of new gangliosides [2,6,7,26], but they are of limited value for unveiling the quantitative ganglioside pattern in an organ if the gangliosides are not quantitatively extracted from the tissue. It is strange that quantitative extraction of gangliosides from tissues has received so little attention. In a previous study we succeeded in increasing the recovery of gangliosides from brain tissue by extraction with chloroform/methanol (1 : 1, v/v), instead for 2 : 1 [19], but we did not make any systemical study of the extracting capacity of the method. Tettamanti et al. [ 121, however, showed that the tetrahydrofuran method, originally elaborated by Trams and Lauter [27] for the extraction of gangliosides could be modified to a very effective procedure for the extraction, separation and purification of brain gangliosides. When one first reads the paper it is easy to get the impression that tetrahydrofuran is a better extraction solvent for gangliosides than chloroform/methanol mixtures, but the present study has shown that it is instead simply the use of more water in the extraction solvent that is important. Before Folch-Pi et al. [28] described the classical solvent mixture chloroform/methanol (2 : 1) for the isolation of brain lipids, chloroform/ methanol (1 : 2 or 1 : 3) were the most commonly used solvents for isolating gangliosides and other sphingolipids [29]. In our own first endeavour to develop a quantitative ganglioside extraction procedure chloroform/methanol (1 : 2) was found to give the best yield of ganglioside sialic acid [30]. Shortly afterwards Bligh and Dyer [31] described a rapid method of total lipid extraction and purification, which was applied to fish muscle. They found the ratio chloroform/methanol of 1 : 2 (v/v) to be best for quantitative lipid extraction, and that the water concentration in the extraction solvent should be high, approx. 20%. The tissue/solvent ratio was 1 : 3 (v/v). When we applied their extraction procedure to brain samples used in the present study we got a low recovery of all lipids, particularly of the gangliosides, due to the low solvent/ tissue ratio.
107
The adult brain tissue is a very high lipid organ and quantitative extraction of all lipids, including gangliosides, was only achieved by repeated extraction with smaller volumes of solvent or by a single extraction with 40 ~01s. solvent/ volume of tissue. We now extract the tissue twice with 20 ~01s. of solvent or, for large scale isolation, use only 10 ~01s. of solvent for the second extraction. The proportion of water is important and the highest ganglioside recovery was found empirically at a water concentration of approx. 20% (Table I). It is noteworthy that the proportion of water is the same as that found by Bligh and Dyer [31] in their chloroform/methanol procedure and by Tettamanti et al. [ 121 in the tetrahydrofuran method. In the beginning we hesitated to use the high proportion of water since we had previously found [32] the contamination of the ganglioside extract with sialic acid-containing glycoproteins and glycopeptides to be greater when fresh brain tissue was extracted with chloroform/methanol (1 : 2, v/v) compared to acetonedried tissue. Tettamanti et al. [ 121 had also shown that the tetrahydrofuran solvent extracted less of sialoglycopeptides than the conventional chloroform/methanol (2 : 1, v/v) mixture. But actual experiments showed that the contamination of the lipid extracts with proteins and peptides diminished when the proportion of water was increased to 20%, and in our experiments with 20% water in the solvent the admixture of sialic acid-containing glycopeptides never exceeded 2% of the total siahc acid content. All our efforts to attach the gangliosides of the total lipid extract directly to an anion-exchange resin were unsuccessful, and it is doubtful whether direct attachment of gangliosides from a tissue extract to DEAE-Sephadex can be recommended in any situation. We have found that for the large-scale isolation of brain gangliosides under strict quantitative conditions, solvent partition, with subsequent evaporation of the upper phase and dialysis is the best method. We got a virtually quantitative recovery of the gangliosides when we made the partition with the solvent ratio chloroform/methanol/water of 1.0 : 2.0 : 1.4 (v/v), with a subsequent repartition (Table II). Under this condition the lower phase is practically 100% chloroform [33]. A portion of other acidic lipids, sulphatides, serine and inositol phosphoacylglycerols, are partitioned in the upper phase, and contaminate the crude ganglioside extract. A similar contamination with the same acidic lipids was found with the tetrahydrofuran method [ 121. These lipids can be easily removed in the subsequent purification steps (Fredman, P., Nilsson, 0. and Svennerhom, L., unpublished data). Since Kanfer and Spielvogel [34] had reported a significant loss of gangliosides at dialysis against water, we tried to remove the contaminating impurities on molecular sieves but we failed to develop an acceptable procedure. This might seem to be in conflict with the recommendation of Ueno et al. [35], who suggested the use of Sephadex G-50 for the separation of gangliosides and low molecular weight substances. They have, however, only applied the method for mixtures of pure gangliosides and free sialic acid. For the purification of crude ganglioside extracts of the methanol/water upper phases we think instead that the method of choice is dialysis against water. In a recent study Ghidoni et al. [36] have arrived at similar conclusions as us about the behaviour of gangliosides on dialysis. The present method was found to yield higher concentrations of gangliosides
108
than any previous method. The ganglioside pattern did not undergo any large change when the recovery of gangliosides increased by lo-30%. The proportion of tetrasialogangliosides was somewhat higher than in previous studies and pentasialogangliosides were isolated for the first time from mammalian brain (Fredman, P., Nilsson, 0. and Svennerholm, L., unpublished data). A direct comparison of the present results with those reported by Tettamanti et al. [12] and Nagai et al. [14] is not possible since they did not study brains of corresponding ages, besides at which they did not describe which part of the brain they used, the proportion of the higher gangliosides increases in caudal direction of the forebrain (Ref. 37; Svennerholm, L., unpublished data). Acknowledgements The work was supported by the Swedish 627). We gratefully acknowledge the assistance
Medical Research Council of Mrs. Birgitta Dellheden.
(3X-
References 1
Sweeley,
C.C.
Mammalian
and
Siddiqui,
B.
Glycoproteins
2
Svennerholm,
3
Li. Y.-T.,
4
Whenret,
5
KoScielak.
I,.,
J.-E.
J.-E.,
Vanier,
(1973)
J.,
Biochim.
Piasek.
A.,
in The
Glycolipids.
M&son,
M&son, J.R.
(1977)
and
and
Li,
M.-T.
Y.-T. and
Biophys.
G6miak.
Glycoconjugates
Vol.
(1973)
H.,
326,
Gardas,
M.I.
Academic
J. Biol.
Svennerholm,
Acta
(Horowitz,
1. PP. 459-540.
Chem.
L. (1973)
and
Press,
248,
Pigman,
W..
New
York
248.
2634
eds.),
740-p742
J. Biol.
Chem.
2636
63-73
A.
and
Gregor,
A.
(1973)
Eur.
J. Biochem.
37.
214.-
225 6
Ghidoni,
R.,
Sonnino,
S.,
Tettamanti,
G.,
Wiegandt,
II. and
Zambotti,
V.
(1976)
J. Neurochem.
27.
511-515 7
Sonnino,
S.,
8
Ishizuka,
I. and
Ghidoni,
R.,
9
Ando.
S. and
Yu,
10
Kuhn,
R. and
Wiegandt,
11
Svennerholm, Problem
12 13
Bonali.
Chem.
F.,
252.
and
19b.
Related
Holmgren,
S.
260.
31,
947
Aspects
of
-956
279-289
256-258
Diarrheas,
J., eds.).
Marchesini,
J. Neurochem.
Acta
62474250
Z. Naturforsch.
Cholera
and
G. (1978)
Biophys.
Karger,
and
Molecular
Basel.
Zambotti,
a
Global
Health
in the press
V.
(1973)
Biochim.
Biophys.
Acta
296,
70 A.,
Slomiany,
PP. 49-75,
14
Svennerholm,
15
Tayot,
Tardy,
and
Svennerholm,
L..
19
Vanier,
Holm,
T. and
Ledeen, Wells,
R.W.. M.A.
Yu,
and
Svennerholm, eds.),
Vol.
M., R.K.
Momoi. Iwamori,
T.,
25
-4ndo.
S.,
26
Ando,
S. and
27
Trams,
E.G.
28
Folch-Pi,
(1972)
Ando,
Methods
29
Klenk,
30
Svennerholm.
31
Bligh,
Nagai,
Y.
Y.
(1978)
Chang,
N.-C.
and
Yu.
and
R.K.
E. and
Lauter,
L.J.
I., Lees,
Rennkamp. L. (1957)
and
(1978)
Dyer,
in
Acta
(1976)
R.K.
(1962)
(Witting,
L.A.,
Chem. Biochim. J.A.
and
Med.
Chem.
Vanier.
Ups,
Stand. M.-T.
Svennerholm.
in
Ellis 13,
Chromatography
Horwood,
of
Chichester
856-858
(1979)
L. (1973)
J. Neurochem.
21,
Clin.
Chim.
J. Neurochem.
Acta 21,
92.
53m~-64
1375.-1384
829-839
2, 1259-1263 Chemistry
(Whistler,
R.L.
and
BeMiller,
London Biophys.
Biophys.
Acta
Anal.
Biochem.
252.
62474250
Biophys. and 62,
J. Biochem.
Acta 528,
441,
60,
F.N.
Z. Physiol.
350-358 (1951) Chem.
l-16 Physiol.
488497
257-267
89,437-450
Acta
LeBaron,
Hoppe-Seyler’s Can.
(1978)
604-611
Acta
Biochim.
(1978)
Meath.
Sot.
(1959)
24,
M.
2. PP. 95-110,
Carbohydrate
Press,
Biochim.
F. (1941)
W.J.
and (1973)
J. Biol. M..
Roumiantzeff, Vol.
Biochemistry
Academic
S. and
Yu,
L.F.
in
Nagai.
and
J.-E.
J.-E.
(1963)
Methodology
IL
ed.),
(1959)
M&son,
Eng,
in Glycolipid
R. Acta
I.T.
J.C.
M. and
J.. Ascoli,
E.G.
and
(1976) Champaign,
R.,
Biophys.
M&son.
Dittmer,
L.
23
(Epton,
G.,
6. PP. 464474,
24
Plan.
Takki-Luukkainen, Hikansson.
M.I.
Society, 1, 42--53
P.,
Biochim.
18 20
Gattel.
L. (1957)
Svennerholm. Miettinen.
Horowitz,
J. Neurochem. M.,
Polymers
16
M.-T.,
and
Oil Chemist’s
Biological
17
21
B.L.
American L. (1956)
J.L.,
Synthetic
22
J. Biol.
in 6.
Tettamanti,
Biochim.
H. (1964)
(1979)
G.,
G. and
H. (1972)
(1977)
(Ouchterlony,
Slomiany, ed.),
R.K.
L.
Tettamanti, 160-l
Galli,
Wiegandt,
37,
912-917
J. Biol. 267,
Chem.
145-153
191,
833-841
J.M.,
109 32
Svennerholm,
L. (1956)
33
Banner,
(1910)
34
Kanfer.
35
Ueno,
36
Ghidoni,
37
Suzuki,
W.D. J.N. K.,
and
Ando. R.,
K.
Chem.
Stand.
Chem.
14,
Spielvogel,
C. (1973)
S. and
R.K.
Sonnino,
(1965)
Acta J. Phys. Yu, S. and
(1978)
694.-696
J. Neurochem.
20,
1483.-1485
J. Lipid
19,
863-871
Tettamanti.
J. Neurochem.
10,
738-761
12.969-979
G. (1978)
Res.
Lipids
13,
820-822
Biochimica et Biophysics 0 Elsevier/North-Holland
Acta, 617 (1980) Biomedical Press
97-109
BBA 57489
A PROCEDURE FOR THE QUANTITATIVE GANGLIOSIDES
LARS
SVENNERHOLM
Key
May 18th,
words:
OF BRAIN
and PAM FREDMAN
Department of Neurochemistry, Psychiatric St. Jiirgen Hospital, S-422 03 Hisings Backa (Received
ISOLATION
Research (Sweden)
Centre,
University
of Giiteborg,
1979)
Ganglioside
isolation;
Ageing;
(Human
forebrain)
Summary In a systematic study of the optimal conditions for the quantitative isolation of gangliosides from brain tissue and their further purification the yield of gangliosides obtained by extraction of the tissue twice with twenty volumes of chloroform/methanol/water (4 : 8 : 3, v/v) was larger than that obtained with all other solvents tested, including tetrahydrofuran/phosphate buffer. The gangliosides were separated from other lipids by phase partition, water was added to the total lipid extract to give a final chloroform/methanol/water volume ratio of 4 : 8 : 5.6. Isolation of gangliosides from the total lipid extract with the aid of anion-exchange resins was not practical as a routine procedure on a large scale. The crude gangliosides extract was freed from low molecular weight contaminants by dialysis against water. This method was superior to the purification on gel filtration media or on anion-exchange resins, which required large columns with selective losses of gangliosides as a result. The present method has been applied to human brain, and the concentration and distribution of gangliosides in the human forebrain in infancy and old age are given.
Introduction Brain gangliosides are a large family of acidic glycosphingolipids common basic neutral tetrasaccharide moiety gangliotetraose to which from 1 to 5 sialic acids [ 11. During the last decade a large number of gangliosides with different carbohydrate moieties have been isolated structure determined. These include gangliosides with an extra
Abbreviation:
NeuAc. N-acetyheuraminic
acid.
with a are bound new brain and their N-acetyl-
98
galactosamine [ 2 1, with N-acetylglucosamine [ 3-51, several fucose-containing gangliosides [ 6,7] and oligosialogangliosides with 3-5 sialic acid residues [8,9]. In some species, particularly in the pig N-glycolylneuraminic acid has been demonstrated besides N-acetylneuraminic acid [6] and in gangliosides with a disialosyl linkage the terminal sialic acid also exists in vivo in the lacton form
[lOI. Thin-layer chromatography has proved to be invaluable in the quantitative study of the major gangliosides of brain tissue, but because of the complexity of the gangliosides, identification of the gangliosides by TLC alone can never be accepted: it requires also isolation of the proper gangliosides and determination of their structures. This is important in the study of the biological function of the gangliosides, particularly as receptors, since the receptor ganglioside can constitute only a small percentage of a ganglioside band at TLC [ 111. Determination of the ganglioside composition of tissues, cells or subcellular particles requires complete extraction of the gangliosides. The optimal conditions for the isolation of gangliosides from tissues have recently been extensively investigated by Tettamanti and coworkers [ 123, and the solvent system suggested by them, tetrahydrofuran/phosphate buffer, has already proved the solvent of choice for the extraction of complex neutral glycosphingolipids from various tissues [ 131. We have used the same principle as they, a large proportion of water in the extraction solvent, and succeeded in improving the extraction of the gangliosides. With the new procedure we have obtained larger yields of gangliosides than with any existing method. It is particularly useful for the quantitative isolation of higher gangliosides, and has been applied by us for inter alia the preparation of tetra- and pentasialogangliosides of human brain (Fredman, P., Nilsson, 0. and Svennerholm, L., unpublished results). Materials Chemicals. Sephadex G-25 fine and G-50 fine, DEAE-Sepharose CL-GB, DEAE-Sephadex A-25, QAE-Sephadex A-25 and DEAE-Dextran were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. The gels were swollen overnight in distilled water and then transferred to Biichner funnels and rinsed with distilled water until the eluate was free from carbohydrates, assayed with the orcinol reaction [ 141. At least 10 1 of water was required for 400 g of Sephadex. The anion exchanges were converted to the acetate form in a column by passing 2 M sodium acetate in water through the anion exchanger until the effluent was negative for chloride. The gels were then rinsed with distilled water and dried by letting ethanol flow slowly through the column until the volume of the gel remained constant. They were then dried by suction on a Biichner funnel. Spherosil DEA and SpherosiI XOB 015, a porous silica gel with a mean pore diameter of 125 nm, particle size 100-200 m, surface area 25 m2/g and pore volume 1 mg/g, were obtained from Rhone Poulence Industries, Division Chimie Fine, Paris, Spherosil-DEAE-Dextran was prepared as previously described [15]. 1 kg of Spherosil was poured into 2.3 1 of a 7.5% aqueous solution of DEAE-Dextran at pH 11.5 and then dried 15 h at 80°C. The DEAEDextran was cross-linked with 1,4-butanediol/diglycidyl ether (Aldrich
99
Chemical Company, Milwaukee, WI). It was used in acetate form. Precoated thin-layer plates, silica gel 60, 20 X 20 cm, were purchased from Merck AG, Darmstadt, F.R.G. Dialysis tubing (20 mm diameter) was from Union Carbide, Chicago, IL 60638. Four N-acetylneuraminic acid (NeuAc) preparations were used for the standardization of the methods: two were obtained from commercial sources, Sigma Chem. Co., St. Louis, MI 63178 and Merck AG, one was a gift from Dr. G. Strecker, Lille, France, and the fourth was prepared in our own laboratory. The molar absorbance differed less than 1% between the four samples when tested with the resorcinol method [16,17]. Tritiated GMl-ganglioside (100 000 cpm/nmol) was prepared as previously described [ 181. Organic solvents and other chemicals were of analytical quality. Tissue material. Human brains from neonatal to old age subjects were obtained by courtesy of Dr. Kerstin Bostriim, Head, Department of Forensic Medicine. Written permission to examine these brains was given by the Royal Swedish Medical Board. Brains from l&month-old cattle were from Scan Vast, Varberg, Sweden. Assay methods. The yield of gangliosides from each isolation was measured with the assay of lipid-bound NeuAc using the resorcinol method. Portions of the lipid extract, containing lo-30 nmol of NeuAc, were evaporated in a small conical tube (10 X 100 mm). Water (0.5 ml) was added and the gangliosides were solubilised by carefully mixing them on a cyclomixer or on a water bath ultrasonicator. To the unknown samples and standards of 15 and 30 nmol NeuAclO.5 ml water was added 0.5 ml of resorcinol reagent [16]. The mixture was heated for 15 min in a boiling water bath and the chromogen was extracted with 1 ml of butylacetate/butanol(85 : 15, v/v) [17]. The solvent mixture was then centrifuged for 5 min at 400 X g and the absorbance of the organic phase was read at 620 nm. The coefficient of variation was 1.0%. The ganglioside pattern was quantified by scanning the TLC plates sprayed with the resorcinol reagent diluted with an equal volume of water. The plates were heated for 5 min at 140°C and the absorbance of each ganglioside fraction was then recorded with a Zeiss KM3 chromatogram scanner at 620 nm (unpublished data). ‘The following solvents and chromatographic conditions were used: chloroform/methanol/0.25% KC1 in water (60 : 35 : 8, v/v) for 2 h, lpropanol/0.25% KC1 in water (3 : 1 and 7 : 3, v/v) for 4 h, and chloroform/ methanol/2.5 M ammonia in water (60 : 32 : 7 and 60 : 40 : 9, v/v) for 2 h. All runnings were performed at 21 * 1°C. Method Extraction of gangliosides from brain tissue. Cerebral tissue (0.5 kg) was homogenized with 1.5 1 of distilled water in a Waring blendor at a maximum speed for 2 min at +4”C. The brain homogenate was poured into 5.4 1 of methanol at room temperature under constant stirring and afterwards 2.7 1 of chloroform was added. The mixture was stirred for 30 min at room temperature and was then centrifuged at 2000 X g for 30 min. The supernatant was cleared by filtration through Celite 535. The brain residue was reextracted once by homogenization in 1.0 1 of water in the Waring blendor and then poured into 4.0 1 of chloroform/methanol (1 : 2, v/v). Centrifugation and filtration was done as before.
100
Isolation of crude gangliosides by solvent partition. The two extracts were combined in a large funnel, and 2.6 1 of water was added to give a final chloroform/methanol/water (+tissue) ratio of 1.0 : 2.0 : 1.4. The solvents were carefully mixed by turning the funnel up and down several times but shaking was omitted to prevent emulsification. When the two phases were distinctly separated (generally within 6 h), the lower phase was removed and the upper phase set aside. To the lower phase (approx. 3 1) was added 1.5 1 of methanol. After thorough shaking of the funnel 1.0 1 of 0.01 M KC1 in water was then added slowly, and carefully mixed with the extract. The funnel was left overnight. The two upper phases were combined and evaporated to dryness after addition of isobutanol to prevent foaming. Purification of the crude ganglioside fraction. The residue of the upper phases was dissolved in 500 ml of chloroform/methanol/water (60 : 30 : 4.5, v/v), and left for 24 h at room temperature. The precipitate was removed by centrifugation. The ganglioside extract was evaporated and the residue dissolved in 200 ml of water. It was dialysed against running tap water for 48 h, and then against two changes of distilled water for 24 h each. The dialysed gangliosides were evaporated and then redissolved in 200 ml of chloroform/methanol/water (60 : 30 : 4.5, v/v). Results
Extraction
a f gangliosides
From our previous studies [ 191 and a large series of preliminary experiments we found the lowest recovery of gangliosides to be that from brain white matter. Another prerequisite for a tissue to be suitable for the elaboration of a new isolation procedure is that it has a relatively high concentration of gangliosides. Therefore, we felt that the most suitable material for the isolation of gangliosides would be one containing both grey and white matter. In the experiments described below we used adult human and bovine forebrains. The homogenized brain tissue was extracted with different solvents, alcohols, chloroform/alcohol mixtures and tetrahydrofuran. We found that the yield of gangliosides was lower when extracted with alcohols alone than with alcohol/chloroform mixtures, and that chloroform/methanol mixtures extracted more efficiently than chloroform/ethanol or chloroform/propanol mixtures. Therefore, in the continued experiments we used only chloroform/ methanol mixtures and tetrahydrofuran. The chloroform/methanol ratio was altered from 2 : 1 to 1 : 4, the water content of the final extract from 5 to 25% (when possible) and the ratio brain tissue to solvent from 1 : 6 to 1 : 40. The recovery of gangliosides was highest when the ratio chloroform/methanol was between 1 : 2 and 1 : 3, and it was 2-5% lower when the chloroform/ methanol ratio was 1 : 1 or 1 : 4 (Table I). In our standard procedure for the isolation of gangliosides we selected the chloroform/methanol ratio of 1 : 2, since the content of contaminants was reduced with the higher chloroform proportion. When the chloroform/methanol ratio was 1 : 2, the optimal water content was 20%. The highest ganglioside recovery was obtained with the largest ratio between tissue and solvent (1 : 40) (Table I), but two extractions with 20 solvent vols./
101 TABLE YIELD
I OF
EXTRACTED RATIOS All
OF
extractions
Solvent
GANGLIOSIDES, WITH SOLVENT were
DETERMINED
DIFFERENT AND
triplicate
(v/v)
AS
MIXTURES
WATER
OF
IN RELATION
TOTAL
NeuAc.
FROM
HUMAN
CHLOROFORM/METHANOL TO
AND
BRAIN VARYING
TISSUE
or quadruplicate. Ratio
tissue/total
volume
water
content
(%)
pm01
Chloroform/methanol
1
2
1
20
4
2.73
Chloroform/methanol
1
4
1
20
4
2.66
Chloroform/methanol
1
2
1
20
9
2.88
Chloroform/methanol
1
4
1
20
9
2.75
Chloroform/methanol
1
2
1
20
14
2.97
Chloroform/methanol
1
4
1
20
14
2.81
Chloroform/methanol
1
2
1
20
20
2.98
Chloroform/methanol
1
2
1
20
20
3.12
Chloroform/methanol
1
2
1
40
10
2.94
Chloroform/methanol
1
3
1
40
10
2.94
Chloroform/methanol
1
4
1
40
10
2.86
Chloroform/methanol
1
2
1
40
20
3.05
Chloroform/methanol
1
3
1
40
20
3.05
Chloroform/methanol
1
4
1
40
20
2.98
Chloroform/methanol
1
2
1
40
25
3.03
twice
NeuAc/g
tissue
tissue volume increased the recovery further by 2-3%. Since it is convenient, when possible, to reduce the solvent volumes in preparative work we decided to use a 1 : 20 ratio of tissue to final extract volume and to make reextractions with half the volume of solvent but unchanged chloroform/methanol/water ratio. In repeated experiments with brain from individuals of varying age, the recovery of gangliosides was 92-96s in the first extract, 4-7s in the second extract and less than 1% in the third + fourth extracts. Assay of the extracts with thin-layer chromatography showed no significant difference in the ganglioside patterns of the extracts. On the other hand, the proportions of non-lipid substances increased with each subsequent extraction. We therefore decided on only one reextraction. In the recommended procedure the recovery of gangliosides from adult normal mammalian brain was estimated to be 99 f 1%. We found some lacton formation of the terminal sialic acid of the gangliosides with a disialosyl linkage and tried to reduce it by homogenization in a neutral or weakly basic buffer. Most of the studies were performed with a phosphate buffer of pH 7.8 with a final buffer concentration up to 0.03 M. The addition of buffer did not reduce the percentage of lactons but rendered the further processing of the ganglioside fraction more difficult, since the dialysis time and the volume of dialysate had to be increased. Homogenization in phosphate buffer instead of water had no effect on the yield of gangliosides or the ganglioside pattern. The buffer thus had no advantage. An organic buffer, Tris/maleate, had similar disadvantages as phosphate buffer. Exaction with tetrahydrofuran was performed as described by Tettamanti et al. [12]. When the brain tissue was extracted with either tetrahydrofuran or chloroform/methanol (1 : 2, v/v) with 5--8% of water [19], chloroform/ methanol extracted slightly more from grey matter, but tetrahydrofuran gave a 10% higher recovery from white matter. With the extraction method devel-
102
TABLE
II
COMPARISON AND All
OF
THE
YIELD
OF
GANGLIOSIDES
EXTRACTED
WITH
TETRAHYDROFURAN
CHLOROFORM/METHANOL determinations
were
triplicate. firno1 NeuAc/g
Tissue
tissue
Tetrahydrofuran/O.Ol (PH Cerebral
grev
Cerebral
white
matter
matter
6.8,
4
: 1, v/v)
M phoshate
buffer
Chloroform/methanol/water (4
: 8 : 3,
3.42
3.53
1.37
1.57
v/v/v)
oped the recovery of gangliosides from mammalian grey matter was 3-10% and that from white matter was lo-15% higher than with the tetrahydrofuran method, which is illustrated by one experiment in Table II. Other advantages of the chloroform/methanol method is a shorter and simpler extraction. Tetrahydrofuran is unstable, and the risk of peroxidation of the solvent and the oxidation of especially phosphoacylglycerol fatty acids, requires rapid handling of the extracts. Isolation of a crude ganglioside extract by solvent partition In preliminary experiments the gangliosides were separated from the other lipids by chromatography on DEAE gels. The procedure described by Ledeen et al. [20] was followed but the load of lipids on the gels was increased. The capacity of the resins tested, DEAE-Sepharose, DEAE-Sephadex, QAESephadex and Spherosil-DEAE-Dextran, was too low to be of interest for large scale isolation of gangliosides from brain tissue. Therefore, all lipid extracts were partitioned, and a crude ganglioside fraction was isolated. When the new extraction procedure with a large proportion of water was adopted there was no need to add extra salt at the first partition. In a previous study [19] we showed that a solvent ratio chloroform/methanol/water of 1.0 : 1.0 : O.~(V/V) gave a higher recovery of gangliocides, particularly from white matter, than the classical Folch-Pi ratio 2.0 : 1.0 : 0.75(v/v). Since we found that the optimal ratio between chloroform/methanol was 1 : 2(v/v) for extraction of gangliosides from brain tissue, we tried to keep the same ratio between chloroform/methanol during the partition. A varying volume of water was added to give ratios between chloroform/methanol/water from 1.0 : 2.0 : 1.0 to 1.0 : 2.0 : 1.4(v/v). When the proportion of water was increased further, irreversible emulsification occurred. The highest ganglioside recovery was obtained with the largest proportion of water, but the differences were small, and chloroform/methanol/water (1.0 : 2.0 : 1.0, v/v) gave 95% of the maximum value (Table III). The yield of gangliosides at the second partition of the lower chloroform phase with methanol/water (1.0 : 0.7, v/v) was not reduced by the salt content of the water, but the salt gave a more rapid and distinct separation of the two phases. Approx. 5% of the total NeuAc was recovered in the second upper phase, but more important was the different ganglioside pattern compared with
103 TABLE
III
GANGLIOSIDE SOLVENT Solvent
CONTENT
FOUND
IN THE
UPPER
PHASE
AFTER
PARTITION
USING
DIFFERENT
MIXTURES ratio
(v/v/v)
l~mol
NeuAc/g
tissue
Chloroform/methanol/water 1.0 1.0 1.0
: 2.0 : 1.0 : 2.0 : 1.2 : 2.0 : 1.4
2.19 2.26 2.31
the first partition (Table IV). Isolation of the remaining gangliosides in the lower phase by anionexchange chromatography is often more rational than to make a third solvent partition. At the second partition the solvent ratio of chloroform/methanol/water was approx. 2.0 : 1.0 : 0.7 (v/v). In small-scale experiments we used larger volumes of methanol/water to secure a solvent ratio chloroform/methanol/water of 1.0 : 2.0 : 1.4 (v/v) but the yield of gangliosides was only negligibly higher and did not warrant the large increase of upper phase volume. When normal human brains were assayed only minor amounts (less than 1%) of gangliosides remained in the lower phase, mainly GM3 and sialosylgalactosylceramide. When pathological brain tissue, containing large proportions of monosialogangliosides, was extracted a third partition should be done (Fredman, P., Nilsson, 0. and Svennerholm, L., unpublished results). Purification
of the crude ganglioside
fraction
In order to exclude evaporation of the upper phase and at the same time remove other lipids, proteins and low molecular weight impurities, serious efforts were made to attach the gangliosides to DEAE gels. Columns with 150 ml bed volumes were loaded with the upper phase of adult mammalian brain extracts, and the leakage of gangliosides was assayed in the effluent. The concentration of gangliosides in the upper phase varied in different experiments between 200 and 300 pm01 of NeuAc/l, and the same upper phase was used for
TABLE
IV
COMPARISON
OF
Ganglioside
Total
GM2 GM1
THE
GANGLIOSIDE
Molar
NeuAc:
percentage
PATTERN
AT
FIRST
partition
Second
58.4
pm01
3.4 8
26
71
4
2
GDla
23
9
GDlb
24
9
GTlb
21 2
2 _
GD3
GQ1
SECOND
of gangliosides
First
1
AND
/ml01
partition
PARTITION
104
the different gels. Spherosil-DEAE-Dextran and QAE-Sephadex had the lowest capacity, and a significant amount of gangliosides appeared in the effluent already after 1 1 of the upper phase. The concentration of gangliosides in the effluent was rather constant, 5-10% of NeuAc in the upper phase, when the columns were loaded further with 3 1 of the upper phase. Only monosialogangliosides appeared in the effluent. DEAE-Sephadex had the largest capacity of the tested gels, but GM1 appeared in the effluent already after 11. The concentration of this ganglioside was, however, very low, only 1% of the gangliosideNeuAc in the upper phase placed on the column, and it remained so low during the load of the column with a further 3 1 (approx. 750 pmol NeuAc) of the upper phase. This means that 1 mmol of ganglioside-NeuAc could be loaded on 150 ml (25 g) of DEAE-Sephadex with a loss of less than 1% of NeuAc. DEAESepharose had the same capacity as DEAE-Sephadex to retain the gangliosides (only loss of 1% ganglioside NeuAc in form of GMl) but after 600 pmol of ganglioside-NeuAc, the concentration of monosialoganglioside rapidly increased in the effluent to 20% of the ganglioside-NeuAc concentration in the upper phase. In view of these results it was evident that the crude ganglioside fraction had to be freed from salts and other non-lipid contaminants before it should be applied to an anionexchange resin. The upper phase was evaporated to dryness in a rotating evaporator after the addition of isobutanol to prevent foaming. The major portions of proteins (peptides) and a portion of low molecular weight substances were removed by redissolving the extract in chloroform/ methanol/water (60 : 30 : 4.5, v/v). The recovery of gangliosides was better than 99% in this step. The gangliosides that remained in the precipitate had the same composition as the dissolved gangliosides. Remaining peptidic material was removed by passing the ganglioside fraction dissolved in chloroform/ methanol/water (4 : 8 : 3, v/v) through a small silica gel column. All gangliosides were eluted with 10 column ~01s. of the solvent. When the evaporated gangliosides were dissolved in chloroform/methanol mixtures with ratios varying from 2 : 1 to 1 : 2 (v/v) but without water, a large portion of the higher oligosialogangliosides, GTl and G&l was not dissolved. Chloroform/methanol/ water (30 : 60 : 5, v/v) gave a lower recovery of gangliosides than the more chloroform-rich mixture used, chloroform/methanol/water (60 : 30 : 4.5, v/v). Two different procedures were used for the removal of salts and other low molecular weight substances in the crude ganglioside extract, chromatography on molecular sieves or dialysis against water. When molecular sieves were used the gangliosides were either dissolved in organic solvent (chloroform/methanol/ water, 60 : 30 : 4.5 (v/v) and the method elaborated by Wells and Dittmer [21] was applied, or they were dissolved in water and separated on Sephadex G-100. With Wells and Dittmer’s method a large portion of trisialogangliosides, and practically a.lI tetra- and pentasiologangliosides were retained on the Sephadex column. When 1 l~mol of crude gangliosides from human brain was chromatographed on 1 g of Sephadex G-25 the recovery of NeuAc was 8590%. Nor separation on Sephadex in water was successful since the crude ganglioside fraction was difficult to dissolve in a small volume of water and the dimensions of the Sephadex G-100 column had to be large but even then the separation was incomplete. These results ruled out the molecular sieve separa-
105 TABLE
V
CONCENTRATION
OF
All
of four
values
Subject’s
are means age
GANGLIOSIDES
IN HUMAN
FOREBRAIN
determinations.
N-Acetylneuraminic (wet)
weight)
Total
lipid
acid
(mmol/kg
NeuAc
fresh
recovery
in upper
phase
(%)
extract
Upper
phase
lipids
2 months
2.83
2.60
92
3 months
2.96
2.78
94
4 months
2.81
2.61
93
61
years
2.31
2.13
92
72
years
2.53
2.39
95
tion method for large-scale isolation of brain gangliosides. Dialysis against water for 72 h resulted in a ganglioside extract with only a negligible salt content and relatively small losses of gangliosides. When 100 nmol/ml water of labelled ganglioside GM1 was dialysed the losses to the dialysate was 5% when the ganglioside was dissolved in 0.5 M KC1 in water or in methanol/water (1 : 1, v/v) but only 3% when dissolved in distilled water. When crude gangliosides of the upper phase in a concentration of 10 pm01 NeuAc/ml were dialysed against distilled water for 72 h, less than 0.5% of total NeuAc was found in the dialysate. The ganglioside pattern of the dialysate was very similar to that of the original crude ganglioside fraction. In the dialysate free NeuAc was approx. 10% of the total sialic acid. Determination
of the concentration
of gangliosides
in human
brains
One half of a forebrain was homogenized and extracted as in the standard procedure. NeuAc and the ganglioside pattern were determined in the total lipid extract and in the upper phase lipids. The recovery of NeuAc, determined from the absorbance in the resorcinol method, was in the upper phase 9295% of that in the total lipid extract. Since the ganglioside pattern was exactly TABLE
VI
DISTRIBUTION All
values
mo., Gang-
OF
are given
months;
yr.,
GANGLIOSIDES
in molar
IN HUMAN
FOREBRAIN
percentage.
years.
% N-Acetylneuraminic
acid
% Ganglioside
lioside 2 mo.
3 mo.
4 mo.
61
yr.
72
Yr.
2 mo.
3 mo.
4 mo.
61
yr.
72
GM3
1
1
1
3
4
2
2
2
6
6
GD3
4
6
5
4
3
4
5
4
4
3
GM2
4
3
4
1
1
7
6
7
2
2
GM1
13
13
15
15
16
24
24
27
28
30
GDla
49
46
45
42
39
18
17
GDlh
8
GTlb
16
GQlb
4
44
20
18
9
9
22
23
17
19
30
31
5
3
5
5
7 10 2
9 10 2
8 11 1
21
21
19
19
2
2
YI.
106
the same in the two extracts the difference in NeuAc absorbance presumably depends on unspecific colour given by substances in the lipid extract. The resorcinol reaction has been criticized because it gives a false colour, mainly for a large number of sugars other than sialic acids, but when the absorbance is read at 620 nm, as in the present study, this contamination is negligible, which is evident from the fact that the same recovery of NeuAc was obtained from the adult brains, which contain several-fold higher concentrations of cerebrosides than the infant brains. The diminution of sialic acid colour occurred during the partition and not during the dialysis, which suggests that the lower phase lipid contained chromogens that reacted with the resorcinol reagent. The ganglioside pattern shows that the proportion of GMl-ganglioside in the human brain is very constant, while GDla and GM2 are higher in infancy than later in life. The portion of GDlb and GTlb increases with age so that in old people most sialic acid resides in ganglioside GTlb. Discussion During the last decade important progress has been made in the development of new procedures for the separation of gangliosides [22-251. These procedures have proved very useful in the discovery and isolation of new gangliosides [2,6,7,26], but they are of limited value for unveiling the quantitative ganglioside pattern in an organ if the gangliosides are not quantitatively extracted from the tissue. It is strange that quantitative extraction of gangliosides from tissues has received so little attention. In a previous study we succeeded in increasing the recovery of gangliosides from brain tissue by extraction with chloroform/methanol (1 : 1, v/v), instead for 2 : 1 [19], but we did not make any systemical study of the extracting capacity of the method. Tettamanti et al. [ 121, however, showed that the tetrahydrofuran method, originally elaborated by Trams and Lauter [27] for the extraction of gangliosides could be modified to a very effective procedure for the extraction, separation and purification of brain gangliosides. When one first reads the paper it is easy to get the impression that tetrahydrofuran is a better extraction solvent for gangliosides than chloroform/methanol mixtures, but the present study has shown that it is instead simply the use of more water in the extraction solvent that is important. Before Folch-Pi et al. [28] described the classical solvent mixture chloroform/methanol (2 : 1) for the isolation of brain lipids, chloroform/ methanol (1 : 2 or 1 : 3) were the most commonly used solvents for isolating gangliosides and other sphingolipids [29]. In our own first endeavour to develop a quantitative ganglioside extraction procedure chloroform/methanol (1 : 2) was found to give the best yield of ganglioside sialic acid [30]. Shortly afterwards Bligh and Dyer [31] described a rapid method of total lipid extraction and purification, which was applied to fish muscle. They found the ratio chloroform/methanol of 1 : 2 (v/v) to be best for quantitative lipid extraction, and that the water concentration in the extraction solvent should be high, approx. 20%. The tissue/solvent ratio was 1 : 3 (v/v). When we applied their extraction procedure to brain samples used in the present study we got a low recovery of all lipids, particularly of the gangliosides, due to the low solvent/ tissue ratio.
107
The adult brain tissue is a very high lipid organ and quantitative extraction of all lipids, including gangliosides, was only achieved by repeated extraction with smaller volumes of solvent or by a single extraction with 40 ~01s. solvent/ volume of tissue. We now extract the tissue twice with 20 ~01s. of solvent or, for large scale isolation, use only 10 ~01s. of solvent for the second extraction. The proportion of water is important and the highest ganglioside recovery was found empirically at a water concentration of approx. 20% (Table I). It is noteworthy that the proportion of water is the same as that found by Bligh and Dyer [31] in their chloroform/methanol procedure and by Tettamanti et al. [ 121 in the tetrahydrofuran method. In the beginning we hesitated to use the high proportion of water since we had previously found [32] the contamination of the ganglioside extract with sialic acid-containing glycoproteins and glycopeptides to be greater when fresh brain tissue was extracted with chloroform/methanol (1 : 2, v/v) compared to acetonedried tissue. Tettamanti et al. [ 121 had also shown that the tetrahydrofuran solvent extracted less of sialoglycopeptides than the conventional chloroform/methanol (2 : 1, v/v) mixture. But actual experiments showed that the contamination of the lipid extracts with proteins and peptides diminished when the proportion of water was increased to 20%, and in our experiments with 20% water in the solvent the admixture of sialic acid-containing glycopeptides never exceeded 2% of the total siahc acid content. All our efforts to attach the gangliosides of the total lipid extract directly to an anion-exchange resin were unsuccessful, and it is doubtful whether direct attachment of gangliosides from a tissue extract to DEAE-Sephadex can be recommended in any situation. We have found that for the large-scale isolation of brain gangliosides under strict quantitative conditions, solvent partition, with subsequent evaporation of the upper phase and dialysis is the best method. We got a virtually quantitative recovery of the gangliosides when we made the partition with the solvent ratio chloroform/methanol/water of 1.0 : 2.0 : 1.4 (v/v), with a subsequent repartition (Table II). Under this condition the lower phase is practically 100% chloroform [33]. A portion of other acidic lipids, sulphatides, serine and inositol phosphoacylglycerols, are partitioned in the upper phase, and contaminate the crude ganglioside extract. A similar contamination with the same acidic lipids was found with the tetrahydrofuran method [ 121. These lipids can be easily removed in the subsequent purification steps (Fredman, P., Nilsson, 0. and Svennerhom, L., unpublished data). Since Kanfer and Spielvogel [34] had reported a significant loss of gangliosides at dialysis against water, we tried to remove the contaminating impurities on molecular sieves but we failed to develop an acceptable procedure. This might seem to be in conflict with the recommendation of Ueno et al. [35], who suggested the use of Sephadex G-50 for the separation of gangliosides and low molecular weight substances. They have, however, only applied the method for mixtures of pure gangliosides and free sialic acid. For the purification of crude ganglioside extracts of the methanol/water upper phases we think instead that the method of choice is dialysis against water. In a recent study Ghidoni et al. [36] have arrived at similar conclusions as us about the behaviour of gangliosides on dialysis. The present method was found to yield higher concentrations of gangliosides
108
than any previous method. The ganglioside pattern did not undergo any large change when the recovery of gangliosides increased by lo-30%. The proportion of tetrasialogangliosides was somewhat higher than in previous studies and pentasialogangliosides were isolated for the first time from mammalian brain (Fredman, P., Nilsson, 0. and Svennerholm, L., unpublished data). A direct comparison of the present results with those reported by Tettamanti et al. [12] and Nagai et al. [14] is not possible since they did not study brains of corresponding ages, besides at which they did not describe which part of the brain they used, the proportion of the higher gangliosides increases in caudal direction of the forebrain (Ref. 37; Svennerholm, L., unpublished data). Acknowledgements The work was supported by the Swedish 627). We gratefully acknowledge the assistance
Medical Research Council of Mrs. Birgitta Dellheden.
(3X-
References 1
Sweeley,
C.C.
Mammalian
and
Siddiqui,
B.
Glycoproteins
2
Svennerholm,
3
Li. Y.-T.,
4
Whenret,
5
KoScielak.
I,.,
J.-E.
J.-E.,
Vanier,
(1973)
J.,
Biochim.
Piasek.
A.,
in The
Glycolipids.
M&son,
M&son, J.R.
(1977)
and
and
Li,
M.-T.
Y.-T. and
Biophys.
G6miak.
Glycoconjugates
Vol.
(1973)
H.,
326,
Gardas,
M.I.
Academic
J. Biol.
Svennerholm,
Acta
(Horowitz,
1. PP. 459-540.
Chem.
L. (1973)
and
Press,
248,
Pigman,
W..
New
York
248.
2634
eds.),
740-p742
J. Biol.
Chem.
2636
63-73
A.
and
Gregor,
A.
(1973)
Eur.
J. Biochem.
37.
214.-
225 6
Ghidoni,
R.,
Sonnino,
S.,
Tettamanti,
G.,
Wiegandt,
II. and
Zambotti,
V.
(1976)
J. Neurochem.
27.
511-515 7
Sonnino,
S.,
8
Ishizuka,
I. and
Ghidoni,
R.,
9
Ando.
S. and
Yu,
10
Kuhn,
R. and
Wiegandt,
11
Svennerholm, Problem
12 13
Bonali.
Chem.
F.,
252.
and
19b.
Related
Holmgren,
S.
260.
31,
947
Aspects
of
-956
279-289
256-258
Diarrheas,
J., eds.).
Marchesini,
J. Neurochem.
Acta
62474250
Z. Naturforsch.
Cholera
and
G. (1978)
Biophys.
Karger,
and
Molecular
Basel.
Zambotti,
a
Global
Health
in the press
V.
(1973)
Biochim.
Biophys.
Acta
296,
70 A.,
Slomiany,
PP. 49-75,
14
Svennerholm,
15
Tayot,
Tardy,
and
Svennerholm,
L..
19
Vanier,
Holm,
T. and
Ledeen, Wells,
R.W.. M.A.
Yu,
and
Svennerholm, eds.),
Vol.
M., R.K.
Momoi. Iwamori,
T.,
25
-4ndo.
S.,
26
Ando,
S. and
27
Trams,
E.G.
28
Folch-Pi,
(1972)
Ando,
Methods
29
Klenk,
30
Svennerholm.
31
Bligh,
Nagai,
Y.
Y.
(1978)
Chang,
N.-C.
and
Yu.
and
R.K.
E. and
Lauter,
L.J.
I., Lees,
Rennkamp. L. (1957)
and
(1978)
Dyer,
in
Acta
(1976)
R.K.
(1962)
(Witting,
L.A.,
Chem. Biochim. J.A.
and
Med.
Chem.
Vanier.
Ups,
Stand. M.-T.
Svennerholm.
in
Ellis 13,
Chromatography
Horwood,
of
Chichester
856-858
(1979)
L. (1973)
J. Neurochem.
21,
Clin.
Chim.
J. Neurochem.
Acta 21,
92.
53m~-64
1375.-1384
829-839
2, 1259-1263 Chemistry
(Whistler,
R.L.
and
BeMiller,
London Biophys.
Biophys.
Acta
Anal.
Biochem.
252.
62474250
Biophys. and 62,
J. Biochem.
Acta 528,
441,
60,
F.N.
Z. Physiol.
350-358 (1951) Chem.
l-16 Physiol.
488497
257-267
89,437-450
Acta
LeBaron,
Hoppe-Seyler’s Can.
(1978)
604-611
Acta
Biochim.
(1978)
Meath.
Sot.
(1959)
24,
M.
2. PP. 95-110,
Carbohydrate
Press,
Biochim.
F. (1941)
W.J.
and (1973)
J. Biol. M..
Roumiantzeff, Vol.
Biochemistry
Academic
S. and
Yu,
L.F.
in
Nagai.
and
J.-E.
J.-E.
(1963)
Methodology
IL
ed.),
(1959)
M&son,
Eng,
in Glycolipid
R. Acta
I.T.
J.C.
M. and
J.. Ascoli,
E.G.
and
(1976) Champaign,
R.,
Biophys.
M&son.
Dittmer,
L.
23
(Epton,
G.,
6. PP. 464474,
24
Plan.
Takki-Luukkainen, Hikansson.
M.I.
Society, 1, 42--53
P.,
Biochim.
18 20
Gattel.
L. (1957)
Svennerholm. Miettinen.
Horowitz,
J. Neurochem. M.,
Polymers
16
M.-T.,
and
Oil Chemist’s
Biological
17
21
B.L.
American L. (1956)
J.L.,
Synthetic
22
J. Biol.
in 6.
Tettamanti,
Biochim.
H. (1964)
(1979)
G.,
G. and
H. (1972)
(1977)
(Ouchterlony,
Slomiany, ed.),
R.K.
L.
Tettamanti, 160-l
Galli,
Wiegandt,
37,
912-917
J. Biol. 267,
Chem.
145-153
191,
833-841
J.M.,
109 32
Svennerholm,
L. (1956)
33
Banner,
(1910)
34
Kanfer.
35
Ueno,
36
Ghidoni,
37
Suzuki,
W.D. J.N. K.,
and
Ando. R.,
K.
Chem.
Stand.
Chem.
14,
Spielvogel,
C. (1973)
S. and
R.K.
Sonnino,
(1965)
Acta J. Phys. Yu, S. and
(1978)
694.-696
J. Neurochem.
20,
1483.-1485
J. Lipid
19,
863-871
Tettamanti.
J. Neurochem.
10,
738-761
12.969-979
G. (1978)
Res.
Lipids
13,
820-822