The Journal of Biochemiitry, Vol. 62, No. 1, 1967
Lipid of Cancer Tissues
II. Neutral Glycolipids of Nakahara-Fukuoka Sarcoma Tissue*
By JUN'ICHI KAWANAMI
{From the Shionogi Research Laboratory,
Shienogi Co., Ltd.,
Osaka)
The isolation and the structure of glycolipids from NakaharaFukuoka sarcoma tissue is described. Glycolipids were purified by
column chromatography on silicic acid, on Florisil and by crystallization
with methanol. Four types of sphingolipid, ceramides, CMH, CDH
and CTH were isolated in a pure state. In contrast to BP3/C3H mouse
ascites sarcoma cells, N.F. sarcoma tissues contained nearly equimolar
amounts of each glycolipid with a trace of globosides. The glycolipids
were cleaved by various methods and the components were determined
by G.L.G. mainly. CMH consisted of about 86% glucocerebrosides and
about 14% galactocerebrosides. CDH was mainly lactosyl ceramide.
CTH was mainly galactosyl (al—4) galactosyl (j31—>4) glucosyl (1-+T)
ceramide. The long chain bases present were 18-dihydrosphingosine
and 18-sphingosine in the ratio of about 4:6 and the pattern of fatty
acid was similar to that of sphingomeyelin (1).
In a previous paper (1), it was reported sarcoma tissue were abundant in glycolipids,
that the phospholipid composition of Naka- the hexose content of the alkali-stable frachara-Fukuoka sarcoma and Shionogi carcinoma tion was determined and compared with the
115 tissue were not significantly different from three tissues described above (Table I). The
that of normal liver, while the neutral lipids results showed that Nakahara-Fukuoka sarshowed different composition between the coma contained over ten times more hexose
tumor tissues and the normal liver. The com- than other tissues. The total yield of glycolipid
position and the structure of tumor glycolipids in Nakahara-Fukuoka sarcoma was about
has not been identified with certainty, although 0.12% of the wet weight of the tumor tissue,
some reports were presented by R a p p o r t
Several classes of sphingolipid were isolated
et al. ( 2 ) and H a k o m o r i and J e a n 1 o z in pure form and characterized as ceramides,
(3) who isolated cytolipin H and a fucose- ceramide monohexosides (CMH), ceramide dicontaining glycolipid from human cancer hexosides (CDH), ceramide trihexosides (CTH),
tissues, respectively. G r a y (4) studied in and two kinds of globosides** (CAH and CPH),
detail BP8/C3H mouse ascites sarcoma cells, respectively. Nakahara-Fukuoka sarcoma was
from which four classes of glycolipid were found to contain about equal amounts of these
isolated and partially characterized. As it sphingolipids, with exception of the globoside
was often observed on thin layer chromato- type of lipids (Table III). CMH was found
grams the lipid extracts of Nakahara-Fukuoka to consist of about 14% ceramide galactoside
—
~:
.
. .
,
and 86% ceramide glucoside. CDH was mainly
* A preliminary investigation was reported at the
8th Meething of the Japanese Conferences on the Biochemistry of Lipids at Hiroshima in June, 1966.
°
'
*• The name globoside is used for ceramide Nacetylhexosaminyltrihexosides.
105
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(Received for publication, February 9, 1967)
106
J. KAWANAMI
HH
HO-^^A-O-CERAMIDE
OH
Fio. 1. Proposed stracture for CTH of N.F.
sarcoma.
TABLE
I
Hexose ConUnt of tht Alkali-stabU Fraction
Per cent of the
wet tissue
(ai glucose)
Liver
Shionogi carcinoma 115
Nakahara-Fukuoka sarcoma
0.005
0.007 ±0.0005
0.050±0.007
MATERIALS AND METHODS
Materials—Nakahara-Fukuoka sarcoma (N.F. sarcoma) from the DS-strain of mouse was supplied
by Dr. K. Yamaguchi of this Laboratory. Normal
fatty acids (H-102, H-104 and H-108 of Applied
Science Lab. Inc., State College, Pa., U.S.A.), hydroxy
fatty acids (18h>, 18: lh 1 , 26h«, 18h«, 18h" of Calbiochem., Los Angel, U.S.A.) and DL-sphingosine and
dihydrosphingosine (YEDA, Research and Development
Co., Ltd., Rehovoth, Israel) were commerical preparations. Fatty aldehydes were synthesized by reduction
of a given fatty acid methyl ester with LiAltL, and
oxidation with CrCvpyndine complex. CTH (bovine
spleen) and globoside (erythrocytes) were gifts from
Prof. Dr. T. Yamakawa (Univ. of Tokyo).
Analytical Methods—All melting points were determined on a Kofler block and were uncorrected.
Optical rotations were measured in pyridine, unless
otherwise specified, with a Rudolf Photoelectronic
Polarimeter, model 200. ORD curves were taken
with a Rudolf automatic recording spectropolarimeter
and the IR spectra were taken on KBr pellets with
a Koken IR Spectrophotometer, model-301. The
estimation of phosphorus and fatty acid composition
was carried out as previously reported ( 1 ) . Hexose
was estimated by the anthrone reaction ( 7 ). Hexosaminc
determinations were earned out by the method of
W i n z l e r (8); siahc acid by the resorcinol method
( 9 ) and nitrogen by the Dumas method.
Identification of Hexosamint by Automatic Amino Acid
Analyser—A Hitachi automatic amino acid analyser
model KLA-2 was used. Determination of hexosamine
was carried out by the method of S p a c k m a n it ai.
(10) using an Amberlite CG-120 column with citrate
buffer after hydrolysis with 2N HC1.
Papir Chromatography of thi Sugar Components—The
hydrolysis of glycolipids was carried out as follows.
The glycolipid (2—4 mg.) was heated with 2 ml. of
3N HC1 for 3 hours at 110°C in a sealed tube. The
hydrolyzate was extracted with n-hexanc to remove
fatty acids, and the aqueous phase was then treated
with Amberlite CG 45 (OH") to remove the HC1. The
neutralized aqueous solution was lyophylized. The
lyophylized material was used for sugar identification by
paper chromatography and for the estimation of hexose
content. The paper chromatography of the sugars
was carried out on Toyo Filter Paper (No. 51A) in
pyridine-butanol-water ( 4 : 6 : 3 , by vol.). The sugars
were identified after spraying with AgNOj-NaOH
reagent ( / / ) and hexosamines were identified with
ninhydrin.
Identification of Long Chain Basts (L.C B.)—L.C.B.
were prepared according to S wee ley and M o s c a t e l l i
(12). They were identified by T.L.C. ( 13) and G.L.C.
(14).
Partial Acid Hydrolysis of tht Glycolipids—The lipid
(20 mg.). was subjected to hydrolysis with 0.1 N HC1
for 30 minutes at 100°C followed by dialysis against
water. After lyophylization of the dialyzate the products
were identified by T.L.C, and isolated by preparative
T.L C. (Silica Gel H).
Thin Layer Chromatography (T.L.C.)—Glycolipid
fractions from chromatographic columnj were monitored by T.L.C. with Silica Gel G, using a solvent
system of CHCl.-MeOH-HjO (65: 25: 4, by vol.). To
render the spots visible, the plates were sprayed with
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ceramide lactoside. CTH was mainly ceramide
digalactosylglucoside. In addition, the CTH
had a dextrorotatory power similar to CTH
from bovine spleen (5) and human kidney
(6~) and its structure was proposed as galactosyl (al->4) galactosyl (jSl-*4) glucosyl (1-»1)
ceramide as illustrated in Fig. 1. On the other
hand, G r a y (4) reported that CTH from
BP8/C3H ascites sarcoma cells contained
galactosyl (y31—»6) galactose and lactose in the
hydrolyzates. In this paper, the isolation and
the structure of these glycolipids are described
in some detail. Although a trace of globosides
(galactosamine-containing glycolipid) were
isolated, their structure could not be determined because of the lack of materials.
Glycolipid of Tumor Tissues
the anthrone reagent ( 7 ) A solvent system of CHCV
MeOH (95: 5, by vol.) was used for ceramides. Thin
layer chromatography of L.C.B. was carried out with
a solvent system of CHCl.-MeOH^tf NIL.OH (40:
10: 1, by vol.), using ninhydrin for the development
of the spots according to S a m b a s i v a r o and
M c C l u e r (13).
Thin layer chromatography of
hexosamines was earned out on cellulose plates (cellulose 400 MN, Macherey, Nagel and Company) with
a solvent system of ethyl acetate-pyridine-water-acetic
acid (5:5:3:1)
107
RESULTS
ly methylated sugars were chromatographed on a 3 m.
column of 5% Ucon LB 55OX on a Gaschrom CLH
at 190°C according to the method o f Y a m a k a w a
and U e t a (17). TMS-derivatives of L.C.B. were
analyzed on a 1.5 m. column of 5% SE-52 on Chamelite
CS* at 214°C according to S wee ley's method (13).
The chain length of L.C.B. was determined by G.L.C.
of the long chain aldehydes obtained by periodate
cleavage of the bases. G.L.C.** of hexosamines was
carried out according to the method of S w e e l e y and
W a 1 k e r -( 18 } {Procedure C} on a 1.5 m. column of
5% SE-52 on Chamelite CS at 193°C.
Proton Magnetic Resonanu (P.M.R)—The
spectra
were measured using a Varian Model A-€0 spectrometer in deuterium oxide solution, containing scdium
2,2-dimethyl-2-silapentane-5-sulfonate (DSS) as an
internal standard.
* Nishio Kogyo Co., 62 Azuma-cho, Meguro-ku,
Tokyo.
** Barber-Coleman model 10 gas chromatograph
was used (glass column).
Preparation of Purified Glycolipids—The crude
neutral glycolipid fraction dissolved in CHC18
was chromatographed on a silicic acid (Mallinckrodt) column (60 g.) and the elution was
made with CHC1,, CHCls-MeOH mixtures
of 9: 1, 8:2, 7: 3 and finally MeOH. Each
fraction was monitored by T.L.C. (Fig. 2).
The characterization of spots in Fig. 2 is given
in Table II which shows the elution pattern.
Each glycolipid fraction from the silicic acid
column was further separately purified by rechromatography on silicic acid and Florisil
to remove contaminating phospholipids. Each
glycolipid purified in this manner was dissolved in hot MeOH and cooled to room temperature which resulted in separation of a
large amount of crystalline material. The
properties of these crystallized glycolipids are
shown in Table III and Fig. 3. Their IR
spectra are also shown in Fig. 4. The total
yield of the neutral glycolipids per w.et weight
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Preparation—The method used for the preparation of the crude glycolipids was based
on G r a y's method (4). Fresh tissue (104.5 g.)
was extracted twice with CHCl,-MeOH (1:1,
by vol.) and then with CHCl,-MeOH (2:1,
by vol.). The three extracts were combined
and evaporated to dryness in vacua. The
residue (5g.) was dissolved in n-hexane and
Gas Liquid Chromatography (G.L.C.)—A Shimazu
dialyzed through a rubber membrane against
Gas Chromatograph model IB, equipped with a hyn-hexane to separate the neutral lipids (2.6 g.)
drogen flame ionization detector, was used throughout
from the phospholipids and glycolipids (2.5 g.).
this experiment. The methyl esters of the hydroxy
The complex lipid fraction (the dialyzate) was
fatty acids were chromatographed as their trimethylsilyl
evaporated to dryness and treated with 0.1 N
derivatives on a 1.5 m. stainless steel column packed
methanolic
NaOH-CHCl, (1:1, by vol.) for
with 5% diethyleneglycoUuccinate polyester on Chro4
hours
at
room
temperature to remove the
mosorb W (acid-washed, sihconized) at 200°C. G.L.C.
diacylglycerophospholipids
as water soluble
of hexoses was carried out as follows
Methanolysis
phosphate esters according to the method of
was performed with 5% HCl-MeOH in place of 3N
H ii b s c h e r et al. (19). The fatty acid methyl
HC1 as in the case of paper chromatography above
esters formed in the reaction were separated
described. Methyl esters of fatty acids were extracted
with n-hexane and the methanol layer was treated
from the alkali-stable lipids by dialysis through
with Amberlite CG 45 resin to remove the acid. After
a rubber membrane against n-hexane. The
evaporation of the solvent, methyl glycosides were
fraction obtained from the dialyzate (425 mg.)
converted to their trimethylsilyl ethera (75). Permecontained 44.4 mg. of hexose as glucose. The
thylation of glycolipids was carried out by H a k o m o r i's crude glycolipid fraction was subjected to
procedure ( 16) uing sodium hydride and methyl iodide
DEAE-cellulose chromatography to give 415
in dimethyl sulfoxide. The partially methylated sugars
mg.
of neutral glycolipid fraction containing
were obtained by methanolysis with 5% HCl-MeOH.
40
mg.
of hexose as glucose (Scheme I).
Both the TMS-derivatives of glycosides and the partial-
108
J. KAWANAMI
SCHEME
I
Flow Shot for tht Isolation of Crude Glycoliptd Fraction
Wet tissues (104.5 g.)
C-M (2: 1)
Total lipid extracts (5.0 g.)
dialysis
mild alkaline
hydrolysis
I
Organic phase
(1.755 g.)
aq. phase
dialysis
I
Inner fluid
(crude glycolipid fraction)
(425 nig.), hexose, 44.4 mg.
(as glc.)
Outer fluid
(released fatty acids)
(l.Hg.)
DEAE-cellulose
Neutral glycolipid fraction
(415 mg.), hexose 40 mg.
(as glc.)
TABLE
II
Chromatography of tht Alkali-stabU Fraction on Silicic Acid
Fr.
1
2
3
4
5
6
7
Wt.
Solvent
C
C-M
C-M
C-M
C-M
C-M
M
(95: 5)
(9:1)
(9:1)
(8:2)
(7:3)
Total
Wt.
(glc)
(ml.)
(mgO
200
200
100
100
200
200
600
109
40
16
40
53
51
132
4. 1
7.75
18.46
4.0
5.3
426
39.61
415 mg. of the alkali-stable fraction was applied to 25 g. of silicic acid
Abbreviations;
C : Chloroform
M : Methanol
CMH: Ceramide monohexosides
CDH: Ceramidc dihexosides
CAH : Ceramide hexosaminyltrihexosidei
Characterization
Neutral lipids
CER, fatty acids
CMH
CMH, CDH
CDH, CTH
CTH, CAH, phosphatides
CPH, phosphatidcs
column.
CER : Ceramides
CTH: Ccramide trihexosides
CPH: Unidentified glycolipid
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Inner fluid (2.5 g.)
(complex lipids)
Outer fluid (2.6 g.)
(neutral lipids)
Glycolipid of Tumor Tissues
109
m>
TI
«•
••
M
Fig 2
Fig. 3
FIG 2 Thin layer chromatogram of the eluates from silicic acid column chromatography
Numbers show fractions in Table II.
Solvent system • CHCl,-MeOH-water (65 . 25 • 4)
Fie. 3 Thin layer chromatogram of the glycolipids from N.F. sarcoma
1, CER, 2, CMH, 3, CDH, 4, CTH, 5, CAH, 6, CPH, M, the mild alkali-stable fraction in which
spots encircled with dotted line were of non-glycohpids such as free fatty acids and phosphatides.
Solvent system CHCl,-MeOH-water (65 : 25 4)
Spray reagent • Anthrone-sulfunc acid
For abbreviations, see Table II.
TABLE
III
Properties of the Glycohpids from N.F Sarcoma
CER
Melting point
[«]D
- Hexose/N- " "
Carbohydrate
glc./gal." ratio
Yield per 100 g
fresh tissue
mg. (about)
83—84
-6.9
CDH
CMH
CTH
CAH
—
146/186
-7.7
^v "1.05
glc gal
—
86/14
1/1.04
—
230/264—266 (d.)
+23 9
—
' "3.05/2
2 65
glc. gal
glc. gal.
gal N
1/1.82
1/1.81
30
31
28
32
180/248—255 (d.)
-8 9
2 0 '
glc. gal
1) Gas chromatography of the TMS ethers.
Abbreviations;
g l c : Glucose gal.: Galactose gal. N: Galactosamine
For another abbreviations, see Table II.
0 8
(d.). With decomposition
CPH
193/268—271 (d.)
+ 18 5
3 13/2
glc. gal.
gal. N
1/1.82
2.7
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I
110
J. KAWANAMI
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Anal. Calcd.: C, 75.63; H, 12.88; N, 2.59.
Found: C, 75.71; H, 12.86; N, 2.49. [a]g=
-6.9 (±2) (c, 1.008). Diacetates were prepared
in acetic anhydride-pyridine and melted at 100
—101°C(MeOH). Anal. Calcd. for C, 3 H 7 ,O 5 N:
C, 73.14; H, 11.79; N, 2.24. Found: C, 74.13;
H, 11.57; N, 2.06. Partial separation of the
di-acetates into two groups (upper and lower
spot) could be achieved by chromatography
on alumina. The benzene-CHCl, (95: 5) eluate
gave a powder with an m.p. of 99—100°C, [a] D =
-9.3 (±2) (c, 1.083), Anal. Found: C, 72.82;
H, 11.95; N, 2.04. The benzene-CHCl3 (9: 1)
eluate gave a wax with an m.p. of 101—102°C,
[«];=»=-16.4 (±2) (c, 1.155). Anal. Found: C,
73.64; H, 11.67; N, 2.38.
Ceramide Monohexosides (CMH)—After silicic
acid chromatography (Table II, Frs. 3 and 4),
CMH were eluted from a Flonsil column with
CHCl 3 -MeOH (8: 2). This fraction could be
crystallized from MeOH as rosette type of
1.600
3200
9O0
leaflets in a yield of 75%. The crystal was
WAVE NUMBER (cm-i)
sintered
at 146°C and melted at 186°C. Thin
Fio. 4. Infra-red absorption spectra of the
layer chromatograms on Silica Gel G afforded
glycolipids from N F. sarcoma (KBr).
three spots, the upper double spot corresponded
For abbreviations, see Table II
to cerasine (mainly glucose) and the lowest
spot to phrenosine (galactose). Also, borate
of tissue amounted to about 0.12%.
Ceramides—Ceramides obtained from the thin layer chromatography showed the CMH
to consist of a large proportion of glucocere5% MeOH in CHC13 eluate were crystallized
from MeOH as waxy cubes, with an m.p. of brosides together with a small amount of
galactocerebrosides (20) (Fig. 6). The molar
83—84°C. The crystallized ceramides showed
a double spot on T.L.C. (CHCl,-MeOH, 95: ratio of nitrogen to hexose (as glucose) was
5, by vol.)- The amide bands in the IR
1:1.05 (Table III). Paper chromatography
spectrum (KBr) were separated into doublets of the sugar obtained from the CMH fraction
(Fig. 4). Hydrolysis of this fraction afforded
revealed no galactose spot but G.L.C. showed
L.C.B. and fatty acids. The fatty acids con- the presence of about 14% galactose and about
sisted mostly of palmitic acid (68%) (Table 86% glucose (Fig. 7). It may be concluded
IV). Hydroxy fatty acids could not be de- that the CMH were a mixture of 86% ceramide
tected. G.L.C. of the TMS-L.C.B. gave two glucoside and 14% ceramide galactoside. In
major peaks. The first peak corresponded to order to examine the fatty acid and base
sphingosine, while the second to dihydro- compositions, the total CMH fraction, before
sphingosine. The area ratio was about 6:4. recrystalhzation, was hydrolyzed according to
Periodate oxidation of the L.C.B. mixture R o u s e r's method (21). Monohydroxy fatty
gave two major aldehydes in a ratio of 4:6 acids and normal fatty acids were detected
(Fig. 5). The one peak corresponded to n- by T.L.C. of the hexane extracts. The major
hexadecanal and the other to rc-hexadec-2- components of the normal fatty acids were
enal (U.V. i ^ * " 216 mp). Assuming that the
palmitic acid (52%), lignoceric acid (17%) and
ceramides consist of a fatty acid with a chain
nervonic acid (16%) (Table IV). The comlength of 16 (Table IV) and of dihydro- position of the hydroxy fatty acids is shown
sphingosine, the empirical formula is C H O
in Table V. As the hydroxy fatty acid methyl
Glycolipid of Tumor Tissues
111
TABLE IV
Normal Fatty Acid Composition of the Clycolipids
from N.F. Sarcoma
CMH
CER
14
23
24
24- 1
52
1
1
1
51
2
2
2
52
3
4
68
•
CTH
3
1
5
6
6
5
1
16
8
17
16
16
22
16
23
Values are expressed as percentages of total
methyl esters.
TABLE
V
Composition of the Hydroxy Fatty Acids m CMH
h*
N F.sarcoma
Rat brain
Rat brain"
18
20
22
3
1
9
3
—
—
40
35
20—25
23
11
13
10—15
24
37
47
59
16
Fio. 5.
L C B.
17
18
CARBON NUMBER
G L C of the aldehyde derived from
10.12; N, 1.58. [«]£=-18.3 (±0.8) (c, 0.552).
Ceramide Dihexosides (CDH)—This fraction
eluted with CHCl 3 -MeOH (8: 2) from a silicic
acid column (Table II, Fr. 5) was separated
ester showed a negative Cotton effect by
from the CTH by a CHCl 3 -MeOH (7:3) eluO.R.D. (in MeOH), it was assigned a D-form
tion from a Flonsil column. The CDH fraction
(Fig. 8). The L.C.B. of the CMH showed the
same pattern as that from the ceramides on thus obtained was crystallized from MeOH
in a yield of 92%. The crystals were sintered
G.L.C. Assuming that CMH consists of a
fatty acid with chain length of 20 and of at 180°C and melted at 227—229°C with dedihydrosphingosme, the empirical formula is composition. The molar ratio of nitrogen to
CMH87O8N. Anal. Calcd.: C, 69.70; H, 11.57; hexose (as glucose: galactose=l: 1) was 1: 2.0.
N, 1.85; hexose, 23.78. Found: C, 69.38; H, G.L.C. revealed hexoses to be glucose and
galactose in a ratio of 1: 1.04. This CDH was
11.29; N, 1.91; hexose, 25.7 (as glucose). [a]f=
not -hydrolyzed-with-0.1 ^ - H C H b r 30mmutes ~
_r-.7.7i±2)_(c, .0.947)
—Penta-acetate—A solution of CMH (51 mg.) (the percent of degradation, 4.7) but for 13.5
in 1 ml. of pyridine was mixed with 1 ml. of hours at 100°C, it was partially hydrolyzed
acetic anhydride and allowed to stand over- (the percent of degradation, 36.6) to yield a
night at room temperature. To this solution, lipid with the same mobility as that of CMH.
ice water was added to effect the crystalliza- The CMH thus obtained was purified by
tion. The crystals were collected by filtration, preparative T.L.C. and the methanolysis of
washed with water and recrystallized from the CMH with 5% HCl-MeOH gave methyl
glucoside as the only sugar component by
MeOH to afford 41mg. of the penta-acetate with
m.p.90—93°C. Anal. Calcd. for C M H ^ O H N : C, G.L.C. On the other hand, the water-soluble
fraction (the outer fluid) consisted of only
66.98; H, 10.10; N, 1.45. Found: C, 67.01; H,
1) Y Kishimoto, and N S Radin, / . Lipid
Research, 1, 79 (1959).
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16
18
18: 1
20
22
CDH
112
J . KA WAN AM I
8 8
TIME
10 9
12
13 1
(minutes)
FIG 7. Gas chromatograms of the methyl
glycoside trimethylsilyl ethers obtained from each
glycohpid
The experimental conditions are described in
"MATERIALS AND METHODS."
I
3.86
FIG 6. Thin layer plates prepared in water
(right) and sodium borate (left).
1 . Galactocerebrosidc (phrenosinc from rat brain)
2- CMH from N.F. sarcoma
3 • Glucocercbroside (CMH from mouse liver) '
Solvent system CHCl,-MeOH-water-conc NH,OH
(280 70.6: 1)
galactose. G.L.C. of methyl glycosides obtained from the permethylated CDH afforded
methyl 2,3,6-tri-O-methyl glucoside and methyl
2,3,4,6-tetra-O-methyl galactoside, but yielded
no methyl 2,3,6-tri-O-methylgalactoside (Fig.
9). The absorption band at 890 cm."1 in the
IR spectrum suggested the presence of a /?linkage (Fig. 4). L.C.B. showed the same
pattern as that from the other glycolipids on
G.L.C. Assuming that the CDH consists of
a fatty acid with a chain length of 20 and
of dihydrosphingosine, the empirical formula
is C 50 H 87 Oi S N-HiO. Anal. Calcd.: C, 64.00;
H, 10.64; N, 1.49; H,O, 1.92; hexose, 38.42.
Found: C, 63.03; H, 10.38; N, 1.43; H S O,
1.89; hexose (as g l c : gal.= l : 1), 36.7. [a]^=
224.5
WAVELENGTH ( m j j )
FIG. 8. O R D curve of the hydroxy fatty
acid methyl ester from CMH
-8.9 (±1) (c, 2.024).
Octa-acelatc—A mixture of CDH (96 mg.)
dissolved in 1 ml. of pyridine and 1 ml. of
acetic anhydride was allowed to stand overnight at room temperature. Ice water was
then added to the reaction mixture under
cooling to cause crystallization. The crystals
were collected by filtration, washed with water
and cold MeOH and recrystallized from
MeOH to afford 101 mg. of the octa-acetates
with an m.p. of 134—135°C. Anal. Calcd. for
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7 6
RETENTION
Glycohpid of Tumor Tissues
llSDjnCi 13
RETENTION TIME ( m i n u t e s )
24.7
FIG. 9 Gas chromatograms of the partially methylated sugars.
A Methyl 2, 3, 4, 6-tetra-O-methyl galactoside
B, E, F- Methyl 2, 3, 6-tn-O-mcthyl galactoside
D, F Methyl 2, 4, 6-tn-O-methyl galactoside
C, F- Methyl 2, 3, 6-tn-O-methyl glucoside
The experimental conditions are described in " MATERIALS AND METHODS "
C68H118O21N: C, 63.08; H, 9.07; N, 1.12.
Found: C, 62.86; H, 8.96; N, 1.08. [«]£=-11.4
(±0.4) (c, 1.036 in CHC13).
Ceramide Trihexosides (C77/)—CTH was
eluted with CHCl 3 -MeOH (6:4) from the
Flonsil column as described above. Fraction
6 in Table II was chromatographed on silicic
acid and Flonsil in a similar manner. The
combined CTH was crystallized from MeOH
to give crystals which were sintered at 230°C,
and decomposed at 264—266°C. The molar
ratio of nitrogen to hexose (as glucose: galact o s e ^ ^ ) was 1:2.65. It was revealed by
G.L.C. that it contained glucose and galactose
in a ratio of 1: 1.82 (Fig. 7). Reaction with
-0.-1 yV-HGl-at 100°e-for 30-min-utes-resulted-ih '
the partial destruction of CTH and the formation of two lipids with Rf values on T.L.C.
identical with those of CMH and CDH. The
partially hydrolyzed glycolipids were purified
by preparative T.L.C. and the methyl glycosides after methanolysis of each glycolipid
were studied by G.L.C. The carbohydrate
moiety of the CMH fraction consisted mainly
of glucose, while the CDH fraction contained
glucose and galactose in a ratio of 1.1: 1. The
result of G.L.C. of methyl glycosides obtained
from the permethylated CDH indicated that
this compound was ceramide lactoside (methyl
2,3,4,6-tetra-O-methylgalactoside and methyl
2, 3, 6-tn-O-methylglucoside).
These results
seem to show that CTH was mainly ceramideGlc. -Gal. -Gal. Further, G.L.C. of methyl
glycosides obtained from the permethylated
CTH revealed the presence of methyl 2,3,6tn-O-methyl glucoside, methyl 2,3,6-tn-Omethylgalactoside and methyl 2,3,4,6-tetra-Omethyl galactoside (Fig. 9). This CTH behaved similarly to cerebroside sulfunc acid
ester from rat brain on T.L.C. but analysis
for sulfur was negative. Absorption bands at
~884~ and 850 cm." 1 "in "the" TRTspectFurrT suggested the presence of a /3- and an a-linkage,
respectively (Fig. 4) (22). The compositions
of the normal fatty acid and bases were very
similar to those of CMH and CDH (Table
IV) and no hydroxy fatty acid was detected
on T.L.C. and by G.L.C. Assuming that CTH
consists of a fatty acid with a chain length
of 20 and of dihydrosphingosine, the empirical
formula is C J B H I O T O ^ N ^ H I O . Anal. Calcd.:
C, 60.13; H, 10.00; N, 1.25; H 2 O, 3.22; hexose,
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8 7
113
114
J. KAWANAMI
48.32. Found: C, 60.02; H, 9.53; N, 1.51;
H 2 O, 2.11; hexose (as glc.: gal.= 1: 2), 51.3.
[a];? =+23.9 (±2) (c, 1.017).
Undeca-acetates—The CTH (63 mg.) was also
acetylated with acetic anhydride-pyridine to
afford an oily product (85 mg.). [a\$ = +245
(±0.7) (c, 0.693 in CHC18).
Isolation of Other Glycohpids
Separation from Phosphatides through Acetyla-
erythrocytes (Fig. 10). The absence of sialic
acid was suggested by the reaction with the
resorcinol spray reagent (5). Gas chromatograms of the methyl glycosides from the
permethylated CPH showed the presence of
methyl 2,3,6- and methyl 2,4,6-tn-O-methylgalactosides and methyl 2,3,6-tri-O-methylglucoside (Fig. 9). The molar ratio of nitrogen to hexose (as glucose: galactose = 1 : 2)
Hydrolysis of the Acetylated Glycohpids—A
solution of the acetylated glycolipids in MeOH
was made up to 0.5 N with yV-NaOH and
allowed to stand overnight at room temperature. After acidification, the reaction mixture
was extracted with CHCl 3 -MeOH (2: 1).
Chromatography of the Hydrolyzates on Silicic
Acid— CTH and CAH were eluted with 30%
MeOH in CHC13 and CPH with 30—40%
MeOH in CHC18 from the silicic acid column.
Ceramide
Hexosaminyltrihexosides
I
{CAH)—
Only a small amount of materials was obtained by the above chromatography. This
CAH had an identical Rj value with that of
globoside from human erythrocytes on T.L.C.
and also contained galactosamine. It was
revealed by G.L.C. that the CAH contained
glucose, galactose and N-acetylgalactosamine
in a ratio of 1: 1.81 : 1.12 as sugar components.
The molar ratio of nitrogen to hexose (as
glc.: gal.= 1: 2) was 2: 3.05. As a result, CAH
appears to be a globoside. Anal. Found.:
N, 1.95.
Unknown Glycohpids (CPH}— CPH from the
column described above was precipitated with
acetone, collected by filtration, washed with
acetone and dried under reduced pressure.
It was sintered at 193°C and melted at 268—
271°C with decomposition. The CPH was
chromatographically distinct from the CAH
and more polar than authentic globosides from
1
CTH CAH
A CPH
2
FIG.
3
II
FIG. 10 Thin layer chromatogram of the
CAH and the CPH
A = globoside I from human erythrocytes.
For other abbreviations see text. Spray reagent;
a naphthol-sulfuric acid. Solvent system ; CHCljMeOH-water (65 . 25 . 4)
FIG. 11. Thin layer chromatogram of sphingosinylohgohexosides
1, 2, and 3 are sphingosinylmono-, di-, and
tri-hexosides derived from the CMH, the CDH,
and the CTH, respectively. Spray reagent;
ninhydrin reagent.
Solvent system; CHCljMeOH-watcr (65: 25: 4).
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tion—Fraction 7 (Table II) was acetylated with
Ac 3 O-pyndine overnight at room temperature.
To the reaction mixture, ice water was added,
and the resulting solution was extracted with
ether. The ether layer was washed to neutrality, dried with Na t SO 4 and evaporated to
dryness. The residue was submitted to chromatography on Flonsil and the acetylated
glycolipids free from phosphatides were eluted
with 5% MeOH in CHC1 3 .
Glycolipid of Tumor Tissues
Ma a-glucoslde
115
due to the anomeric hydrogens of the sphingosinyl trihexoside obtained from CTH is
shown in Fig. 12. Both sphingosine, measured
in deuteriochloroform as a reference, and
sphingosinyl dihexoside derived from CDH
did not show any signals at the r 4.5—5.2
region. On the other hand, sphigosinyl
trihexoside from CTH exhibited a signal at
r 5.04.
5.J
5.0
4.5
ppm. p)
Fio. 12. PMR spectra in deuterium oxide
of sphingosinyl trihcxoside from CTH (STH) and
sugars, showing the resonance lines due to the
anomeric protons.
was 2:3.24 and of hexosc to hexosamine was
3:0.77. Anal. Calcd. for C,oHuoOIISN,.5H,0:
C, 54.67; H, 9.18; N, 1.82; H,O, 5.86. Found:
C, 53.52; H, 8.92; N, 2.09; H,O, 5.56; hexose
(as glc: gal.= l: 2), 43.5%; [a]r?=4l8.5 (c,
1.151). As seen in Fig. 4, the IR spectrum
was similar to that of the aminoglycolipids
{23). Although it is assumed that CPH is a
kind of globoside from the above results, work
is under way to provide the large amounts of
CPH necessary for a more detailed study.
P.M.R. Study—P.M.R. spectra of glycolipidperacetates were too complex to determine
the configuration of the terminal anomeric
hydrogen because of the complexity due to
the ceramide moiety. Accordingly, glycolipids
were converted to sphingosinyl oligohexosides
according to the method of T a k e t o m i and
Yamakawa(Af KOH-BuOH) (24). Purification of the sphingosinyl dihexoside and
trihexoside was achieved by chromatographic
fractionation on a silicic acid column (Fig.
11). The signal in the proton magnetic spectra
The hexose content of the crude glycolipid
[ fraction from N.F. sarcoma has been found to
be ten times more than that of normal liver
and twice that of BP8/C3H ascites sarcoma cells
reported by G r a y (4). This fact has facilitated the isolation and characterization of
glycolipids. Three different glycolipids were
isolated in pure form and the presence of
comparatively large amounts of ceramides was
also revealed. The similarities in fatty acid
and long chain base composition among the
neutral N.F. sarcoma glycolipids indicated
that they might be metabolically related to
each other. However, it appears to be peculiar
that only CMH contained hydroxy fatty acids.
In contrast to the fact that CMH from liver
contained only glucose and CMH from brain
only galactose, it was of interest that tumor
CMH {4) as well as kidney CMH (6) contained both glucose and galactose. The CDH
from N.F. sarcoma was similar to cytolipin H
(2) and to CDH from erythrocytes (25) and
bovine spleen (5), in regards to the optical
rotation. In addition, the structure of the
carbohydrate moiety was lactose. Thus, the
structure of CDH was concluded to be lactosyl
ceramide. The CTH was similar to CTH
from human kidney (6) and from bovine
spleen (5) in regards to the optical rotation.
The terminal galactose was more easily cleaved
with dilute acid than that from the CDH.
Since the methylated sugars obtained from
permethylated CTH were methyl 2,3,6-tri-Omethyl glucoside, methyl 2,3,6-tri-O-methyl
galactoside and methyl 2,3,4,6-tetra-O-methyl
galactoside, the structure of CTH was assumed
to be galactosyl (l->4) galactosyl (1—>4>glucosyl
ceramide. The instability of the terminal
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DISCUSSION
116
J. KAWANAMI
for the identification of methyl 2,3,6-tri-O-methyl
galactoside and for very useful discussions. The author
is grateful to the members of Analysis Room of this
Laboratory for elemental analyses and rotation- and
spectral measurements.
REFERENCES
(/)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(75)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
The author is indebted to Dr. H. Otsuka for his
stimulating interest in this work and to Professor Dr.
T. Yamakawa, University of Tokyo, for gifts of globoside from erythrocytes and CTH from bovine spleen,
(23)
(24)
Kawanami, J., Tsuji, T., and Otsuka, H . , / .
Bwchem., 59, 151 (1966)
Rapport, M.M., Graf, L., Skipski, V.P., and
Alonzo, N.F., Cancer, 11, 1125 (1958); 12, 438
(1959)
Hakomori, S., and Jeanloz, R . , / . Bio/. Chtm.,
239, PC 3606 (1964)
Gray, G.M., Bwchtm. J., 94, 91 (1965)
Makita, A., and Yamakawa, T., / . Biochem., 51,
124 (1962)
Makita, A., / . Bwchem., 55, 269 (1964)
Roe, J.H., / . Biol. Chem., 212, 335 (1955)
Winzler, R.J., " Methods of Biochem. Analysis, "
ed. by D. Glick; Interscience Publish. Inc., New
York, Vol. U, p. 292 (1955)
Svennerhokn, L., Biochim. el Bwphys. Ada, 24,
604 (1957)
Spackman, D.H., Stein, W.H., and Moore, S.,
Anal. Chem., 30, 1190 (1958)
Trevelyan, W.E., Procter, D.P., and Harrison,
J.S., Nature, 166, 444 (1950)
Sweeley, C.C., and Moscatelli, E.A., / . Lipid
Research, 1, 40 (1959)
Sambasivaro, K., and McCluer, R.H., / . Lipid
Research, 4, 106 (1963)
Gaver, R.C., and Sweeley, C.C , J. Am. Oil
Chemist's Soc, 42, 294 (1965)
Sweeley, C . C , Bentley, R., Makita, M., and
Wells, W.W., J. Am Chem. Soc, 85, 2497 (1963)
Hakomori, S., / . Bwchem., 55, 205 (1964)
Yamakawa, T., and Ucta, N., / . Exptl. Med.,
34, 37 (1964)
Sweeley, C . C , and Walker, B., Anal. Chem., 36,
1461 (1964)
Hubscher, G., Hawthorne, J.N., and Kemp, P.,
J. Lipid Research, 1, 433 (1960)
Kean, E.L., J. Lipid Research, 7, 449(1966);
Young, O.M., and Kanfer, J.N., J. Chromatog.,
19, 611 (1965)
O'Brien, J S., and Rouscr, G., Anal. Chem., 7,
288 (1964)
Spedding, H., Adoan. Carbohydrate Chem., 19, 23J
(1964)
Svennerholm, E., and Svennerholm, L., Biochim.
ct Bwphys. Ada, 70, 432 (1963)
Taketomi, T., and Yamakawa, T., / . Biochem.,
54, 444 (1963)
Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 11, 2016
galactosidic linkage to acid, the dextrorotation, infrared absorption spectrum (850 cm."1)
and P.M.R. study (r 5.04) seem to show the
presence of an a-linkage. Accordingly, the
conformation of the CTH was proposed as
galactosyl (a\—A) galactosyl (/31->4) glucosyl
ceramide (Fig. 1). In contrast to this result,
it was reported that the CTH from BP8/C3H
ascites sarcoma cells contained galactosyl
(/SI—>6) galactosyl (/SI—>4) glucose as the sugar
moiety (4). Although it was suggested that
the CAH and the CPH were a type of globoside, their structure could not be determined because of the lack of materials. The
presence of nearly equimolar amounts of sphingosine and dihydrosphingosine was peculiar.
P.M.R. Study— Whether the glycosidic linkage in disaccharides is an a or /3 can be
determined from Hudson's rule or enzymatic
hydrolysis studies. However, it is difficult to
apply these principles to such complex compounds as glycolipids. van derVeen(26'),
R a o and F o s t e r (27) measured the P.M.R.
spectra of a series of glycosides of D-glucose
and D-galactose, and succeeded in correlating
the spectra to the glycoside configuration.
Thus, in the present study, P.M.R. investigations were carried out to determine the
configuration of the trihexoside moiety of the
CTH. Proton magnetic resonance spectra of
fully acetylated glycolipids were too complex
to assign definitively the peak of the anomeric
protons because of the presence of the ceramide
moiety. Therefore, P.M.R. spectrum of the
long chain base was measured, in which it
was found that the hydrogen atoms on C-l,
2, and 3 of the long chain base (sphingosine)
did not overlap with the /3 (equatorial)-anomeric hydrogen of oligosaccharides. Further,
P.M.R. spectra of the sphingosinyl oligohexosides derived from glycolipids were measured.
Based on comparison with these reference
compounds (26), it was concluded that the
sphingosinyl trihexoside from the CTH contained an a-linkage.
Glycolipid of Tumor Tissues
(25) Yamakawa, T., Yokoyama, S., and Handa, N.,
J. Biochm., 53, 28, (1963)
(26) van der Veen, J.M., J. Org. Chem., 28, 564
117
(1963)
(27) Rao, V.S.R., and Foster, J.F., / . Phys. Chim.,
67, 951 (1963)
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