STUDIES ON THE LIPIDS IN BRAIN OF BLACK RIGHTWHALE IN THE NORTHERN PACIFIC OCEANHIDEO TSUYUKI* AND UHEI NARUSE**INTRODUCTION

fhere have been many works on lecithin contained in the brain of human andmammal living on land as well as on lecithin in the egg of hen. The works on lecithinin the brain of human and mammal were reported by Yokoyama & Suzuki (1932 ab)
md Levene & Rolf (1921, 1922). Reviewing the works on lecithin in the brain)f sea mammal, Igarashi & Zama (1955) studied on lecithin in the brain of sperm
whale (Physeter catodon) and Alaskan plolack (Theragra chalcogramma).
According to the works of lgarashi & Zama (1955), the characteristics of the.ecithin in the brain of sperm whale was as follows : Iodine value 71.5, Nitrogen:ontent 1.68%, Phosphorus content 3.61 %, Choline content 14.4%. Further the)resence of palmitic, stearic, oleic and eicosatetraenoic acids was confirmed and the)resence of C 20 , C 22 , and C 24 acids as saturated fatty acids and C 20 monoenoic
lcid as unsaturated fatty acids were presumed as the component fatty acids of:he lecithin.
In the present work, the writers have studied the properties of the acetone;oluble lipid and phospholipids in the brain of the black right whale in the Northern
Pacific Ocean.
It is pleasure that the writers express here their sincere thanks to Dr. Hideo'.)mura and Dr. Akio Shionoya for their kind advices and thanks to Mr. Seiji Ohsumi
who had kindly them the frozen brain of the black right whale.
EXPERIMENTS AND RESULTS

fhe operation such as the distillation and concentration of solvents were taken inltmosphere of nitrogen gas. Iodine value was measured by Wijs' method, N:ontent by micro-Kjeldahl method, P by method of Beveridge & Johnson (1958),~thanolamine and serine by method of Levine & Chargaff (1951), choline by Glick's
method (Glick, 1944) and inositol by method of Bohm & Picharz (1954).
l. Material
Material is the frozen brain of the black right whale, Eubalaena glacialis (male, bodyength: 15.1 m, presumed age: more than 9 years old) which was caught in the:outhern sea of Kojak Island in the Northern Pacific Ocean (N: 55°53'-54', W:

l54°4'-6').
*Department of Food Engineering, College of Agriculture & Veterinary Medicine, Nihon University,49, 3-chome, Shimouma-cho, Setagaya, Tokyo.
~*Dept. of Fisheries, Coll. of Agr. & Vet. Med., Nihon Univ.
173

174

H. TSUYUKI AND U. NARUSE

2. Preparation of Acetone-Soluble Lipid and Phospholipids
The acetone-soluble fraction was 5 times extracted from 500 g of the frozen brairwith 5 weights of acetone, and the obtained acetone-soluble fraction was 3 time
extracted with 5 weights of ethyl-ether. Then ethyl-ether was distillated off fronthe ethyl-ether extract and the acetone-soluble lipid was obtained from the 500 !
of the frozen brain. The properties of the acetone-soluble lipid are shown ii
Table 1.
TABLE I.

PROPERTIES OF ACETONE-SOLUBLE LIPID IN BRAIN
OF BLACK RIGHT WHALE

Yield

N~

g
%*
6.3
1.26
Phosphorus Nitrogen
(%)
(%)
0.03
0.02

1.4765Choline
(%)
0.01

Acid
value
2.6
Ethanolamine
(%)
0.01

Iodinevalue
142.1
lnsitol(%)
trace

Sapon.
value
183. lSerine(%)
0.02

Unsapon.
matter(%)
1.37
Sterol**(%)
18.62* % of acetone-soluble lipid from the frozen brain.
* * % of sterol in unsaponifiable matter by digitonin method.

The residue obtained after extracting the acetone-soluble lipid from the brai1
and the residue after extracting ethyl-ether-soluble fraction from the acetone-solubl,
fraction were mixed, and the mixture was 5 times extracted with 5 weights of chloro
form-methanol solution (2 : 1). The extract was 3 times treated by the washing oFolch, Ascoli, Leas, Mexth & Lebaron (1951), and the chloroform layer wa
separated and then chloroform was distillated off from the layer. Thus the phospnolipids were obtained. After all 2.1 g of phospholipids was obtained from th;material (Fig. 1). The properties of the phospholipids are given in Table 2.
TABLE 2.
Yield
%*
0.42

Nitrogen
(%)
1.81

N/P**

Choline/P**

Serine/P** Ethanolamine/P**

1.08

0.31

~

g

2.1
Inositol(%)
0.82

PROPERTIES OF PHOSPHOLIPIDS IN BRAIN

Phosphorus
(%)
3.72

0.24

Choline(%)
4.68

Ethanolamine
(%)
4.11

Serine
(%)
3.03

0.55

* % of phospholipids from the frozen brain.
* * molar ratio.

3. Component Fatty Acids of Acetone-soluble Lipid and Phospholipids
Mixed fatty acids were prepared from the acetone-soluble lipid and phospholipid
in the brain of the black right whale by alkali-hydrolysis.
The mixed fatty acids obtained from the acetone-soluble lipid were separatec
into two portions, solid and liquid fatty acids with lead salt-ethanol fractionatio1of Hilditch (1956) (Table 3). After synthesizing the acetal-ester of the solid fattacids with mono-brome-acetone, the acetal-ester of solid fatty acids were dissolved and heated in ethanol, and a little excess volume of 2N-HC1 methane
solution contained 2,4-dinitrophenylhydrazine were added to the above solutior:

LIPIDS OF BLACK RIGHT WHALE

175

Then 2,4-dinitrophenylhydrazone of acetal-ester was educed by cooling thissolution at the room temperature, and separated with filtration. Finally 2,4-dinitrophenylhydrazone of the acetal-ester was identified with paper chromatography
as its 2 % benzene solution by the method of Inoue, Hirayama & Noda (1956 a).
The results are shown in Table 4. The other hand the liquid fatty acids were con-'T'"1Extract wifh 5 weights of acetone 15 times)

Residue

Acefone exfract

Extract with 5 weightsof ethyl-efher 13 times J

Residue

Acefone-soluble lipid

Extract with 5 weighfs ofchloroform/mefhanol 12: l I. solufion 15 times I

ResidueldiscardJ
Folch's washing

Chloroform soluble
fraction(3 times}

Methanolsolution!discard I

Phospholipids

Fig. 1.

Extraction and fractionation of lipids from brain of balck right whale.

verted into the methyl ester of the fatty acids by the usual method. Mercuric
acetate was added to the methyl ester of the fatty acids in methanol by method of
Noda, Hirayama & Inoue (1956), and the liquid fatty acids were indentified with

176

H. TSUYUKI AND U. NARUSE

paper chromatogaphy as their mercuric acetate complexes of methyl ester (Table

5).
TABLE 3.

FRACTIONATION OF MIXED FATTY ACIDS OBTAINED FROMACETONE-SOLUBLE LIPID
Iodine value
Kinds of fatty acids
Neutr. value%
151.2
Mixed fatty acids
191.8
Solid fatty acids
38.6
200.2
10.3
Liquid fatty acids
61.4
183.4
200.3

TABLE 4. RF VALUE OF 2,4-DINITROPHENYLHIDRAZONES OF ACETAL ESTER
OF COMPONENT SATURATED ACIDS OF ACETONE-SOLUBLE LIPID
Acid
SampleAcetone soluble lipid
Standard

Myristic
0.35

Palmitic
0.29
0.29

Stearic
0.22
0.23

Arachidic
0.15
0.16

Behenic
0.10
0.11

Paper: Toyo No. 2. Moving phase: methanol-acetic acid-tetralin (l"O: 2: 1).
Stationary phase: Tetralin. Development: Ascending at30°C. Detection: N/2 KOH
in ethanol.
TABLE 5. RF VALUE OF MERCURIC ACETATE COMPLEXES OF
METHYLESTER OF COMPONENT LIQUID ACIDS OF ACETONE-SOLUBLE
LIPID
Acid
SampleAcetonesoluble lipid
Standard

Zoomaric
0.26
0.25

Oleic
0.14
0.16

Eicosenoic
0.09
0.09

Erucic0.05
0.05

Linoleic
0.51
0.52

Stationary and moving phase were the same as shown in Table 4.
0.2% dinitrocarbazone in ethanol.

Linolenic
0.70
0.71

Detection :While para-bromophenacyl bromide were added into the methanol solution
contained 20mg of the mixed fatty acids (Table 6) separated from the phospholipids,
and the solution was heated for two hours in water bath and then cooled, 2N-HC1
methanol solution contained 1 % 2,4-dinitrophenylhydrazine was added into theabove cooled solution, and the solution was set on for 3 hours at room temperature.
TABLE 6.

PROPERTIES OF MIXED FATTY ACIDS OBTAINED FROMPHOSPHOLIPIDS

%
62.38

Neutralization value
155.3

Iodine value
190.1

Thus para-bromophenacyl-ester-2,4-dinitrophenylhydrazine of the fatty . acids
was synthesized. This ester was extracted with ether. After the extracted solution was washed with water, the water in the solution was removed with anhydrous
sodium sulfate and the ether solution was divided into two portions. After eachportion of the ether solution was concentrated to 1 ml, anhydrous methanol solution
contained mercuric acetate was added to one portion. The solution was heatedfor 40 minutes at 40°C and cooled. Then 1 ml of benzene, lOml of water and one

177

LIPIDS OF BLACK RIGHT WHALE

drop of acetic acid were added into the above solution, the benzene-ether solution
was produced on the upper layer. This solution was called as Sample B. To anotherportion, benzene, water and acetic acid were added by the same method mentioned
above except mercuric acetate, thus sample A was prepared. The sample A and
B of complexes of fatty acids were treated with paper chromatography of Inoue,
Hirayama & Noda (1956 b.) Rf value of the sample A and B are shown in
Table 7 and 8.
TABLE 7.

RF VALUE OF SAMPLE A OF MIXED FATTY ACIDS OBTAINEDFROM PHOSPHOLIPIDS BY BROMOZONE METHOD.
Acid
SamplePhospholipids
Standard

Myristic
0.34
0.35

Palmitic
0.27
0.29

Stearic
0.23
0.23

Arachidic
0.18
0.17

Behenic
0.12

Oleic
0.25
0.26

Paper: Toyo No. 2 (40 X 40 cm). Stationary phase: Petroleum hydrocarbon (b.p.
140-170°C). Moving phase: Methanol-Acetic acid-Petroleum hydrocarbon (5: l: l).
Ascending at 30°C. for 5 hours.
TABLE 8.

RF VALUE OF SAMPLE B OF MIXED FATTY ACIDS OBTAINEDFROM PHOSPHOLIPIDS BY BROMOZONE METHOD
Acid
SamplePhospholipids
Standard

Zoomaric
0.55
0.57

Oleic
0.51
0.51

Eicosenoic
0.46
0.47

Erucic0.41
0.39

Linoleic
0.73
0.72

Linolenic
0.68
0.69

Paper, ascending method, stationary and moving phase were the same as shown in
Table 7.

4.

Silca Gel Column Chromatography and Silicic Paper Chromatography of
Phospholipids
Phospholipids in the brain of black right whale were fractionated with silica gelcolumn by Rhodes' method (Rhodes & Dawson, 1960). At first, 40 g of silica gel(Kanto-kagaku Co.) being suspended in methanol, the silica gel-methanol solution
was poured into glass column fulled with glass-fiber for chromatography (30 X
1.7 cm), and set until the silica gel was sunk down. Then the solution was flowed
down by opening the glass cock of the column. Directly before the surface of thesolution reached to the top of silica gel column, the column was washed with2 volumesof chloroform. When 2-3ml of chloroform was remained on the top of the silica
gel column, the cock was closed. Next, the chloroform solution contained 100 mg
of phospholipids was added into the column mentioned above. Various phospholipids were eluted with various methanol-chloroform solvents (Table 9). The
properties of each phosphlipid are shown in Fig. 2 and Table 10.
Morever, the phospholipids were treated with silicic paper chromatography(Rhodes & Dawson, 1960) to separate each phospholipid. After the filter paper
for chromatography (Toyo No. 51) was immersed in sodium silicate solution(sodium silicate: water/I : 1) for 5 minutes, the paper was suspended in air for 5
minutes and the excess solution was removed from the paper. Further the paper

178

H. TSUYUKI AND U. NARUSE
TABLE 9. SOLVENTS FOR FRACTIONATION OF PHOSPHOLIPIDStS im
Kinds of phospholipids
Chloroform : Methanol
Volume(ml)~she<
25Polyglyceroric acid90: 10ern1
15Phosphatidyl serine and Phoshatidyl ethanolamine 1
80: 20ared
Phosphatidyl ethanolamine50
75: 25·eatt
Monophosphoinositide25
60:40
Phosphatidyl choline
50:50
75
Sphingomyelin and phosphatidyl choline
10: 9025

TABLE 10.

PROPERTIES OF EACH FRACTION OBTAINED FROM PHOSPHOLIPIDS
BY SILICA GEL COLUMN CHROMATOGRAPHY
Fraction

Component
Phosphorus (%)
Nitrogen (%)
Choline (%)
Ninhydrin
Inositol
N/P*
Choline/P** Molar ratio

1
3.31
0.23

0.15

2
3.72
1.62

3
3.70
1.61

4
3.34
0.39

5
3.36
1.66
12.40

6
3.67
3.34
11.30

+

+

+
+

+
+

+

0.95

0.96

0.25

0.99
0.87

2.01
0.83

f(µg)

!5

20

25

30

35

f-90: 10.fso:2of---1s: 25----:-60:.10-:- s o :25Fig. 2.

15

50

25

75

T.be

so--: o: o~~ !~~:orc!o,~:-,,,·~or.ol]
i

i

25

ml

Silicic column chromatography of phospholipids.

RF VALUE OF PHOSPHOLIPIDS BY SILICIC PAPER
CHROMATOGRAPHY
ComponentStandard
SamplePhosphatidic acid
0.72
Phosphatidyl ethanolamine
0.56
0.54
Phosphatidyl serine
0.52
0.51
Phosphatidyl choline
0.33
0.33
Sphingomyelin
0.23
0.22
Inositol phosphatide
0.20
0.18
Development: di-isobutylketone: acetic acid: water (40: 25: 5), ascending at room
temperature for 12 hours.
Detection : all lipids-rhodamin 6G, phosphatidyl choline and sphingomyelinmolibdic phosphate, phosphatidyl ethanolamine and phosphatidyl serine-ninhydrin,
inositol phosphatide-Elson-Morgan reagent.

TABLE 11.

\s

:ig1
an
se1
oi
li
c

LIPIDS OF BLACK RIGHT WHALE

179

was immersed in dilute HCl solution for 30 minutes. Then the paper was 5 times
washed with water in order to remove the HCI. After setting the paper in air
overnight, the paper was dried for one hour at ll0°C. The silicic paper was prepared by the process above mentioned. The result of chromatography which wastreated with this silicic paper was shown in Table 11.
DISCUSSION

As has been shown in Table 1, the acetone-soluble lipid in the brain of the blackright whale contained a trace of nitrogen, phosphorus, choline, ethanolamine, serine
and its acid value and unsaponifiable matter content were lower. Therefore it
seems that the main constituent of acetone-soluble lipid is glyceride, and the contents
of free fatty acid and unsaponifiable matter are little. The yield of acetone-soluble
lipid was 1.26% to the frozen brain of the whale, and was comaratively lower. The
content of sterol in unsaponifiable matter was 18.62 %. The paper chromatography
by the method of Inoue, Hirayama & Noda (1956 a) and Noda, Hirayama &Inoue (1956), indicated that palmitic, stearic, arachidic and behenic acids as saturated fatty acids, and zoomalic, oleic, eicosenoic, erucic, linoleic and linolenic acids
as unsaturated fatty acids were contained in the acetone-soluble lipid (Table 4, 5).
M yristic acid was unable to be found in the aceone-soluble lipid in the brain.
The yield of phospholipids obtained from the brain of the whale was 0.42 %.
The properties of the phospholipids were as follows : choline content 4.68 %,ethanolamine content 4.11 %, serine content 3.03% and inositol content 0.82%(Table 2). According to the bromozone method of Inoue, Hirayama & Noda (1956b), component fatty acids of the phospholipids seemed to be myristic, palmitic,
stearic and arachidic acids as saturated fatty acids, and zoomaric, oleic, eicosenoic,
erucic, linoleic and linolenic acids as unsaturated fatty acids. However, the presence ofbehenic acid was not able to be found in the phospholipids.
When the component fatty acids of the acetone-soluble lipid and phospholipids
were compared, the presence of palmitic, stearic and arachidic acids were found inboth lipids in the case of saturated fatty acids, but myristic acid was not containedin the acetone-soluble lipid and behenic acid was not able to be found in thephospholipids. Considering of the component unaturated fatty acids, their component acids were common to both lipids and the presence of zoomaric, oleic,eicosenoic, erucic, linoleic, linolenic acids were observed in the case of both lipids.
Perhaps, various component unsaturated fatty acids seemed to be contained in phospolipids in the whale brain. The results of silica gel column chromatography
by Rhodes' method indicated the presence of phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl choline, sphingomyelin and inositol phosphatide
was observed in the phospholipids. But phosphatidic acid was not able to foundin the phospholipids (Table 11).

180

H. TSUYUKI AND U. NARUSE

SUMMARYI) The characteristics of the acetone-soluble lipid and phospholipids in thebrain of black right whale in the Northern Pacific Ocean were studied.
2) The yield of acetone-soluble lipid obained from the frozen brain was 1.26 %and sterol content in unsaponifiable matter was 18.62 %. The results of paper
chromatography by the methods of Inoue, Hirayama & Noda (1956 a) and Noda,
Hirayama & Inoue (1956) indicated that the presence of palmitic, stearic, arachidic,
zoomaric, oleic, eicosenoic, erucic, linoleic and linolenic acids in the acetonesoluble lipid.
3) The yield of phospholipids obtained from the frozen brain was 0.42 %.
The results of the bromozone method indicated the presence of myristic, palmitic,
stearic, arachidic, zoomaric, oleic, eicosenoic, erucic, linoleic and linolenic acids inthe phospholipids.
The results obtained by the silica gel column chromatography and silicic paper
chromatography indicated the presence of phosphatidyl ethanolamine, phosphatidylcholine, inositol phosphatide, polyglyceroric acid, phosphatidyl serine and sphingomyelin in the phospholipids.
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