;.
FISHERIES RESEARCH BOARD OF CANADA
NATIONAL RESEARCH COU;,:;',
Translation Series No. 658
OTTAWA
CANADA
PHOSPHOLIPIDS OF AQUATIC ANIMALS -
By Koichi Zama
FISHERIES RESEP.RCH BOARD OF CANADA
Library
Lor.ry
V1.ft STREET,
1707
P. O. PC.X 4'29,
HALIFAX, NOV. 's SCOTIA
From: Bulletin of the Japanese Society of Scientific
Fisheries, Vol. 30, No. 8, pp. 660-672, 1964.
Translated by: T. Murayama
Bureau for Translations, Foreign Languages Division,
Department of the Secretary of State of Canada
Fisheries Research Board of Canada
Technological Research Laboratory,
Halifax, N. S.
1966
!.; cl
2.23
c
MAR -
,
.
SI:CRÉTARIAT D'ÉTAT
DES TRADUCTIONS
DEPARTMEe OF THE SECRETARY OF STATE
BUREAU FOR TRANSLATIONS
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DIVISION
TRANSLATED FROM
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CANADA
TRADUCTION DE
INTO
Japanese
••■
English
SUJET
SUBJECT
Bulletin of the Japanese Society of Scientific Fisheries.
v.30, no.8, p.660-672 1
AUTHOR ••• AUTEUR
Zama, Koichi
TITLE IN ENGLISH ••- TITRE ANGLAIS
Phospholipids of aquatic animals.
TITLE IN FOREIGN LANGUAGE
•■
TITRE EN LANGUE éTRANGèRE
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DATE COMPLETED
REMPLIE LE
Jan., 1966.
2, Feb.,1966
{-0.;
.
Bulletin of the Japanese Society of Scientific Fisheries Vol.30, No.8, 1964
2) Phospholipids of Aquatic Animals
Zama, Koichi
Among lipids which are called conjugated lipid in living organisms,
the lipid containing phosphoric acid is specified as phospholipid. And '
they are classified into two groups according to their construction, i.e.
one is glycero-phospholipid constructed with L-a-Glyerophosphoric acid
as basal comelent, and the other is sphingo-lipid whose basealcomponent
is sphingosine (D-Erythro-1,3-dihydroxy-2-amino-4-trans octadecene).
Situation of Phospholipids in Organisms
Phospholipids contain both
hydrophobic groups (fatty acid residues) and hydrophilic groups (phosphoric
acid residues or basic groups) in one molecule and they accumulate at
interface area between water and fatty subtance layer.
It is considere4
that hydrophilic groups may be combined with protein by electrostaticielyor by hydrogen bond and one part of 'hydrophobic groups seem to form socalled lipoprotein by Van der Waals Force.
The major parts of phospholipids in serum exist as a form of a- orplipoprotein, and phospholipids in organs or muscles also exist as a complex
such as lipoprotein.
In cell, lipoproteins are aklo located at nuclei,
mitocondria or microsomes, e.x. lipoprotein contents of nucleus in
liver tissue are reached to 20-30 % of wet weight (about 10 % of its lipoprotein is phospholipid and cholesterol 1 )).
Cell membrane is, also,
a kind of lipoprotein, it isolates the inside water of cell from outside
and regulates transportation of substances.
It is difficult to extract conjugated lipid with benzin or ether,
directly, since the conjugated lipid combine with protein in the dase of
lipoprotein.
Combinations between protein and lipid in lipoprotein molecule
seems to be some kinds of coordination hond, elctrostatic bond, or
coupling with auxiliary valency and as proteins which combine with lipid,
they are phosphoprotein, nucleoprotein or simple sprotein.
-1-
Distribution of phospholipids
There are many reports about phosphOJaviller 2 ), Kaucher 3 ) /
lipids contents in land animal organs.
and the auther 5 ) have been reported about the distribution
Matumo4)
of phospholipids in aquatic animals, especially in fish organs. (shown
in Table 1)
Table 1. Phospholipid content of fish tissues, as per cent of wet weight.
Fish
_ Tissue
Sardine
Herring
Tuna
Groper ,
Shark
Liver
0 . 3 8 – 0.43
Salmon
Cod
Ordinary muscle
Herring
Herring
Phospholipid.;
(%)
:I
0.49
;
2.63–'2.85
1
2.05
.I
;
0.16-1.50 il
0.30
■1
i Dark colored
muscle
Fish
Various species Brain
Phospholipid
(%)---- ---9.4-29.4*
Various species Spinal cord
25.6-39.3*
Herring
Milt
1.29
Herring
II Salmon
Roe
1.30
4.00
Intestine
0.41
0.47
1.00
0.35-0.80 . Sardine
;i
0.90-0.97 ;: Tuna
2.58
Tissue
:I Snoek
11
,
* Per cent of dry weight
The components of phospholipids are also shown in Table 2 1
although they are not absolute data because analytical methods adopted
for each phospholipid were not yet completed one and also physiological
condition of samples used for was not same, as it is very dificult to
-66 0
adjust.
Table 2.
-
Components of fish phospholipids as per cent of total phospholipids.
— ----------------Fish
Tissue
Lecithin
Cephalin
Sphingomyelin
I
Cod
; Flesh
77
12
11
;
Salmon Flesh 58 42 .:: nil
Tuna
Flesh
,
41.8
48.3
',
9.9
Various siiecies*
Brain
27-39 (33)
41-59 (52) ; '2-24 (15)
Various species* , Spinal cord
28-36 (32)
24-52 (43) : 15-40 -(25)
* Average values for seven species, in parentheses.
One can suppose by these results that brain and spinal cord etc.
have high phosPholipid contents when compared with other organs in fish
as same as in mammals, and muscle phospholipids contains considerable
amounts of lecithin, on the other hand, phospholipids in brain and
spinal cord are rich in sphingomyelin.
Differences in such components
might be depend on differences of mechanisms between each organ.
Extraction of Phospholipids
Phospholipids has some kinds of
combination with protein in organisms, therefore, it is necessary to
split off phospholipids moiety from combined substance before extraction,
-2-
•
It is very difficult to extract phospholipid in
lipoprotein by benzin
or ether but if ethahol added ether is used for extraction or lipoprotein
is pretreated with hot ethanol before ether extraction amounts of extracted
phospholipid should be increase.
Macheboeuf 6 ) studied the effects of
ethanol on the amount of extracted phospholipid using lipoprotein Cenapse
which prepared from horse serum, and he suggested that lipoprotein
should be extracted by ether after penetration of ethanol into combined'
water and dehydration occurred.
In the case of extraction of phospholipid from large amount of
tissues, benzin or ether extraction after aceton pretreatment have been
used. Effects of aceton in this case should be that water, neutral fat,
sterin, carotinoid etc. are removed by aceton and at the saine time
combination between phospholipid and protein are broken by aceton.
(However, during the aceton pretreatment of tissues, a part of phospholipid was also extracted by mutual solubility between lipids 7 ).)
Therefore, it is better to extract using special solvent which is choiced
accoring each phospholipid wanted to avoid mixing of other kinds of
phOspholipids.
In the case of direct extraction of conjugated lipid
fraction from fresh tissue, ethanol-ether, methanol-ether or chloroformmethanol ( 1 s 1 - 4 : 1 ) have been used. However, if the combination
between protein and phospholipid is more rigid, use of mixed solvent
such as chloroform-methanol-10 N HC1 ( 2 s 1 s 0.02 ) are recommended.
If the solvent such as chloroform-methanol system is used for extraction,
none lipid substances such as amino acids, urea or sugars ect. are also
extracted.
Therefore, it is necessary to use some technics for
elimination of ne lipid. The method adopted by many workers Fo1ch's
washing method 8 ) or cellulose column chromatography introduced by
Bevan 9 ).
Purification of Phospholipids The isolation and purification of phospholipids are doing by combination of suitable methods after fraction by
organic solvents.
-3-
(1)
Purification by solvents.
Each phospholipid has different
solubility to organic solvent as shown in Table 3, therefore it is -661possible to fractionate using these solubility difference.
(cf. Series
of Experimental Chemistry edited by Japan Chemical Society V01.23, P 490.)
Table 3. Solubility of phospholipids in various solvents.
j
Lecithin
Lysolecithin
Phosphatidylethanolamine
Phosphatidylserine
Cardiolipin
Inositolphospholipid
Sphingomyelin
Acetone Ethanol Ether I Chloroform Pyridine Acetic aciel
+
+ 1
— 1
+ I
+
+
+
+.
+
!
i
I.
,
.-+
i
+
+
—
+
I
+
+
+
+
—
-
1
1
I
- ;
+
,•
.•
.
!
+'
+
:
+. i
+
+
+
+
+
I
-
I
i.+
-
+
+
Fo10h10 ) has done further fractionation of cow's brain cephalin
fraction by using chloroform-ethanol mixture.
Sinclair 11 ) has purified
lecithin by low temperature treatment of ethanol solution of egg-yolk
phospholipid.
(2)
Purification by Double salt.
Phospholipid forms double salt with
cadmium chloride or barium chloride and solubility of these double salts
show remarkable difference from those of original phospholipid.
Purification method used such solubility difference was adopted for
purification of lecithin by McLean and Levene et al. previously, and
after that Panghorn 12 ) had isolated and purified lecithin from egg-yolk
and moreover cardiolipin and lecithin from cow heart by using this method.
(3)
Purification by column chromatography.
This method is progressed
by Rice 14) and Hanahan 15 ) since Taurog 1 3) had used magnesia adsorbent
for fractionation of liteer phospholipid in
1944.
Hanahan 15 ) got
purified lecithin from egg-yolk, liver and other tissues by using
This method gives good results for
alumina column chromatography.
purification of so called cholin containing phospholipid such as lecithin,
sphingomyelin,
but it is very difficult to get good recovery of
cephalin fraction ,
moreover, oxidation of phospholipid or formation
of Lyso-compounds were observed so often during operation 16 ).
4
Recently, silicate column chromatography which has considerably less
fault has been progressed by many research workers 1 7). When conjugated
lipid is fractionated by silicate column chromatography, usually conjugated
lipid is extracted with chloroform first of all, next with chloroformmethanol mixture and at the last with methanol.
Rhodes and Dawson 18 )
got result about relationship between compnents of extracting reagents
and conjugated lipid extracted, as it is shown in Table 4, but it is
not complete
one.
Table 4. Fractionation of conjugated lipids on silicic acid.
Eluting solvent
(Me0Ii
Eluted materials
in CHCI3)
.
5-10
10-20
20-40
50
Unidentify
Polyglycerophosphoric acid
Phosphatidic acid, Phosphatidylserine Phosphatidylethanolamine
Monophosphoinositide, Phosphatidylcholine, Lysophosphatidylethanolamine
Lysophosphaticlylcholine, Sphingomyelin
For example, if acetal phospholipid is included in it, component ratio
of phospholipid is fractuate and bad effects of air were observed during
preparation of phospholipid, moreover there are some gaps on the
eluating condition for the difficultiness to get same conditionated
silicate gel at every time.
In some cases, it is impossible to get
sharp fractionation by mutual interferences between phospholipids.
In
such a case rechromatography is required and it .is necessary more or
less empirical technics for selection of ratio of eluting solvent or
condition of charge of adsorbent.
In the case of slicate column chromatography of phospholipid,
_eluate becomes acidic gradually.
This may be caused by ellimination
of alkaline-earth metals which had combined with phospholipid during
the chromatography. To avoid this effect, some one used following
methods, i.e., pretreatment of silicate with sodium bicarbonate 1 9) or
• 2Q), or acid treatment 11 ) of phospholipid before desalting.
ammonia
Moreover, there are some reports to get good fractionation of phospholipid using cellulose 22) , florisil 23 ), Mg-trisilicate 24 )-column
chromatography.
Ion exchange resin was not used for isolation of phospholipid
but for isolation of partially hydrolysates of phospholipid to determination
of structure.
5
4, •
-
But recently application of ion exchange cellulose column chromato•
graphy to fractionation of lipid is discussing 25)
(4) Purification by counter current extraction.
Application of
counter current extraction for purification of phospholipid in fish meat
or others was tried
26) , but one can not expect to get good results by
using this method.
(5)
Purification by dialysis.
There is a report 27 ) that lipid
in cellophane bag was dialysed against some kind of solvent for purification
of phospholipid.
Above mentioned purification methods have both good points and
bad points each other, so it is difficult to get a good isolation and
purification of each phospholipid by only one procedure.
(1)
Paper chromatography.of phospholipids.
It was proposed to -662--
apply of paper chromatogrphy for analysis of phospholipids by Bevan 28)
after that this method was checked by Huennekens and Hanahan 29) ,
Hack 30 )
and Marinetti. Hara et al. 32) got a good results using nitric
acid treated paper, erhammer et al. 35 ) used Formalin treated paper,
Lea et al. 34 ) and Marinnetti et al. 35 ) used silicic acid treated paper
36)
and Dieckert et al.
used silicic acid treated glass fibre filter.
Zipper et al. 37 ) adopted paper electrophoresis and Douste-Blazy et al.
diviced a method which combined paperchromatography and counter CUrrent
extraction 38)
However, the methods adopted by many workers are that paper is
treated with silicic acid and diisobutylketone-acetic acid- water is
used as a solvent. ( By Marinetti et al: 35 )) Chloroform-alcohol mixture
is also used as a solvent.
Procedure for development of spots is
shown in Table 5.
Lipids
Table 5. Detection reagents for phospholipids.
: Rhodamine B or 6G
: HANES-IsITERWOOD'S
: Ninhydrin
LEVD;E-CHARGAFF'S
Choline
6%-HgC1 2 aq. Fuchsine-S0 2 or 2, 4-DNPH
Acetal-lipids
Cardiolipin etc.: Nile Blue
Amino-Base
(2)
Thin layer chromato-
Thin Layer Chromatography of phosphO.ipies.
graphy was introduced for analysis of phospholipids as like as other
natural substances since its rapidness, sharpness and effective ( if
For
compared with column chromatography or paper chromatography
the analysis of phospholipids, silicate gel mixed with suitable amounts
of Ca-sulphate is used for thin layer.
Selectioh of solvents is in
accordance with paper chromatography. But it is necessary more advance
for the amounts of Ca-sulphate in silicate gel or choice of solvents.
Thin layer chromatography and also paper chromatography should be
applied to not only qualitative analysis but also quantitative analysis
after more investigation 39)
For development of spots can use saine procedure with paper
chromatography, in addition it stronger reagent such as sulfuric acid
-
can apply.
(3)
Direct analysis.
Table 6. Analytical Methods ( I).
FISKE-SUBBAROW's, KING's or LIEB's method
: Micro-KJELDAHL procedure
Amino-N : VAN SLYKE's or LEA-RHODES' method
Ester-Group: RAPPORT-ALONZO'S method
Glycerol
: 13LIx' or RAMSAY'S method
Aldehyde
WITTENBERG's or REUPOLD's method
bd. Value : WiJs' or YASUDA's method
Choline
(4)
Table 7. Analytical Methods (II).
• GLICK's or APPLETON's method
Serine, Ethanolamine
NOJIWA-UTSUGI's or BURMASTER's
Inositol
Glycerophosphoric Acid:
Sphingosine
•
Eit)Hm's or WOOLEY's method
BURMASTER's or OLLEY'S method
MCKIBBIN-TAYLOR'S
Determination of Hydrolysates.
method
method
In the case of analysis for each
constitutive components in phospholipids, condition of hydrolysis
should be consider first of all.
Ohb can choice suitable condition
for hydrolysis if each component is analysed individually and this
is not so difficult but if the analysis of each component should be done
at the same time, it is very difficult to find out an ideal condition.
Usually, ild.rochloric acid is used for determination of cholin, serine,
ethanol amine and inositol,
sulphuric acid for sugars, pottasium
hydroxide-ethanol for fatty acids and for sphingosine barium hydroxide
and hydrochloric acid hydrolysis are used together.
Physiological meanings of Phospholipids
Living organisms contain
about 70 % of water and the majority of substances present in organisms
are water soluble.
All of the reactions performed in organisms are
proceeding in aquatic phase.
Therefore, it should be exist some
special mechanisms for translocation or metabolism of water insoluble
fat in organisms. Phospholipids may be related to this mechanisms.
The major parts of bleaod lipid are neutral fat, phospholipid, cholesterol,
cholesterol eSter and a little of lipid-like substances. And majority
of such lipids are soluble state in blood plasma as a form of lipoprotein
and move into any tissues.
In blood plasms, there is a particle, -663--
called kiromicron, this particle is considerable increase when
fat is feeded and sometimes blood plasma becomes turbid but after a
few hours it becomes clear as fat particles are disperse into fine
particles. Although this mechanism depend on the action of Clearing
factor 40 ), phospholipids are also an important factor for this mechanism
and phospholipids seem to act as a stabilizer for kiromicron. Artom 41)
àha
Zilversmit 42 ) reported that lecithin accelerates oxidation of
fatty acids in living organisms.
Another physiological role of phospholipids is
as a cell
constitutive substance, namely a large amonnts of phospholipids located
in mitochondria and microsomes.
Moreover, succinic oxidase or ATP-ase44 )
are inactivated by the action of phospholipase,
therefore one can
suppose the important role of phospholipid for their activities.
In addition to these, cephalin fraction concernsto the reaction of
blood coagulation 1 5) and a kind of phospholipid,e.g. cardiolipin,
has a function as antigen for syphilis serum reaction •4 6),.
Biosynthesis of Phospholipids
Biosynthesis or metabolism of phospho-
lipids are investigated using radio isotopes such as 32P or 14 C and
velocity of uptake or turn over ratio were compared in each animal
tissues.
Although there are many reports about metabolism of phospho-
lipids in animal organs ( Chargaff 47 )), generally speaking, 3 2P
-8-
e
which is fed as inorganic phosphate through the mouth or intraperitioneal
injection is incorporated into phospholipids in every tissues. Incorporation
rate differs in each tissues
and
also it depends on the feeding methods.
For example, if 3 2P is fed through the mouth, a large aMounts of 3 2P
accumulate in liver and small intestin and in the case of intrapertioneal
injection, 32P- incorporates into organs in following order, i.e., liver,
spleen, kidney, lung and small intestines.
Distribution of 313 which is incorporated into phospholipid fraction
was traced using many kinds of phospholipid fractionation methods.
Sakagami et al 48 ) got a result shown in Table 8 using 3 2P and rat liver -664
Specific activities of phospholipids in rat
liver after intraperitoneal injection of p32,48)
Specific actiity
Phospholipids
30min. 12 hrs.
3x1o8
Lecithin
110 x
Phosphaticlylethanolamine
13
103
Phosphatidylserine (Phosphatidic acid) 125
102
Inositol phospholipid
17
102 -1
Table 8.
as sample. Namely, in the early stage,
incorporated 32 P was found in
inositol phospholipid first of all, next in phosphatidylethanolamine,then
in phosphatidylserine and lecithin so on, bit as time advances it becomes
almost same level.
Specific activity of 32 P in blood plasma is lower than that of
liver in early stage after 32P feeding but after 12 hours it becomes almost
same to that of liver. Therefore many investigator have considered that
liver is a main source of blood plasma phospholipids.
And also many workers had been done using tissue slices 49 cjr
tissue homogenates in same methods. These results showed that 32P was not
incorporated into lecithin or phosphatidylethanolamine directly but it Ilies
incorporated into only phosphatid or inositol phospholipid..
Although
many informations about phospholipid metabolism had been reported, the
concrete explanation for biosynthesis mechanisms could not be done.
However, Kornberg et a1 5°) and Kennedy et al 51) showed biosyhthetic
pathway of glycerophospholipid using enzyme systems and Zabine and Mead 52)
Brady et al 53 ) and Kennedy et al 54 ) also showed the pathway of sphingomyelin
as shown in Fig. 1 9 6 )•
Palmitic acid
' Choline (ethanolamine)
7->ATP
(1g P /IP -1--
. Mg+ 41 (Choline phosphokinasc)
Dihydroxyacetone phosphate
DPNH + Hi.
(•Glycerophosphate
dehydrogenase
ATP (UTP)
Mg+ +
PN + Co-A
I
(Glycerokinase) (m n +)
DPN+
Palmityl Co-A
ADP
Phosphoryl -choline(-ethanolamiqe)
Glycerol
Palmital
(Phosphorylcholine cyfidyl transferase
Phosphorylethanolamine cytidyl transferase)
I
Pyrophosphate
Cytidine diphosphate-choline (-ethanolamine)
ADP (UDP)
a-Glyceropt osphoric acid
7.•---2 Coenzyme A-activated NH3
fatty acids
I
I
Corophosphate
.
(Serine
Dihydrosphingosine
in-H2
v in
1 CFlaav
00
2-0--P-0--Y-0CI-I2CH2N(CH3)3
CH
6-
a-Phosphatidic acid
Sphingosine
Co-A-activated
fatty acid
i(Phosphatidic acid
ffl lycerylkinase)
ig
ATP
phosphatasc)
D-1,2-Dig1yceride2
(-1- H3PO4)
CTP
4"
.
Cytidine diphosphate diglyceride.
mg++y
(mn++)y
es;
Plasma!
monoglyceride
r
Ceramide
(Yhosphorylcholine
ceramide transferase)
Mn++
o.
- Inositoll
Phosphzttidylinositol (-1- CMP)
1.7
f.
Lecithin (+ CMP)
(phosphatidyl ethanolamine)
Choline plasmalogen (+ CMP)
(ethanolamine plasmalogen)
Sphingomyelin (+ CMP)
Fig. 1. Enzyme systems that can synthesize phospholipids in vitro and possibly in vivo".
Grp
Ct)
cy
Enzyme which hydrolyse
Enzymatic decomposition of Phospholipids
classified into phosphophospholipid is called phospholipase. The are
by their reaction mechanisms.
lipase A, B, C, D and lysophospholipase
This enzyme liberates one fatty acid from lecithin
Phospholipase A.
(1)
Reaction site of this enzyme
with the formation of a*lysolecithin.
discussion, following decision have been
was not clear, but after long
-666
gotten that phospholipase A might 4ttack to H-position.
-.
Table 9. Phospholipases.
Property
Name
Distribution
Ph-E—>'Lysoph-E+FA (1 mol.)
Lysophospholipase
Phospholipase B
Penicillium notatum
Lysolec -4 GPC+FA (1 mol()
Aspergilliis oryzae
Lysoph-E —>GPE+FA (1 mol.)
Mammalian tissues
Lec —›GPE+FA (2 mol.)
Pancreas, Intestinal mucosa,
Human plasma
Rice, wheat bran
Senatia plymithicum
Ph-E ---> GPE+FA (2 mol.)
Aspergillus oryzae
Penicillium notatum etc.
Liver, Brain,
Snake venom
a-toxine of Clostridium perfringens
(WELcHn) etc.
Lec —>PC+Diglyceride
Phospholipase C (or D)
_
Scorpion venom
Snake venom
Bee venom
'Mammalian tissues
Aspergillus oryzae
Rice germ
Lee --+Lysolec+FA (1 mol.)
Phospholipase A
.
Sphm --›PC+Ceramide
Lec Ph-A+ Choline
D (or C) Ph-E-->Ph-A+Ethanolamine
Phospliae
Cabbage
Carrot
Cottonseed
Ph-S-->Ph-A+Serine
want to introduce some historic review about this problems.
Trie auther
Hanahan 55 ) supposed that there are two passway as shown in Fig. 2...when
F1,COCOR
R'000t:'1.1
11S-0-Ph -Choline
Fuon
wouié•
11,CC/COR
moéu
ir;é..o-ph-choune
i
NMnOi. 37C, 8
i
hrs.
C0011
KM,10,,
arc.
8hrs.
11,COCOR
(.1-0
lf,è-0-1 1 11-Choline .
WOCO(.:11
11,é-0- Ph C.:bonne
•I 2 N.11C1, 1e°, 2
..
..
II:é.0-1'11.Clniline
hrs.
2 N-IIC1. 913°C. 9 11rs.
Ç.0011I
110C11 0
+ In0011
.11.C-0-. on
4:Hi
4 . ri,onne
Or
Ph Chutine
(
coo)
n
noq-u-én
ou n,Con
11,(10 1 1
é-0'
RC001f
-
0
° - ?'- 011
1 1C1
CI T
luo,)
Fig. 2. The Site of action of phospholipase A on lecithin.
-1- RC0011
Cludine
L.
phospholipase A reacts with lecithin.
And he got 3-phosphglyceric acid,
fatty acids and cholin as products, therefore he had decided that
passway A was reasonable (namely this enzyme reacts at a'-position.
Marinetti et al 56 ) used 3 2P labelled lecithin prepared from
rat liver as substrate and snake venom as enzyme and method adopted was
almost same with Hanahan. By their results, 35 % of 3 2P was present in
ketolesolecithin and 60 % of 3 2P was in lysolecithic acid, moreover
2-and 3-phosphoglyceric acid, phosphorylcholin, and orthophosphoric acid
were identified in following hydrolysate. His opinion is that
phospholipase A
reacts with both a'and p position.
Tattrie 57 ) got Œ!43-diglyceride by the action of phospholipase C
( same with phospholipase D)
onto lecithin and prepared triglyceride by
the addition of known fatty acid. He compared fatty acid which was
liberated from the triglyceride by the action of pancreatic lipase with
fatty acid which was liberated from lecithin by the action of phospholipase A.
( These experiments were planned from the facts that pancreatic lipase
hydrolyses only primaly ester of triglyceride.)
By the results of
these experiments he had suggested that phospholipase A
position.
Saito agreed with this results, recently.
reacts with Ç3And what phospho-
lipase A also reacts to 13-position of phosphatidylethanolamine was recognized.
Lysolecithinase and phospholipase B.
Contardi and Croon found
that if lysolecithin was incubated with old extraction of rice bran,
its hemolytic action disppeared with the formation of glycetophospholylcholin.
After that, this enzyme, present in the rice bran extracts, have been
called lysolecithinase or phospholipase B.
Fairbairn 58) found that
the enzyme come from Penicillium notatum reacted with lysophospholipid
but did not to phospholipid. Uziel, Hanahan 59) also have gotten same
results. Therefore there were some doubts about whether phospholipase B
and lysolecithinase were saine enzyme or not.
Dawson 60) found that
when raw lipid prepared from rat or sheep liver was suffered an action
of enzyme prepared from Penicillium notatum, the raw lipid liberated
fatty acids quantitatively, faster than lecithin.
- 12 -
But if purified
lipid instead of raw lipid wao used,
the enzyme could not attack the lipid.
He traced this activating rnotor of enzyme and he suggested that it
was monophosphoinositid and crxrdiolipin-like phospholipid.
Therefore
one enzyme which reacts with only lysolecithin is called lysolecithinase
( lysephospholipase ) and another enzyme which has additional special
Both two enzymes seem presence in
properties is called phospholipase B.
Penicillium notatum.
Table 10.
Enzyme
Phospholipase B
Properties of phospholipase 13 and lysolecithinase.
•
Opt. PH •
3.1-6.7
Inhibitor
Other characteristics
Ca2 +, Mg2 +, F-
Activated with cardiolipin etc.
Inactivated for 10 min. at WC.
from Bacteria
CN— , Heavy metal,
3.5-4.4
Lysophospholipase tissues of animal and plant. F—, CH2ICOOH,
Inactivated at PH 4.5, 41°C.
Ether, Ethanol
6.0-6.2
(3)
Phospholipase C and D.
Definition of these enzymes are reversed
in point of their reaction position sometimes. There are few reports
about properties of
these enzymes.
Ester linkage between two molecules
of fatty acids which are included in substrate molecule is necessary to
react with these enzymes, so these enzymes might not react with lysolecithin
or glycerophosphorylcholine etc.
(4)
Other phospholipid hydrolyzing enzymes.
Phosphatidic acid phosphatase
splits off phosphoric acid and diglyceride from phosphatidid acid, it
exists in spinach or animal tissues.
Lysolecithin migratase which is found in Penicillium notatum, rice
embryo or pancreatin etc. makes transmutation of fatty acids in lysolecithin
molecule.
Glycerophosphorylcholindiesterase decomposes glycerophosphorylcholin into phosphoglyceric acid and cholin. It has been found in -668
Serratia phymuttica, liver etc. There is a report about the presence
of inositolphospholipid splitting enzyme.
Researdh Situation about phospholipid in aquatic animais.
It is
very unsatisfiable situation about strudy of phospholipid in aquatic
animals when compared with that of land animals. Only investigations
were that of phospholipid contents in tissues of aquatic animais, mainly
fish, properties of isolated phospholipid and behaviour of the lipids to
organic sdrients.
- 13 -
It is necessary to consider about extraction methods of lipids
from tissues for the study of their properties. Lovern et al 61),
Nagakura et al 62) and the author had investigated about conditions for
extraction of lipid from fish meat. Namely, Lovern and Nagakura had
investigated the contents of free- and boundaly-lipid in fish meat after
extraction with benzin, ether, ethanol, chloroform, chloroform-methanol-HC1
successively in above order from fresh fish meat.
The author also had
compared the amounts of lipid extracted by several kinds of solvents from
fish meat.
Extraction with chloroform-methanol solvent system after pretreatment
of fish meat with aceton was the most effective.
From above mentioned
fact, phospholipids seem to be presence as a form of lipoprotein but there
are few reports about lipoprotein in aquatic animals.
011ey 63 ,) observed
using lipoprotein in cod meat that strength of linkage between lipid and
protein, nature of the lipid, localization of lipoprotein in tissue,
decomposition of phospholipids after death, so on and he concluded that
phospholipid has a weak conjugation with protein rather than exist as a
free state. Moreover ha had found from the results of silicate gel
column chromatography and paper chromatography.that phospholipids was
constructed with mainly lecithin, phosphatidylethanolamine and few of
phosphatidylinositol, polyglyceropbospholipid a4phingomyelin, 50-60 %
of such phospholipiexist in muscle fibril and remains are in particle of
muscle fibre, phospholipids were decomposed at the rate of 30 mg/100 g/Day
after death at 20°C and its decompostion rate did not change even at
-70C.
Young et al 64) investigated about properties of lipovitellin
prepared from salmon egg. Asano et al 65 ) reported that poisonous componients
of Nagazuka egg was a kind of lipoprotein.
Isolation Of phospholipid from aquatic animals, study of its property,
and comparison between constitutive fatty acid components of neutral fat
anehospholipid (mainly lecithin) have been done by many workers 66 ) •
-
14
-
According to those reports, there are obvious differences between
fatty acid componet of phospholipid and that of neutral fat, moreover
constitutive fatty acids of phospholipid differ in each organ more or
less even in same body. The author suppose that meaning of such differences
between fatty acid coniponents in each organ would be clarified according
to the progress of analytical method of fatty acid and isolation method
for phospholipid.
About the fatty acid components of cephalin --- As cephalin itself
is not a single substance but a mixture of phosphatidylethanolamine,
phosphatidylserine, other aminophospholipidand inositolphospholipid,
it is desirable to fractionate it into each fractions and to investigate
fatty acid components.
But these are very difficult problems since
the complete analytical method does not establish yet.
The author 5 )
and Klenk 67) have gotten pretty pure phosphatidylethanolamine and
phosphatidylserine, and got a result shown in Table 11 by the comparison
of their constitutive fatty acid.
Table 11.
Source
r
Composition of the fatty acids of cephalin fraction of brain.
Phospholipid
Saturated acid
Unsaturated acid
Ci6 C18 C20 C22
(1( brain
Ph-S
Ph-E
3.2 32.7 -9.7 9.7 1.5 0.9
Rorqual brain
Ph-S
Ph-E
30.5
16.9
C16 C18
!
1
•
C20
C22 C24
-- 51.6 7.1 5.4 -2.3 34.7 15.1 24.4 1.7
-- 59.1
66.1
1.5
7.8
-- - 8.9
9.2
--
--
As a general, phosphatidylethanolamine contains a plenty of unsaturated
fatty acids than in phosphatidylserine, although there are some deviation
according to each animals.
About glycerophospholipid
It is interesting problem that
wilat kind of fatty acids are distributed in a' or
p
position in glycero- -668----
phosphate. Pancreatic lipase, phospholipase A and D were used to solve
this problem. Brockerfoff et al 68 ) have investigated the position of
conjugation between fatty acid and triglyceride or lecithin. Considerable
amounts o
igher unsaturated fatty acids were distibuted in 8-position
as shown in Table 12, and he explained that above results seem to be
related with metabolism of essential fatty acids.
- 15 -
Sphingomyelin is distibuted to brain, spinal cord and éther organs.
There are some invesigations about properties and constitutive fatty acids
in sphingomyelin isolated from aquatic animals, but there are no considerable
difference from that of land animals.
The author et al 69 ) found that
existence of sphingomyelin containing dihydrosphingosin in sphingomyelin
fraction prepared fro4perm-whale and this is interesting fact in the point
of biosynthetic passway of sphingomyelin.
One can suppose the presence of new phospholipid which could not
exist in land animals because environmental conditions of aquatic animals
are differ from that of land animals. Hack 70 ) investigated phospholipid
in aquatic animals by paperchromatography.
Rapport et al 71 ) also reported
about contents of acetalphospholipid in sea urchin and other ten kinds of
aquatic animals. Lovern et al 26 ) predicts the presence of new phospholipid
in cod and haddock meat.
The author et al 72) 73) identified a phospholipid
which containing threonine isolated from salmon and tunny meat, and
Rosenberg et al 74 ) also found the presence of threonine and ethanolamineItasaka et al 75) 76)
phosphoric abid in meat of fresh water fish.
reported that the existence of lysine containg phospholipid in sexual gland
of ikechyogai and peptid containing phospholipids in setashijim1( a kind of
Corbicula astrata). Hori 77 ) reported about sphingomyelin-like phospholipid which contained amino acid instead of cholin.
Bergmann et al 78 )
isolated acetalphospholipid and sphingomyelin-like phospholipid from
porifera and sea anemone. Lindberg 79 ) isolated glycollecithin from
egef sea urchin. Uno et a1 8 °) and Trusov 81 ) reported about phospholipid
contents in each organs of salmon which are captured in river
at spawiing-time.
Green 82 ) reported that consumption of phospholipid during spawning-time
are less than that of neutral fat in masunosuke at colombia river.
Table 12.
Fatty acid distribution in a- and a-position of triglycerides and
lecithin mol per
cent for each position.
Source
Position
- Co-crriVii---- —
Tr iglyceride
{
f
Lecithin
_
Cod flesh
Triglyceride
Lecithin
a)
14 : 0 16 : 0 16 : 1
a
A
a
l P
-----------Fatty acid
16 : 20 18 : lb) 20 : 1 22 : 1 20 : 4 20 : 5 22 : 6
5
10
8
3
13
27
43
14
23
<1
18
<1
133
3
6
35
10
20
9
15
9
2
5
6
4
1
3
—
—
1
1
2
10
5
23
1
11
4
35
5
9
3
8
25
21
50
21
15
12
18
6
22
19
14
24
8
4
8 . 6
1
1
1
3
—
—
1
4
3
3
6
13
6
19
3
15
L
f
l
f
a
P
a
16 : 2, 16 : 3 and 17 : 0.
b) with 18: 0 as a minor component
13
4
4
5
Properties of phospholipid in salmon blood were investigated by Okamura 83 )
and the author 84 ) but it is still obscure about their physiological
meanings.
There are many reports about phospholipid contents in sexual glad of
Sexual glad contains
fish and properties of the isolated phospholipid.
considerable amounts of phospholipid if compared with other organs,
therefore, it might have important role for the activity of sperm or the
process of embryonic stage of egg. It had been discussed with neutral
fat and sterin etc. related to these problems.
Glover et al 85 ) found
that sterin synthesis showed rapid increase just before the hatching in
embryonic stage of Salmo salar egg but at the same time phospholipid
exhaustiônualso was shown.
The author et al 86) also identified that
in the course of embryonic stage of salmon egg, consumption of lecithin and
neutral fat did not always run parallel. Moreover, the authors shawed
some changes in basic groueeere occured during embryonic stage of
Tarabagani egg. Yamagami et al 87) showed some changes in phospholipid
during embryonic stage of seaurchin and salmon trout egg from the results
of silicagel column chromatography and paperchromatography.
Mon i 88 )
showed considerable decrease in phospholipid when seeburchin sperm was
incubated aerobically.
He had reported that phospholipid was decomposed -669-
into glycerophosphorylcholin and two molecules of fatty acids by the
action of phospholipase A and B in the early stage but acetalphospholipid
did not show any change in embryonic process of egg and in sperm.
Hartree et al 89 ) reported opposite result, namely he showed acetalphospholipid
was main substrate for above enzymes in sheep sperm.
According to Rothschild
respiratory substrate in mammalian sperm are 9arbohydrates but in the
case of seaurchin sperm, it is mainly phospholipid in cell 90) •
It is
very interesting to see the relationship between phospholipid and embryonic
stage.
Loverm 91) investigated changes in lipid componehts of the cod stocked
in ice, and he observed that hydrolysis of phospholipid accompanied with
production of free fatty acids by autolysis and esterification of free
cholesterin, but intermediates such as lysophospholipid, phosphaticid acid
-
17
-
or diglyceride were not observed and rate of hydrolysis of lecithin and
phosphatidylethanolamine were almost equal.
On the other hand, he have
assumed some intermediates between free cholesterin and esterified
cholesterin. Moreover, 011ey 92) had compared the decompostion rate of
phospholipid at various temperature, and also investigated about liberated
fatty acids or denaturation degree of protein during storage of grozen
fish meat. Miwa 93) reporteliabout changes of phospholipids during the
process of work up of fish meat.
Cardin et al 94) says that 77 % of
fatty acids in phospholipid are spplit off during the process of manufacture
of salt storage cod fish but in this case mainly higher unsaturated fatty
acids such as C20, C22 and saturated fatty acid such as C16 aresplited off
selectively. Dyer et ai
) observed the production of free fatty acids during
the storage of cod fillet at 10°F and -10 °F.
Production rat000f free
fatty acids are slower at the lower temperature and usually taste and
extractability of actomyosin becomes worse according to formation of
fatty acids but if kept at -10°F, such phenolpena could not be observed
for a long time of storage.
The origin of free fatty acid might be
phospholipid rather than glyceride.
Anyway, what phospholipid
are
decomposed by the action of phospholipases in fish meat during storage
or manufacture of fish meat products and stocked fish meat are become
worse by produced fatty acid are important problems which should be
solved in near future.
-18-
.)•
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