Bulletin of the Japanese Society of Scientific Fisheries
`
`48(12), 1803-1814 (1982)
`
`Molecular Species of Fish Muscle Lccithin*"*’
`
`Koretaro TAKAHAsH1,*“ Tsugihiko HIRANO,“ Kozo TAKAMA“ and
`Koichi ZAMA“
`
`(Received May 19, 1982)
`
`Afsatisfactory separation of the molecular species offish muscle lecithin was made by modifying
`the lecithin into diglyceride acetate for high performance reversed phase liquid chromatography
`(HPLC). The molecular species of each chromatographic peak was determined by fatty acid
`composition and by total acyl carbon number analysis subsequent to separation, using a thin
`layer chromatographic technique with silver nitrate impregnated silica gel plates (Ag"'-'1'LC).
`After plotting the relative retention time (RRT) of each molecular species from soybean, egg
`yolk, chum salmon, big-eyed tuna, Aiaska pollack, and carp muscle lecithins semilogarithmically
`against the total acyl carbon number or the number of total double bonds of each molecular species,
`a set of parallel straight oblique lines was obtained. Thus we can express the molecular species
`in matrix relation by giving a variable integer x for the acyl carbon number and a variable integer
`y for the number of double bonds of each fatty acid in the molecular species. By using the cor-
`relations between the RRTS and the corresponding molecular species on semilogarithrnic plots,
`it was concluded that an identification of molecular species from fish lipids could be done by RRTs
`from HPLC.
`
`Lecithin has been studied by many workers
`concerning lipid changes during storage or pro-
`cessing of fish products.“ However, these works
`were done mainly by analyzing the lipid composi-
`tion or fatty acid composition, which might not
`be enough to elucidate the mechanisms of deterior-
`ation of foods caused by the changes in lipid.
`Therefore, a new method for determining molecular
`species of lecithin is necessary not only in the
`field of biochemistry, but also in the field of food
`chemistry.
`Recently, HPLC method has been introduced
`by CSIIIMA et al.*‘’ in order to analyze fish pho-
`spholipids which might have the most complicated
`molecular species composition among biological
`sources.
`
`The purpose of this study was to develop the
`method of Osrrmp. et ul.” to determine the in-
`dividual molecular species of fish phospholipid.
`
`Experimental
`
`Total lipids were obtained from the fish muscle
`tabulated in Table 1, according to the method of
`BLIGH & DYER. Soybean lecithin was purchased
`from Wako Pure Chemical
`Industries, Ltd.,
`Osaka, and egg yolk lecithin was kindly supplied
`by Asahi Chemical Industry Ltd., Tokyo. These
`total lipid and crude lecithin were subjected to a
`silica gel column chromatography which has been
`done successfully by LANDS et al.” for the puri-
`fication of lecithin. Eluates were monitored by
`TLC. And lecithin of more than 95 ‘X, purity
`were collected.
`
`Preparation of Diglyceride Acetate from I.ecithz‘n
`Pure lecithin was dephosphorylized with phos-
`pholipase C (Clostridium perfringcns), according
`to the method of R1-:NK0NEN.“’ Diglyceridc was
`prepared by preparative TLC from the depho-
`sphorylized lipid. The developing solvent was
`n-hexane/diethyl ether (1 : 1, v/v).
`Acetylation was performed by adding an appro-
`priate amount of acetic anhydride to the solution of
`diglyceride in pyridine, and by standing it for 12 h
`
`Preparation of Lecithin
`"
`*1 Molecular sisecies or ‘Marine Animal Lipid-I.
`*9 Presented at the Japanese Society of Scientific Fisheries Meeting, Tokyo, April, 1982.
`*3 Laboratory of Food Chemistry I, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041,
`3ll:i1§ifi7<’%‘-'7l<fi.3£5r‘-l":‘i3)-
`Japan (FL'Efi;E;1CEl5 '
`rTv‘il‘a‘l?‘.§ifi
`' Ef'a'l9Y:'-'3
`ll "la<ltt'*’i,¥$E2Y.;€>’li)-
`*4 Nikkan Denshi Ltd., llakodate, Hokkaido 041, Japan
`(iiiflflkfz :
`*6 T. Osnnvm, S. WADA, and C. KOIZUMI: Presented at the annual meeting of the J apanesc Society of Scien-
`tific Fisheries, Tsu, Japan, October, 1981.
`
`°°°°°1
`
`Petition for Inter Panes Review
`Of U.S. Patent 8,278,351
`Exhibit
`
`ENZYMOTEC - 1028
`
`000001
`
`

`
`1804
`
`TAKAHASIII, HIRANO, TAKAMA. and ZAMA
`
`Table l.
`
`Fish examined.
`
`Species
`
`body length and
`weight
`
`Locality of catch
`
`Date of catch
`
`65 cm,
`
`3 .5 kg, U)“
`
`72 cm, 4.5 kg,
`
`(1)
`
`ll0 cm,
`
`20 kg,
`
`(1)
`
`44 cm, 610 g,
`
`23 cm, 175 g,
`
`(10)
`
`(5)
`
`The offing of Akkeshi,
`HOkk€lid0
`The Moheji River,
`Hokkaido
`Purchased from the
`Mflfkfit
`The Uchiura Bay.
`Hokkaido
`Cultured
`
`June 1980
`
`Nov. 1981
`
`—
`
`"Dec. 1981
`
`Sep. 1980
`
`Chum salmon (Summer)*’
`Ortcor/iynclms keta
`Chum salmon (Fall)‘”
`Oncorhync/ms kem
`Big-eyed tuna
`Parntltunrlus abems
`Alaska pollack
`Tlzeragra chalcogramma
`Carp
`Cyprinus carpia
`'0 "#1 AREE?”E~iIt;7»E‘l}{ZiliZiL{;i’usea.
`
`0
`
`*0
`
`at room temperature." The resulting diglyceride
`acetates were purified by the method of prepara-
`tive TLC by using the solvent n-hexane/diethyl
`ether
`(75:25, v/v). Finally,
`they were filtered
`through a 0.45 M type FP-45 Fluoropore filter
`(Sumitomo Electric Industry, Ltd., Osaka) and
`subjected to HPLC.
`
`HPLC Fractionation of the Molecular Species of
`Diglyceride Acetate Derived from Lecithin
`The diglyceride acetates were fractionated into
`major molecular
`species on twin 8 X250 mm
`l.iChrosorb RP-18 columns wich were connected
`in
`series. A Hitachi Liquid Cltromatograph
`Model 638-50 (Hitachi Ltd., Tokyo) equipped
`with a Shodex RI detector Model Sl:‘—ll (Showa
`Denko Ltd., Tokyo) was used. The eluting
`solvent used was isopropanol/acetone/methanol/
`acetonitrile (l: 1: 3: 4, v/v). Diglyceride acetates
`were dissolved into 5 volumes of tetrahydrofuran,
`and 25 /ll of these solutions were applied to the
`column under room temperature (lower the better)
`and at a flow rate of 1.5 ml/min.
`
`Identification of Molecular Species of Each Peak an
`IIPLC
`
`Peaks on HPLC chromatograms were numbered
`in sequence of elution. The fatty acid composi-
`tion of each collected predominant peak was
`analized by gas chromatography. The analytical
`conditions for fatty acid were as follows.
`Gas chromatograph: Hitachi 063, Column:
`Unisole 3000 (Gasukuro Kogyo Ltd., Tokyo),
`glass column 0.2 X200 cm, Column temp: 220°C,
`Detector: FID, Detector temp.: 2S0"C, Injection
`temp.: 280°C, Carrier gas: N2, flow rate: 20 m1/
`min.
`
`Methyl esters of fatty acids were prepared ac-
`
`cording to the method of CHRISTOPHER and
`GLASS described by PREVOT and MoRmu5'r.“> An
`aliquot amount
`(less than 20 mg) of lipid was
`dissolved in 1 ml n-hexane and 0.2 ml ofmethanolic
`
`2N—NaOH solution was added. After shaking
`this mixture, it was stand for 20 s under 50°C and
`then 0.2 ml of methanolic 2N-HCI solution was
`
`added. The n—hexanc layer was collected and
`then concentrated. Methyl esters prepared were
`subjected to a gas liquid chromatographic analysis.
`The small peaks which have critical pairs were
`first
`subjected
`to Ag+-TLC. The developing
`solvent used was benzene/dicthyl ether
`(8: 2,
`v/v). The band obtained by Ag“-TLC were then
`eluted with diethyl ether containing dotriacontane
`which was used as an internal standard, and
`then applied to fatty acid analysis and total acyl
`carbon number analysis. The analytical condi—
`tions for total acyl carbon number analysis were
`as follows.
`
`I-litachi 063, Column:
`Gas chromatograph:
`OV-10l
`(Gasukuro Kogyo Ltd., Tokyo),
`steel
`column 0.3 X 50 cm, Column temp.: 300~330“C,
`programcd as 1°C/min, Detector: FID, Detector
`temp.: 340°C, Injection temp.: 345°C, Carrier gas:
`N2, flow rate: 60 ml/min.
`
`Hydrolysis of Diglyceride Acetate Derived from
`Lecithin by Pancreatic Lipase
`Fraction of the molecular species of 16:0 in
`position 1 and 22: 6 in position 2 was collected
`by HPLC. This lipid (less than 5 mg) was then
`suspended by shaking vigorously in a mixture of
`1M tris-I-lCl buffer, 13118 (1 ml), 2.2% CaCl,
`(0.1 ml), and 0.05% taurocholic acid sodium salt
`(0.25 ml) at 40“C for 1min. Then, 40mg of
`pancreatic lipase (Calbiochem, San Diego, Calif_
`92112) was added to the mixture and the reaction
`was proceeded for 4min at 40°C by shaking it
`
`000002
`
`000002
`
`

`
`Molecular Species of Fish Muscle Lecithin
`
`1805
`
`Vil'B0r0usly. The reaction was stopped by adding
`1 ml of ethanol and 1 ml of 6 N-HCL The hydro-
`lysate was extracted with diethyl ether and purified
`b)’ using a preparative TLC with n—hexane/diethyl
`ether/formic acid (80: 20: 2, v/v) as developing
`solvent.
`
`Results
`
`1—-3 show chromatograms of diglyceridc
`Figs.
`acetate derived from each lecithin sources on
`
`regularly com-
`HPLC. Chromatography was
`pleted in about 2 h. The diglyceridc composition
`of each collected predominant peak was easily
`determined by fatty acid analysis, as shown in
`Table 2. This table shows the results on big-
`eyed tuna lecithin as an example. Peak number 7
`in Fig. 3-E is obviously ascribed to the diglyceride
`acetate composed of 16:0 and 22:6. We have
`considered that 22: 6 is bound in position 2 of the
`molecule since this peak was the most predomin-
`
`Table 2. Determination of lecithin molecular species
`of major component*
`
`Fatty acid
`
`_ W7 V7
`if E’iCB.l(WlTl‘l‘lHlbCI'
`——~~-—-———?—————-.- —»—
`6
`7
`5
`
`15: 0
`16:0
`17: 0
`18:1
`20: 4
`20:5
`22: 4
`22:6
`
`25.6
`
`25.9
`
`23.3
`
`25.1
`.
`
`'
`
`Molecular species
`" Example of big-eyed tuna.
`
`g
`
`48.4
`
`4.1
`
`3 .7
`
`43.8
`.
`
`‘
`
`8
`
`trace
`53.8
`trace
`trace
`trace
`trace
`trace
`46.2
`.
`
`I
`
`2
`
`ant, and it is said that highly unsaturated fatty
`acids such as 20: 5 or 22: 6 are usually dominantly
`bound in position 2.‘‘"”)
`In addition,
`after
`pancreatic lipase hydrolysis, only a trace amount
`
`.t.l.
`
`"7
`
`__]
`
`NJ
`40
`
`50
`
`80
`
`100
`
`170 min
`
`Fig. 1.
`
`HPLC chromatograms of soybean and egg yolk lecithin.
`B: Egg yolk.
`A:
`Soybean.
`
`000003
`
`000003
`
`

`
`1806
`
`TAKAHASHI, HIRANO, TAKAMA, and ZAMA
`
`
`
`Fig. 2.
`HPLC chromatograms of chum salmon muscle lecithin.
`D: Captured in fall.
`C: Captured in summer.
`
`of 22: 6 was detected in the free fatty acid fraction
`(this fraction represents the fatty acid in position
`1), although more than 70% of the lipid was hy-
`drolized (determined by a densitometric method).
`Peak number 6 had the same combination as
`
`that in peak number 7, whereas 18: 1-20: 5 were
`considered as contaminants of peak number
`5. Nevertheless in this case, 22: 6 was considered
`to be bound in position 1 in the molecule. Peak
`number 5 had an almost even amount of fatty acids
`of 16:0, 18: 1, 20:5 and 22:6. Among these
`fatty acids, 16:0 as well as 22: 6 were regarded
`as contaminants from peak number 6, considering
`that peak number 6 is larger than peak number 5.
`It was concluded that peak number 5 was the
`combination of
`18: 1
`and 20: 5. Molecular
`
`species from other biological sources were also
`determined in the same manner. The small peaks
`which have critical pairs were first subjected to
`Ag+-TLC and separated according to their degree
`of unsaturation.
`In most of the samll peaks
`in the first half of the HPLC chromatograms of
`
`fish lecithin, only one band appeared on Ag+-
`TLC plate. The complex combinations of mole-
`cular
`species were identified in the following
`manner. An example of determination of mo-
`lecular species in the small peaks with critical
`pairs appeared in the first half of the HPLC chro-
`matogram of chum salmon (captured in fall)
`is
`shown in Table 3
`(also see Fig. 2-D).
`Small
`peak number 1 is obviously a combination of 20: 5
`and 16: 1. Small peak number 2 is also a com-
`bination of 20: 5 and 16: 1 with 10% unidentified
`contaminants. The difference in retention time on
`
`HPLC between these two peaks was attributed
`to the differences in binding position of the fatty
`acid.
`It was considered that small peak number 1
`had 20: 5 in position 1, whereas small peak number
`2 had it in position 2 in the molecule, since mo-
`lecular species which have a highly unsaturated
`fatty acid in position 2 are likely to elute later
`than the one which has the same fatty acid in posi-
`tion 1. Small peak number 3 is a combination
`of 20: 5 in position 1 and 14:0 in position 2, and
`
`000004
`
`000004
`
`

`
`Molecular Species of Fish Muscle Lecithin
`
`1807
`
`
`
`20
`
`BU min
`
`
`
`40
`
`80
`
`aumin
`
`~—\__.
`EU min
`
`Fig. 3. HPLC chromatograms of big—eyed tuna, Alaska pollack and carp muscle lecithin.
`F: Alaska pollack.
`G: Carp.
`E: Big-eyed tuna.
`
`also 16:1 in position 1 and 20:5 in position 2
`since this peak is composed of two combinations
`of total acyl carbon numbers of 34 and 36 respec-
`tivcly, However,
`the latter molecular
`species,
`i_c, 16; 1-20: 5, are considered to be contaminants
`from the previous peak. From the fatty acid
`composlllon and total acyl carbon number, three
`molecular species were presented in small peak
`number 4,
`i_c, combination of 14:0 in position 1
`and 20; 5 in position 2 for 63.3°.,; combination
`of 16: 1 in position 1 and 20: 5 in position 2 for
`57%; and combination of 22: 6 in position 1 and
`
`16:1 in position 2 for 31.0%. Among these
`molecular species, 16: l~~20: 5 can be considered
`as contaminants from the two previous peaks.
`In small peak number 5, three molecular species,
`i.e. 14:0 in position 1 and 20:5 in position 2;
`22:6 in position 1 and 14:0 in position 2; and
`16:1 in position 1 and 22:6 in position 2 were
`identified in the same manner as that
`in small
`
`In this case, molecular species
`peak number 4.
`of 14: 0-20: 5 was considered to be a contaminant
`from the previous peak.
`In the case of small
`peak number 6, molecular species of 14:0 in
`
`000005
`
`000005
`
`

`
`1808
`
`TAKAHASHI, HIRANO, TAKAMA, and ZAMA
`
`Table 3. Determination of lecithin molecular species of minor component“
`
`—-
`.._..
`Fatty acid
`
`. :.-:_ W2;-re
`- A»
`------—
`
`1
`
`-.—
`
`WM‘
`53.0
`47.0
`
`I
`
`I
`
`95.0<
`
`'"fX; 0
`16:1
`20:5
`22:6
`
`_CarIi6n number*22
`34
`36
`38
`
`"(Ea-rbon number”
`34
`
`--
`
`--
`
`2
`
`V
`
`2
`
`I
`
`47.7
`52.3
`
`90.0
`10.0
`
`—
`
`-
`
`3
`
`"21 .3
`28.3
`50.4
`
`2
`
`42.6
`57.4
`
`2*“
`
`-*
`
`—
`
`5
`
`~
`
`34.6
`19.6
`15.6
`30.2
`
`32.2
`41.7
`26.1
`
`W" ~—~—v
`---~ —
`
`—
`~-
`
`~-' ~
`6
`
`44.6
`9.7
`8.3
`37.9
`
`10.8
`89.2
`
`4
`
`30.6
`19.0
`36.2
`14.2
`
`63.3
`5.7
`31 .0
`
`20:5‘
`16:1‘
`l6:1i 95'°< [20:51 90-0
`
`36
`38
`7'4, in caehapeak
`'1 Example of chum salmon (Fall).
`
`20:5
`i14;oi 42:6
`16:1
`20:5! 57:4
`
`14:0
`|20;5i 53:3
`16:1
`i20:5
`5-7
`31.0
`
`14:0
`i2o;5i 32-2
`22:6
`;14:o. 4‘-7
`
`26.1
`
`14:0
`i2O:5i 102
`14:0
`22:6i 89-2
`
`‘*2 Total acyl carbon number.
`
`position 1 and 20: 5 in position 2 were consi ered
`to be the contaminants from the previous peak.
`By analyzing the data in this way, molecular
`species of all other small peaks were identified.
`As illustrated in Fig. 2-D, the HPLC chromato-
`gram of diglyceride acetate of fish muscle lecithin
`can be divided into four molecular species groups,
`that is, 1: molecular species composed of highly
`unsaturated fatty acids such as 20: 5-22: 6, 22: 6-—
`22: 6 and 20: 5-20: 5,
`III: molecular
`species
`composed of generally found fatty acids such as
`16:0 or 18: l with combinations of 20: 5 or
`
`22:6, that is, 1620-20: 5, l6:0~22:6, 18: 1-20: 5
`and 18: 1-2226, and II and IV: others.
`In all
`marine fish examined, groups I and III accounted
`for more than 70 % of the molecular species which
`were determined in the following manner.
`All HPLC chromatograms were traced on sec-
`tion papers, and the area of the identified peaks
`were measured, though there was some limitation
`in accuracy caused by the overlapping of the
`peaks.
`The results for fish muscle lecithin are sum-
`merized in Table 4. Fish from marine sources
`
`contain a significant amount of highly unsaturated
`fatty acid combinations such as: 22: 6-22: 6,
`22: 6-20: 5 and 20: 5-20: 5. All
`fish lecithin
`
`The RR'I‘s of all peaks were determined by
`dividing the retention time of each peak by the
`retention time of 16:0 22: 6.“ In the case of
`
`soybean lecithin, 16: 0-1822*" was used as a
`reference peak and the RRTS were reculculated
`against 16: 0-22: 6“ by using the RRTs data in
`egg yolk lecithin. The RRT of each molecular
`species is summerized in Tables 5-8.
`
`Discussion
`
`We have plotted the RRT of each molecular
`species semilogarithmically against
`the partition
`number
`(PN) defined as PN:C’” 2d*‘ since
`PN is proportional to the logarithm of RRT.‘”
`A general expression for this can be written as:
`
`PNoc log (RRT)
`
`(1)
`
`After plotting the RRT of each molecular species
`semilogarithmically,
`a similar correlation was
`obtained, that is:
`
`PNoc log (RRT) +219
`
`(2)
`
`(2) is similar to (1), and if we formulate (2) into an
`equation
`
`PN-=P - log (RRT)-L AQ
`
`(3)
`
`examined contain 16: 0-22: 6 as the predomin-
`ant molecular species.
`
`where P becomes the slope of the oblique line, and
`AQ becomes the intersection on the ordinate.
`
`*1 16:0 invpositiohwl ad 22; 6 in position 2
`*2
`16: 0 in position 1 and 18:2 in position 2
`£1: Total double bonds.
`” C: Total acyl carbon number,
`*4
`
`000006
`
`000006
`
`

`
`Molecular Species of Fish Muscle Lecithin
`
`1309
`
`Table 4. Molecular species of fish muscle lecithin
`
`\
`
`Alaska pollack
`V;
`1
`
`23.9‘;
`W
`
`245%
`
`Carp
`.” 7
`
`244%
`'
`
`_
`
`5
`
`0
`.
`1”/“
`
`I
`
`'2
`
`4.3°o
`
`'
`
`,
`
`0
`6.5 0
`/
`
`0
`2.5/,
`
`_
`
`WWW
`1-3/°
`
`1.0%
`
`21%
`— —
`1
`"
`.9A
`
`.
`30.3 0
`""1231 '
`‘T 16:01
`A 2o;5
`16'5’3
`A 20:51
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`°
`1331
`13‘ ‘I
`_?.2.ffJ____ 2,- 32..‘_6_L_.. _.
`22; 51
`O
`.
`1
`16:0,
`8.6/,
`13;?
`9.0%,
`—~—~—~»——
`WW- __..~
`- _,, _V, __.__
`-
`.
`.
`-
`6-3%
`2822‘
`8-6%
`-
`K 7250i
`4
`H 8-zml‘
`‘*3 0
`13;:
`20:5
`.
`161°‘
`.
`3-9/»
`16=1
`$2
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`.
`2.
`2226
`‘~l6:O‘
`0 ——:~———
`18:1
`4'5/’
`1351‘
`2 97
`14-0‘
`2035
`'
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`2021
`W 4 ~"'16"(*)~”"‘—'
`_
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`2.47
`20: 5
`22.5
`‘’
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`5-13-"1"? " '_ '5
`22: 6
`3
`3
`1.9””
`I
`.20. 4
`/
`18-1
`14_a"% "‘-~ ~- —~--—:
`'5
`22: 6
`1 3‘
`2.0%
`.
`22.6
`18:2l
`20: 5
`X
`.
`20:5
`22.6
`1.9 0
`_.. .
`._.._ .22
`14; 0
`16:1
`22:6.
`‘22: 6
`— —~~ — ~
`1“
`16:1
`.
`20:5
`22:6
`£81‘
`14. 0!,
`20; 5
`20:5,
`- V 1-
`&0
`16:1
`116:2‘
`20:5
`18:]!
`
`9-3% _.
`—‘
`
`5 4.7
`'
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`
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`4-6/2
`
`2-74
`. 2
`
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`
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`
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`
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`
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`
`1.7%
`
`1.7%
`
`Big—eyed tuna
`16:0
`22.6
`"E2; 6
`22:6
`..... .2.
`22- 6
`165 o
`13:1
`22: 6
`-
`A2325)
`22: 5
`22:6
`
`C uEI11=:fi)m°n
`16:0
`22:6
`22: s
`2216
`. W2
`20; 5
`22: 6
`.
`16:0
`20: 5'
`18:
`20: 5
`22:;
`22:6,
`22: 6‘
`22: 6
`45%
`15:0
`16:0
`. ’ ;
`’
`15.0
`44/»
`i2;8
`13:1
`_-
`.12,,,,2
`_"2___“.1,.
`14:0
`20:5
`22:6,
`20;5
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`1
`1620
`1420‘;
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`22:5
`20: 51
`"“""““““”
`22: 6
`20:5
`0
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`18:}
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`’ ””" ‘““”“"”’
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`2.94
`20; 5
`.
`2.2
`20: 5
`“/° _.____‘H,;_:,._,___,__ _.
`
`18:1
`.,
`"
`20:4
`20:5
`_2..»____..2.-_-
`22 6.
`20:5
`'16:1
`22; 5
`22: 5
`22-5
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`1
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`"'6'
`2
`mi
`17:1
`20:5
`22:6
`14:0
`205 5
`2035
`=18_f1___.__
`1&1
`""’#f2‘
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`22;5
`0'9/’
`22:6
`-_.-._;._..._ _ 18:1
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`.,
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`
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`
`16:
`22.2]
`16:
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`A 20I%
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`_.."__-
`22: 6
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`22.6
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`9
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`12/51
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`18.8/.,
`
`10'933
`.1
`‘°"“
`
`18 30/
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`
`V
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`
`5-54
`-
`,
`
`35%
`
`3 10/
`‘
`°
`
`_
`
`3.8%
`
`419/
`'
`"
`
`
`
`_.
`
`4V———~-»»
`omers
`13.6%
`-— *-'
`
`—
`
`,
`
`£2
`M-
`16:0
`20:4
`22~ 5
`16:0
`16:0
`,
`22' 52,
`0:4
`32. 6
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`53:
`.
`2 “‘“
`22; 6
`18:0!
`__,__
`18:1
`1(:0
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`16.0
`18:1
`V-3)‘;
`1620
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`
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`
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`
`0
`22:5
`22. 6
`0-94
`14. 0
`
`
`-——»~w«—_
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`Others
`14.7%
`-
`5
`5
`~
`othm 16 7=y
`m~—-~»+—.» - 1__ Oyflfrjm 3-5/3 2- 5 - —-5-‘
`
`_
`
`:
`
`2-
`
`H# Others
`VIE’ —§a?o:cOn)pongnt in gach peak.
`
`9.7%
`
`Thjs equation (3) suggests that PN is not ‘only a
`function of RRT alone, but also a function in—
`eluding AQ_
`I
`Back to the definition of the PN, that IS, PN-r:
`C—2d, the left member in (3) can be changed as:
`C~——2d:P-log (RR'I')el-AQ
`(4)
`50 RRT can be expressed as follows:
`
`27 -2 22 2‘
`
`R
`
`fig,
`
`(5)
`RT:10
`If we put AQ as Qll-oz, equation (5) may be
`written as:
`__
`P
`RRT 1OcA_r_.€_(QJiQ
`~
`
`..
`(Q is a minimum fA
`‘
`° %g)
`where P and Q are invariables and a is a variabic
`
`000007
`
`000007
`
`

`
`»
`114.1
`
`7
`122.2
`132.0
`
`134.9
`~
`143.7
`
`47.0
`
`4
`50.4
`54.4
`-
`52.6
`1'
`59.2
`
`”
`
`72.4
`r
`,,
`1,
`79.0
`II
`
`W
`
`175.7
`7
`7
`1
`191_-7
`II
`
`33_5
`
`2023
`
`”
`121.8
`7
`_
`
`__
`
`”
`295.1
`
`18:0
`22: 6
`15:0
`18:1
`
`'
`
`183 1
`g
`'
`22: 4
`
`'
`18: 0
`13‘ 2
`1850
`
`__>_ ___i
`_ " V
`N
`'
`Abbreviations:
`PN: Partition number, MS: Molecular species, ‘RRT: Relative
`retention time.
`
`26
`28
`
`1/
`«I
`0
`
`3
`n
`~
`1/
`
`1
`
`28
`
`31
`7
`.
`30
`32
`I]
`
`1/
`II
`
`34
`
`" "
`
`Ms
`1g;'2“‘
`‘I813
`-
`
`'
`
`.
`l§;%
`16: 0
`183
`-
`
`.
`
`13:0
`18:3‘
`17:0
`18:2
`-
`
`12:11
`_
`
`mu
`26
`28
`~
`
`7
`30
`
`.
`,
`
`31
`32
`~
`,
`
`1;
`
`13; 0
`18: 2
`16: 0
`16:0
`
`M
`27.6
`37.6
`39-2
`
`42.0
`51.4
`
`55.2
`60.0
`
`62.8
`73.8
`»
`79_5
`
`II
`
`iinr
`70.4
`95.8
`9”
`
`107.1
`131.0
`
`140.7
`152.9
`
`160.1
`188.1
`»
`202_9
`
`II
`
`83.6
`113.2
`,,
`
`213.0
`1
`288.6
`34
`,
`,,
`—"” “aim
`"z{1?1§£éJ1a111$x§:
`PN: Partition number, MS: Molecular species, RRT: Relative
`retention time.
`g is used as the reference peak.
`
`‘
`
`‘
`
`range of Q1. By letting C and d be invariables,
`RRT becomes a function of (1 which is:
`RRT=/(co
`
`(7)
`
`.
`.
`Back to equat1on (4), by putting AQ, as Q14 (.1,
`the following equation can be obtained.
`
`C~2d=P-log (RRT) l--Q, +11
`
`(4')
`
`If we let d be invariable and rearrange the ex-
`pression for C, (4') will become:
`
`C=‘P "103 (RRT)+a + Q2
`
`(Q2 = Q1+2d)
`
`(3)
`
`On the other hand, if we let C be invariable and
`rearrange the expression for d, (4') will become:
`
`(1
`.. J1.
`d~ 2 10g(RRT)——2-~I~Qa
`
`<9)
`—§(Q.—c))
`These two functions, i.e. (8) a11d (9) have a devia-
`tion which are a for the former and -—a/2 for the
`
`1810
`
`TAKAIIASI-II, HmANo, TAKAMA, and ZAMA
`
`Table 5. Relation between relative retention time of
`HPLC and molecular species of soybean lecithin
`
`Table 6. Relation between relative retention time of
`
`HPLC and molecular species of egg yolk lecithin
`_ 13*
`T
`MS
`R3
`- .’€“T.-
`16: 1
`28
`18:2
`41.2
`100.0
`18:2
`”
`13:2
`”
`”
`14:0
`”
`18:2
`”
`”
`16: 0.
`,,
`22.6
`16:0
`18,3
`18:1
`20.4
`16:0
`20.4
`16*’
`22.4
`16: l
`18.1
`léié
`.
`
`latter. Alfa is considered to be a factor that has
`a slight effect on RRT, and may be due to the
`positional isomers such as between 1 and 2 posi-
`tion in the molecule, or by the large differences
`in number of double bonds between the two fatty
`acids in the molecule. This leads us to the concept
`of a matrix since it
`is convenient
`to distinguish
`the positional isomers or the bias in the number
`of double bonds between the two fatty acids in the
`molecule. So in order to simplify the equation,
`we can induce the following equations from (8)
`and (9) respectively under the conditions of:
`
`000008
`
`000008
`
`

`
`Molecular Species of Fish Muscle Lecithin
`
`1811
`
`Table 7. Relation between relative retention time of HPLC and main molecular species of fish
`muscle lecithin
`
`9
`
`Chum salmon —7
`(Summer)
`
`salmon
`(Fall)
`
`Big-eyed tuna
`
`W/glaska pollackrm _— Carp
`
`MS
`
`PN/RRT
`
`MS
`
`PN/RRT
`
`MS
`
`PN/RRT
`
`MS
`
`PN/RRT
`
`MS
`
`PN/RRT
`
`20: 5
`20: 5
`20: 5
`22:6
`22: 6
`22:6
`14:0
`2226
`16: 0
`20:5
`18:1
`2216
`22:6
`1620
`16: 0
`22: 6
`
`20
`27.3
`20
`40.7
`20
`44.2
`24
`69.7
`26
`92.2
`26
`92.2
`26
`97.1
`26
`100.0
`
`20: SI
`20: 5
`20: 5!
`22:6
`22: 6
`22:6
`18:1
`20:5
`20: 5
`16:0
`16:0
`20:5
`18:1
`22:6
`22: 6
`16: 0
`16: 0
`22:6
`
`20
`37.5
`20
`40.4
`20
`44.1
`26
`87.0
`26
`89.5
`26
`91.9
`26
`91.9
`26
`96.7
`26
`100.0
`
`20: 5
`22: 6
`22: 6l
`22:6
`20: 4
`22:6
`22:5
`20:5
`22:5
`22:6
`18:1
`22:6
`16:0
`20:5
`22: 6
`16:0
`16: 0
`22:6
`16:0
`22:5
`
`20
`41.2
`20
`44.6
`22
`55.2
`22
`55.2
`22
`60.0
`26
`93.5
`26
`93.5
`26
`96.8
`26
`100.0
`28
`134.9
`
`-1
`if
`7
`1
`7 M
`A'isi;.en-;{£.§ns?
`PN: Partition number. MS: Molecular species. RRT: Relative retention time.
`
`20: 5
`20: 5
`20: 5
`22:6
`22: 6
`22:6
`18:1
`20:5
`20: 5
`16:0
`22:6
`18:1
`16:0
`20:5
`18:1
`22: 6
`22: 6
`16:0
`16:01
`22:6
`16: 0‘
`1821
`14: O4
`20:1
`
`20
`37.2
`20
`40.4
`20
`44.3
`26
`86.7
`26
`89.8
`26
`89.8
`26
`92.0
`26
`92.0
`26
`97.1
`26
`100.0
`32
`216.6
`32
`216.6
`
`20: 5
`22: 6
`22: 6
`22:6
`16:1
`20:5
`18:2
`20:5
`16:1
`22:6
`18:2
`22:6
`14:0
`22:6
`18:1
`20: 5
`16: 0
`20:5
`18:1
`22: 6
`16: 0
`22: 6
`16: 0
`20:4
`
`20
`42.5
`20
`46.2
`24
`62.5
`24
`62.5
`24
`67.7
`24
`67.7
`24
`67.7
`26
`88.3
`26
`93.7
`26
`93.7
`26
`100.0
`28
`124.0
`
`C’:
`
`x
`C2
`
`
`
`(1,
`dz
`
`
`
`(Cg, d, and dz are invariablcs)
`
`(c,, c, and d2 are invariables)
`
`d’-_:
`
`q
`C1
`3'
`
`c, d,
`c/=p,-1og(RRT)+q.
`
`(8')
`
`(9')
`
`d’:-:=p2‘1og (RRT) +02
`
`by analyzing the RRT data of each molecular
`species.
`_
`After plotting the RRT of each molecular species
`from the source examined against the total acyl
`carbon number or the number of total double
`bonds of each molecular species, the RRT plots of
`molecular species laid almost on a straight line by
`giving a variable integer x for the carbon number
`and a variable integer y for the number of double
`bonds of each fatty acid in the molecular species,
`when we express the molecular species in matrix
`relation. The oblique lines were parallel to each
`other as shown in Fig. 4. By applying these cor-
`relations between the RRT and the corresponding
`molecular species under the condition of matrix
`for the determination of molecular species, it was
`
`suggested that an unidentified molecular species
`of Iecithins could be identified from RRT on HPLC
`even if it has the most complicated composition of
`molecular species such as fish muscle lecithin.
`While this manuscript was
`in preparation,
`another method for HPLC analysis of phospholipid
`molecular species was published by I’AT'1‘oN et 11].”)
`To our surprise, the sequence of each molecular
`species in elution on IIPLC, when the RRTS
`were plotted against the total acyl carbon number
`from our biological material, were the same as
`common molecular species of rat liver which was
`analyzed by PATTON er 11].”) despite the fact that
`the analytical conditions were significantly dif-
`ferent. This suggests that, although there are
`differences in retention time or in RRT among
`the different conditions on HPLC,
`the sequence
`in elution of each molecular species might be
`unchangeable. This
`leads us
`to a conclusion
`that in HPLC,
`the sequence in elution might be
`controlled by a fixed correlation, that is matrix
`relation. By accepting this idea, we can expand
`this matrix concept to triglycerides. Details will
`be reported at a later date.
`
`000009
`
`000009
`
`

`
`1812
`
`TAKAHASHI, HIRANO, TAKAMA, and ZAMA
`
`Table 8. Relation between relative retention time of HPLC and molecular species of fish muscle
`lecithin
`
`Chum salmon
`(Summer)
`
`I
`
`salmon
`(Fall)
`
`Big-eyed tuna
`
`Ala8k61 5611651;
`
`cafif
`
`*1»/1s 7
`
`a'\>—A
`
`20:
`
`18:
`
`16:
`18:
`16:
`16:
`18:
`22:
`18:
`20:
`18:
`18:
`16:
`18:
`
`PN/PLRT
`24
`61.0
`24
`61.0
`24
`64. 2
`24
`65.9
`24
`65.9
`24
`69.9
`28
`115.7
`28
`115.7
`28
`120.3
`28
`125.0
`28
`137.5
`28
`137.5
`30
`137.5
`30
`137.5
`30
`150.0
`30
`150.0
`30
`150.0
`28
`150.0
`32
`207 .4
`32
`207.4
`34
`301 .9
`34
`301.9
`
`15 25
`
`MS
`
`16:
`20:
`18:
`20:
`14:
`20:
`16:
`22:
`I8:
`22:
`14:
`22:
`22:
`16:
`16:
`20:
`16:
`22:
`20:
`20:
`18:
`20:
`20:
`22:
`18:
`16:
`I8:
`18:
`14:
`18:
`16:
`16:
`16:
`18:
`18:
`22:
`18:
`16:
`20:
`14:
`16:
`20:
`
`01
`
`I18:
`18:
`
`_Vs
`— 20:4
`22: 6
`22:5
`20: 5
`22:5
`22:6
`16:1
`20:5
`14: 0
`20:5
`16:]
`22:6
`22:6
`114:0
`15:0
`20:5
`17:1
`22:6
`14:0
`22:5
`15:0
`22:6
`20:5
`18:1
`18:1
`20:5
`20: 5
`16:0
`22:6
`18:1
`16:0
`22:5
`16:0
`20:4
`
`17:0
`22:6
`20:1
`20:5
`
`PN/RRT
`22
`50.6
`22
`50.6
`22
`52.8
`24
`60.8
`24
`64.8
`24
`65.8
`24
`67.4
`25
`77.8
`25
`82.3
`26
`82.3
`25
`84.4
`26
`87.2
`26
`90.2
`26
`90.2
`26
`90.2
`28
`121.9
`28
`126.6
`
`27
`126.6
`28
`126.6
`
`MS" A PN/RP:T
`18:3
`22
`22: 6
`48.7
`20:4
`22
`22: 6
`48.7
`22:5
`22
`20:5
`48.7
`20:5
`24
`16:1
`59.1
`20: 5
`24
`18:2
`59.1
`16:1
`24
`20:5
`60.8
`18:2
`24
`20:5
`60.8
`20:5
`24
`14:0
`62.2
`14:0
`24
`20:5
`64.2
`22:6
`24
`16:1
`64.2
`16:1
`24
`22:6
`67.1
`22:6
`24
`14:0
`67.1
`14:0
`24
`22:6
`69.5
`16:1
`26
`22:5
`77“.8
`15:0
`25
`20:5
`77.8
`17:1
`25
`22:6
`77.8
`20: 5
`26
`18:1
`82.2
`
`22: 5|
`16:0
`16:0
`22:5
`
`28
`116.6
`28
`119.9
`
`PN/RRT
`24
`65.0
`24
`66.9
`24
`71.1
`24
`71.1
`26
`71.1
`24
`74.0
`25
`78.4
`24
`78.4
`25
`78.4
`25
`78.5
`25
`82.7
`25
`84.7
`26
`84.7
`26
`88.2
`26
`91.3
`26
`91.3
`28
`116.7
`
`27
`116.7
`28
`116.7
`
`22:6
`16:1
`18:2
`22: 6
`14:0
`22: 6
`22: 4
`22: 6
`16:1
`20:4
`20: 4
`22: 5
`22:6
`17:1
`22:5
`22:5
`17:2
`20:4
`17:2
`22:5
`17:1
`22:6
`15:0
`22:6
`20:5
`18:1
`18:1
`20:5
`22:6
`18:]
`20:5
`16:0
`20: 4
`16:0
`
`17:0
`22:6
`18:1
`22:5
`
`16:0
`20:4
`20:1
`20:5
`18:0
`20:5
`18:1
`16:1
`14: 0
`18:1
`16:0
`16:1
`
`28
`124.6
`28
`124.6
`28
`133.3
`30
`137.2
`30
`142.9
`30
`142.9
`
`28
`20:1|
`140.6
`22:6
`28
`18: 0
`140.6
`20:5
`30
`18:1
`143.6
`16:1
`28
`18:0
`152.5
`22:6
`30
`14:0
`152.5
`18:1
`30
`16:0
`152.5
`16:1
`32
`24:1
`265.7
`20: 5
`Abbreviations:
`PN: Partition number, MS: Molecular species, RRT: Relative retention time.
`
`16:0
`20:4
`22: 5
`16:0
`22:6
`18:0
`18:0
`22:6
`18:1
`18:1
`18:1
`16:0
`16:0
`18:
`1
`
`28
`124.6
`28
`130.0
`28
`143.2
`28
`148.1
`32
`189.6
`32
`206.1
`32
`215.5
`
`0000010
`
`PN/RRT
`22
`50.7
`22
`52.3
`22
`54.5
`22
`56.4
`26
`79.4
`26
`79.4
`25
`79.4
`25
`79 .4
`26
`82.7
`26
`82.7
`28
`111.7
`28
`111.7
`28
`115.8
`28
`119.3
`30
`133.8
`28
`133.8
`30
`138.4
`30
`138.4
`30
`148.4
`30
`148.4
`28
`148.4
`30
`179.4
`32
`195.3
`32
`210.1
`
`
`
`
`
`‘—‘C7*-‘r—-50O\OV-‘C190Mr‘'-"-‘G'\‘-‘-#0UIO-5*-2NOUI>—«.1310-5*-*
`
`
`
`
`
`
`
`0000010
`
`

`
`Molecular Species of Fish Muscle Lecithin
`
`1813
`
`x0
`‘ilii . Sim
`3i. Eilii
`x 1
`0 41.
`‘Z12’; 21.13123,
`
`“i2
`
`Zilii
`hid
`312,25 2,
`1512);
`
`91233
`mizii
`
`hie
`
`3|1§
`
`‘Ola?
`nizii
`
`|[1ii
`7l2ii
`“[232:
`13123;: E
`
`
`
`30
`
`32
`
`38
`L0
`.,.36_., _
`734“
`Total Acyl Carbon Number
`22v
`‘922 6
`
`
`20 y
`22 6,
`1 1:5 Tél, ml
`70 y
`16 0,
`241%?) ti,
`25]
`2*='|i§
`1H y]
`16 0|,
`
`29}
`Milli
`
`21.3.8
`
`
`
`22y160
`20y
`L40,
`
`231
`2|,
`ll,
`231
`0
`1.16 0
`V1116 y
`
`700
`500
`A00
`300
`
`200
`
`retention
`
`Relative
`
`100
`
`70
`
`50
`1.0
`30
`
`20
`
`
`
`31
`
`27 25
`
`30
`
`
`
`9
`8
`12713
`10111
`or
`‘E 6
`*0 “T‘2““§">;
`Number of Total Double B0ndS
`
`12.
`
`15 16-17
`
`Fig 4 Relation between relative retention time and total acyl carbon number and relation between
`relative retention time and total double bonds on HPLC of lecithin.
`
`Acknowledgements
`
`The authors wish to thank J. CI-IIGIRA of Asahi
`Chemical Industry Ltd. for the supply of egg yolk
`lecithin‘ and W. NAGA'FA for reading the manu~
`script.
`
`References
`
`in “Quality of Fish” (ed. by Japan.
`1) K_ TAKAMAZ
`Soc, Sci. Fish.), Suisangaku Series No. 4, Kosci-
`sha-Koseikaku, Tokyo, 1974, pp. 138-144.
`2) W. M. LANDS and P. HART:
`J. Am. Oil Chem.
`Soc., 43, 290-295 (1966).
`
`3)
`
`4)
`
`5)
`
`6)
`
`7)
`
`8)
`
`9)
`
`0000011
`
`J. Am. 01'] Chem. Soc'., 42, 298-
`
`Iipids, 2, 2l7—224
`
`Rev.
`
`I-‘se.
`
`0. RENKONENZ
`304 (1965).
`A. Kmcsrs and 1.. MARAII
`(1967).
`A. F. Piuzvor and I-'. X. Mououm:
`Corps Gras, 23, 409- 423 (1976).
`K. ZAMA, T. MARUYAMA, and K. TAKAHASHI:
`Bull. Fac. Fir/1. Hokkaido Univ., 27, 181-190
`(1976).
`K. TAKAHASIII, K. ZAMA, and T. MATSUOKA:
`Bull. Fae. Fish. Hokkaido Univ., 29, 378-385
`(1978).
`K. TAKAHASHI, F. CABLING, and K. ZAMA2 Bull.
`Fac. Fish. Hokkaido Um'v., 29, 386-391 (1978).
`A. Kuxsis:
`in “Progress in the Chemistry of Fats
`
`0000011
`
`

`
`1814
`
`TAKAIIASHI, HIRANO, TAKAMA, and ZAMA
`
`and Other Lipids"(ed. by R. T. HOLMAN), Vol. 12,
`Pergamon Press. Oxford, New York, Toronto,
`1972, pp. 105-111.
`10) R. WOOD and F. SNYDER: Arc/1. Biachem.
`Biophys., 131, 478-494 (1969).
`
`11) S. WADA, C. KOIZUMI, A. TAKIGUCHI, and J.
`NQNAKA: Yukagaku, 27, 579-584 (1978).
`12) G. M. PATTON, J. M. FASULO, and S. J. Ronms:
`_
`J. Lip1'dRes.,23, 190-196 (1982).
`'
`
`0000012
`
`0000012