Bulletin of the Japanese Society of Scientific Fisheries
`
`48(12), 1803—1814 (1982)
`
`Molecular Species of Fish Muscle 'Lecithini‘h’“2
`
`Koretaro TAKAHASHI,“ Tsugihiko HIRANO,“ Kozo TAKAMA“ and
`Koichi ZAMA’”
`
`(Received May 19, 1982)
`
`Aisatisfactory 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"'—'l'LC).
`After plotting the relative retention time (RRT) of each molecular species from soybean, egg
`yolk, chum salmon, big-eyed tuna, AEaska 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 semilogarithmic plots,
`it was concluded that an identification of molecular species from fish lipids could be done by RRTs
`from HPLC.
`
`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 all.” for the puri-
`fication of lecithin. Eluates were monitored by
`TLC. And lecithin of more than 95 ‘Z, purity
`were collected.
`
`Preparation of Diglyceride Acetate from Lecithin
`Pure lecithin was dephosphorylized with phos-
`pholipase C (Clostridium perfringcns), according
`to the method of RHNKONEN.” Diglyceridc was
`prepared by preparative TLC from the depho—
`sphorylizcd 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 Stacie; of Marine Animal Lipid-I,
`*2 Presented at the Japanese Society of Scientific Fisheries Meeting, Tokyo, April, 1982.
`*3 Laboratory of Food Chemistry 1, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041,
`ittfiifii’ixififfififil-
`Japan (Fifilfiiéjtfill - fil‘a‘li‘é‘fii
`' EFa'ifiE—:
`U Mtfi¥$fit®iil
`*4 Nikkan Dcnshi Ltd., Hakodate, Hokkaido 041, Japan
`(545E153;- :
`u T. 05mm, S. WADA, and C. KOIZUMI: Presented at the annual meeting of the J apanese Society of Scien-
`tific Fisheries, Tsu, Japan, October, 1981.
`
`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 OsmMA 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 OSHXMA er al.“ to determine the in-
`dividual molecular species of fish phospholipid.
`
`Experimental
`
`000001
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`
`ENZYMOTEC - 1028
`
`000001
`
`

`

`1804
`
`TAKAHASHI, HIRANO, TAKAMA. and ZAMA
`
`Table l.
`Fish examined.
`
`
`
`
`’Méén' body length and
`Date of catch Species Locality of catchweight
`
`”Chum salmon (Silthiherfil _ _
`65 cm,
`3 .5 kg,
`(1)*2
`The offing of Akkeshi,
`June 1980
`Omar/lynch": keta
`Hokkaido
`Chum salmon (Fall)"‘1
`The Moheji River,
`Oncorhynchus keta
`Hokkaido
`Big-eyed tuna
`Purchased from the
`Parntlmnnus abems
`Market.
`Alaska pollack
`The Uchiura Bay.
`leeragra chalcogramma
`Hokkaido
`Carp
`Cultured
`Cyprinus carpia
`’1 1 Male.
`
`72 cm, 4.5 kg,
`
`(1)
`
`110 cm,
`
`20 kg,
`
`(1)
`
`44 cm. 610 g,
`
`(10)
`
`23 cm, 175 g,
`
`(5)
`
`'2 Nos. of individual used.
`
`Nov. 1981
`
`—
`
`Dec. 1981
`
`Sep. 1980
`
`at room temperature.“ The resulting diglyceride
`acetates were purified by the method of prepara-
`tive TLC by using the solvent n-hexanc/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
`Diglyccridc Acetate Derived from Lecithin
`The diglyceride acetates were fractionated into
`major molecular
`species on twin 8 X250 mm
`LiChrosorb 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 SE‘ll (Showa
`Denko Ltd, Tokyo) was used. The eluting
`solvent used was isopropanol/acetone/mcthanol/
`acetonitrile (12 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 ofli'ach Peak on
`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.: 250"C, Injection
`temp: 280°C, Carrier gas: NZ, flow rate: 20 ml/
`min.
`
`Methyl esters of fatty acids were prepared ac-
`
`cording to the method of CHRISTOPHER and
`GLASS described by PREVOT and MORDRET.“ An
`aliquot amount
`(less than 20 mg) of lipid was
`dissolved in 1 ml n-hexane and 0.2 ml of methanolic
`
`2N—NaOH solution was added. After shaking
`this mixture, it was stand for 20 3 under 50”C and
`then 0.2 ml of methanolic 2N-HC1 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'l-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.
`
`Gas chromatograph: Hitachi 063, Column:
`OV-lOl
`(Gasukuro Kogyo Ltd., Tokyo),
`steel
`column 0.3 X 50 cm, Column temp: 300~33OUC,
`programed 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 fi’om
`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-HCl buffer, 13118 (1 ml), 2.2% CaCI2
`(0.1 ml), and 0.0570 taurocholic acid sodium salt
`(0.251111) at 40”C for 1min. Then, 40 mg 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
`
`

`

`_ “if“,
`LP‘CakwlTl‘l‘lfller
`Fatty acid
`—»a~-r--————————~- ,._
`6
`7
`5
`
`15: 0
`trace
`16:0
`53.8
`17: 0
`trace
`18:1
`trace
`20: 4
`trace
`20:5
`trace
`22: 4
`trace
`22:6
`46.2
`
`48.4
`
`4.1
`
`3 .7
`
`43.8
`
`25.6
`
`25.9
`
`23.3
`
`25.1
`
`
`
`
`3% 3'23 gMolecular species lg g
`
`
`" Example of big-eyed tuna.
`
`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
`
`Molecular Species of Fish Muscle Lecithin
`
`1805
`
`Table 2. Determination of lecithin molecular species
`of major component”
`
`Virg0rously. The reaction was stopped by adding
`1 ml of ethanol and 1 ml of 6 N—HCl. The hydro-
`lysatc was extracted with diethyl ether and purified
`by using a preparative TLC with n-hexane/diethyl
`ether/formic acid (80: 20: 2, v/v) as developing
`solvent.
`
`Results
`
`Figs. 1~-3 show chromatograms of diglyceridc
`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. 3-13 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-
`
`
`
`
`
`Fig. 1.
`
`HPLC chromatograms of soybean and egg yolk lecithin.
`B: Egg yolk.
`A:
`Soybean.
`
`000003
`
`000003
`
`

`

`1806
`
`TAKAHASHI, HIRANO, TAKAMA, and ZAMA
`
`
`
`
`30
`
`IOU min
`
`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: 1~202 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
`
`60
`
`EU min
`
`80min
`
`
`
`20
`
`40
`
`50
`
`x
`an min
`
`Fig. 3. HPLC chrornatograms of big-eyed tuna. Alaska pollack and carp muscle lecithin.
`F: Alaska pollack.
`G: Carp.
`E: Big-eyed tuna.
`
`also 16:] in position 1 and 20:5 in position 2
`since this peak is camposed of two combinations
`of total acyl carbon numbers of 34 and 36 rap-ec-
`tively. However,
`the latter molecular
`specreS,
`i.c. 16: 1—20: 5, are considered to be contaminants
`from the previous peak. From the fatty acid
`composition 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
`5.7%; and combination of 22: 6 in posmon 1 and
`
`16:1 in position 2 for 31.0%. Among these
`molecular species, 16: 1-20: 5 can be considered
`as contaminants from the two previous peaks.
`In small peak number 5, three molecular species,
`i.c. 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: 07-20: 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“1
`
`"I
`-.
`":—
`h
`I W
`”70136512 number
`7
`7
`
`Fatty acid
`-------
`.
`—
`~
`—-—r-
`-
`-.. ~
`~-
`1
`2
`3
`6
`
`
`4
`
`5
`
`21.3
`WAN ‘
`w14:0
`28.3
`47.7
`53.0
`16:1
`20:5
`47.0
`52.3
`50.4
`
`22:6
`
`30.6
`19.0
`36.2
`14.2
`
`34.6
`19.6
`15.6
`30.2
`
`44.6
`9.7
`8.3
`37.9
`
`barb—oh number”,
`34
`36
`38
`
`95.0<
`
`i
`
`.
`
`10.8
`32.2
`63.3
`42.6
`57.4
`5.7
`41.7
`89.2
`
`31.0
`26.1
`
`90.0
`10.0
`
`Vaarbon r1umber"‘2
`34
`16:11
`2015‘
`16:11 95'°< 120:51 90-0
`36
`38
`
`
`74, in each peak
`'1 Example of chum salmon (Fall).
`
`‘2 Total acyl carbon number.
`
`20:5
`114:01 42‘6
`16:1
`20:51 57'4
`
`14:0
`120: 51 63-3
`16:1
`120:5
`5'7
`22: 6
`16:11 31.0
`
`14:0‘
`120:5 32-2
`22:6
`114:0: 41-7
`l6: 1
`
`22:61 26.1
`
`~14:0
`20:51 10-8
`14:0\
`22:6 89‘2
`
`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, I: molecular species composed of highly
`unsaturated fatty acids such as 20: 5—22: 6, 22: 6-7
`22: 6 and 20: 5—20: 5,
`III: molecular
`species
`composed of generally found fatty acids such as
`16:0 or 18: 1 with combinations of 20: 5 or
`
`22: 6, that is, 16: 0—20: 5, 16: 0~222 6, 18: 1—20: 5
`and 18: 1—22: 6, 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."1
`In the case of
`
`soybean lecithin, 16: 0~18:2"‘2 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 (PM defined as PN2C’” 2d“ since
`PN is proportional to the logarithm of RRT.“
`A general expression for this can be written as:
`
`PNoc 10g (RRT)
`
`(1)
`
`After plotting the RRT of each molecular species
`semilogarithmically,
`a similar correlation was
`obtained, that is:
`
`PNoc log (RRT) +AQ
`
`(2)
`
`(2) is similar to (1), and if we formulate (2) into an
`equation
`
`PN=P - log (RRT) :1 AQ
`
`(3)
`
`examined contain 16: 0~22z 6 as the predomin-
`ant molecular species.
`
`where P becomes the slope of the oblique line, and
`AQ becomes the intersection on the ordinate.
`
`16: 0 in 6665106371 ad 22:61n position 2
`1:
`16: 0 in position 1 and 18:2 in position 2
`*2
`d: Total double bonds.
`*3 C: Total acyl carbon number,
`*4
`
`000006
`
`000006
`
`

`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`'
`
`2.
`
`419/
`-
`a
`
`"'
`
`“
`
`3 7V
`'
`"
`
`.,
`1.14
`
`-
`
`--
`
`3.8%
`
`‘
`
`.77.
`
`a
`2.54
`
`1.7%
`
`"""H
`1,
`1'“ - -
`
`1.0%
`
`.
`
`Molecular Species of Fish Muscle Lecithin
`
`1309
`
`D
`14.71.
`
`__
`u
`6.1/
`-=
`
`__
`
`104%
`
`18 30/
`
`1658
`18-1
`.
`
`7
`
`If
`
`4.2%
`
`0
`4.5%:
`
`_-
`
`3 1 y
`‘
`°
`
`Table 4. Molecular species of fish muscle lecithin
`
`
`Chu __/
`_..,w..__, ——-»h:~r
`'_],.......
`.
`‘rrw:».#* :=-—— ’= 4; W 7-~—*‘
`fi“»~
`---
`:z»
`7 z 774_
`(3mg?!)
`C uirlgzfi)mon
`Blg—eyed tuna.
`\
`Alaska pollack
`Carp
`
`16:
`a
`16:0
`6‘
`16:0
`:1
`7716:0‘
`0
`1 ‘7 7
`22:6
`18-8/1
`22:6
`30-3 °
`22:6i
`23-9 o
`2&2
`244%
`22:21
`41-2/7
`
`16:
`'
`22: 6
`W22:6
`“:7
`16:01
`"W”;
`’ _
`A 20:(si
`22: 6
`22: 6
`16-54
`A 20: 51
`A i035)
`103%
`
`245%
`18:1
`_
`18'1l
`18.1
`_
`.......
`$22.6.
`22:2
`94% _, 39.18;...2. - 22361
`22: 6
`—‘
`22- 61
`i 637—. _
`.
`160‘
`‘
`8 6°/
`1
`'
`"
`22.6
`.
`_
`9.04,
`-
`,
`.
`o
`5 4‘7
`-V-1ff_0_l _-_H-
`18'1_-- _--_,-_
`22:6
`20:5I
`16-()'
`0
`4
`20:51
`20:5
`0
`18:1
`'
`°
`16:0
`'
`°
`22:6}
`6-3/1
`20:4‘
`8.6%
`22:61
`5-14,
`22:6
`A20:5
`
`-- "1811‘
`4
`16:70.
`1
`22: 6
`22: 6‘
`i
`_
`22: 5
`_
`in
`20:5
`6-3 a
`18:25
`16:0
`45%
`16:0
`87%
`22:6
`“/0
`
`,.----...._._
`‘
`i
`-
`li‘fiiirVV
`A 'Nifi—f”
`16; 0'
`20:5
`..
`16:0
`0
`20:5
`..
`22:6
`1.
`,
`16:0
`4'14
`5-54
`22:6
`“A
`22:6
`3-9/0
`18:1
`16-1
`
`..,-.......
`,.
`.
`-
`--.-
`_
`.-
`-
`.
`_ _— ..-.
`18:0'
`14:0
`0
`20:5
`16:0
`116:0
`22:6
`—~— —.
`22:6,
`2-2/‘1
`20: 5
`35%
`20:4
`18:1
`
`0
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`
`A Major component in each peak.
`
`This equation (3) suggests that PN is not only a
`function of RRT alone, but also a function in—
`cluding AQ-
`.
`Back to the definition of the PN, that is, PN»:
`C—Zd, the left member in (3) can be changed as:
`
`C~—2d=P-log (RRT) l—AQ
`
`(4)
`
`So RRT can be expressed as follows:
`
`04221 —AQ
`RRT:10—'P—
`
`(5)
`
`If we put AQ as Ql-l-a, equation (5) may be
`written as:
`
`C "3d— (0111:)
`
`RRT:1(}" P'V’
`
`(Q1 is a minimum of AQ)
`(6)
`where P and Q are invariables and a is a variable
`
`000007
`
`000007
`
`

`

`1810
`
`TAKAIIASHI, HmANo, TAKAMA, and ZAMA
`
`Table 6. Relation between relative retention time of
`Table 5. Relation between relative retention time of
`HPLC and molecular species of soybean lecithin
`HPLC and molecular species of egg yolk lecithin
`
`PN
`Ms
`RT
`' fine
`PN
`Ms
`RT
`RRT
`
`16:1
`18:2
`18:2
`18:2
`i322
`16:0
`22:6
`16:0
`18:3
`:3};
`16:0
`20.4
`16:0
`22: 4
`16: l
`18.1
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`18:2
`.18: O
`22:6
`15:0
`18:1
`16:0
`17:1
`
`17:0
`18: 2
`
`18:0
`20' 4
`18: 1
`181 1
`18' 0
`225 4
`
`
`
`28
`7
`,,
`
`26
`
`28
`r/
`a
`30
`
`n
`”
`
`r/
`
`28
`
`31
`
`'1
`
`”
`
`30
`
`32
`
`”
`
`,7
`II
`
`hi
`
`26
`28
`7
`
`II
`
`30
`.
`,
`31
`
`32
`7
`
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`
`'
`
`I
`
`34
`
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`
`1822“
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`18:0
`18: 2
`16:0
`16:0
`18:0
`18:1
`
`18:2
`20:0
`
`7
`
`27.6
`37.6
`39.2
`
`42.0
`
`
`
`51.4
`55.2
`60.0
`62.8
`
`73.8
`r
`
`79.5
`
`”
`
`83.6
`
`113.2
`
`[I
`
`70.4
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`99.9
`
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`
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`152.9
`160.1
`
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`r
`
`2029
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`”
`
`213.0
`.
`288.6
`
`”
`
`‘
`
`‘fi
`H "
`'Kfiléiiatiégz ‘
`PN: Partition number, MS: Molecular species, RRT: Relative
`retention time.
`“5:01 _
`22: 6 15 used as the reference peak.
`
`a
`
`range of Q1. By letting C and d be invariablcs,
`RRT becomes a function of a which is:
`(7)
`RRT=f(a)
`Back to equation (4), by putting AQ1 as Q14 01,
`the following equation can be obtained.
`
`C~2d=P-log (RRT) +-Q1 +11
`
`(4’)
`
`If we let (1 be invariable and rearrange the ex-
`pression for C, (4’) will become:
`
`Ce‘P'log(RRT)+a+Qz (Q2=Q1+2d)
`
`(8)
`
`On the other hand, if we let C be invariable and
`rearrange the expression for d, (4’) will become:
`
`a
`.. J:
`d-..
`2 lemme—71o
`1
`<
`(9)
`710—0))
`These two functions, LC. (8) and (9) have a devia—
`tion which are a for the former and —-a/2 for the
`
`41.2
`,7
`Ir
`”
`
`47.0
`II
`50.4
`54.4
`-
`53.6
`"
`
`59.2
`
`"
`
`100.0
`7
`”
`
`7:
`
`114.1
`7
`122.2
`132.0
`
`134.9
`,
`
`143.7
`
`”
`
`72.4
`
`175.7
`
`If
`
`”
`
`”
`
`79 0
`'
`”
`
`II
`
`”
`
`”
`
`191 7
`'
`
`”
`
`83.6
`1v
`
`202.9
`II
`
`
`
`g?
`18: 0
`:
`i: 3
`
`”5‘1
`121‘8
`18; 1
`34
`_ 1 V
`_.
`1'-
`__
`Abbreviation;k WW!“
`PN: Partition number, MS: Molecular species, IRRT: Relative
`retention time.
`
`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 ditferences
`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
`
`
`fl
`
`Chum salmon _7 Chum salnio;
`(Summer)
`(Fall)
`
`Big-eyed tuna
`
`
`iii/gasket pollackim —— Carp
`
`
`MS
`PN/RRT
`MS
`PN/RRT
`MS
`PN/RRT
`MS
`PN/RRT
`MS
`PN/RRT
`
`20: 5
`20
`20: SI
`20
`20: 5
`20
`20: 5
`20
`20: 5
`20
`20:5
`27.3
`20:5
`37.5
`22:6
`41.2
`20:5
`37.2
`22:6
`42.5
`20: 5
`20
`20: 5|
`20
`22: 61
`20
`20: S
`20
`22: 6
`20
`22:6
`40.7
`22:6
`40.4
`22:6
`44.6
`22:6
`40.4
`22:6
`46.2
`22:6
`20
`22:6
`20
`20:4
`22
`22:6
`20
`16:1
`24
`22:6
`44.2
`22:6
`44.1
`22:6
`55.2
`22:6
`44.3
`20:5
`62.5
`14:0
`24
`18:1
`26
`22:5
`22
`18:1
`26
`18:2
`24
`22:6
`69.7
`20:5
`87.0
`20:5
`55.2
`20:5
`86.7
`20:5
`62.5
`16: 0
`26
`20: 5
`26
`22:5
`22
`20: 5
`26
`16:1
`24
`20:5
`92.2
`16:0
`89.5
`22:6
`60.0
`16:0
`89.8
`22:6
`677
`18:1
`26
`16:0
`26
`18:1
`26
`22:6
`26
`18:2
`24
`22:6
`92.2
`20:5
`91.9
`22:6
`93.5
`18:]
`89.8
`22:6
`67.7
`22:6
`26
`18:1
`26
`16:0
`26
`16:0
`26
`14:0
`24
`1620
`97.1
`22:6
`91.9
`20:5
`93.5
`20:5
`92.0
`22:6
`677
`16: 0
`26
`22: 6
`26
`22: 6
`26
`18:1
`26
`18:1
`26
`22:6
`100.0
`16:0
`96.7
`16:0
`96.8
`22:6
`92.0
`20:5
`88.3
`16:0
`26
`16:0
`26
`22:6
`26
`16:0
`26
`22:6
`100.0
`22:6
`100.0
`16:0
`97.1
`20:5
`93.7
`16:0
`28
`16:0l
`26
`18:1
`26
`22:5
`134.9
`22:6
`100.0
`22: 6
`93.7
`1620‘
`32
`16:0
`26
`18:1
`216.6
`22: 6
`100.0
`14:01
`32
`16: 0
`28
`20:1
`216.6
`20: 4
`124.0
`
`
`
`
`
`
`
`Abbreviations:
`PN: Partition number. MS: Molecular species. RRT: Relative retention time.
`
`C’=
`
`x
`c2
`
`
`
`(1,
`d2
`
`
`
`(C2,
`
`(11 and dz are invariablcs)
`
`(c1, c2 and (12 are invariables)
`
`(1’:
`
`y !
`C1
`
`(12
`c2
`C’=p1-log (RRT)+q1
`
`(8’)
`
`(9’)
`
`d’::=p2‘log (RRT) +qz
`
`by analyzing the RRT data of each molecular
`species.
`.
`After plotting the RRT of each molecular spec1es
`from the source examined against the total acy]
`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 spcc1es
`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 PAT’I‘ON er (11.1”
`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 PAI‘TON er 11].”) despite the fact that
`the analytical conditions were significantly dif-
`ferent. This suggests that, although there are
`ditferences 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 clution 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
`
`Big-eyed tuna.
`Aiaéké 5611651;
`Chum salmon
`V (Shii—rn‘salmon
`
`(Summer)
`(ball)
`
`_fi
`PN/RRT
`MS
`PN/RRT
`— 20:4
`22
`18:3
`22
`22:6
`50.6
`22:6
`48.7
`22:5
`22
`20:4
`22
`20:5
`50.6
`22:6
`48.7
`22:5
`22
`22:5
`22
`22:6
`52.8
`20:5
`48.7
`16:1
`24
`20: 5
`24
`20:5
`60.8
`16:1
`59.1
`14:0
`24
`20: 5
`24
`20:5
`64.8
`18:2
`59.1
`16:1
`24
`16:1
`24
`22:6
`65.8
`20:5
`60.8
`22:6
`24
`18:2
`24
`14:0
`67.4
`20:5
`60.8
`15:0
`25
`20:5
`24
`20:5
`77.8
`14:0
`62.2
`17:1
`25
`14:0
`24
`22:6
`82.3
`20:5
`64.2
`14: O
`26
`22:6
`24
`22:5
`82.3
`16:1
`64.2
`15:0
`25
`16:1
`24
`22:6
`84.4
`22:6
`67.1
`20: 5
`26
`22: 6
`24
`18:1
`87.2
`14:0
`67.1
`18:1
`26
`14:0
`24
`20:5
`90.2
`22:6
`69.5
`20: 5
`26
`16:1
`26
`16:0
`90.2
`22: 5
`77.8
`22:6
`26
`15:0
`25
`18:1
`90.2
`20:5
`77.8
`16:0
`28
`17:1
`25
`22:5
`121.9
`22:6
`77.8
`16:0
`28
`20: 5
`26
`20:4
`126.6
`18:1
`82.2
`17:0
`27
`22: 5
`28
`22:6
`126.6
`16:0
`116.6
`20:1
`28
`16:0
`28
`20:5
`126.6
`22:5
`119.9
`20:1
`28
`16:0
`28
`22:6
`140.6
`2024
`124.6
`18:0
`28
`20:1
`28
`20: 5
`140.6
`20: 5
`124.6
`18:1
`30
`18:0
`28
`16:1
`143.6
`20:5
`133.3
`18:0
`28
`18:1
`30
`22:6
`152.5
`16:1
`137.2
`14:0
`30
`14: 0
`30
`18:1
`152.5
`18:1
`142.9
`16:0
`30
`16:0
`30
`16:1
`152.5
`16:1
`142.9
`24:1
`32
`265.7
`20: 5
`Abbreviations:
`PN: Partition number. MS: Molecular species, RRT: Relative retention time.
`
`
`
`
`
`
`
`Carpw
`
`~ivis —
`
`
`
`.1:
`
` HOHr—acoxoV-‘ONONH”Hat“heLIIO#HNO[In—4ANAH
`
`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
`
`PN/iiRr
`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
`
`l5 25
`
`MS
`
`16:
`20:
`18:
`20:
`14:
`20:
`16:
`22:
`18:
`22:
`14:
`22:
`22:
`16:
`16:
`20:
`16:
`22:
`20:
`20:
`18:
`20:
`20:
`22:
`18:
`16:
`18:
`18:
`14:
`18:
`16:
`16:
`16:
`18:
`18:
`22:
`18:
`16:
`20:
`14:
`16:
`20:
`
`
`
`O1
`
`I18:18:
`
`MS,
`22:6l
`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
`22: 5
`16:0
`22:6
`18:0
`18:0
`22:6
`18:1
`18:1
`18:1
`16:0
`16:0
`’18:
`1
`
`
`
`PNl—RRT
`24
`65 .o
`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
`28
`124.6
`28
`130.0
`28
`143.2
`28
`148.1
`32
`189.6
`32
`206.1
`32
`215.5
`
`0000010
`
`0000010
`
`

`

`Molecular Species of Fish Muscle Lecithin
`
`1813
`
`l1? gl. 2[1}: El h: it. 3h}; gl, 4l1ii l. 5h? gl Elli; ll
`lllli gl
`
`9l2l§ gl, "1lec gl, ”lzh kl. ”l2: gl ”l2: l
`7l2i§ 2, 8]}; 2,
`1
`1
`x 5
`5
`”12:2: 5l,. 15h); el, 1Blzo 4|, ”l2? sl.
`1
`an 5
`18122 6
`
`
`
`
`- —
`AZ
`
`30
`
`32
`
`-v~ ~_
`44
`
`700
`500
`400
`300
`
`200
`
`100
`
`70
`
`50
`1.0
`
`g 30
`20
`
`8 g
`
`100
`
`70
`
`~22
`
`30
`20
`
`% ‘0
`"‘
`0
`1
`3215.88
`H
`52300
`200
`
`
`
`LO
`’"34“ "7360 _ 3a
`Total Acyl Carbon Number
`22
`18
`,.
`_
`20
`.
`20
`19§§2|l§§%|,20l22%, 2’lzo z, 22'22 El 23hr; 5,
`
`
`'
`.
`0
`2433 ll,
`25K? 2%,
`lbllgl
`27I18 ll, 28V ’
`
`lH .
`1.8
`.
`25llia ll 30l16 l
`
`
`
`..
`
`27
`
`30
`
`.
`
`25
`2928
`
`10 ""'"""— ""‘
`8
`1253113767
`Number of Total
`
`9
`12"13
`to '11
`Double Bonds
`
`14
`
`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. CHIGIRA of Asahi
`Chemical Industry Ltd. for the supply of egg yolk
`lecithin; and W. NAGATA 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. E. M. LANDS and P. HART:
`J. Am. Oil Chem.
`Soc., 43, 290—295 (1966).
`
`3)
`
`4)
`
`5)
`
`6)
`
`7)
`
`8)
`
`9)
`
`J. Am. ()1'] Chem. Soc, 42, 298~
`
`0. RENKONENZ
`304 (I965).
`A. KUKSIS and 1.. MARAIZ Lipids, 2, 217—224
`(1967).
`A. F. PREVOT and 1". X. MORDRET: Rev. 1-‘se.
`Corps Gras, 23, 409- 423 (1976).
`K. ZAMA, T. MARUYAMA, and K. TAKAHASHI:
`Bull. Fae. Fir/1. Hokkaido Univ., 27,
`181—190
`(1976).
`K. TAKAHASHI, K. ZAMA, and T. MATSUOKA:
`Bull. Fae. Fish. Hokkaido Um'v., 29,
`378—385
`(1978).
`K. TAKAHASHI, F. CABLING, and K. ZAMA: Bull.
`Fac. Fish. Hokkaido Univ., 29, 386—391 0978).
`A. KUKSIS:
`in “Progress in the Chemistry of Fats
`
`0000011
`
`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. Pan-r014, J. M. FASULO, and S. J. ROBINS:
`.
`J. LipidRes.,23, 190—196 (1982).
`'
`
`0000012
`
`0000012
`
`