Biosci. Biotech. Biochem., 59 (8), 1389-1393, 1995
`
`Platelet-activating Factor (P AF)-like Phospholipids Formed during Peroxidation of
`Phosphatidylcholines from Different Foodstuffs
`
`Tamotsu TANAKA,* Akira TOKUMURA,** and Hiroaki TSUKATANI
`Faculty of Pharmaceutical Sciences, The University of Tokushima, Shomachi, Tokushima 770, Japan
`Received July 4, 1994
`
`Previously, we reported that induction of peroxidation of synthetic phosphatidylcholines (PCs)
`containing a polyunsaturated fatty acid by Fe 2 + -EDT A in the presence of ascorbate resulted in the formation
`of four types of PCs with an sn-2-oxidatively fragmented acyl group, which had platelet-aggregating
`activity due to interaction with platelet-activating factor (P AF) receptors. These PCs were compounds
`with a short-chain monocarboxylate, w-hydroxymonocarboxylate, dicarboxylate, and dicarboxylate
`semi aldehyde residue, respectively. In this study, we investigated the P AF -like lipids formed during
`peroxidation ofPCs from hen egg yolk, salmon roe, sea urchin eggs, and krill in an FeS0 4 /EDTA/ascorbate
`system. The platelet-aggregating activities of these oxidized PCs were all inhibited by FR-900452, an
`antagonist of P AF. The activity of oxidized krill PC, which was equivalent of 89.8 ± 8.8 pmol 16: 0-P AF I
`/lmol of starting PC, was about 5 times those of oxidized PCs from salmon roe and sea urchin eggs, and
`about 50 times that of oxidized hen egg yolk PC. The P AF -like phospholipids that had different combinations
`of long-chain alkyl or acyl groups with one of the above four types of short-chain acyl groups were identified
`by gas chromatography-mass spectrometry. The results indicated that foodstuffs that are rich in I-D-alkyl-2-
`docosahexaenoyl-sn-glycero-3-phosphocholine are potential sources of compounds with high P AF -like
`activity formed by deleterious lipid peroxidation.
`
`Peroxidation of phospholipids contammg polyunsat(cid:173)
`urated fatty acid (PUF A) is known to result in formation
`of secondary degradation products such as short-chain
`fatty aldehydes. These compounds have been shown to be
`produced by chain scission of the PUF A moiety via phos(cid:173)
`pholipid hydroperoxides formed as primary products.l)
`However, until recently, less attention has been paid to
`the chemical nature of other classes of cleavage products
`retaining a phospholipid backbone, although oxidized
`phospholipids have been reported to have significant
`biological effects such as enhancement of atherogenesis 2 )
`and augmentation of macrophage growth-stimulating
`activity.3) Itabe et al. 4
`) reported that PCs with an azelaoyl
`group were generated as cytotoxic compounds by the
`oxidation ofPCs containing linoleate with oxyhaemoglobin.
`Furthermore, Smiley et al. 5
`) demonstrated that some
`oxidized PCs expressed platelet-activating factor (PAF)(cid:173)
`like activity (neutrophil adhesion to vascular endothelial
`cells), and identified PCs with an oxovaleroyl group as
`active components. Using gas chromatography-mass
`spectrometry (GC-MS) and fast atom bombardment-mass
`spectrometry, we identified four types of PCs with an
`sn-2-oxidatively fragmented acyl group formed during
`peroxidation of different synthetic PCs with a PUF A by
`Fe 2 + -EDT A in the presence of ascorbate. 6, 7) These were
`PCs with an sn-2-short-chain monocarboxylate (MC),
`dicarboxylate (DC), dicarboxylate semialdehyde (DCsa),
`or w-hydroxymonocarboxylate (HC) group. Furthermore,
`
`we showed that oxidized PCs had platelet-aggregating
`activity via PAF receptors and that this activity was mainly
`due to PCs with a short-chain monocarboxylate group.7)
`In this previous study, oxidized 1-0-hexadecyl-2-docosa(cid:173)
`hexaenoyl-sn-glycero-3-phosphocholine (GPC) was shown
`to have a much higher platelet-aggregating activity than
`oxidized 1-0-hexadecyl-2-arachidonoyl-GPC or oxidized
`l-palmitoyl-2-docosahexaenoyl-GPC. This was found to
`be because the PAF-like phospholipids derived from 1-0-
`alkyl-2-docosahexaenoyl-GPC had suitable structural re(cid:173)
`quirements for induction ofPAF-like activity: an sn-l-alkyl
`ether linkage and an sn-2-acyl group of shorter chain length
`(Fig. 1).
`
`DC
`
`DCsa
`
`MC
`
`HC
`
`Fig. 1. Structures of PAF-like Phospholipids Formed during Peroxida(cid:173)
`tion of PCs from Different Food Stuffs.
`DC, DCsa • MC, and HC indicate short-chain dicarboxylyl, dicarboxylate semi alde(cid:173)
`hyde, monocarboxylyl, and co-hydrocarboxylyl group, respectively.
`
`* Present address: Department of Food Science and Technology. Fukuyama University, Fukuyama 729-02, Japan
`** To whom correspondence should be addressed.
`Abbreviations: AA, arachidonic acid; BHT, butylated hydroxy toluene; DC, dicarboxylate; DCsa, dicarboxylate semialdehyde; DHA, docosa(cid:173)
`hexaenoic acid; EPA, eicosapentaenoic acid; FR-900452, I-methyl-3-( 1-[5-methylthiomethyl-6-oxo-3-(2-oxo-3-cyc1openten-l-ylidene)-2-piperazinylJ(cid:173)
`ethyl)-2-indolinone; GC-MS, gas chromatography-mass spectrometry; GPC, sn-glycero-3-phosphocholine; He, w-hydroxymonocarboxylate; MC,
`monocarboxylate; PAF, platelet-activating factor; PC, phosphatidylcholine; PUFA, polyunsaturated fatty acid; tBDMS, tert-butyldimethylsilyl;
`TIC, total ion chromatogram.
`
`RIMFROST EXHIBIT 1014 page 0001
`
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`

`1390
`
`T. TANAKA, A. TOKUMURA, and H. TSUKATANI
`
`It is known that many marine animals eaten by humans
`are rich in 1-0-alkyl-2-acyl-GPC8) and that some aquatic
`animals contain abundant docosahexaenoate (DHA).9) So,
`there is a possibility that deleterious lipid peroxidation of
`such compounds from marine animals might result in the
`formation of oxidatively fragmented PC with potent PAF(cid:173)
`like activity. In this study, we examined this possibility
`using krill, salmon roe, and sea urchin eggs.
`
`between the lipid-phophate contents before and after alkaline hydrolysis.
`The amount of 1-0-alkenyl-2-acyl-GPC was calculated from the difference
`in the lipid-phosphate-contents in the lower phase before and after acid
`hydrolysis.
`
`Fatty acid analysis of 1-O-alkyl-2-acyl-G Pc. Krill PC (16 J.lmol) was
`hydrolyzed with phospholipase C from Bacillus cereus and the resultant
`glycerides were acetylated as described previously. 13) The diradyl acetates
`were separated by TLC,13) and the fatty acid composition of l-O-alkyl-
`2-acyl-3-acetyl-glycerol was measured by GLC as described above.
`
`Materials and Methods
`Materials. The reagents used and their sources were as follows: butylated
`hydroxy toluene (BHT), imidazole, and 6-(p-toluidino )-2-naphthalene(cid:173)
`sulfonic acid from Wako Pure Chemical Co. (Tokyo); perdeuterated acetic
`anhydride and silica gel 60 TLC plates (0.25 mm thickness) from Merck
`(Darmstadt, Germany); Bacillus cereus phospholipase C from Sigma
`Chemical Co. (St. Louis, MO), and fatty acid methyl ester standards for
`GLC from Nu-Chek Prep. (Elysian, MN). FR-900452 was kindly donated
`by Fujisawa Pharmaceutical Co., (lbaraki, Japan). I-O-Hexadecyl-2-
`perdeuterated (d3 ) acetyl-GPC was prepared by treatment of 1-0-hexa(cid:173)
`decyl-2-lyso-GPC with perdeuterated acetic anhydride as described
`previously.lO) A lipid extract of krill (Euphausia superba) was a generous
`gift from Itano Refrigerated Food Co. (Tokushima, Japan). Eggs of a sea
`urchin (Hemicentrotus pulcherrimus), ripe roes of salmon (Oncorhynchus
`keta), and hen eggs were obtained commercially.
`
`Preparations of PCs. Samples of 2 g of salmon roe, sea urchin eggs, and
`hen egg yolk were homogenized in 12 ml of water at 4°C. The homogenate
`was mixed with 22.5 ml of chloroform-methanol (1 : 2, by vol.) containing
`0.01 % BHT, and 7.5 ml of chloroform was added to the mixture for phase
`separation. After centrifugation at 2000 rpm for 5 min, the chloroform(cid:173)
`rich layer was withdrawn, and the remaining aqueous layer was again
`mixed with 15 ml of fresh chloroform and centrifuged. The chloroform
`extracts were combined and evaporated to dryness under reduced pressure.
`The residue was dissolved in 25ml of 80% ethanol and mixed with 10mi
`of n-hexane for phase separation. After centrifugation at 2000 rpm for
`5 min, the upper phase containing triglycerides, cholesterol, and BHT
`was removed, and the lower layer was evaporated to dryness. The phos(cid:173)
`pholipids in the lower phase were fractionated by Sephadex LH-20 column
`chromatography with a solvent system of chloroform-methanol (1 : I, by
`vol.). The PC-rich eluate was collected and dried, and the residue was
`dissolved in a small volume of ethanol containing 0.01 % BHT and stored
`at -20°C. Just before use, PC was separated from BHT in this stock
`solution by TLC with a developing solvent system of chloroform(cid:173)
`methanol-28% ammonium hydroxide (65: 35 : 5, by vol.).
`Krill PC was purified from the crude lipid extract of krill in the same
`way by Sephadex LH-20 column chromatography and TLC.
`
`Fatty acid analysis. PCs (1.6 J.lmol) were transmethylated, and the
`resultant fatty acid methyl esters were analyzed in a Hitachi 263-70 gas
`chromatograph with a fused silica capillary column (J & W, DB-225,
`30 m x 0.242 mm i.d., 0.25 J.lm-thickness). 6) The column temperature was
`kept at 120°C for 1 min, then raised to 220°C at a rate of 10°C/min, and
`kept at this temperature for 25 min. The carrier gas was helium, and the
`temperatures of the injection port and flame ionization detector were both
`set at 250°C. Fatty acid methyl esters were identified by comparing their
`retention times with those of authentic standards.
`
`Analysis of subclasses of PCs. The percentages of PC subclasses were
`measured by successive degradations of PC with mild alkali and acid. I I)
`Briefly, native PC (3.2-16.1 J.lmol) was dissolved in 15ml of 0.1 M meth(cid:173)
`anolic KOH and the mixture was stirred for 4 h at room temperature.
`After neutralization of the solution with 1 N HCl, 12.5 ml of water and
`15 ml of chloroform were added for phase separation and the mixture was
`centrifuged. The lipid-phosphate content of the lower phase containing
`both 1-0-alkyl-2-lyso-GPC and I-O-alkenyl-2-lyso-GPC was measured by
`the method of Chalvardjian and Rudnicki,12) Next, the lower layer was
`dried and the residue was dissolved in 28.5 ml of a mixture of chloroform(cid:173)
`methanol-water (7.5: 15: 6, by vol.) acidified with 5N HCl and stirred at
`room temperature for 20min to decompose 1-0-alkenyl-2-lyso-GPc. To
`the reaction mixture, 7.5 ml each of chloroform and water were added for
`phase separation follwed by centriguation. The lower phase containing
`only 1-0-alkyl-2-lyso-GPC was used for determination of lipid-phos(cid:173)
`phate. 12 ) The amount of diacyl-GPC was calculated from the difference
`
`Peroxidation of PCs. PCs were peroxidized with FeS04/EDTA/ascor(cid:173)
`bate, and the oxidatively degraded phospholipids were analyzed by TLC
`as reported previously.6) Briefly, PCs from various samples (2 J.lmol) were
`suspended in 8 ml of water acidified to pH 4.0, and the suspensions were
`sonicated for 15 min in a probe type sonicator (200 W, 9 kHz). The resulting
`liposomes were incubated with FeS04 (25 J.lM), ascorbate (50 J.lM) and
`EDT A (25 J.lM) at room temperature for 2 h. Lipids were extracted from
`the reaction mixture by the method of Bligh and Dyer 14) after acidification
`of the aqueous phase with 1 N HCl, and were separated by TLC in a solvent
`system of chloroform-methanol~28% ammonium hydroxide (65: 35: 8,
`by vol.). With the aid of authentic PC (l,2-dipalmitoyl) and lysoPC
`(l-palmitoyl), the plate was divided into a zone corresponding to standard
`PC and a zone from the origin to the area below the standard PC, which
`was deduced to contain the PAF-like phospholipids. The lipids were
`recovered from the PC zone of silica gel (PC fraction) by the method of
`Bligh and Dyer l4
`) and from the active phospholipid zone of the silica gel
`(active phospholipid fraction) by the method of Bligh and Dyer l4 ) after
`acidification of the aqueous phase.
`
`Measurement of platelet-aggregating activity. The active phospholipid
`fractions by the oxidative degradation of PCs were dispersed in saline
`with 0.1 % bovine serum albumin. Samples of 50 J.ll of the dispersion were
`added to 250 J.ll of a suspension of washed rabbit platelets, and platelet
`aggregation was monitored as described previously. 7) In some experiments,
`the platelets were treated with different concentrations of FR-900452 for
`I min before addition of the dispersion. The platelet-aggregating activities
`of the active phospholipid fractions were calculated as pmol equivalents
`of PAF (C I6 :0 ) per I J.lmol of starting PC using a calibration curve for
`synthetic PAF (C 16 :0).
`
`GC-MS. PAF-like phospholipids derived from several PCs were hydro(cid:173)
`lyzed with phospholipase C and the resultant glycerides were converted to
`tert-butyldimethylsilyl (tBDMS) derivatives as described previously.6.10)
`The tBDMS derivatives were fractionated by TLC with a solvent system
`of n-hexane-ethyl ether (90: 10, by vol.) after treatment with ethereal
`diazomethane. 6) The regions of silica gel in the range of R f 0.04--0.80, in
`which the tBDMS derivatives of glycerides from PAF-like compounds
`migrated, were scraped off and tested by GC-MS as described previously. 6)
`The PCs containing a short-chain monocarboxylate group in active
`phospholipid fractions were measured by the peak areas on the total ion
`chromatograms (TIC) using 1-0-hexadecyl-2-perdeuterated (d3 ) acetyl(cid:173)
`GPC as an internal standard.
`
`Results and Discussion
`Analysis of subclasses and fatty acids of various pes
`In this study, we used krill, salmon roe, and sea urchin
`egg PCs as potential marine sources of PAF -like lipids, and
`hen egg yolk PC for comparison. The subclasses and fatty
`acid compositions of PCs prepared from these foodstuffs
`are shown in Tables I and II, respectively. Krill PC was
`rich in both alkylacyl-GPC subclass and DHA as a PDF A,
`suggesting that it contains abundant 1-0-alkyl-2-docosa(cid:173)
`hexaenoyl-GPC. GLC analysis found that alkylacyl-GPC
`from krill had the following fatty acid composition: 14: 0
`(2.1 %), 16: 0 (10.6%), 16: 1 (5.2%), 18: 0 (4.5%), 18: 1, n-9
`(10.0%),18:2, n-6 (1.3%),18:3, n-6 (0.4%),18:3, n-3
`(0.4%),20:3, n-6 (3.7%),20:3, n-3 (2.9%), 20:4, n-6
`(0.3%), 20: 5, n-3 (30.8%), 22: 6, n-3 (21.8%), unknown
`(6.0%). Sea urchin egg PC was rich in alkylacyl-GPC, but
`its DHA level was very low. Neither salmon roe PC nor
`
`RIMFROST EXHIBIT 1014 page 0002
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`

`Platelet-activating Factor-like Lipids
`
`1391
`
`hen egg yolk PC contained a significant amount of alkyl(cid:173)
`acyl-PC, but the DHA content of PC in salmon roe was
`much higher than that in hen egg yolk.
`
`Platelet-aggregating activities of oxidized PCs
`We peroxidized PCs prepared from hen egg yolk, salmon
`roe, sea urchin eggs and krill in an FeS04/EDTA/ascorbate
`system and measured the platelet-aggregating activities
`of the active phospholipid fractions containing oxidative(cid:173)
`ly fragmented PCs with an sn-2-short-chain acyl moiety.
`All these phospholipid fractions induced aggregation of
`washed rabbit platelets. FR-900452, an antagonist of PAF,
`inhibited the platelet-aggregating effects of these fractions
`to similar extents in a concentration-dependent manner
`(data not shown), suggesting that the observed platelet ag(cid:173)
`gregations were mediated via PAF receptors on the plate(cid:173)
`lets. The activity of oxidized krill PC was about 5 times
`those of oxidized PCs from salmon roe and sea urchin
`eggs, and 50 times that of oxidized hen egg yolk PC (Table
`III).
`
`GC-MS of P AF-like phospholipids formed during peroxida(cid:173)
`tion of PCs
`To identify the PAF-like phospholipids formed during
`per oxidation of PCs from various foodstuffs, we took the
`
`Table I. Subclass Composition of PCs from Food Stuffs
`
`PC
`
`Diacyl
`
`Alkylacyl
`
`Alkenylacyl
`
`Hen egg yolk
`Salmon roe
`Sea urchin egg
`Krill
`
`99.2±0.2
`9S.S±0.2
`57.5 ± 1.1
`77.0 ± 1.2
`
`%
`O.S±O.1
`1.2±0.2
`41.5 ±0.3
`23.0± 1.2
`
`<0.1
`<0.1
`1.0 ±O.S
`<0.1
`
`Values are means ± SE for four experiments.
`
`Table II. Fatty Acid Composition of PCs from Food Stuffs
`
`Fatty Acid
`
`Hen egg
`yolk
`
`Salmon roe
`
`Krill
`
`Sea urchin
`egg
`
`14:0
`16:0
`16: 1
`18:0
`18: 1 (n-9)
`IS: 1 (n-7)
`18: 2 (n-6)
`lS:3 (n-3)
`18:3 (n-6)
`20: 1 (n-6)
`20: 1 (n-3)
`20: 2 (n-6)
`20: 2 (n-3)
`20: 3 (n-6)
`20: 3 (n-3)
`20: 4 (n-6)
`20: 5 (n-3)
`22: 5 (n-3)
`22: 6 (n-3)
`Unknown
`
`a
`
`-
`36.0±0.4
`1.4±0.1
`11.1 ±0.3
`28.5 ±O.l
`0.3 ±0.03
`16.2±0.9
`
`1.3±0.4
`20.7 ± 1.7
`1.6±0.4
`1O.9±0.3
`16.3 ± 1.6
`1.9 ±0.2
`
`%
`
`1.3±0.1
`22.8± 1.6
`l.l±O.1
`l.0±O.3
`5.2±O.5
`4.7±O.2
`1.9±0.03
`0.9±0.04
`0.8 ±O.l
`
`2.3±0.4
`
`3.7 ±0.4
`0.5±0.5
`
`15.3 ± 1.2
`4.4±0.6
`22.2± 1.2
`5.5 ±0.5
`
`0.5±0.3
`4.5 ± 1.0
`0.4±0.2
`32.9 ± 1.0
`0.8±0.4
`19.4±1.4
`1.6±0.4
`
`l.9±0.2
`7.7
`1.1
`2.1 ±O.2
`2.2±0.1
`O.6±0.1
`4.S±0.9
`1.2±0.3
`
`8.0 ± l.5
`3.5±0.5
`19.1±1.8
`7.4±0.5
`l.0±0.3
`
`15.1 ±0.8
`17.0±1.0
`
`1.0 ±0.3
`7.3 ±2.7
`
`active phospholipid fractions separated by TLC of the
`oxidized PCs and separated them by GC-MS as described
`in Marerials and Methods. Figures 2-A, B, C, and D show
`total ion chromatograms (TIC) of the tBDMS derivatives
`of glycerides obtained by the phospholipase C hydrolysis
`of the active phospholipid fractions of hen egg yolk, salmon
`roe, sea urchin egg, and krill PCs, respectively. Based on
`previous findings on mass spectrometric analysis of P AF(cid:173)
`like phospholipids derived from synthetic PC6,7) and in a
`bovine brain lipid extract, 1 5 17) the peaks on TICs were
`identified as shown in Table IV. Several major peaks on
`the TIC shown in Fig. 2-A were assigned to the tBDMS
`derivatives of diacylglycerols containing MCs : 0, DC9 : 0, or
`RCs: 0' Since the chain lengths of the sn-2-acyl moieties in
`major PAF-like phospholipids were found to depend on
`the double bond vicinal to the ester linkage of PUF A in
`the parent PC,6,7) the PAF-like phospholipids containing
`MC 8 : 0, DC 9 : 0, and RC 8 : ° are thought to be formed during
`peroxidation of PC with a linoleate moiety, which is a major
`PUF A in hen egg yolk PC (Table IV).
`The PAF-like phospholipids formed during peroxida(cid:173)
`tion of slamon roe PC were separated into three groups
`(Fig. 2-B, Table IV). One included I-long-chain acyl-PCs
`with MC 3 : 0 , DC4 : 0 , and HC 3 : 0 , and was regarded as
`the oxidation products of PCs having a PUF A with a
`double bond vicinal to the ester linkage at positions C4
`and Cs such as DHA. The second consisted of PCs with
`MC4 : 0 , DC s:o, and HC4 : 0 , which were deduced to be
`generated from PC having a PUF A with a double bond
`vicinal to the ester linkage at positions C s and C6, mainly
`eicosapentaenoic acid (EPA). We detected several species
`of PCs with short-chain monounsaturated hydroxymono(cid:173)
`carboxylate groups in oxidized salmon roe PC.
`Both I-O-alkyl and I-acyl type PCs with a DC s :0, MC4 : 0 ,
`or RC4 : 0 were detected as major PAF-like phospholipids
`by GC-MS of oxidized sea urchin egg PC (Fig. 2-C, Table
`IV). These were probably derived from both PCs containing
`arachidonate (AA) or EPA with a double bond at positions
`Cs and C6. The major PAF-like phospholipids formed by
`oxidation of krill PC were classified into two groups:
`I-O-alkyl and I-acyl PCs with MC 3 : 0 , DC4 : 0 , and DCsa4 : 0
`derived from PCs containing DRA, and l-O-alkyl and
`I-acyl PCs with MC4 : 0 , DCs :o, DCsas :o, and HC4 : 0 de(cid:173)
`rived from PCs containing EPA (Fg. 2-D, Table IV).
`We failed to assess PAF-like activity of individual
`molecular species of PCs in these foodstuffs, because the
`separation was unsatisfactory. A previous study on
`synthetic PCs containing a PUF A demonstrated that
`oxidized PCs with an ether linkage at the sn-l position
`showed much higher platelet-aggregating activities than the
`
`Table III. Platelet-aggregating Activities of Active Phospholipid Frac(cid:173)
`tions Obtained by Oxidation of PCs from Various Food Stuffs
`
`PC
`
`Platelet-aggregating activity
`
`Hen egg yolk
`Salmon roe
`Sea urchin egg
`Krill
`
`Equivalents of C 16 : 0 PAF as pmol/J1mol PC
`1.7 ±0.6
`17.8 ± 1.9
`17.2±4.0
`89.8±8.8
`
`Values are means±SE for three experiments. "Not detected.
`
`Values are means ± SE for three or four experiments.
`
`RIMFROST EXHIBIT 1014 page 0003
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`1392
`
`A
`
`T. TANAKA, A. TOKUMURA, and H. TSUKATANI
`
`B
`
`10
`
`20
`
`40
`Time (min)
`
`60
`
`20
`
`40
`Time (min)
`
`60
`
`c
`
`13
`
`10
`
`0
`
`13
`
`19
`
`12 J ,,16
`\
`
`w£ 10 \/15V'
`
`~y
`
`18
`
`30
`Time (min)
`
`40
`
`50
`
`10
`
`20
`
`Time (min)
`
`30
`
`40
`
`Fig. 2. GC-MS Fractions of Phospholipidic Secondary Products Generated during Peroxidation of Egg Yolk (A), Salmon Roe (B), Sea Urchin Egg
`(C), and Krill (D) PCs with Fe2 + -EDT A in the Presence of Ascorbate.
`The materials in peaks were identified as shown in Table IV.
`
`Table IV. Molecular Species of PAF-like Phospholipids Formed by Oxidation of PCs from Food Stuffs
`
`Peak No.
`
`Hen egg yolk
`
`Salmon roe
`
`Sea urchin egg
`
`Krill
`
`16 : 0/3 : 0 (M C)
`16:0/4:0 (MC)
`18:0/3:0 (MC)
`16:0/4:0 (DC)
`Unknown
`16: 0/8 : 0 (MC)
`16:0/5:0 (DC)
`16: 0/3: 0 (HC)
`18 : 0/4 : 0 (DC)
`16:0/4:0 (HC)
`18 : 0/8 : 0 (M C)
`18 : 0/5 : 0 (DC)
`18:0/3:0 (HC)
`16 : 0/8 : 0 (DC)
`16: 0/9: 0 (DCsa)
`16: 0/9: 0 (DC)
`Unknown
`16: 0/7 : 0 (HC)
`16: 0/8: 0 (HC)
`18 : 0/9 : 0 (DC)
`
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`29
`30
`31
`32
`
`16:0/3:0 (MC)
`16:0/4:0 (MC)
`14:0/4:0 (DC)
`18:1/3:0(MC)
`18:0/3:0 (MC)
`18 : 1/4 : 0 (M C)
`16: 0/4: 0 (DCsa)
`18:0/4:0 (MC)
`16: 1/4:0 (DC)
`16:0/4:0 (DC)
`Unknown
`16: 0/5 : 0 (DC)
`18: 0/4: 0 (DCsa)
`Unknown
`16:0/3:0 (HC)
`18: 1/4:0 (DC)
`18: 1/4:0 (DC)
`18:0/4:0 (DC)
`Unknown
`Unknown
`16:0/4:0 (HC)
`18 : 1/5 : 0 (DC)
`18: 0/5 : 0 (DC)
`16: 0/6 : 1 (HC)
`18: 1/3:0 (HC)
`18: 0/3 : 0 (HC)
`16: 0/7: 1 (HC)
`18:1/4:0(HC)
`18: 0/4: 0 (HC)
`18:1/6:1 (HC)
`18: 0/6 : 1 (HC)
`18: 0/7 : 1 (HC)
`
`0-14: 0/3: 0 (MC)
`0-14: 0/4: 0 (MC)
`14:0/4:0 (MC)
`0-16:0/3:0 (MC)
`0-16: 0/4: 0 (MC)
`Unknown
`16: 0/3: 0 (MC)
`0-14: 0/4: 0 (DC)
`16:0/4:0 (MC)
`0-14: 0/5: 0 (DC)
`0-16: 0/4: 0 (DCsa)
`16:0/4:0 (DCsa)
`0-16: 0/4: 0 (DC)
`0-16: 0/5: 0 (DC sa)
`16: 0/4: 0 (DC)
`0-16: 0/5: 0 (DC)
`16: 0/5: 0 (DCsa)
`Unknown
`16: 0/5: 0 (DC)
`Unknown
`16:0/4:0 (HC)
`18: 1/5:0 (DC)
`
`14: 0/4: 0 (MC)
`0-16: 0/4: 0 (MC)
`16:0/3:0 (MC)
`16: 0/4: 0 (MC)
`0-18:0/4:0 (MC)
`14: 0/5: 0 (DC)
`18:1/4:0(MC)
`18:0/4:0 (MC)
`16: 0/4: 0 (DC)
`0-16: 0/5: 0 (DC)
`Unknown
`20:2/4:0 (MC)
`16:0/5:0 (DC)
`20: 1/4:0 (MC)
`Unknown
`Unknown
`0-18: 0/5: 0 (DC)
`Unknown
`18 : 1/5 : 0 (DC)
`18:0/5:0 (DC)
`Unknown
`Unknown
`0-18: 0/4: 0 (HC)
`20 : 2/5 : 0 (DC)
`20: 1/5:0 (DC)
`20: 0/5 : 0 (DC)
`
`Labels in parentheses indicate the kinds of short chains depicted in Fig. 1. 0-14: 0, 0-16: 0, and 0-18: 0 indicate tetradecyl, hexadecyl, and
`octadecyl residues, respectively.
`
`RIMFROST EXHIBIT 1014 page 0004
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`

`

`Platelet-activating Factor-like Lipids
`
`1393
`
`corresponding oxidized I-acyl PCs, and that the activities
`of oxidized PCs were mainly due to MC-PCs. 7
`) From these
`findings, active components in the oxidized PCs were
`suggested to be MC-PCs formed from alkylacyl-GPC.
`Since MC-PCs with an sn-l-0-hexadecyl group were the
`predominant species, we measured the yields of MC-PCs
`with an sn-l-0-hexadecyl residue and sn-2-short-chain
`monocarboxylate moiety in oxidized krill and sea urchin
`egg PCs as their tBDMS derivatives by GC-MS. 1-0-
`Hexadecyl-2-deuterated acetyl-GPC was used as an internal
`standard. As shown in Table V, the yields of 1-0-hexadecyl-
`2-propionyl-GPC and 1-0-hexadecyl-2-butyryl-GPC were
`0.0035% and 0.0049% (or 0.0076%), respectively, of the
`starting krill PC. The yield of 1-0-hexadecyl-2-butyryl(cid:173)
`GPC was 0.02 % of the starting sea urchin egg PC, but no
`significant amount of 1-0-hexadecyl-2-propionyl-GPC was
`detected. The platelet-aggregating activities of synthetic
`1-0-hexadecyl-2-propionyl-GPC and 1-0-hexadecyl-2-bu(cid:173)
`tyryl-GPC were 1.16 times and 0.02 times that of PAF
`(C 16 : 0 ).7) Consequently, the PAF-like activities of these
`two phospholipids formed by oxidation of krill PC are
`equivalent to those of 40.6 pmol (PC with an sn-2-pro(cid:173)
`pionate) and 1.0 pmol (PC with an sn-2-butyrate) of
`16: O-PAF (Table V). The sum of the estimated values
`accounted for about half the observed platelet-aggregating
`
`Table V. Quantitative Analysis of l-O-hexadecyl-PCs Containing a
`Short-chain Monocarboxylate Group in Active Phospholipidic Fractions
`Obtained by Oxidation of PCs from Krill and Sea Urchin Egg
`
`% of starting PC
`
`1-0-Hexadecyl-2-
`propionyl-PC
`
`1-0-Hexadecyl-2-
`butyryl-PC
`
`Krill
`
`Exp.1
`( 74.9 pmoW
`Exp. 2
`(101.0pmo1t
`Sea urchin egg
`( 8.9pmo1)U
`
`0.0035
`(40.6pmo1)b
`0.0035
`(40.6pmo1)b
`_d
`
`0.0049
`(l.Opmo1)"
`0.0076
`(1.5pmo1Y
`0.020
`(4.0pmo1)'
`
`fraction
`a Platelet-aggregating activity of active phospholipid
`(equivalents of C 16 : 0 PAF as pmol/JLmol PC) measured by the
`bioassay.
`b,c Platelet-aggregating activity of 1-0-hexadecyl-2-propionyl-GPC
`and 1-0-hexadecyl-2-butyryl-GPC (equivalents of C 16 : 0 PAF as
`pmol/JLmol PC), respectively. Values were estimated from the
`yields and platelet-aggregating activities of synthetic 1-0-
`hexadecyl-2-propionyl-GPC and 1-0-hexadecyl-2-butyryl-GPC.
`d Not detected.
`
`activity (74.9 pmol). Thus the fraction of oxidized krill PC
`may also contain some other PAF -like phospholipids that
`were not identified in our GC-MS analysis. The PAF-like
`activity of 1-0-hexadecyl-2-butyryl-GPC formed by oxida(cid:173)
`tion of sea urchin egg PC is equivalent to 4.0 pmol of
`16: O-PAF (Table V). The remaining activity would be
`mainly due to 1-0-octadecyl-2-butyryl-GPC.
`In conclusion, we demonstrated
`the formation of
`PAF-like phospholipids during peroxidation of PCs from
`different foodstuffs. The PCs derived from foodstuffs that
`have high contents of both alkylacyl-GPC and DHA, such
`as krill PC, were sources of phospholipids with potent
`PAF-like activities. However, the occurrence of PAF-like
`lipids in some stored foods is still speculative and requires
`further investigation.
`
`Acknowledgments. We thank Masaoki Toujima for technical assistance
`and Eiji Yamashita, Itano Refrigerated Food Co., for supplying a lipid
`extract of krill. This study was supported in part by a Grant-in-Aid for
`Scientific Research on a Priority Area from the Ministry of Education,
`Science, and Culture of Japan.
`
`References
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