Fmm C6133 991»-«E750
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`Order IV“ D 5085315090418-4514
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`Tue Nov 16 13:52:30 2004
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`Page 2 of 10
`NOV 16 ‘‘EM EENBPM
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`M0
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`1»43
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`ARTlCLE
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`Molacular Species Composition of the Major Diacyi
`G-lycemphospholipids from Muscle, Livm. Retina
`and Brain of Cad 0(Gadus momma)
`
`ILV, Bait" and LII. Dick
`NERC UM ofi Akuuath: Elmhaamlsfrrg, Sarto! of Natuml Sciancet. aw-may 0' 5‘fir%g. Sflrllng FKQ ALA. Scofkzsnd
`
`This mnlmular sptzarim mmpmifion M pfuamhnlidylchulim
`{PC}, phmapfiatidylaizhunu amine (PE}~nJ1:A!ll phoaphntidyi»
`Marine (1335 fmm white muscle, liver, Retina and brain of
`cod {fiadus morhzm} were dwermiued by high-
`perfatmance liquid chm Lagmphy 43! the rape-ctive
`lflwdlintzyliglyceml 3,5-dicnitrobauzayl darivatinream 1%.
`minimum of 39 tlilmyl Bxmcfies wan idsumtified. In muacie
`and livsar mmuamd fmuay mufidmokyunutumtad fatty acid
`CPUFA3 and mm ounmtnmbxd {stay a¢:iui£N.TF‘A mulmulusr
`special amm predominant, pnrticuluiy lE:DI2fl:5 and
`15:0»‘22:& in PC, 1a:nm:a and lB:1!22:>fi in l”Eand 18:flP23:fi
`1 d lB:1$fl:6 in PS. Didncmnhexaannyl spades ware ma»
`jar mmpunenta uf PC, PE and PS fmm retina. mmprhr
`ing 25$. ‘$1.5 and 503% at the tespcvztiva tubal». I3-iv
`domamhmzwmoyl xerprmiu ware aim abundant in PE mud
`PS fmm
`ammming {M 13.8 a.nd24.£}‘}€a of tha twtnh.
`mipwtiveiy. Di!”-‘UFA amenities were important. in muacle.
`tutaiiing 21.2% in PC and 38.3% in PE. PC Inn
`81?.
`tissues bad the lsu-gasi: amaunts of spades mntainlng
`only saturated or monounsaturated fatty nddm. ac.-
`munting for 59.3% :1! PG from brain, imziuding 12.8% at
`1&:u24:.1 plan z4:1us;1.
`Lipids 3'6‘. fiES5~E73 (19911.
`
`The main mtructurnl giycaruphoahalipida {mm vertebrate
`naural tissue contain Inga amounts of émuaahmcnamic
`acid HJHA; 22:61:-3). Pulyunaacurntad fatty acidn {PUFA§
`am very umacioualy mtaimfi by nemni tiwua £1}. and We
`«:1: thaw gtaussmtimna wt’
`cicmrlvaizmn. am raquimti
`to reduce aligmfiawmy the ma PLIFA cement. mi brain in
`ran (23 and brain and retina. at rhesua man bays 3]. Dephr
`than »of 15-0 PUFA in brain and ratirm results in Iwucw
`visual and cagnitiwa abilities in thaws‘ fiankeya 14%.
`Howmmr, the pm;-ise mie cf 22;6n~3 in
`minus in
`unknmmza.
`Early atmmpts to mpmmm phuaphnh.ipid1u mm bavine
`md uutasr Mgmentau mflfli mmrding to the ciagwu of am
`saturation. by adsorption thin-layat chmmabography
`{TLC} {E}, and later by nrgentation TLC 1%}, showed the
`pmsance uf fraction» cnmzmnlng mare than six dmhle
`bands par nwlecule.
`analyses of the: fatty Adda
`i.nFl;§msd PE‘: frummagflflflafmawmi £2:*6uw3 mbemkamh
`the s:n~;1 and mwfl jgumitimm of the gkyvonridm and it was
`dedumd that; diPUf-‘A apaaciaa ware pzwanté (71. The
`
`l"!‘o when wrwapandmm nhwid be addrmaa-d.
`Abhfflvifiunnfl EHT. humyrhuuadi twrimxymluaxw; GLC, ga.u»—Iiqu§d
`cfuflrmmwwhyz H PIJC. high;-:me.r"f¢x11ranz:acvaTtiu1;uv’s¢:¥ dwarvruam aptw;
`HFRR. mfiuwpmfmmmm £h£m:~Mym armramamawphy; MU -A. mu-
`nuunautuzaud Mfauy
`amid: m. phmphuidylchulina: PE,
`phmzphatitiylethanalaminzz; Pl. phanphutidylin-Mimi: PS,
`phosphatifiylsariua; PU!-‘A. paigmnsamratad fatty acid; ROS. rod
`cuter aegmmt: SI-‘A, satuwtad fatty Mid: TLC.
`thimluyer
`chmmnmgmphy; VLCPUFA, wary long chain ?U‘F‘A. Molmdu
`npmgévas am abbrnviawd M foflawo: o.g.. m:0.‘32:E» PC Ea
`1-;m1rni¢a:~y§~f2-aliovlzimflhuxatmn-mmv1«$w«gIymn'ar3-ph¢3agah9nhulinn.
`
`daevalcmmmt. at metzhodxs far analyzing ma molecular
`spacing mmmmition of giyamnphoaptmlipida by high
`perfarmanw liquid ch:-amnmgmphy (HPLE3 directly {8}
`or by ‘prepasing derivvatiws (9-12}. or by gnrliquid
`CHIOIHWDO
`hy (GL8) 01 wlatile derivaziwa (13) has
`revmalad the
`fly camplnxity at" the lipid wmp-auants of
`memhrana muctmw. and mm techniquea have new haem
`applimi
`to mummy ayuwmas.
`‘I'M pmmma atif MFUFA
`mulmulm speaciea in mg twins new wnfirmml by din-em
`analyaia 114;. while aualysw af glyeemphnapholipida mm
`the brain and mtina of tmut ahowed large amounts of
`di22:5n~3 arpmizea, especi
`3: in; phnsphatidyiahaline {PG}.
`phasphm.ifi3.rEatl1ano1n:mina{'PE} and phnaphatidylaerine
`(PSI Emm mishw am! i1:PE and P3 fmm brain um“ Smafl
`«mmuum of MMJFA agmzim «Jim haw been fsmmzd in new
`mural tiwuaw including rm; {11}, mt mm (16),
`fish muncla {W and and ma fim). Marine flak: we pmticsr
`Karly dab in ma I-‘UFA (ISL and this papa: presents
`malamlar speden analyses cmf PG. PH and PS cf foul:
`tisaues oi and {Gadus morima}. The resuka: from two
`neural timum. brain anal mama. are mmpaxwi with thaw
`{mun liwsw ma muaclu.
`
`MATERIALS mm METHODS
`
`Cod (Gadus ms-whua} {$00-$00 3 weight) wmw obtained
`from the Marine Station (Miilport an the I.-‘§r’€h of Clyde.
`Sfmmtlandb. mainmimzud in u aaarwamr nquiurium at 10-14°C
`cm a dim: of aimppuad «quid am! used within Wu Mmke uf
`cnpzum. ¥"iah mm klllad by daxmpitacinm. and tha eym.
`liver and fillfl at muscle rammed and frozen at --'!0“'G
`until required. Brain was rammed and uaeadimxnsdiahely.
`Phnsphofipase C from Eamiflus cenms was obtained
`hum Boehringer Carpal-atian Euundan) Ltd. imwm. East
`Suamax. Engiwdl. Butylmzexi hydrmymluexw £BH’I‘} was
`fmm Sigma fl Cu mania. Uaraet. Englmdl. 3.fi~
`mnit v-ob-anmwszhlwide my {mum Mdxich C"b:mu‘.ca1 Ca
`{fiminghum flmaet, Enghmd} and mm rs-cnmtallized
`from carhm tetrachloride haiure uaa Standazdi lipids were
`obtaifie-ti fmm Sigma and {mm Nuflhek Pray (Elysian,
`MN}. as dataiied in I15)‘ TLC and high»-petfarmanoe thin-
`laymr chrumawgrap
`(Hl§"1"}Le(33
`ma wam mated with
`milieu gel sat: (Mack.
`axmnmddz-. ormamm Analar mda
`glacial mania mid, carlxcm gatréwchlczride. pmgan~2~n1 and
`pyridine mm purchased from BI)!-I Ltd. {Paula Do:-net...
`Engnncu. Aiintl-nu anlwntau£HPI.£'g!'adavmehnm
`Ra Mm fwhllhrh
`Baeblmshke. Scotland),
`Ultruphem E335 and Ultrwspham uttyi HVPLC oalurnma
`(26 K 0.46 cm. 5 nmicmn particle aim! wem obtained fmm
`A1mNBockmsm(I:'m:knxm Immmmta UK. Mat. High
`Wgmombes. Hunks. lilnglandg.
`uxtmction and purifimttan ufiiptda. All s9¥vmt.s. apart.
`{mm those used for TLC and HFLC. mnmbwd 0.01% (WM
`B!-IT. Sampiaw more stored at -2fi"C undar mflzmgan be
`Human ptepamfiiva
`Tim brvainaa Wm tukan from 20 and. cm mamfingea 112-
`
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`Frnm (513) 991-4760
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`Carder # 0S0?B$3lfiUP04184514
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`Tue him: 15 13:52:30 2004
`
`Page 3 nf 10
`NEW 1% '%4
`32:49PM
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`slash
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`MN. BELL AMI} LR. DICK
`
`mcwad and tha mmninimg tiwtw (8.3 g9 hamnganimd in
`10 voiumm «emf nmamtannfamthanol {$321, vim using at
`Pnlytzron timuaa dismptw. The mtinaa {mm 4%?! frozen and
`eyes (5.? g mane} and 20 chapped fimen liverfl £89 5] mm
`such homog,'esruiz.ed in 10 vniumes of ch1m'afn:m:Tmethano1
`{2:1. HM, and finely shipped (mum muacie ffllats {211 g}
`fmmé 20 fiah warm homumenizaed in 5 vulumea of anlwsnm.
`Aim: homngmaixation mm Jflpid was aamrmmd by the
`mathod of Falah at all 09;.
`Neutral lipid was firs: mmmmd mm the brain and liver
`extracts by ysrevparative "LC in haxanefdiethari etheriacetic
`acid t'I0:30:1, vfvafv}. Jhmphalipida were fluted from the
`origin with cihlemfannlmathamolfwatex (5:53, WMVL driéd
`by minty mmparatia n undvar vacuum at. am‘: and finafly
`undar an Mmam cf nitmgmz. Phuzsphmfipid glass»; mam
`aapammd by TLC in chinrnfumumnthannlfwnmftria
`ethylamina i30;3fi:6:35, by V03} 1120} and the Hprids detected
`under UV light after awaying with (L1% (WIV)
`2:'.T'~d1ch!nmfl‘moreacein in methanoi containing 0.01%
`{WW} BET. Phéoaphnflpida mm alutnd {mm we Silica gal
`with threw *&G~mL wrmhm Q! chlitnm armhnufltnmauwamr
`eszhrzt, «NM and dried ma imam 2','3'+I)inh1crmfhmrwcein
`was remnwti from lipids by emtracuon with a salution of
`2% (wfw Pi}-§C'D,,. PC and PS were further purified by
`‘TLC in chiamformfacemnefnrmthanoflauemic acidfwvator
`t10:4:2;2:L by ml} [2L), and tietectaad and mzztmcwd as;
`warm M pahamxpholipida ww finally chm.-had Em‘ purity
`by HFTLC inn. memhyl mamwpmpmwzmiimmmuionni
`m.e:hanoU0.E§% {WW} KC} ifi5:25:25:l0tfi.. by ml} £232}.
`Lipids ware date-cmd by swaying with 3% {WM copper
`acelzane. in 3% {WW} phosphorht acid and charring at 16D"C
`for 15 min.
`Fatty acid mmwl waters Wm prepared by mwrificntiun
`in 2 ml, af 1% QWI mnmntmmd mglfuxir: Mid Sn 0 manna
`at 50°13 Eur 3:5» hr under
`The fatty Mid com11osi~
`‘Lion of the phaaphalipids was determined by GLC of fat:
`Ly acid mathyi when in a Packard 4336 chmmamgraph fit"
`ted with a GP Wu 5208 fusmd aflicn cnpilkry mhuznn {E0
`in K 0.32 mm Ld.) (Chmmpamk UK.
`Landau,
`England? W3}. and with a Cl? $0503 mm siiimu capillaxy
`momma W3 )6;
`(3.34 mm i..d.} Qfllnromxzrsmké ma). wing
`hydmgen as carrier gas.
`Prepamntiam af 3.5-dinitmbenzayl dedmtiwa. O e-mg
`portions of phosphnlripid were hydraijmad with
`phovsphnhipam C uaing an we phase system {:1 1 mL dinthyi
`ether and 1 ml; 0.1M mdium bnrate buifaza Mi 7.5. M:
`mam mmpatamw undar
`far 6» 131: :25}, M max: and
`nf the imnbatima 1.2~dim':yIg%y¢-Bmln were eumracted and
`purified by TLC in haxaneafiriiethyl e*thm-mcetic acid
`¢60:50:1,
`vfxrs’-A E17}. Mklkamyl-2-ucylglycamla and
`1~n1kyI—$~acyigh«wro1s wen sseparabe-d at this atnga
`Amtruwzs oi pmmphalipid mmafining after pmzmholigsaae
`£21“ cugmmion mm lam thm 2% at the: awning mamm.
`mm dafivutimé in my
`idlm with
`1.9 ,. -. _mb=a;w:m1 ahlmtda an: we hr 15 min under
`nitrogen ‘estimated and washefl‘ as described by Thkamura
`fit all
`({2}, Tim pu-ity of the product was checked by
`HPTLC in hemnafdiehhyi &thaz<m*acaLie swirl t'i'£3r:3(}:1, wm.
`Sepamrion ca,‘ molecular aymim. The L.2—dim:yl'3~dini~
`tmbmzoylvsaeglywmla Wm zmpamtd by HPLC at
`19-21% am rsveraa phase miunrms using a fire Unicam
`‘H310 pump (Pm Unicam L£d.., flambridgaa, England} and
`three iaa-xzratic eoivant syafexrm An 0133 calmmn was
`mad with mnthmollpmpan-2&1 195:5. VM. flaw rate 1.0
`
`mlxmm: and mm£nmiLrilMpmpnn»2~ol (80:20. W}. flaw raw
`M) mhlmin £32};
`a G8 rzeiumn was: mama with
`methmavwntexfwmmnihrfla tfiflzéafl. VMVJ, flaw mm 1.2
`mlfmln (173. Paaka wan: detectmd at 254 run with a Pya
`Unicam 4020 datncmr and quantified using a Smmadzu
`C~H£A wcnrding mtegrnmr{Anaa:hen1. Luann, Engiand}.
`Pieaks were identified tram plate of lag“, imlatwe 2-etan~
`tfmn times. am‘; am. the afimtiw carbon numhm on the
`arm poaitimw <3!‘ mm glymride an dmcrib-ed by fiunmn at
`ml {8} and fihkamum at at
`$3.2} using 16:0§£2;fi as a
`reiezmce peak. Dridmoan!1mmana5rEg1ycero!{Nu<Ghak Prep}
`saw also avaiinbia for direct m arisen of nsmniéqzm times.
`We main n-3 PUFA-containing mulacular upwiéas were
`i»dI§n‘t.iTiud dimafiy by GLC mf flaw may «aid mathcfl eumra
`43% while: Twzimflzl plum 2021:1021 and m:ma4:1 plus:
`2«i:1&’1B:1 bad prwimaly ham aymhmimd i 15}. Each sam-
`yla was chromatagraphed three times in each {If the sol-
`wmt. systems and the atandarzi dervlatinna calculated.
`Wham final peak amass mm mlmxlmted by Rnbt-l."&£»“Ei02't. than
`atvwdmd dmiatluna of than c=mmributing peaim warn
`mzidked to giw» aim final anm. fimmta am giwmn hot cum
`dascimaél place fin-r clarity. The log“, {RRT X 1151} at all the
`mwlecular spencim in early solvent syaham are given in the
`appendix.
`
`flkfitfl
`
`in muscle $FMPUFA. «pacing wm mm mm: ahumdmt
`aims. comprising mar 50% of PG and P3 with almost.
`equal arnounts of 1E:IDe‘2D:5 ami 1§:0J’22:6 in PC and mainly
`18:0/22:6 in ?E‘:«‘ {Table 1}. The I out abundant:
`MUFMPUFA apeciaa in each aims and a majm: wmpcr
`new car PE and ?S was lfizlfifitlzfi. {MP1} FA spacsea Wm
`aim abundant
`in PG {21.2%} and PE 038.3%}.
`pmiaxvminantly ‘22;N20:l5 plus $0:fi£‘t22.*~6 in “C and ‘M:fi:‘fl2:6
`in PE. Anothnr nine diPUFA 5;:-emits ware found. Only
`anmll amounts of spacing containing only saturated and
`mmawmatmated. fatty uziwis were prvaaem 9% which
`mmlatl was was xmat ahundemc mmprming fiA%~ at PC.
`The xxaoleaxzmzlasx ape-ties comgasmtiun at PC; FE and BS
`tmm Hm was kwadly aizrxilar ta that of muscle {Table 2}“
`$FfiJPUFA were: izha most. ahunciant wpexziea in PC and PS.
`anti MUFAIPUFA species in PE.
`In sdi
`three
`giycmnphnapholipzixda 161112225 was a major upsniw com»
`prising a quarter «:17 the» PE and i8:1!22:Ei wan abundant;
`in W3 and P5. In “P5. 1S:0f3’2:6 was 4;: mini“! spwim but
`was much mm ubunaant than in H3 fmm mumm; }El:1.~‘2fi:6
`mad l8:¢J!22:6 were equally abunfimc in PS from fiver. In
`PS from liver 2fl:1d‘22:6 was aha & major species Mable
`2%. IzTa'fldiI.inn_ liver contained up to nine diP U1-‘A species
`totaling 13.1% in PE with 22:6-atomaining speciw domi~
`mam.
`wntaining 0:11)? Mtumted and mmwun-
`mzmmwd iamzy mm mm again git‘ mimr iznwmamaza with
`lE:W18:1 mmpzining 3.4% of Pi;
`Iiiiflizflupadut-a:amnstahAandnntinPC. PE.md PS
`fmm retina accounting fur 99.3. ms and 59,-m, zvspec-«
`tiwiy [Table 3}. Qfihar diPUFAfiapeu:iaa ware mt mmm gm.
`pawmm In PC». 13~‘W32‘-E. 16203102] and 15:fift'220:6 warn
`the only ouxmfipemms premmt at meatzer them 5.1%., mm
`aipwtfi Wwtfig 9.0% mm alga pmaomt in PE
`and were probably my long chain PUFA«mntairuIng aw
`
`thaonly M53? $909163 prwaat morntlmn 2%, whilia
`in PS 18:D!2E:fi was agar; nhundam. mum 3:. Species cum-
`
`cam£253.InPE.15:w22:s.xammaand1s;om.-.5at5-6%
`
`Lupzms Vat m. Na a aw;
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`Fmm (613) 991-«2:?‘6fl
`
`0 rziier" a‘ 050353 1fiflF“U41.B45 14
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`Tue Nmr 16 13:52:30 2004
`
`Page 4 0f 1!)
`WV 18 “M WEMQPM
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`Sofia?
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`“Speciem in italics have dmflned and and saw’! fatty acid distributions. -nthewisa it was
`assumed that um mm; mturatad futy new win an the 55-1 pnrsit-on. Duly mm fatty
`acid mmbinutiun in; man far thaw swam with who wqumbr‘ fllmly pmiuanal immers.
`Vflrcucrimmud «Mela: are minim cant}:-tmenta hum‘! an fatty mid mmpaaibion 453% The
`emml am: givm M m 1 nmmmrd deviuum mzmied w an xwamat decimal mm am
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`H:i'!fi 8:1. IB:Eh"20:ll.
`l&:I..n‘18:1. l8:1fE0:1. 24:1.-flkfi, 24:1./22.12 w:u24;1.
`«kw. Nut mamccm
`cm-. «cm ‘ah.
`
`prising a’:.aI2.‘a3r' BEA and JMUFA. tnmflnd just 21% and 5.9%,
`mapactivaelyr. in PE and P3 (mm mtina.
`N3 {mm mm brain shqwasdi the moat; mmydlm ctompami»
`cian of any of the glyczmphmpholjpicla axamlnedg a
`minixnum mi 54 species were found,
`inciuding swan
`unknowns which mtafled 040% 'l'Ih'ni!la 4}. At; least 10
`species mnmining 24:0, 24:1 and 26:1 {any muidn tam}
`
`ling 163% mm found. um must impnruamt M which M18
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`Order # 050B531fiDPD4184S14
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`Tue Nov 16 13:52:30 2004
`
`HOV
`
`Page 6 of 10
`15 ‘B4
`B2=4‘3F'f'1
`
`56‘?
`
`MOLECUAH SPECIES 01- CO1] GLYCEROPHOBPHOLINDS
`
`TABLE 3
`
`Pliimolfibi
`
`PS[mol“.H
`
`Mulacnln‘ Sp-eciu Carnpanllhnn 0! Fhoaphatidylchollne, Phnsphntidgrlathnnolaminu and
`Fhnsphntidylsainc from God Rn-fins‘
`Species
`PCtmo1%1
`d.iFUFA
`20:fil22:6
`323920
`22512246
`Total
`
`0.1 :2 0.1
`29.3 :1: 0.0
`0.2 3 0.0
`00.2
`
`1.7 :2 01
`11.5 :I.: 1 T
`1.0 ‘.1: D 1
`14.6
`
`0.4 ‘.1’: 00
`59.7 :1: 1 4
`0.4 1 0 0
`60.5
`
`SFNPUFA
`II‘i.flr'.'¢$l'.5
`10022.6
`.lfi.'G'20.'4
`.l&CbW.’5
`I.‘.£fl'22'0
`.l8:01‘20."l
`Total
`
`MUFNFUFA
`I8:.l4?0:5
`.l8:lrZ’2‘6
`an-ma-4
`T01-8.1
`
`SFNSFA
`Total
`
`SFAJMUFA
`16:0!13:1
`1B:0J'1B:1; (16:0f2D:1I
`Total
`
`MUFAIMUFA
`18.‘!/18:1,‘ l16:lJ'20:1I
`Total
`
`Unknowns
`U1
`1.12
`
`2.0 i 0.5-
`28.2 :1: 0.1
`1.7 t 0.3
`0.5 :|: 0.1
`9.1 :1: 1.1
`0.5 1': 0.0
`38.5
`
`0.6 1': 0.1
`1.? .3 0.5
`0.2 1: 0-0
`-I-.1
`
`'0.'T :t 0.1
`5.0 It 0.2
`0.0 i 0.0
`0.8 :1: 0.0
`5.0 i 0.1
`1.0 :1: 0.0
`14.4
`
`1 2 1': 0.3
`5 B .1: 0.0
`-9
`
`9.0
`
`0.1 1: 0.0
`1.7 :9: 0.1
`0.4 i 0.0
`1.9 :t 0.5
`18.2 :1: 0.1
`1.6 :1: 0.5
`22.2
`
`0.4 :1: 0.1
`5.9 i 0.4
`1.1 1 0.1
`11.3
`
`0.1‘
`
`tr‘
`
`0.5
`
`13.4 t 0.1
`1.3 3 0.1
`15.3
`
`1.3 3: 0 1
`2.8
`
`5.1. 1'. 0.2
`1.1 1': 0.1
`
`0.3 :5: 0.0
`0.5- .1: 0.0
`0.3
`
`0.3 :1: 0.0
`1.6 .1: 0 2
`3.3
`
`0.6 :1: 0.0
`1.3
`
`2.6- :1: 0.1
`3.1
`
`——
`-—
`
`--
`—
`
`“Species in italics have flflned 55-1 and em? fatty acid diauihutinna, otharwiaa it was
`assumed Lhar. the mast saturated latty acid was on the 311-! position. Only ona fatty
`acid combination is given Iur those apocies with two equdly likely positions] isomers.
`Brackated species are minor compolumcs based on fatty acid composition data. The
`errors are g-ivan as 1: 1 standard deviation rounded to the nearest: decimal place lane
`text). The following molecular ape:-..es were also detected as (0.9% In at least one of
`the glyccrophnspholipids: 20:5r20:& 22512226. 22:5'2‘R‘-'5. I¢.0r22~6, J6.13flL'5. 1&'0’22:5.
`1 8:1 /2 0:4.
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`!8'J.£E2.-5 2(1:L'2.2:6'. 20.-1.225 14:01’16:0. 16:Of18-:0. 18-rflilfltl. 16:1!1B:l. 18:1.’20:1. BO;1.fll:.l.
`18211245.
`5-. Hot. detected.
`7-‘tr. <0.1%.
`
`almllu: in
`13:n(lB:fl. PE: and PE ham hum wan
`ualnnositlnn be those tram muscle. Kw and mm. Dime
`was abundant. in both classes, especially PS {24.[}%},, while
`18:fl!22:Ei.
`lB:1!22:fi and 1B:O!22:fi were the other major
`species in PE, and 18:O!'22:6 and 13:l!22:6 in PS.
`
`mscussmfl
`olvix 3 most of the
`The method used here is capable of
`molecular species pm-ant in glycemp zospholipids fro .
`fish tissues. However. kn-odes containing‘ fatty acids which
`
`an piano: in vary small amounts {(0.5% were not always
`detactad. 13., 16:|n-3 and 18:31:-El. Iaoumm-3 of unaamratad
`fatty acids are not separated. e.g.. n-7 and n-9 MUFA or
`n~3 and n-6 PUFA. Analysis by GLC showed that 22:5 was
`the 11-3 isomer. 22:4 the n-6 iaocnar and 20:4 pmdorninanlly
`the n-6 isomnr: 20:-in-3 was a very minor componanl. in
`most samples. The clunmatogmphlc ajmbama could not
`separate d;iPUF'A species conléalulug diffeuant latny acids
`but. the same total carbon number and double bonds Very
`long chain fatty nc.id-containing species were also difficult
`to resolve. and the mlumn and chmnmtosrinhy candltlozza
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`‘B4
`BE’.=dE9PN
`
`MN‘. BELL Mam .I.R. DICK
`
`TABLE 4
`
`Mumlwuimr 39::-aim Gampmiuwm M Phoagphaiimrlchuliua. Phmghammyhtmmiamime and
`
`Fbuwhamdylacfine {mm Cad Brain“
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`PC4mni%3
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`—-91
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`awumfl that than most uwrctod fatty acid. was an the amt gmicion- Only cm: Fatty
`mid mmbinauion is given fur 3.1105: specks with turn aqwnlljc iikeiy positional immwro.
`Bnmkamd ltpaurinfl we mimr cmnp-nnema based on fatty acid cnmpaniliun data. TM
`arm am given us :9: I wmdard dnvmmn rczumtlnd he use mums: dwrimnl plasma flaw
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`34:15:13 lflalfflfll, Efifiifikl. 18:flR4.:1 + ¢1a:m*2s;1t. 20-.1.i‘flI:i. + mzllfllkl. + Rflflfifl
`34.'fl?fl°5 + 25:l!2G.'£ 24.‘0?2‘2‘.'€ + 25:11'M.'5.
`b‘-, N03’. di:e'tacLezL
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`1
`
`{lime r If 05D3S31fi:|DiP041B-45 14
`
`Tue Nov 1&3 13:52:30 2064
`
`Page 8 of 10
`NW 15 ‘B51 m:2:a'am
`
`5?]
`
`MQLECULAR SPECIES OF CGD KELYCERGPHGEFHOLIPEDE
`
`28:1.*PUFA.
`used mum nut sapsu-am aim 24:fl!PUFA
`Palm fmmfl in PC? from brain flhble at Tim twn mlwmt.
`sysmma uwdé with the C113 mlmsmrs can segmmm mtmmsa
`posinimai mmzrrxera. a.g., mamzo fmm mwzosa but met
`16:0!18:1 from 18:1I16:G. The pmsumption that must
`saturated fatty acids are in-catad at :he aft-l position of
`they glynerai maiety is. they-efare. supported by the results
`inhll casein where the mathod can distinguish positional
`imzrrmm.
`Marina flesh am partiazuimiy rich in M3 F’U¥'”fis. mp-miauy
`dmosuhexmnmln acid (183. Tim present mmflta aalmw am
`very unaammned mtum 0f the moleunisr species of
`glycamphaspholipids item cod. PE was the most un-
`saturnmd glycerophosphnelipma in all axcsept brain.
`while PC was the moat amturamd and mntained the
`shortest ammge chain length. both ‘dun tau :1 lm-mt gum»
`puruicm af '2‘££;fin~3 containing speetiw. PC imam fish brain
`mntaim 3. rrmazh lmgar am-aunt of SF}&- and MUFA~
`containing: species than giycarophosphulipids from other
`fish tifisuflfi Of particular note is the lama amount at
`1B:1f24:1 pins 24:1:*1B:1, which was found in hunt (9! and
`ii: evan mum abundant in and. Simza cad hrain contains
`1.0% mi 22:1 fatty Mid. 2e;im2:1 may he 30 minar comm»
`mint. uf this» peak. fiimail ammmts of other aagamim mm‘.ain«
`1113 very Iamg chain fatty maids (2410. 28:1} were also fmmd
`in and brain PC, raising the question 0f the special nme
`0 of these mnletzular
`spccifis in the atructuza and function
`of hiomemhmnes in this tissue
`
`The incrwaed mphiatsmatinn of nnalytiml techniques
`in mcxam yawn has land to aim-yiincaxr-ary sf 6iԤ?UPA ape-aim
`in an wmmmedly Image number turf tiwum ouch am
`erythmqmm (1110 and mates £16}
`fmm mt. Earflxex
`molecular fig-acies anaiyses of fish muscle iefiiktlflns found
`appreciable amounts of diPUFA species {mainly 226f22:6,
`20:J5."2E;6 and 22:5{22:8i in chum salmon Cfncorhynmfius
`hem.
`‘bigaayefi tuna Pbrazahannua aiming and Alaska
`pallack Tiwmmra ckdlwgmmma. but rum. in mean: Cyprinus
`cmpia 19); am prtamrnz mauim mnfirm nhasm fmdirlgxi. The
`pmaent away also found smuali amounts cf d.i|'E‘UFA
`species in PB, PE‘. and PS fmm cod liver
`F8 from ma
`muscle, whit-aé the Large muaunt of d.i2i’;6 in PE from and
`mumtba im the gent-eat. pmpo:rtian of this naziecular spaciaa
`yet naportaesfi in a nnnmaursl tisauam
`In bmrina mtina Waunasmmated fatty maids mm fuund
`in an Imus‘. 43% of Pfi %% M? PB: and 24% af P123 (6). and
`PE from frag mama mmainaedi about 513% «MPUMFA apecims,
`mainly 22:$n«3f22:6n«~3 Q14}. Tissues from mafine fish mt»
`€9.51} prsduminantly n-3 PIEF;-‘L with very Ettla n-6 PUFA,
`and themfem diPUFA apatite: in fiah mum am almost m~
`birely
`22‘:fi(fi2:Ei.
`TM amnunw of
`dizmfi
`glynzamphaaspholipida fmmxd in cod retina am the Mghmt.
`«mum: in the mcina mf any apmies and! am mahsmntially
`higher than them found in hunt mtina 115?. Hmrmnar, we
`found that PI
`um retina of trout £15} and cod (av;
`does not mmmain dIPUl-‘A species. The maia::u1a.r species
`of PI are amuafly dominated! by 1B;D.’C,n PUFA apeciw
`with 1E;0i2iMn~6 a majm up-wigs in the wtina ai Fmh
`4715.27’) M in tiaauws fimm wmrwtrial anirnala £011.26), but
`1fi»:0!20;5n~$e $9 pmduminmxfi in fish hmira £15,127}. PI is
`thmeiore much more
`aaturatad than other
`glycsmphmpholipids in retina.
`God are visml feed:-"3 with the liver beim g a major dnpat
`for stomga cf neutral lipid, but other atrateguea exist fur
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`term of nanleacuiar apecfmtien of phflflpholipsids in the
`maum mi and dnsvcxibed hem thamiom may not he
`mprwammtiw mi all spams. Matty lnbommry a§:miE.s.m with
`{ah am ather animals ham uhw; that mm? lipid
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`neural tiamma wf essential polyumaturnmd fatty acids in
`mamrnnla I1-31:. Tha pmaaance of large: nnmunta of d.i22:E
`glycamphmpholipidn in Mam mtiuaa at an wiacie range at‘
`animal apmisas suggmcs a. spmziadl rule for them mmeculaa.
`It. has been pmpmed than tram unique lipidzs play a atmw
`mml min by inmramting with rhudapain in the R08 mam-
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`PTJFA in mtim imm a vagina}: at
`apmims found
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`11%}. The latter study also mum extremely high levels of
`22:61:-3 in and retina and sugwted a pmponéexance of
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`we arr? yaaitimx Up .0 a
`Bf PC from hmwirm RG8
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`mg VLCPUFA,
`the unknowns in PC from cod
`brain were dlffvamnt and did nut appear in fit this pattern.
`‘The calm of mrsicular fatty name in hiammnhmww stmr:
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`apecificity indimati g defineé roles for usome of thesis
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