From £613} 352-32-47
`
`Grain r f!‘ D5085 31839041345 5 3
`
`Tue Hm 16 14:05:01 Zmai
`
`HOV 11$
`
`Page 2 mi‘ 8
`’fi~r1
`ECSHBEPM
`
`
`
`0 ARTICLEL
`Lipid Cvamposifiun uf the Pineal Organ from Rainbow Trout
`(0ncwnynchu$ mykiss)
`
`FLJ. Hma.am.n='. my. saw, at nnaun. Sarnartr’ and J. Falcon‘
`*H.ER,C. Uaimf fiuquatia Bindwamiaw Dapanmaant at Ebtogicm and Malawlur saunas, uréwrméty at Sanding, swam Fl-{'9 am. Smaam, Unitad
`Klrlgdorn and ‘Labormoim do Meumbbbgie at da Natmarkiocninoiogfin Gemiahua, EJFLA YZNPJ5 290. Fflcuim dune Sdenoaug Ema: Puma-m
`Godfiflt. Fmnm
`
`
`
`
`
`T1-m llpid «imposition ofihe pineal organ fmln the rain‘
`how tx-out iflncarhy Jkuamuahlasl was date mined in mi»-
`tnhliah whafihmr the immiwmment nf this my-gun in the
`control an!’ mimmilnn rhytitma in railawlad by Ipeuifln
`nuixmtatmns of lipid ammpuuition. Lipid rmmprhud sum
`out than mama mm; weight mm! tmimylulymmlw Mum mm
`Imafior
`fipi/ii class
`remnant
`(f;I£"J‘£a at’ semi lipid). Phat»
`phafldylchoiine (PC) was tho principal pulls: lipid. and
`smaller pampmrtiuns of other phnnphrmpids and cholvaaw
`tern! wens also present. Pmuunxaln-gen: trihulad 11%
`ant’ the wlhxmnlmnminm gbrcemgthwyrhnlipidu CEGPJ. Na
`mmbmaidm mam cluarfiuttam. Thu [am amid mmproumon
`at tfincylgiarcmnlu was ya-Mmlly «similar to that of tarsal
`lipids in which Imturnuad, monounsaturated and
`polynmmmrawed fatty acids {PUFM wane gnmaant in M-
`most equal pmupnrtiuna. Rush of the pale: lipid clnwaa
`had a spnmifluw tam! maid wmpaflfinn. with than amuptinn
`M phwphatiaflyflmrmiuid (P1). In wlxinh amam-am cznmprinad
`21.4% as!’ than mu fatty nfidl. 221:6»-3 wan Wt pflnuml
`PUFA in 931 lipid alums. The prop-at-(inn at’ iB{}:6n-3
`na-var amzeenfled H.036 of She: fatty nnlds in any Hpid dust.
`The pmdominmnt molecular apeclmu 41! PC were
`1fl'.0I23:‘fin—-3 and 1G;I1r'18:l, which ncxaounheafi Mr % and
`”!£8J5%. rmpeemlvely. at the mm Iuealeculsr matinee cf thlu
`phuuphomanm. Phmauhummylafltuu alumina ¢iE‘E) mu»
`mined. than highmt law! can! ¢t.s.:m:am.-:1 { 13%} «(may phm»
`phoIipld.‘T%auam was also «LEVI: and’ this mnieculu specimen
`in phaaphati-dyluflnn {PS} and 4.1% in PE. In PE, the
`species 165133225, 1fl:L¥EE;6 and l8:(u’2Ra8 mtaflcd 45.1%.
`while In PS 18:l1!§‘.2:6 unmounted fur 48.9% of um total
`molecular mwiw. Tim must abundant: mnlecular
`qzn-emima of F1 was 18.13/2fl:a4n«fl €$’f.$%).
`’I‘1:pnz.~ fiwid mmpwwim
`tin» 0! than gmimml urgnm at tmut, and psxrtiuulawly than
`molecular apmias caznpoaitiann ad‘ P1, in man similar to
`the aompuaiiiun of
`vfl rating than that uftha brain.
`phi! 28, 331-317 (19941.
`
`In fiah, an in all vartabxatexa, the retina of the aya and
`the pineal azgtan of the brain are esscnfriai mmpunenm
`of the cirtadian system that measures the period and
`phase 91' the dafly light»-dark cycle and intimately mn-
`tmls rhytlhmiax pmww/as (1.2),. In keeping with this role.
`the pineal Mam affiush mnmina phummmpmr cells that
`display elm mmn~mnnt1mu1 nmlngiu Wm: the phommw
`—.—-r-—-
`
`""b_wImm can-espnundence shouid be addressed.
`Abbreviatiumu; EGF’. ethmnlmfina glyaumphmsaéxalipidu: HTLJC,
`high~parfwrmMma liquifl c'la.mmmmgrW Y: HI-“T‘3»{"3, high~pmfnr~
`mmm: thinularyazsx vczlmromatagrapuhy; 3313. gmhmuplzntfischrlahulinag FE.
`yhmphmmdylwmmnkmina; PG. phmphaudylgiywtw; FL phuw
`phnfidylinmimt PS* phoupimnfltiylurlnng PUFAI, wiyunnuumad
`fatty acids; SM. sphingomyoling ‘EKG.
`I’. "ugrfigiyaamh. Mu!-emalar
`up-azi am ahhmwriatad as 1‘mmwa:~m.g., 16:m’2*.2:6PG iau l~pnl:niloy1-
`3~dmosahm:swnwl~an—g1y:aru-a~phmphmho!ine.
`
`captors at’ the mining (3,4). The plxomrwepmr cells {from
`hath tissues: tramalnm the lighirdark information into an
`neural nutput mf axcinatory neumtrnnamiwr 4(5) and u
`neurohonnmnai csmzput in film mm: cu" melammin (iii In
`acidition tn haimg mulummmngw mus, gm» gahutommpm
`tor calls ofthe pineal cxrgan an aim mumeflecmra and
`can Lranaduce information supplied by athar era
`31
`{Each BE lelnnp-Qrflture)
`am‘
`i! “Eerngl
`icalechalmami mg-5,
`adenc-sine, atamidei factors. Wlmcreaa retina! melatu nm
`was prmfamnfiafly in an auimminalpnrnwine mumwr,
`the melaumin mwzwetecl by the pimwl mntritzusm Iaxpmly
`has the circulamug levela and may be inmlvad in the con»
`ml of seaaonai events, parlitulnrly reproduction, in war-
`mbratea {MES-3)‘.
`'.l"he amacwral phmsphnlipids at’ the zmina am! brain
`turf vartebriltee, including fmh, am krmwn m mmmin high
`lwnalg <3!“ the pa1;mm&a.Luratm:} fatty acid (PTJFAE %2:6n~»3
`E‘£1,!{)}* Within the relzix-a, the E‘E:6n-£3 ia apparently com
`canbrated in the me: brancus nuwr segments of the pha-
`twrece-ptnr rad edit: E11}, and these cells iaaiaéted from
`fmg retina e.xi':ihit a selactive uptake of 22:6n«3 in uitm
`(12). A raquimxnmxt for 22;6n«~3 in the visual pmwaw has
`ham damonmmmd in stadium wiéfli rmwhtam wrimatm
`and prflarm human infanta, whivzzh have ahcswn that the
`visual acuity‘ is afibctcd by deprivation of 2R:$n—3 during
`pmcnnml deveiapment ofth-2 infant (13). Pemxidafion of
`2‘s2;6n~3 is one 3!" the primary events ubaermsfi in inher-
`mad or traummimlly indumd prhomrazasaptar deg&a.rmIa-
`than (9.1%
`Di~—22:6n-3 phmphm1.ipida am known to me major com-
`pncments of rod cuter segment membrarteu in frag and rat
`£15518}. Hanan: mrmlysas of the» phuaphnlipiaiai of the
`bzain and retina at’ trout and and have shown that di-
`fifizfinwfl maiescular apaciets am maxim’ cmnatituenhs of
`phaaphatidjrlfiméine (PC). yhmphaxidyflmzhmmalamirw
`{PE} and phaayhamdylwrina {ml in Lhasa tmnursm and
`that flu’ is; particularly tum of the retina, where the
`amounta are nxmsiderably higher than thaw Found in
`terrestrial mammals E1'i',18-3. 111%‘ ixnportance uf 22;6n-3
`in me vimml pirucstma of fish in also indicated by the ab»
`mwation that ma pmparrticvn at’ d.i~22:6n~3 in the phcww
`phnlipid of the drweluping was» at’ herring iamvae in-
`cmuu with up an the rods use rwmimd intn the phanto-
`receptor popuiation (19). The resmlts of them; nutritional
`atudjes and the: presence mi‘ iame amounm of d.i—22:B
`phumpholipid in the photomcx:-pww ml’ the retina auggexm;
`that 22:fin~3 has an mmntial mm uniqtm role: in the pri~
`wary mmnta amwuxinned with aw abaorptiun and trans-
`ckzctinn of phnmm. This mlghk In of cnmial importance
`fw the phntnperlodic control 6f the producticm at‘ mes-
`sages, such an melatonin.
`
`
`
`warms. ‘mi. :33, m. *5 mm}
`
`AKER877|TC00070477
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`smug 3:; Want mrtvi Jmmm
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`C23-mrrlght 8'31 mm by AGES Pmas
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`L4
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`000001
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`

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`From (5133 952-3247
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`Order ii 051385310090-$184553
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`Tue Nov 15 14:05:01 2004
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`Page 3 of 8
`NOV 16 'B4
`B3=Z2F'l"1
`
`.':!12
`
`‘R.J. HENDERSON ETAL
`
`y analogy with the retina, it is probable that 22:61‘:-3
`is a major PU “A of the lipids in the photoreceptor calla
`of the fish pineal organ. To examine this hypothesis, we
`determined ir the present study the detailed lipid
`our position of the trout pineal with particular attention
`to the FUFA content and molecular species of phosphd
`lipida. As far as we are aware, the detailed lipid compo-
`aidun of the pineal organ from fish has not been re-
`ported previously and only a limited amount of informa-
`tion is available on the lipids of the organ from
`r arnmala (20,21). Information gained from the analysis
`of the trout pineal organ is of basic impnr once as mam-
`malian pinealocytes are accepted as being phylog'erseti-
`cally derived from the flab pineal photoreceptor cells (2).
`
`lllA'l'EFi‘.|Al.S AND M 11-IDES
`
`Fish and pineal organs. Rainbow Lmut (Onoorhyncliua
`myliiaa) of average weight 500 g were obtained from a
`com nercial fish farm (Piaciculture Ballet, Angouleme.
`France} where they had been maintained under natural
`conditions of water temperature and photoperiod. One
`hundred fish were killed by decapitation. Pineal organs
`were removed immediately from the fish, {moon in liquid
`nitrogen and stored at -8013 until taken for analynia.
`Chemicals and solvents. Phospholipaae C from Endl-
`lus cercus was purchased from ‘Hoehringer Corporation
`fLordon) Ltd. (Lewes, East Sussex, England). Oxalyl
`chlo do and anthracene 9—carboxylic acid were supplied
`by Aldrich Chemical Co. {Gilli ig am, Dorset, England).
`All other chemicals and biochemicalo were purchased
`from Sigma (Poole, Dorset, Eng and], ard solvents of
`high.—porfor'rnance liquid chromatography (HPLC) grade
`were obtained from Rathbuni. Chemicals (Walkerbum,
`Peebleeatire, Smtlandl.
`the
`Lipid’ exiraction and analysis. After thawing,
`pineal organs were weighed and transferred to a canon.
`in-gluaa homogenizar. The organs were homogenized in
`3'3’ mL of chloroforxnlrncthanol (2:1, volifvol) essentially
`as described by Christie (22) to extract lipids. Solvent
`was removed under a. stream of nitrogen and the result»-
`ing lipid extract‘. desiccated overnight under vacuum in a
`preweighcd tube. The tube and contents were then
`reweighed to obtain the weight of the lipid extract. which
`was redissolved in chloroform/methanol (2:1, vol.-"voll
`and stored under an atmosphere of nitrogen at ~7{l'C be-
`tween aualyaea.
`To establish the lipid claaa composition, aliquots of
`lipid extract were subjected to high-performance thin-
`layer chromatography {V
`I-’TLCl alongside authentic
`standard! uliufl halunehliathyl ethudglacial acetic acid
`cannon. by will u the developing solvent for the an
`-
`ration of neutral lipid clauses and methyl acat-ateJpro-
`pa -2-olfchlorofonnfmethanoll'0.25% (whfvoll aq. KC-l
`(25:25:25:l0:9, by vol) for the separation of polar lipids.
`To confirm which polar li id classes were present,
`aliquots of total lipid were also subjected to two-dimcn
`aioual I-lP'l'L«C. The polar lipid developing solvent aya-
`tern described previously was used for development in
`the firat dimersion and chlorcforrnlacetonefmethanolr’
`acetic acidfwater (10:-1 :2:2:1, by vol} was employed for
`
`development in the second dimension. Developed chro-
`matograma were visualized with copper acetate in phoe-
`phoric acid (23). Lipid class composition was quanti-
`tatad by double-development. HPTLC coupled with scan-
`ning denaitometry, as described elsewhere (24).
`Ea ' rates of the relative amounts of the plaamologen
`arid the diacyl for
`a of eLha.uola:mi,ne glycorophc-apha-
`lipids 036?) were obtained y acid hydrolysis of Lho ion-
`lated EGP in em: on an T
`plate followed by chro-
`matography and quantitative phosphate staining as de-
`scribed by Bell and Dick (25).
`For the analysis of fatty acid composition, individual
`lipid classes were separated by two-dimensional TLC on
`20 x 20 can glass plates coated with silica gel G 60 (0.25
`mm Lhickl using the solvent systems described here. The
`aeparabad classes were visualized by spraying the chro-
`matogram with 0.1% 2','l'—dicl1lorofluoreacein in metha-
`nol containing 0.01% butylated hydroxytoluere and by
`viewing in der ultraviolet light. Tr-lacylglycorolo (TAGa)
`were ‘pill ‘find by redeveloping the chromatograro in the
`reverse direction of the second development using
`hexonefdiothyl etherlglacial acetic acid (8(J:2D:2, by vol)
`after removal of the individual polar lipid claaaea. The
`fatty acids of the separated lipid classes were converted
`to their methyl -;-rater-a on the adsorbent by acid-cat-
`alyzed trarsaaterification (223. An aliquot of total lipid
`was also aubjecbad to the aame procedure. The re culling
`fatty acid methyl esters: were purified by HPTLC and re-
`covered from the adsorbent with haxaneldiethyl other
`i1:1. volfvol).
`Fatty acid methyl esters were analyzed on a Packard
`439 gas chromatograph equipped with a fused ailica cap-
`illary column (50 In >6 0-22 mm i.d.} coated with FFAP
`phaae (S.G.E.. Milton Keynes, United Kingdom]. Saar
`ple applicatio was by un—column injection, and hydro-
`gen was used as the
`"en gas. During the course of an
`analysis, the oven temperature waa programmed to in-
`crease from 50 to 225'C. Samples were also analyzed
`using an Omogawax 260 fused silica column (30 Ill x
`0.25 in
`i.d.. Bupelchem UK. Ltd.. Essex. United King-
`domlwith the oven temperature programmed from 50 to
`260°C. Fatty acid components were identified by refer-
`ence to a wall-characterized fish oil fatty acid mixture,
`and the unsaturated nature of components was con-
`firmed by re-—aralyaia of samples after catalytic hydro-
`genation over H02. The separated components were
`quantitatad using a recording integrator linked to
`e
`chmmatograph.
`Analysis ofmolecular species. A 500-313 portion oftotal
`lipid was separated into the component pboapholipids
`by HPTLC alongside 20 pg of a cod retina total lipid
`standard using methyl
`acetatolpropan-2-olfcbloro—
`fo
`’ethanolA'J.25‘iB(\vtfvn1laq. KCI (25:25:25:1D:9, by
`vol) as the developing aolvent. The standard spots and
`the edge of the bands of pineal organ lipids were visual-
`ized by exposure to iodine vapor, and the bands of ad-
`aorharit. containing PC, PS, phoaphatidylinoaitol (PI)
`and EGP, were scraped from the plate. The phospho-
`lipida were hydrolyzed on the silica with phoapholipaae
`C using a two-phase system of 1 mL diethyl ether and
`1mL ol'0.1 M sodium horate buffer, pH 7.5 at room text»
`
`IJPIDS, Vol. 29, no. 5 (19941
`
`000002
`
`AKER877|TC00070478
`
`000002
`
`

`
`firon (613) 952-3247
`
`Order # 05085310DP04184553
`
`Tue Nov 16 14:05:01 2004
`
`Page 4 of 8
`NOV 16 '04 03=B2F"l"l
`
`313
`
`lJ.PlD COMPOSITION OF TROUT PINEAL ORGAN
`
`
`
`by EPG (9.9%). No choline plaomalogono were detected,
`whereas othanolaxrjna plaamalogeno accounted for 11%
`of the total EPG fraction. PI and PS each nocounted for
`less than 5% of the total lipid, and both phoaphat.idyl—
`glycerol (PG) and apingon1yolin{SM) were present at less
`than 2%. No cardiolipin or cerebrnsidaa were detected.
`Palrnitic acid (18:03 comprised 233% of the fatty acids
`in the total lipids and was the most abundant fatty acid
`in the pineal organ (Table 2]. The rromounsaturnted
`1B:1n—9 and polyunsaturated 22:61:-3 aocounted for 17.6
`and 12.4%, respectively. of the total fatty acids, Overall,
`saturated, monounsaturated and polyumnturated fatty
`acids accounted for similar proportions of the total lipid
`fatty acids. The fatty acid oompoaition ol'TAGs (Tlsibla 2)
`was generally similar to that of the total lipid although
`the proportion of llB:2n—6 {l2.3%} was notably higher,
`and that of16:0 lower, than in total lipids.
`-
`
`TABLE 2
`
`Fatty Acid Gornpoaifton {writ} of Total Lzlpld and Lipid
`Classes from 'l‘ruuI. Piunal Organ“
`Ar.-yl chain
`"mm: lipid
`14:0
`3.9
`15:0
`0.9
`‘l.6:0DM.A
`-—
`16:0
`33.?
`10:1n«9
`""
`16:1n-T
`5.3
`17:0
`0.6
`18:ODMA
`—-
`18j.1n—llDMZA.
`-
`Ill: ln—'¢"DMA
`—
`18:0
`7.2
`18-:1n—9
`17.6
`18: 1n-7
`4.0
`lB:2n—6
`9.3
`18:31‘!-3
`L2
`20:1:-v-9
`2.5
`20:2-n-6
`0.6
`2D:3n—l:l
`0.4
`20:-In—8
`1.6
`20;3o»3
`-—
`20:-£n—3
`0.9
`2015:‘:-3
`3.5
`22:1n-'11
`1.1’
`22:31:-0
`0.3
`32:4n-:6
`0. l
`22:.'5n~6
`—
`22;5n-5
`1. 2
`22;Bn—8
`12.4
`21: 111-9
`0.7
`Unidtnflfiad
`0.9
`
`TAG
`3.5
`0.3
`--
`17.4
`""'
`3.0
`0.4-
`--
`—
`——~
`4.2
`18.5
`3.7
`12.3
`2.0
`3.1
`1.2
`0.2
`0.6
`0.2
`1.4
`3 .9
`2.0
`0.3
`0.3
`0.2
`1.5
`12.6
`0.1
`1.4
`
`PC
`0.8
`0.3
`-—
`34.0‘
`0.5
`1.3
`0.3
`-—
`—
`—
`5.4
`18.3
`2.4
`1-8
`—-
`0.4:
`0.4
`0.3
`2.0
`-—
`0.3
`4.6
`—
`—
`--
`0.2
`1.3
`24.6
`-—
`0.9
`
`EGP
`0.2
`0.9
`0.6
`10.7
`-—
`0.?
`0.3
`0.-I
`2.2
`0.?
`10.2
`10.3
`3.9
`3.4
`0.3
`1.4
`0.9
`0.4
`7.2
`—
`0.6
`4.3
`—-
`—
`--
`0.4
`2.2
`36.0
`0.4
`0.?
`
`.
`
`36.3
`32.6
`81.2
`.lE-1.2
`12.0
`
`Total saturated
`ToLal monounsaturated
`Total ‘
`"A
`'I‘ot.a| n-8
`Total 11-6
`
`3.57
`6.55
`1.44
`1.60
`____
`n~3!'n_—§_
`‘EGP, othsnolamino glyocrnphoopholipida; PC, phoophalidylv
`choline: TAG, h-iacylglyuorols; FUFA, polyunsaturated fatty acids;
`DMA. dimethylaoetals.
`
`25.8
`35.’!
`37.!
`21.9
`15.2
`
`40.6
`23.0
`35.5
`30.8
`4.’!
`
`23. 3
`19.6
`56.2
`43.9
`12.3
`
`I
`
`:..'pe1"al:|.|l.'e under nitrogen for 2 h (26). At the end of the
`incubation period, 1,2—dirndylgiyoerols were extracted,
`‘-dried down under nitrogen and finally desiccated under
`1-“nun; for 1 h. 9~A.'l1'l2l'll‘D}'l chlofde was prepared from
`9-anthraoene corboxylio acid and oxalyl chloride as de—
`'scribed by G-otao at al. (27). The diradylglyoerola were de—
`' rivatized and purified by a modification of the method of
`‘1‘al-:m:n..ra and Kira (28) as described elsewhere (19).
`' The 1’-O-alk-.1’-e.nyl—2-acyl derivatives were removed
`during the final HPTLG purification step. The 1,2—din-
`cyl-3-anthrooylim.-glyoerols were separated by HPLC at
`19-—20'C on an ODS column (25 :4 0.46 cm. 5 pm particle
`size; Beclunan Instrnmenla U.K. Ltd... High Wyoombe,
`Bunkinghar shire, United Kingdom) using a Pye Uni-
`cam FU4010 pump (P3.-o Unicorn Ltd... Cambridge,
`England} and two isocra ‘c solvent systems, methanol?
`pr-opan-2-ol E-hi, vollrol) at a flow rate of 1.0 mlfmin,
`and acetonilrila/propan-2-ol (7:13, volfml) at n flow rate
`of 1.0 mL»‘min as described by 'I‘akam.ira and Kim {28}.
`Peaks were detected using a Waters 470 scanning fluo-
`rescence dotector {Milfiporo UK Ltd., Edinburgh,.Soot-
`land} with excitation and emission wavelengths of 330
`and 460 nm, raapeotively, and quantified uaing a Shi-
`madzu CRSA recording integrator [Anach.em, Lumn.
`United Kingdor-I}. Poaka were identified by their mela-
`tive retention time using 16:0f22:6n-3 as a reference
`peak. l}i—docosahexaenoylglycerol (di-22:6n~3; Nu-Che.k-
`. Prep, Elyaiari, MN} was also available for direct. com-
`parison of retention times, as were a range of samples of
`known co"nposii.ion from previous studios (17,1B,29J.
`Each sample was nhro om-graphed three times in each
`solvent system and the standard dmiationa calculated.
`Whore final peak areas won: calculated by subtraction,
`the standard deviationn of the contributing peaks were
`added to give the final error.
`
`RESULTS
`
`The lipid content and lipid class composition of trout
`pineal organ are presented in Table 1. Around 4.9% of
`the wet weight of the pineal orgar was lipid. of which al-
`“nost half (47%) was in the fun of 'i'.AG. PC was the
`major polar lipid present (16.5% of total lipid), followed
`
`TABLE 1
`
`Lipids of Thou! Pineal Organ: Lipid Clan Golnpoaitlnn"
`
`‘ii: Thin] lipid
`3.8 t 0.3
`47.0 n 2.1
`3.4 :2 0.8
`5 7 1 0.4
`l 5 1: 0.1
`9.9 3: 0.6
`1.4 1 0.1
`3 2 3: 03
`3.-I : 0.3
`16.5 1 0.8
`
`
`
`H‘ ‘
`
`Lipid clams
`Chnlzdteryl mm
`TH:
`ioseola
`Free ntty acids
`Cholesterol
`Diacylglyoarnla
`Ethmolamino glycorophuopholiplda
`Phnaphotidylglyoaml
`Phosphatidylinooitol
`Phoaphatidylsarine
`Plmaphatidylcholine
`Sfliinggmyelink
`"Vn1uun am moans 2 SD of thin: dotarminntioam.
`
`
`
`000003
`
`UF'lDS. Val. 25, no. 5 (1594)
`
`AKER877|TC00070479
`
`000003
`
`

`
`r-‘mm (5133; 952424;?
`
`flrflar ii D5CI853l90P041B45S3
`
`Tue Nmr 16 14:05:01 2004
`
`NOV 1E:
`
`Page 5 of 8
`‘W3
`9330RPM
`
`314
`
`TABIJE5
`
`RJ- HENDERSKDN ET AL.
`
`Fatty Mimi Cmnxpmitiqn {cw-1%§‘ M Ligsid
`Pineal flwgm“
`
`mm Twuc
`
`Acyl chait:
`14:0
`15:0
`16:0
`1fi:1n~9
`)‘-.6:ln~'7
`1750
`18:0
`18:1n-9
`lB:1n-7
`lB:2n~6
`18:3r!-3
`2o.~1w
`WIIZED-6
`2101331-6
`20:“-6
`2.D;3n-3
`Eflrdn-3
`§20:!En»3
`flS::Ln«3.1
`23: 1114!
`22:4n~S
`22:5n—fi
`?.2;5u~3
`fl:6n~8
`24:0
`34:1n«9
`Unidwntifla-£3
`
`Tbial uturafiaci
`‘Thu! mna.mm$a£,u.r:ted
`Thu! PU!-‘A
`‘Tami n~3
`Total WE
`u»a.vn~a
`
`P1
`0.3
`01
`31.0
`--—~
`--
`$11.?
`33.8
`5.3
`‘L3
`1.5
`G. I
`{W
`133
`0.3
`2?.4
`-M
`G, I
`33?
`W
`---
`0.5
`-—v
`0.43
`7.0
`W
`03¢
`(SW
`
`«£33
`‘$27.2
`-$3.0
`1.33
`20.9
`£3 M
`
`P5
`0.3
`0,7
`3.0
`w
`9.1!
`'14
`38.2
`5.5
`2.0
`IA
`(N
`1.2
`0.4
`—-
`1.0
`--
`--
`L3
`-~=
`-
`0.3
`0.4
`3.0
`29.4
`-—-
`»-»
`0.0
`
`P40
`0.8
`0.3
`K033’
`W
`11.10
`0.5
`5.42
`I 5.0
`302
`3.8
`M
`1.4
`L1
`M»
`1.8
`--
`005
`L6
`-M
`-—
`0.9
`--
`UK?
`!&8
`W
`-~
`1.1
`
`SM
`0.46
`0.4
`19.2
`«-
`43.5
`0.6
`9.5
`3.9
`1.9
`23
`w
`MI
`L0
`1.0
`0.5
`-—
`0.2
`03
`-W
`0.6
`~-
`-
`0.2
`19
`0.3
`45.9
`1. 3
`
`31.8
`1'???
`42.5
`58.?
`»&=i.1
`15.5
`33.?
`81.1
`3-5.2
`4.2
`33.5
`34.1
`11.7
`3&6
`dd.
`
`
`LT36.3?! am
`
`‘H3, phmphatiwaliylglyueroig PI, pfimphuhidxlinmitifl; F5. phat»
`phal.id,r}se:rirw; SM. sphiugazwmzmiim PUFA, Vobrunaaturated fatty
`acids.
`
`in manounamzumt-ed fatty acids and PUFA which mm-
`priaad 44.1 anti 37.1%, rwwmzfivaly. uf thaw mm fatty
`aamzzids in thin phmpholipid Wabim 3}“ The levela ml“ 18: 111»?
`mui 18:11::-":‘(1’?.fl and 10.2%} in PC} wane: highm” than in
`my other lipid (inns, and, of
`the phnphalipids PG
`had the highest mutant cnf Iarfln-6. In SM mature than
`half (58.2%) at the fatty mzida. were mnnmrmnturawd,
`mainly dam tn the pmaemm at!‘ an wary Imigh ymporhion of
`24:1 (4.fi,‘3%r}. FUFA mmprimti uvnly B:$“%~ ml“ the éflltvi fatty
`Mida (Thbia 3}.
`In all
`lipid classes, 20:5n-3 was present. in small
`amounts am! nmter azcaadaaci 6.0% of that mmp-cznenl
`fatty acids, The long chain mazxoennic fatty acid
`22:1n~11 ohmarved in mm Hpid wan concentrated in
`Tfifi. wlmm it mcctounmd fur mmund 2% at‘ the fatty
`adda. GT M1 the lipid clamw, TEMZ} aim mnmmad film
`bigfmaté Emmi vi" 14:0.
`The prixlcipai mulecular spectias of PC, PE, PS and P7
`are presented in Table 4. Tam malaculm: species pre-
`dnminated in PC, narrualy 1a:w22:6u4t nné l6:0!18:1.
`which aawuntmi for 33.13 and 23.5%», rnapuctivxeiys, of the
`total moiewlar epeaciss-,3 cef this: piamphnupld. 1h-P‘U'l“4‘L. di-
`mmrp.md amt di.-mmmmammtad spwim mash mm-
`prinadIeisthanfi%ofthemtm%PGandthemntantnf
`mnnn1u'ma€umt:ed»PUFA spacial
`totalled 4.9%, within
`which 1B:ZU22:Eu~3 was thin xuaéur‘ mm neat. Miculecular
`speciea
`22:61;-3 mam particwnrly abundant in
`P3. Di~22:6n-»3 wmprimd 13.0% cm” the total. and
`m:I1l»’2‘2:6 amt! 1fl:lJ22:fi warm bath prmlem‘. at; luwmla af
`mam than 15%. in PE, 18:!3r"L‘2:£$n~-3 amounwzi far almost
`half (43.9%) of the total molecular species, and
`1£~:0!22:6n-3 was the only ether species present‘ at a. level
`nf greater than 10%.
`‘I121: mum. abundant molecular
`aperias of PI wan 1$:(Wfl:4n-8 which wmunted far 3‘I.B‘EEa.
`Amthax apeaim cuntajning flafldmefi,
`l6:0A’20fi4n~"$, mm»
`gwimd 14.6% at” the mmlwuimr apecaiem £321‘ Pl mid
`1B:iW0:5n«3 nccuunhm for 10.6%, the highest bevel For a
`species cnntaizxing 201511-3 nhsenred in any 91’ Lha phos-
`gshnlipida examined.
`
`%Gl|$$%GN
`
`Each. of than palm lipid clwwa had a apaciafic (‘arty am-id
`composition. In PC, 16:9 and 22:5n-{1 wm-ca the: principal
`co ponuanta and together accmunted fmr nearly 60% :31"
`the total fatty acids pmsemt in this phnsphnlipid
`("I‘hhla 2‘), In mntramz, 16:0 wmpriaed nniy 10.7% of the
`fatty maids in E01‘. whereas Elm Ifltvttl of 22:6;1-3 (36%)
`mm the highwt; nbaawmi in any lipid mum. in 4; amae-
`qusncze, the WM! mmmnt of PUFA in EGP‘ 653.3%) Wm
`the higfmt at an up-id etaanu. Only the E001’ fraction
`pa-aclunessi diflwi-hr! are-Ema haw tranamgthyimm of 1-0.
`Elk-1'—EnyI iimked. ether chain:-L
`PI was unique nmnng the lipid alumna in that 20:4n~6
`was a major mmponent and mmuntefi for %7A% of him
`fatty mcitzla, whareaa “2.‘2:6w~.3 mzampriwd mnlgr
`'3'.0%«
`(Thble 3}. A5 a cnnsequancve. the overall mm at" ma to
`11-6 PUFA in P1 was the Iuwgst of any lipici dass. The
`aaturataad fatty acid 13:0 was also a main: fatty acid in
`PI. P3 was cimractorized by a high mnmnt. of 18:0 and
`22:fin»-3, ccmplefi with a low mznwm. at‘ 16:02; PG was rich
`
`Mflmugh the pineal urgsm in an adjunct: at‘ him brain, it
`in known bu haw smlvad from as wall-diflemnfimqd phu»
`tamceptive organ that "Le fimquenfly considered to he a
`fimctianal third eye in Iowa: vertebrates K3}. in fact, In
`date, the praperfiafi eatabliahad far the pineai photorw
`mpmm have hem mtendnd w film retinal phmsomwpwm
`mud vice mama (3% ‘Hum mafia: dmlarence betwmen the
`ymaal and mtizm mlmaa to than neumnal organization.
`which is nlmple in the pineal but complex in the retina.
`‘mun. puma! phntancnptare main unntmwt with second
`m-der neumns mat: send their amns to brain uenters.
`fhetinal phalsammptom, an the inner hand, am in wmzuct
`with bipolar mm; that, in tum, wmmuAuic:ma with gnu.»
`align axella and mmmerous inmmmum that am pmemnt
`ibmrimuntal, amncrinna.
`interp»&;-dfann calla}. Gauss»
`quantly, the ratia af phnwnceptum to other neurons is
`much higher in the pi aal t.ha.n in the retina, and the
`tipiid oompaaitian of the pinm} urgau can be axpecwd bu
`mflecrt that of ma phuwrewgtm mils.
`
`LJPIDS. Vol. 29. M, 5 (1994)
`000004
`
`
`
`1.31%.-'JI\l\\3xvus“,~.“rtpw‘~«*l-“—V§1.m«n.A,.v:
`
`AKER877|TC00070480
`
`000004
`
`

`
`I
`
`_r»f'gm (5133 952-«$24?
`
`02!‘-mar
`
`1? DS‘0853100P04154$53
`
`Tue Nov 10 34:05:01 2004
`
`
`
`TABLE; 4
`
`LIPID QQMPQMTIQN GE‘ TROLW PI HEAL QRGAN
`
`Mans:
`NOV 18 ’(§r-‘X
`
`IE: of 8
`E’S333E12FM
`
`31$
`
`Mmdmuiar flpummu M Plump-hnlipma ltimm ‘imam Wmauii mam“
`
`fig-v:w:ien
`Hi~PUF'A
`20:5Q2:6
`££:—£fl$t:«$
`E2€:$'11¢:fi
`3ammm:&{PUFA
`I-i:&?3:6
`1‘.G:0is'f§0;5
`l‘5:0r‘ER;&
`16:fir"".»0.«i
`¥.0:W£2:5
`m:mm;s
`lfixfllflflzfi
`18.00024
`18;0flfl:5
`Mmamwwatwmwh
`1E:L’32:Ifi
`ifiiiffiflifa
`lflzmafi
`l3;UQO:ti
`1H‘.1#'§€i$:5
`lliaewmnted
`J'L5«.IWlJEe;0
`Smwauwmcvmunwmmtu-d
`i0:‘W13;1
`za:rma:1
`Dianonumuncnnnizwi
`18;%.<‘l.BA:1
`0.8 :1: (L1
`0.6 ‘.1: 0.2
`1.2 3 0.1
`000» 1: (L0
`
`20:1fIB£:]L
`M
`-*
`0.3 X: 0,0
`m _M_
`mama warn
`Wflum ma mom» and we mum at mpfimm dmmrminnfinuu a: 3 $1’), It was amumzzafi that Mm mm aammuad futty
`1ma§mc~& mm the uzml paufimm Mammal” was-c:.s"mI amwmnim mmw fimttsgx acid; wr Stay maid immamr Wm mm. wmhml {mm the
`wmrm mwiuia with m mammng WLESW3 mm. §.“fl.f£éu»6. M3,, phmw
`mm mmmmmm Mnlinmtm mmmms
`phatimtfiszhnimaeg
`mhmphatmylmmmdamm; PE. phmnhafiwdwinmiwk 5%‘, p&xmphmdy'lm’n~im; PU?A, pmlymnmmvntmi
`famv mm
`
`0.5 an 0133
`4.1 3: 0.53
`1.0 2:: 0,1
`
`L2 3: 0.1
`8}’! 1 0.4%
`33$ 1:’ 1.0
`M: an {Lit
`3*} at 00
`L2 :1: (W
`3.? 1: on
`‘L5 x 0.1
`LE: I 0.32
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`0.5 1: IL},
`0,0 as 0.1
`MD at 1.?
`v-
`0.5 x 0.1
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`0&3 3: 0.1
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`130.0 as 1.0
`2.5» :3‘. 0,2
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`1.0 at 0.0
`13.0 as 0.0
`4.6 1: 099
`
`3.8 1:01
`45,2 :1 0.}.
`30.1 1 0.1
`2,1 m; 0.1
`1.0 an (‘M
`33? 3 0&1
`9.6 x 0.3
`2.? 10.1
`0.8 1: 0.2
`
`0.5 3: 0,1
`3.7 3: 0,};
`104 :4 0.1:
`2¢6.'1:0m7l
`2.11 2 (H
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`9.3. 1‘. 0.0
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`1.13 1: am
`0.2 4: 0.1
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`0.3 1 01}
`100 3; DJ}
`$3 1 CH.
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`0.? :2 03
`10.8 :2 0.3
`iififl :1: 0.3
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`3.1‘ 3: 03
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`1.3 :1: 01
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`0&3 :2: 0!)
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`-«
`0,3 an 0.0
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`~£.£ 3 0.33%
`103 2: 85$
`H0 at M:
`0.0 3: 0.31’
`101% at 0.1
`‘M it 0.5
`37.8 K 1.0
`0.3 i (L)
`
`0.0 :5; 0.0
`0.? 3: 0.0
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`(11203
`w
`
`w»
`
`6.0 as 0.3
`0.3 1: (I0
`
`The iipifi mutant at‘ we tmut pineal (7-t,9% 9!‘ wet
`waight) in almont axnntly halfway between that founfi for
`the brain and retina (63.8 and 3.1%, map-edivuly} of was
`«same species (10). Huwwm; the level of TAG in than lipid
`at the pinea! 001%) in cmfiisiarably higher than the 8%
`and 30% fwmml am than Lima at film brain mat mtim, but
`ia xwvamhefimm zzimm tan mm mm. The mm uf ‘TAQ in
`bmim and rmaixm uf tmut mm known tn baa Mghar than
`in the same tinamea mm and (3.0). An imfluemwimg faac.1.m'
`may be the fact ‘that: the want mmmimad Wm fumm
`Ssh» It in wall kzxmwfl that immmni fiah, irmluding mat,
`haw higher lipid mutants in their mu mm their wim
`wunmrmm (ML. ‘mvmtlm the
`aims mutant a!‘
`neural tiuwa Mm hetwawm MI and mm fish at
`whehhm higfx TAG luavelaa am 9. specific feature: bf than
`Mpida of twat neural timws mmains ta he eambliahadk
`A momma {mature and“ um “main uf fish and mimaw in
`
`geniwal is than high mmxmrtimn us!" my in 0% mm} lipid
`(15.13 1}» In mm am and taunt, the: E1€§PW‘G mm in brain
`Rigid is uppmnimawly 1:1 (103. The ham! at‘ EGP fin ma
`pineal
`iipid is \nnt.ahiy lmmer
`than aim:
`and‘ PC,
`EGP/PC ratio basing 0.021” “I'M: value is closer to firm
`037:1 ubuerww fur the rmtiu at‘ these twco phwphnfipixlw in.
`
`the mtirm :39’ mm mm mad czmi (10). Wm athanciiamine
`plnsrnmlugem mmxant of ma pineal in much Lamar than
`that offiah brain 636.43% afE*Z.7‘r‘P} (25) and claw: but that
`at" retina K-515% :31’ E301’; E811. MN... unpumixhmi dam).
`The ahvmenca mi’ aarshrmidea in $123 lipids sf mm mum! in
`am: mum typaiml MI retina than ‘bmin. am is» the» law
`amemnm cpl‘ athmuhamma wimmiagem €31}. Tha ib-
`mnm «sf ¢a»r«iiniip&n in erigrsifimmt amaunm in pimgsfi
`lipids £13 unusual an min ijeyfid alum in as mmmmm mmm»
`mat ofnaiwchuudrml mamhmma and ham man taunt! in
`lipid ezitrmttmi mm mm Fmh raisin» am! brain (10);
`1119 total maid and component lipid dam-an at the
`amam, with him mmyuon 0!‘ SM. warm ::h.arm:~
`tau-ind by M3 wnmnta uf”Pi.¥i“A. Navm-thelens. film wuw
`mm; «mum in ma Mal fipsd oftmut ox-gm £‘a:L2*3%») is
`Immr than thus wrrmmanclmg mines of 419% and mm,
`mpartezfi far film brain and mama, mapecmrafljy, ma‘ thca
`mama wpeciuk £10). and aim mnmdmmhly lam than him
`PUFA wn1teu!:M3.6%1af mm lisxrié fmm rm pinm} £20}.
`1%: diam-ihufim 57!" fatty mam among the varimm iipird
`ciamaaa canfarma to wall uwtmhlinhed pa
`an. FM‘ mmw
`me,
`the:
`lung-azhain mamarmic fatty acid §l2:1n~11 ac»-
`cunmi unly in "M0. TM ahmnmwofflfia fatty acid, which
`
`000005
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`MHB3. ‘A1 20, :1)» 6 (135%)
`
`AKER877|TC00070481
`
`
`
`Mm
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`
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`
`Tue Nov 1%» 14:05:01 2004
`
`MIN 1%.?»
`
`?ag;e 7 of 8
`'84 BBIEEPM
`
`315
`
`R..I. HENTDEREWN ETAL
`
`nrirginams in talmmid zmnpianktmu. in ms mmman faature
`91‘ fish phrmphnlipids (32). Tha PUFA lfitfinwfi waa aim
`r:.o:n.fi.ned an TAII2, It is nambln that tha mm: lipid of
`pineal giant! vtantaina higher levels of am fatty acifl
`ilmn brain
`the rat (20), alitlmugh it. remains to be eaw
`tablished whether the 132331-5 is apecrmmity associated
`with TAG. The prwenne 01' high levels: rm! 10:0 in PC is a
`charmtarimzia feature at‘ this phasphulipid.
`including
`that extrmzwd [mm imut brain and rating (10) and
`bovine pineal curgan (21). We EGP of neural tissues are
`knmlm to be specifically enriched in 2316a-3 (31), and
`this was aim at nmable faawre nhaarued in the present
`atudy uf the: E6? fmm than in-ant pineal. Wm value fam-
`22:6n.-3 in E0»? (36%) is slightly higlwr than 135:: concern.
`in I-"‘..£'3~P Mam tmut. brain (31%). but leaa than than of thib
`retina EGP £-46.8%) of the same specias (10). Interest-
`ingly, the 22.‘8n‘-3 conbants ant” P0, P1 and PS found here
`in the pmeai are all intennadiata between: those found
`in thfl brain aumzl in him retina.
`‘Thus PI :35" Eh-at pimml urgam mnformmi '40 {ha vwu ms-«
`mblishmi pmdlnzm in fish mange, whawby N baa as:
`higher content of :Z0:¢n—6 than mlmr phmphnlipida and
`is conaewquemfiy ch aractaerizedik hy a law ratia cef 11-3 in n~E
`PUFA (32). The actuni mutant of 2l}:4n-6» in the pineal
`P‘! (27.4% sf total fatty acixitaiv is markedly higher than
`that mpawe-ti for trout brain (10.2%) and mtina {14..$%}
`and for the same biauuea at «sand £4,040 and 1fi.5%, respec-
`tively) (10). The results xuggaat that in temua of tha
`L*O:4n-6 content of PI thus pfinwl exceadfl retina which, in
`turn, exceeds brain. In the ran, the level {If 20;¢n~6 in the
`tubal lipida {mm the pixzaal cnrgan in also higher Llmn
`that fwmd in brain lipida (2%. Thia ixs wnuriaumt with
`tlw idem mmrding to whixzh ashumnrunadumimn might aw-
`tivatc phoaphalipase A-2 with the subsequent funnalion
`of eimaanuida E133). An inwawement Bf eiwaanoids de-
`rived fmm 20:4tn~6 in the kightrdependent. control
`an!‘
`meluhmin pmvciudtion in thamfnam inrlicntawi.
`Corwiatam with the: mxemll fatty acid mmpmitmn. mt”
`EGP. PE mntsined the highwt level (L‘3.£3%} af cli~«22:5
`molecular ipeciaa of Lbs phoapholipidn examined. How-
`ever, thn vaiua was less than that aha-enred §i‘£"E‘H'.i.OUfl1)" in
`trout. brain (14.9%), and cxmmiderably lean than that in
`trout retina (41.3% (1‘?). Likewise. di~2.‘2;fi moleuzular
`apaciaa of P0 mud P8 ware WW abundant in the pineal
`man the mama, and 01133 1:-imul PC had a ixigmr di~22:6
`cunt.-ant
`than the mrreawwdingé phnsphnlipids from
`brain. The athkesr molecular species were ail as expected,
`with 16:B»'22:6 and l6:0f'1S;3. dazuminating ?C, 161032216,
`18«:1fl2:$ and 18:(1.'fl2:fi mash compriaing 9.fi~—~20.1% af
`PE. and lflaiémlzfi being mm: pmrclamjrmnt Ffl epatziea. In
`coulsaam, 31 mm mm in lszmwm m be mlanimiy impowm
`[mm with mam. tn mam-3 oqnmning mntwculu
`species; whiie C20 PUFA species are abundant (11218).
`In lzmut, retinal PI wan gwredominanfly 10:0.-'20;-in-6
`(40.1%) and brain PI 18:m“2vG;l§n-3 (42.-3%} fl W. This: ti?»
`we apewificity in the '*uaIe»:u01a0r speciaa commmitinn of PI
`was ¢0n.fin~xm~:£ in 00421, in wiiiah 1B:0!%0:4n~»£S was the we»
`daminant apecim in liver and! me ($16.?
`49.1%, m»-
`srpectivelyi and 13:;L»‘2Q:«In-6 was the max: must abun-
`dant (13,29). In brain.
`lS:{if20:5n-3 and }.&:1)'.i0:5n-3
`mmpried omr half the PI :1’ 13) w1't,h arachidanyl spaciea
`
`umns. Va, 2% no. 5 (1994;
`000006
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`mmmng manly 16.3%. whemmu in ramaa w:nm:e,
`13:0/20:4, 1&:G1‘22‘:$ and Rflzflffiflrfi wan rim mm! ahun»
`tiamt spezciea, in that ordar, totalling 84.5% at P1 (18).
`The PI from want. pineal an-gan thus cloaahr reuse blea
`that of trout retina with 1»B:#3fE£Mn~6 prednmimmz. The
`13:0-V20:5n~3 species an clmraewristic MP1 {mm trout
`and and brain wmprirml may $0.895: crf smut pixrum PI.
`Thug? althczugh it ia aituawti adjacent tn {me brain marl in
`very zzloaaly ammiatad with Ilka brain, thfl pmmt in alu
`must, ‘dentin! to retina in terms MP1 composition. Fur-
`bhemmre, no 18:IL!24:I wan found in the PC. of the pineal
`cmrgfln. wharwa this mniecular apeciea mmprises be-
`twmen 9 and 13% of flab brain PC (17,131
`Mthnugh ILM mm lipid of mm mm: pixmxi arias: has an
`Inwar mutant mi’ mm-.3 than thm. found. in brain Cb!’
`retina, the lmrei in nevertheleaa will higher than that usu~
`My observed in Esther non~1neru1-3.! tissues, SL1-Eh as liver or
`muzscle E32)-. Fnrinherrnnm. the Eflizfin-3 conten