`
`
`
`
`
`TM 11de cumpoaition arm pineal organ fmun the ram
`
`how trout {flmrhy mamas) was date mined to snap-
`ubliah whathmr the inwhmment of ”13% man in the:
`control and“ mimadhn rhythm» ink mmm‘ by “initial:
`Mutauuns 0! “grid uampwiflon. Lipid mmprhud {3%
`
`at 111m W WM weight mmi mimymlywmh W m»
`
`
`
`Mar lipid! class
`regent HWK: af total lipid). Phnm
`
`phfltidylchai‘ina (PC) was thin principal pulls: livid. Ind
`smaller mparfians of ether phonphmipids and choluw
`tam! wuss also mus-em. Phxnmlmm mtrihulad “1%
`
`at" me Mmlmufina ngmgthMyhfilipidfl (EGPJ. Na
`mam we"! mm “mm [any mm Minna—swan
`
`of tfincylgiammlq we“ manually ahnflar to that of mm}.
`
`lipids in which natmnwd, monounsaturated and
`polyunsaturated fatty acids {PENN Ware gamut in M-
`mosf. equal pampmflm Em}: of the: palm:- lipid chasm
`had a mama: fatty fluid maflfim. With firm umpfinm
`M phwphamflyfimwiw {91}. m whim man-m mmpfisui
`23.4% «if ma WM fatty amid» amen-a van that [imam
`PWA in 931 lipid chum. The mmrfiun at mm-a
`newn- fiancee-ind 6.0% of She: {may uldn in my Hpid dun
`
`The predominant man-1mm special a! PC were
`lemmas; and “£1821, which accountant Mr 3&2 and
`
`“28.65%. mmmflwly. at the m HAM/aw!” WW cf this
`
`phmphonpmw PMmhmMyhfihm «lemma WE) mu»
`mined. than highmt law! a! «Emma-a {13%} «(my 9110*
`phoflpld.‘Tham was also *‘M «231' this mm M“
`in phasphafidyluflna {PS} and 4.1% in m. In PB, the»
`species 1.653122%, lflflflflfl and [8.1% mulled 45.1%,
`while In PS 1&W22:6 amalgamated I'm 48.9% of “In mm
`molwulm mwiw. Tim must abundant: malecuhr
`sped“ aim was lfiWfiuafl (TWML 111.2: mm mmmim
`trim: a! flaw minim! mm a!“ Mat, and pmimmly than
`
`
`molecular apmias campoamcm M P1, in mm similar to
`
`the compmifiun of
`a retina than that aftha brain.
`
`
`
`phi! 28, 331.317 (1994!.
`
`
`
`In fiah, an in all vartabmwfi, the retina cf the new and
`
`the pineal {1:31am of the brain are esscnfiai mmpunenm
`0f the cirtadian system that: measures the period and
`phase «91' tha daily lame-dark cycle and ultimately mn-
`tmla rhythmic pmwm (1.2),. In keen‘mg: with this role.
`ch» pm: mam affifih mam-aim phatomapmr cells that:
`display 421m Manhunflmfl naming!“ with the phom
`w—r-n—
`
`V'b whom mwesmndenm shwid be addressed.
`Abbrefiafiana; EGP. ethmnlmmm filymmphmvhafipiedu; HTLJC,
`high~wfumw Maui/ti chmmMgrw 3r: mm. highpmfim
`maxim thimhyu mmmamgmphy; 9C, pheaphnfifiyhhulim; FE.
`yhmphmfidylwmmumma; PG. phmphaudylgiym; FT. phuw
`phniidylinmima Pa phmphutitiyluflm; mm, wimmamd
`
`Emmy with; 5M. uphingomyolin; HG. t. "Mgiymmh. Mulmlar
`apam’n an abbreviated as mum-133mg“ 16:11’22H5PG m lwpnkniloyL
`2~¢mahamnwl~m~glywniauphmphmhofim.
`
`
`
`
`
`
`
`wptors of the mung {3,4}. The phomrmpmr sells firom
`bath tissues: translate the lightt—dark infomation into a
`
`neural nutput mf mammary neumtramm'ttar 4(5) and a
`neumhonnmai mutput in film fawn m" mermm (3; In
`amnion m [maxim mtdtjmmnw mus, {Am iahmommw
`
`tar cells Mme pinaal organ an aim muMaflectam and
`
`
`can Lranaduce informatixmn supplied by athar eta
`a!
`
`
`
`{much as temp-amt“) m is mum] {catechnltwaminem
`adenosine, atamida} factors. Whereas retina! melam niu
`mm pmfamnfiafly in an aumnalpmafim mumm
`the Mammy; mmde by the pinata} mmribuim 15mm"
`m the circulating lwela and may be involved in the con——
`firm! of seamnai events, parfitulnflx reproduction, in war-
`mbratea {3,6—8)‘.
`The emu-tum} phnaphnlipids at‘ the retina and! brain
`M yartebr‘ama‘ including mm, am lmmm m mmmin high
`lambs of the wlmgatumwfl fatty acid MFA} $23114}
`£19.13} Withn the retina, the fififimfi in apparently com
`
`
`
`mnmted in the me brancus mm:- segments 0f the pha-
`
`twreoe-ptur mfi calls (11}, and these cells iaaia‘ted from
`fmg retina exhibit a selacfive uptake of 22:6nn3 in MIN
`(12). A rmtfizmam fur 22fiw3 in the visual 9W3 has
`
`Mean damomtmmd in Mudiw mm 11¢me wfimama
`and preterm human infanu. whim have 311mm that fihe
`visual acuity i3 aflmtcd by deprivation 01'22 :Sn-fl during
`pmcnaml deveiapment affine infant (13). Pemidafion 01'
`22:6m3 is one $47 the primary events ubaeweé in inher-
`fiwd or haumnfimlly indumd fihoturmapmr daganmw
`him (9.1%
`Di~22£n-3 phmpmlipifla m knowu to m: major coma
`mutants of rod cuter segment membramu in frag and rat
`
`{15,16}. Hanan: analyses of the» phosphnfipifia of the
`brain and retina (of trout and and have shown that di-
`2fizfin-3 mniawhr annuities am madm- mmtuenha of
`phmphmidylcvhmme (PC), phwphatidylmhmammim
`WE} and phwyhmidylwrim {MB} in Lhasa tiwum and
`that thin ia particularly tnm of the retina, when: the
`amounts am nunsiderably higher than thaw Found in
`
`terreatrial mammals (11,13). We importance uf 22:6n-3
`in («he vimml pmcaa of fiat: 115 also intimated by the at»
`”Nation that m pmpartim at“ di~2226nva in films phmaw
`phafipfid of mm dweloping gym «1' herring inflate in-
`
`
`uam with m‘ a: the rods in mind mm the phanto—
`mepmr popuiafian (19). The faults of them; nuaritianal
`atudias and tha presence wf iame amouma of {ii-22:6
`phogpholipid in We 12%mepr at“ the retina WWW;
`MK 2225an has an nmdal and “:qu miss in film: pri~
`mm event» amwinmd with m abaommun and trima-
`«fixation of phnhmn, This mm In of cruciai important»
`{w the phnmpefladic mnuml 6f the productim af mes—
`sages, such an melatonin,
`
`Frqwgm (613} BSZwsBZ-i?
`
`Grain r S! 05085 31839041343 5 3
`
`Tue Hm 16 14:05:01 2mm
`
`NOV 1E:
`
`Page 2 uf 8
`“3&1 @WWM
`
`
`
`
` . ARTICLEgmww
`Lipid Ccmposifim 0f the Pineal Organ from Rainbow Trout
`(Onwmynchw mykiiss)
`
`311
`
`m. Hendamm“. my. new, m: mam Sarge-m" and J. Falcon”
`
`MERE. umw Aquatic BMaMw Damnmm a! EH99“! and Muimww Saunas, may at Surfing. 3mm F149 M. 8mm, United
`Kingdom and Wait» do Mwmhbbgie at ads mmmmm Wm, UM {WEE 290. Famine dung Samoan. 3:50:22 Mien
`German Franm
`
`L
`.9
`
`WIQM @ mm by ADCS m
`
`000001
`
`“Whig a mu “WW? 4..me
`
`
`
`mung. was, :29, m. 2; {m4}
`
`AKER877ITC00070477
`
`000001
`
`

`

`
`
`[Eran (5133 952-3247
`
`Order # 050853100904184553
`
`Tue Nov 15 14:05:01 2004
`
`Page 3 of 8
`NW 16 '84 23:22PM
`
`312
`
`
`‘RJ. HENDERSON ETAL.
`
`,i
`i'i
`‘3
`
`gi
`
`.3
`
`i j
`
`
`development in the soccnd dimension. Developed chro-
`matograms were visualized with copper acetate in phos-
`phoric acid (23). Lipid class composition was quanti-
`tatad by double-development. HP‘TLC coupled with scan-
`
`ning donaitomctry, as described elsewhere (24).
`
`
`
`
`
`Ea rates of the relative amounts of the plasmaiogen
`
`
`
`
`
`
`
`ard the diacyl for
`a of elhaoolamine glycorophospho»
`
`
`lipids (EGP) were obtained by acid hydrolysis of Lhoiso-
`
`
`lated EGPin still: on an
`plate followed by ch no-
`matcgraphy 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 cm glass plates coated with silica gel G 60 (0.25
`mm thick) using the solvent system described here. The
`
`separated classes were visualised by spraying the ctu-o—
`
`matogram with 0.1% 2',7'—dichlorofluorescein in metha-
`
`
`nol containing 0.01% butylated hydroxytoluer'e and by
`
`
`viewing or der ultiaviolet light. Tn'acylglycorols (TAGs)
`
`
`
`
`were on 'fied by redeveloping the chromatograro in the
`reverse direction of the second development using
`hexaneldiothyl etherlglacial acetic acid (80:20:2, by vol)
`
`after removal of the individual polar lipid classes. The
`
`fatty acids of the separated lipid classes were converted
`
`
`to their methyl actors on the adsorbEnt by acid-cot—
`
`alyzed trar'sastarification (22}. An aliquot of total lipid
`
`was also subjected to the same procedure. The re suiting
`
`fatty acid methyl esters were purified by HPTLC and re-
`covered from the adsorbent with hexaneldiethyl other
`(1:1, volfvoll.
`Fatty acid methyl esters were analyzed on a Packard
`
`439 gas chromatograph equipped with a fused silica cap-
`
`illary column (50 In x 0-22 mm i.d.} coated with FFAP
`
`
`phase (S.G.E.. Milton Kcyneo, United Kingdom]. Sam'-
`
`
`
`
`ple applicatio was by Lin—column injection, and hyt‘ro—
`
`
`
`gen was used as the
`"a: gas. During the course of an
`analysis, the oven temperature was programmed to in-
`
`crease Eroru 50 to 225'0 Samples were also analyzed
`
`
`using an Omegawax 260 fused silica column (30 Ill
`
`
`
`0. 25 m i.d.. Bupelchem UK. Ltd" Essex. United King—
`
`
`
`domlwith the oven temperature programmed from 50 to
`2.60°C Fatty acid components were identified by refer-
`
`ence to a woll-character'zed fish oil fatty acid mixture,
`
`and the unsaturated nature of components was con-
`
`firmed by re—aralysia of samples after catalytic hydro-
`
`
`
`genation over H02. The separated components were
`
`
`
`quantitatod using a recording integrator linked to
`e
`
`chromatograph.
`Analysis ofnwleculor species. A SOD—pg portion oftotal
`
`lipid was separated into the component phospholipids
`
`by W alongside 20 pg of a cod retina total lipid
`
`standard using methyl
`acotatolpropan-z-olfcbloro—
`
`
`
`to
`’ethanoli‘llflfi‘b (Wvol) sq. KCI (25:25:25:10:9, by
`vol) as the developing solvent. The standard spots and
`
`
`the edge of the bands of pineal organ lipids ware visual-
`ized by exposure to iodine vapor, and the bands of ad-
`sorbent, commuting PC, PS, phosphatidylinoaitol (PI)
`
`
`and EG‘F‘, were scraped from the plate. The phospho-
`lipide were hydrolyzed on the silica with phospholipase
`
`C using a two-phase system of 1 mL diothyl outer and
`
`
`1 mL 0111.1 M sodium borate buffer, pH 7.5 at room tem
`
`AKER877ITC00070478
`
`
`
`
`
`
`
`y analogy with the retina, it is probable that 22:611-3
`ia a major PU ”A of the lipids in the photoreceer cells
`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 spacios of phospho
`
`lipids. As far as We are aware, the detailed lipid compo-
`
`
`sidun 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 animals (20,21). Information gained from the analysis
`
`of the trout pineal organ is of basic impm .snce as mam~
`
`
`malian pinealocytes are accepted as being phylogeneti-
`
`
`Cally derived from the fish pineal photoreceptor nails {2}.
`
`
`HAT‘B‘UALS AND M m
`
`
`
`Fish and pineal organs. Rainbow trout (Oncorhynchus
`
`myhiaal of average weight 500 g were obtained from a
`
`
`
`com nercial fish far’n (Pisciculture Ballet, Angoulomo.
`
`
`
`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, boron in liquid
`
`nitrogen and stored at —80'C until taken for analysis.
`Chemicals- and solvents. Phospholipaae C from Eucli-
`
`
`lus cereus was purchased from ‘Hoehringer Corporation
`
`
`fLordon) Ltd. (Iewes, East Sussex, England). Dulyl
`
`
`
`chlo do and anthracene Q-carboxylic acid were supplied
`
`
`
`by Aldrich Chemical Co. (Gilli xg am, Dorset, England).
`
`All other chemicals and biochemicols were purchased
`
`
`from Sigma (Poole, Dorset, Eng and], 3rd solvents of
`lugbvporformance liquid chromatography (HPLC) grade
`were obtained from Rathbum. Chemicals (Walkerburn,
`
`Poeblcsstire, Scotland}.
`the
`Lipid extraction and analysis. After thawing,
`
`pineal organs were weighed and transferred to a Teflon-
`
`ill-glass homogcrizar. The organs wens homogenized in
`37 mL of chloroformlmcthanol (2:1, volfvol) essentially
`as described by Christie (22} to extract lipids. Solvent
`was removed under a Btflmm 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, who”
`and stored under an atmosphere of nitrogen at ~70'C be-
`tween anal yaea.
`To establish the lipid class composition, aliquots of
`
`
`lipid extract were subjected to high-performance thin-—
`
`
`layer chromatography { PTLC) alongside authentic
`
`
`
`standards using haaaue’diothyl ethon’glacial acetic acid
`
`
`(sauna. by roll or the developing solvent for the as
`-
`
`ration of neutral lipid classes and methyl acetate-{prov
`
`
`pa -2-olfchlorofomifmethanolf0.25% (wh’voll sq. KC]
`
`(25:25:25:10:9, 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—dimer!
`
`
`
`clonal HPTLC. The polar lipid developing solvent sys-
`tem described previously was used for development in
`
`
`the first dimersion and chloroformlacetonefmethsnolo’
`
`acetic acidfwater (10:4 12:2:1, by vol} was employed for
`
`
`
`
`IJF'lDS, Vol. 29. no. 5 {1994}
`
`000002
`
`000002
`
`

`

`
`
`firm (613) 952—3247
`
`
`
`Order it 050853100904184553
`
`Tue Nov 16 14:05:01 2004
`
`
`Page 4 of 8
`NOV 16 ’84 03:02?“
`
`313
`
`
`
`
`LLPlD COMPOSITION OF TROUT PINEAL ORGAN
`
`
`
`
`by EPG {9.9%}. No choline plasmalogeno were detected,
`
`
`whereas emanolarrjne plumalogena accounted for 11%
`of the total EPG fraction. PI and PS each accounted for
`
`less than 5% of the total lipid, and both phosphatidyl—
`
`
`glycerol (PG) and apingomyelin (SM) were present at less
`than 2% No cardiolipin or cerebroaidea were detected.
`
`Palmitic acid (16:0) comprised 23. 7% of the kitty acids
`
`
`in the total lipids and was the most abundant fatty acid
`
`
`in the pineal organ (Table 2]. The rronounaaturated
`18:1:1-9 and polyunsaturated 22:6:1-3 accounted for 17.6
`
`and 12.4%. respectively. of the total fatty acids, Overall,
`saturated. monounsaturated and polyunsaturated fatty
`acids accounted for similar proportions of the total lipid
`fatty acids. The fatty acid oompoaition ofTAGs ('Ihbla 2)
`
`was generally similar to that. of the total lipid although
`
`the proportion of 18:2n—6 {12.3%} waa notably higher,
`and that of 16:0 lower, than in total lipids.
`'
`
`
`
`I
`
`
`
`
`pasture under nitrogen for 2 h {26). At the end of the
`
`incubation period. 1,2-diradylglyoerols were extracted
`7driecl down under nitrogen and finally desiccated under
`
`
`
`vacuum for 1 h. Q-Anthroyl chlor do was prepared from
`9—mthracene carbowlic acid and oxalyl chloride as de—
`
`scribed by Goto et at. {27). The diradylglycerola were do
`
`' rivetized and puifled by a modification of the method of
`
`
`‘I‘akaniua and Kilo (28) as described elsewhere (19)
`, The 1-O.alk—1’-eny12-acyl derivatives were removed
`during the final HPI'LC purifimtion step. The 1.2-dia-
`
`cyl-S-anthmcyl-an-glycorola were separated by HPLC at
`
`
`1EL—20'C on an ODS column (25 x 0.46 cm. 5 pm particle
`
`size; Beckman Instruments UK. Ltd... High Wycombe.
`
`
`Buckinghzu shire, United Kingdom) using a Pye Uni-
`
`
`
`cam P114010 pump (Pye Unicorn Ltd... Cambridge.
`
`
`
`England} and two isocra 'c solvent systems, rrnithanollr
`propan-Z—ol {4:1, vollvol) at a flow rate of 1.0 mIJmin,
`and acetoniu'ile/propan—2-ol (7:3, volhml) at a flow rate
`
`of 1.0 mlfnfin as described by “Ramos and Kilo {28}.
`Peaks were detected using a Waters 470 scanning fluo-
`
`rescence detector {Elliptical UK Ltd. Edinburgh..Seot-
`
`
`land} with excitation and emission wavelengths: of 330
`and 460 nm. respectively. and quantified wring a Shi-
`
`
`madzu CRSA recording integrator (Annulment. Lump.
`
`
`
`United Kingdor-I}. Peaks were identified by their mele-
`tive retention time using 16:0f22:6n-3 as a reference
`
`peak. Di—docosahexaenoylglycerol (di—2216m3; Nu-Chek-
`. Prep. Elyaian1 MN} was also available for direct com-
`
`pariaon of retention times, as were a range of samples of
`
`
`known composition from previous studies (17.18.29).
`
`
`
`Each sample was chro autographed three times in each
`
`solvent system and the standard. deviations calculated.
`Where final peak areas were calculated by subtraction.
`the standard deviationn of the contributing peaks were
`added to give the final error.
`
`
`TABLE 2
`
`
`
`Fatty Acid Gorapoaliton {m3 of Total. Lipid and Lipid
`
`Classes from Trout Pineal Organ"
`
`_ Any} chain ..
`Thtal lipid
`TAG
`PC
`EGP
`14:0
`2.5
`3.5
`0.8
`0.2
`15:0
`0.9
`0.3
`0.3
`0.9
`16.00153
`-—
`--
`-—
`0.0
`16:0
`33.?
`17.4
`34.0
`10.7
`16:1m9
`——
`--—
`0.6
`~—
`163an
`5.3
`3.0
`1.3
`0.?
`17:0
`0.6
`0.4-
`0.3
`0.3
`18:0DMA
`——
`-
`-—
`0.4
`183041.00“.
`—
`—-
`-—
`2.2
`lii: ln—TDMA
`—
`——~
`—
`0 7
`18:0
`7.2
`4.2
`5.ii
`10.2
`13:1n-9
`17.6
`18.5
`18.3
`10.3
`18:.ln-‘i'
`4.0
`3.7
`2.4
`3.9
`18:2n—6
`9.3
`12.3
`1-8
`3.4
`18:311-3
`1.2
`2.0
`—
`0.3
`20:1:1-9
`2.5
`3.1
`0.4:
`1.4
`20:2n-6
`0.6
`1.2
`0.4
`0.9
`20:3nrtl
`0.4
`0.2
`0.3
`0.4
`20:4n—0
`1.6
`0.5
`2.0
`7.2
`20.3on3
`-—
`0.2
`-—
`—
`ammo
`0.9
`1.4
`0.3
`0.6
`20:5!1-3
`3.5
`3.9
`4.6
`4.8
`22:1n-11
`1.7
`2.0
`—
`--
`22:1n—0
`0.3
`0.3
`—
`—
`32:4n-v6
`0.1
`0.3
`-
`---
`22:511-6
`—
`0.2
`0.2
`0.4
`22:5n—5
`1.2
`1.5
`1.3
`2.2
`22.8n-8
`12.4
`12.6
`24.6
`36.0
`24: Ln-Q
`0. 7
`0.1
`—
`0.4
`Unidentified
`0.9
`1.4
`0.9
`0.?
`
`_
`
`Total saturated
`36.3
`25.8
`40.6
`23. 3
`
`
`ToLal monounsaturated
`32.6
`35.7
`23.0
`19.6
`
`
`
`”A
`’lbtal ‘
`81.2
`37-!
`35.5
`56.2
`'l‘otnl 11-3
`19.2
`21.9
`30.8
`43.9
`Total 11-6
`12.0
`15.2
`4.7
`12.3
`3.57 milling .... 1.60 1.44 6.55
`
`
`
`
`
`‘EGP. ethanolamina glycerophoepholipida; PC. phmphatidylv
`
`choline: TAG. hincylglycorols; PUFA, polyunsaturated fatty acids;
`DMA. dimefliylaoetalc.
`
`
`
`RESULTS
`
`
`
`
`The lipid content. and lipid class composition of trout
`pineal organ are presented in Table 1 Anmnd 4. 9% of
`
`
`
`
`the wet weight of the pineal orgar was lipid of which al-
`
`
`
`most half {47%} was in the {on of“'AG. PC was the
`major polar lipid present (16. 5% of total lipid), followed
`
`
`TABLE 1
`
`
`
`
`Lipids of Khan! Pineal Organ: Lipid Clue Composition“
`
`Lipid clans
`H 3‘
`‘32 Thin] llpid
`Gunnery! eaten
`3.8 t 0.3
`
`on n 2.1
`tile
`ice
`Free atty acids
`3.4 2: 0.0
`Cholesterol
`5 1' :t 0.4
`Diacylglyoarnla
`l 5 t 0.1
`Ethanolemino glycorophuopholipida
`9.9 I: 0.6
`thphatidylgiyoeml
`1.4 :I: 0.1
`Phosphatidylinoaitol
`3 2 3: 03
`Phosphatidylcerine
`3.4 a: 0.3
`Phosphatidylclioline
`16.5 a: 0.8
`Sphing’gmyeiinm
`"Value“ are moans : SD of three dctarminatimm.
`
`000003
`
`UPIDS. Val. 25. no. 5 {1594}
`
`AKER877ITC00070479
`
`000003
`
`

`

`firm (513:) 952424?
`
`flrder all 050853100P04134553
`
`Tue NW 16 lefizfll 20M
`
`Page 5 M“ 8
`NOV 18 'EM 0330850“!
`
`314
`
`TAMI!
`
`R1. HENDERSKDN ET AL.
`
`uwmxamé,wc,‘gwnw.mu.'..«mm«
`
`in mmaunaatmmd fatty acids and PUFA which mm-
`priaad 44.]. NM 311%, mwmfimly. uf mm mm fatty
`acids in mm thulipid {Tabla 33* The lessen: m? 18:11:»?
`and 18:11:14 am and 10.2%} in PG were highw than in
`my other lipid glass, and, of all the phnlphelipids PG
`had the highasn mutant af Iafln-G. In 3M mama than
`half (58.2%} a! the fatty midi: were mnnmnmturawd.
`
`mainly data m We prewum (if a weary high ymporfian of
`24:1 (dfiifl‘lhl. FUFA mmmfiwfl timely 09% ml“ 022: 0M fatty
`Marla (Tabla 3}.
`In all
`lipid classes, 20:51:54 was present in small
`amounts am! mum “caudal 6.0% of that mmpcmenl
`
`fatty acids, The lung chain manoennlc fatty acid
`22:12:14]. ohmwed in total lipid wan; wacmtrated in
`Tfifi. wlmm it accounted fur mmd 2% 0f the fatty
`mm 01" all the lipid chum TAG m ammmd um
`biglmat lewd 0!" 14:0.
`The principal malecular spasms af PC PE PS and PT
`
`
`are presented in Table 4, Tax: molecular spams pre-
`dominated :11 PC unruly 1%:m2Snail mu} 155mm
`which mmmd Pm 33 $3 and 2a5% rwmcfiwm at flu:
`total malmlar maths-,3 cef {hi3 pfiwphollpld. DWUWL, di-
`mmmd amt dlmmammm swam malt mm—
`prinadleuflmnfi‘fiyofthemmmwdmgmmtof
`
`mannmafmhedwpUF‘A spasm totallsd 3.9%“ within
`which lflzm2rfia~3 was Hm xmm‘ cum neat. Malemlar
`
`spacial; whining 22:6:1—3 mam partlcwuly abundant in
`pa Di~22zfimfl wmprimd 13.0% a” the mm. and
`mumsés and 1%:mgrfi wam hath pment at; lamb al'
`mam man lfi‘fiz. in PE! 18:0!222fiw-3 ammm far almmt
`half (43.9%)
`of“
`the total malacular 3min, and
`1‘5:0J'22:6n-3 was the only ether species presam‘ at a. level
`all greater than 10%. Tim mum. abundant mlecular
`apeflas 01‘ PI mm 1$:mn:4n$ which amounted {car 313%.
`mm: maxim cuntaining 28:42:”!«6.
`lfifllfiflflmlfi, mm»
`grim 14.6% at” the mmlmulmr apmiaa (if Pl
`find
`IBWfinoa mauled for 10.5%, the higheat level For a
`spefies containing 20:511-3 nbsewed in any 91’ that phea-
`ghnlipida examined»
`
`
`
`W M
`
`flmugh 0m pineal urgsm in an adjunct: «:6 film brain, it
`in brown m haw mmlved From a wall-djflemntiumd phur
`mmceptive argsn that "Le fimquenfly considered to he a
`functianal third eye in Iowa: vertebrates {S}, In fact, to
`date, the graperfiea eatablialmd far the pineal photon»
`mptnm have been unbridled w the wfinal Wprw
`Md via: new (3}, Thug mafia: Maren”: belwmn aha
`951M311 and mm mlntaa to Kim neumnal omaniaatlnn.
`which is almple in the pineal but complex in the retina.
`film. plum planar-capture mafia contact with second
`arden- neum that; send their nuns to brain uentersa
`Retinal phatamwwwm, on the ”thin- hand, am in wntucl
`with bipolar mills that, in mm, ammunimm with gm»
`glam will! and numemm inmmm that; am gramm-
`{humontaL amncrina,
`interpwxlfbrm calla}.
`(lunacy
`
`mun tbs mtla af phawnmplaum to other neurons is
`
`
`much higher in the pi aal than in the T813113, and the
`lipid compaaltian of the pineal man can be matted m
`reflect that a! ma phutmewpmr mils.
`
`AKER877ITC00070480
`
`mm mm Cumwaitim (m; M mam mam rm. Mm
`Pimnl firm“
`
`Acyl chain
`14:0
`15:0
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`{6:11:13
`17:0
`18:0
`18:1n-9
`15:1nw?
`1622116
`18:3m3
`2021:1«9
`WIRED-6
`2:013:16
`20:41:45
`Ethan-3
`203113141
`20:36:10
`film“
`23:11:43
`221mm.
`22:5n—fi
`22fiu~3
`22:6an
`34:0
`$421109
`Unidmliflm
`
`PI
`0.3
`0 a
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`33.3
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`
`31.8
`1'17
`42.5
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`'1th uturai'aci
`58.32
`drill
`15.5
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`Thu] mmmmalunted
`833'
`81.3
`3-5.2
`#33
`Thu! PUFA
`£2
`3&5
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`133
`W n~3
`W
`m 33.48
`20.9
`Total m6
`
`mama Mill {l M 63$! Lara
`
`
`
`
`‘30. phmphafidylglywoi; FL phmphmdxlinmml; PS. phmu
`phaudxlsefina; SM. sphiugmmyallm PUFA, polyunsaturated fatty
`acids.
`
`Each. of that»: palm lipid flames had a 3mm: (“arty mid
`
`mmmaificn. In PC, 1620 and 22:6n~fl wax-ca tha principal
`co panama and together amunted far nearly 60% 91'
`the tonal fatty acids 131-135th in tha yhasphnlipifl
`fThhla 2‘}, In mntram 16:0 wmpfiaed nnly 10.7% of the
`fatty maids in E011. whereas than law! of 2mm (36%)
`was thus: 312$th ubaawad in my lipid wlxam m a came-v
`qusnw, the mm! mum at PUFA in EGP {53.2% was:
`the hlgfiw: at all lipid mum Only the E0? fraction
`winners! {Imam}! are-am» haw tranmfihylnllm of 1-0.
`alk-l’—enyl linked ether chasm
`PI Was. umque amnng the: lipid clwsea in that 20:4n~6
`mm; a major mmponexxt am! amounted EM WAVE) of 0m
`fatty unifiafi whamas “Eaflwil campfiwd Md} [0%
`(Table 3}. A5 a consaqumcve, the overall ratio at" 1141 to
`11—6 PUFA in Pl was the Iuwgst of any lipid dass. The
`
`anturatad fatty acid 13:‘0 mm also a maim- f‘aatty acidm
`PI P3 was chamcmfized by a high mammal. of 18: 0 and
`22:fin~3, cwplafi with a low mutant at 1&9; PG was rich
`
`LIPIDS. Vol 29, m, 5 (1994)
`000004
`
`000004
`
`

`

`__ Mm (”613) 9523247
`
`
`Twm 4
`
`flrdfir # 050853100P04154$53
`
`Tue Nov m 14:05:01 2004
`
`Mm:
`NOV 16: ’aa
`
`if; 01'“ E
`03133321301
`
`
`LIPID WWMTIQN 0F TROLW PI HEAL flRflAN
`
`31 fl
`
`
`MNMW &W M thphmnpma Mm Wm M 0mm“
`
`Men
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`152133210
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`1192:1005
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`10200130
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`(Mi 1: (10
`1.2 a 0.1
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`Wflm m mum and m mum at m510mm amminnunm a: x $13 It mm mam that mm W saturated fatty MM“ mm
`imam :m the anl {uranium Mammy 16W Whining: mimw fatty mm mm any mid”mm“: firm mt mm {mm the:
`W mmnmm WW mums swimming 13M0 Mam with m mat-timing 2.0551750 and 33030 W phw
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`1.0 :t 0.9
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`4.6 8 (M
`
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`#3 I 0.}.
`363.1 1 40.3
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`9.15 x 0.3
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`£63101
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`
`The iipifi mutant if we mm pineal (-13% 3:! Wm;
`waight} in almmt mmiy Way between that found far
`the brain and retina {6.8 and 3.1%. mammal?) of” this:
`Mme. spades (m). Hawwm, the: level oi? TAG in than lipid
`{if the pineal {’W%) in mmiéssmbly higher Khan the 831
`and 30% {mum in this iipid at“ film brain maxi mum, but
`i3 mwflhaima 1:19am in m mm. The im 0f 139.0 in
`braim mm mm M" mu!» am him-m m M highar than
`in the same flames Mm cad (10). An influe‘msfimg {Mar
`may in the fact mm; W mm mmimd warm fax-mm
`
`fish» It. in we“ Imam that [W fink, minding mm;
`haw; mm: lipid, gunman :11 their fies!) than mix wfici
`mammal (30} mm the
`aim mum qi‘
`mural Linnea Mm him“ M! an: m fish at
`whammy higii TAG 11.5st m a specific feature: of than.
`Mpida 0f twat- neuml than“ remains m be wmbliaham
`A main“: {Mime M m “mm 0f firm and mm in
`
`annual is mm high: mmmrfim as!" E0?!” in W mm} iipid
`{153,3 1:2» In 8mm mi msi warm. the: EMPW Tahiti in Main
`iipid in uppmflmawly 1.21 mm The Wm! at“ E6? in aha
`Emmi fluid is mum? lawer than that 0? PC, rim
`EGPIPG ratio basing 0.021, This value is closer to 1110
`037:1 thinner-wad far the mm at" these two phwphnfipixw in
`
`the whim 09' mm twat amt and {10). Way. athanalamine
`
`pimalngem mutant a!“ this pimal in mush. WWI“ than
`that offiah brain (36.43% MEGP} (25) and dam in that
`at" retina (415% 31’ EMF; MI. MN. unpuhiimai mm).
`The WW: :3!“ mmhrmidea in film lipids 9f 10?“: mum! in
`aim mum twink} MI whim than 13min. M £5 thaw law
`amwm (if athmuimim nimmlagam @331} This:
`tab.
`mam «if mnfiiwfipm in Significant mm in 33mm}
`lipids is unusual m min tiyid aims in a mmu mums»
`mat mindwchmdhnd mmhmma and ham Men [mud in
`lipid BMW Emu: bath th retina am! brain (10);
`”Hm total {mid and mmpmmnt lipid dmm at the
`3mm! mam, with him minim of SM. warn inflame»
`mm by m when“ ufWFA. Namflheim. film can“
`WM anUFA in ma mm 0W1 nftmut organ {310290} is
`Inmr than flm wmapondmg mines of" 4M and 00.10%
`mmmfi fear him brain and mm, respectiwiy, mi film
`W 99pr £10)“ and 5m mnaidmmhiy 1mm than the:
`mm maxim! {43.6%} 61‘ mini ma {mm rm pinmi {20}.
`
`Thu dimer-Emmy» 97f {any Mich mm the: variwa fluid
`
`
`aim :0an in wall mfibfiahed pa
`mu FM mm
`we, than 1131’!ng mmanniu fatty acid 30:11:14}, we»-
`cmma :1an in TAG». TM abmmiofflfia fatty acid, which
`
`000005
`
`MEWS. m 20. m 6 (13M)
`
`AKER877ITC00070481
`
`000005
`
`

`

`gamma..-.amm.—m.aw¢__..-A._
`
`
`H.,-..._....._m__.m._._;._.,~.
`
`firm “(6135 952~5242
`
`flrder 9‘ 05085 3100P041345 5 3
`
`Tue. Nov 1% 14:ll5:01 2004
`
`NQV 1E!
`
`Page 7 of a
`‘84 QBIEEF’M
`
`315
`
`
`RJ. HEMBEREWN ETAL
`
`prism-Mm m talmnnid mmplankmu. is n mmmon literature
`01‘ fish phmphnlipids (32). The 191.11%. lfitfinwfi waa aim
`confined in Tm. It is nambln that. film mm}. lipid of
`pineal gland mntaina higher levels cf this fatty gem
`than brain m the rat {2ft}g almaugh it remains to be leap
`tab-fished whether the 18:331-6 is apeclllmlly associated
`with TAG. Th3 preaenm pf high levala M 1&0 in PC ill a
`cshamulamua l‘wmm (if this phmsplmlipw.
`including
`that extraqu [mm mm: brain and rating (10) and
`bowine pinafll argan (21). 13m 1920? of nwral unsung am
`lmnwn to be spadfically enriched in 22:63:43 (31), and
`this warm aim at nuLable faamm nhuwed in the present
`amid}! nf that EL}? fmm aw hunt pineal. Th3 value far
`22:6m3 in £2035" (36%) la alightly higlmr mm m mnwm
`in HQ? Mm tmut. brain (MW, but. lease: than that. at“ will
`retina EGP {46.8%} of“ the same speciag (1m. Interest-
`ingly, the 22333.3 contents at? P0, P1 and PS found here
`in the pineal an: all intermediate betwean than found
`in than brain Mill in film retina.
`This Fl a5“ ELM: pinml urgam mnfpr'mml m that: me” an»
`mbliahud pattern in {in}; wastage, whamby Pl hats at
`higher content of 20:4.11—6 than mlmr phmphnlipida and
`is consequently ch master-212d by a law ratia Elf 11-3 in [NE
`PUFA (‘32). The actual mutant. of 20:4.n-fi in the pinaal
`Pl [214% {31’ total fatty with]! lag markedly higher than
`that mwm fur trout brain (10.2%} Md mfim {14.53%}
`and for the: same blames 01‘ ml MA. and 12.5%, rupee-
`tivelyl {10% The results want that in mg of tlm
`20:4n'6 tenant of P] the pineal exceeds retina which, in
`tum, 9':de brain. In the ran, the level pf MAIL-6 in the
`mm 11de Ham the pineal aman in also high” than
`that {mud in brain liplda {20}. Thins: ip malaiem will;
`this idem mm m which phpmtmnmlwtim might M‘-
`tlvatc phoaphalipase A? with flu. subsequent formation
`of eimaanm‘da €33). An invalvement cf eiwaanoids (ls-
`rived {mm 20:41:14,;
`in 1.115 lightrdflpendent. mntml ml“
`melamnin pmtluction i5 thamfnm inflimlwi.
`maximum with that Meg-all fatty acid mmpmltim pf
`EGP. PE mislmd the highwt level (new of cllwllfirfi
`molecular apwlea of the phwpholipida examined. How-
`ever, 1:13.: valua was less than that observed firm-£11181); in
`mu: brain {H.9T9), and mmiderably lam: than that in
`trout mfim (41.3%} (1?).
`likewise. divflfi molemlar
`apaciaa of P0 we! P3 ware laws abundant m the pmaal
`than the mafia. and only pimal PC had a higher divwfll
`cunbent
`than the mneanding‘ phosphnlipids from
`brain. The {1mm molecular speciaa were all as expected,
`With 16:0?2216 and lfiflflfifl duminating m, ISMZIG,
`MAME“; and 18111323.”: aim-h wmpriaing ”RE—30.1% 9f
`PE and 1.13:W:6 being 11.1%meth m spams In
`
`mums... P: m m ll mm m be mlmimly impom
`[shad will-4 mmmmam-a mmlmm ymnloculu
`species. while Cm: PUFA species are abundant {11218}.
`In trout, retinal PI waa pmdominanlly Iflzflflméu-fi
`
`140.1%) and brain PI 18:fl&fi;fin-fi (42.3%} {1'7}. This: tip»
`
`aux: amcificity in the malwulpr minim mmpwltim of PI
`was cpnfimm in cod, in whim 1820mm4n~$ WM the: pm»
`dnmimmt apedfls in liver well me (36.? and 49.1%, m-
`mpectlvelyl and 18:]!20Mn-6 was the mm must abun-
`dant (13,29). In brain. 18:{W£}:5n-3 and lazmfilzfin-fl
`mmpfiled mam half the PI (13) with araclfidanyl spacial]
`
`UWDS. Vol. 2% m. 5 (1994}
`
`000006
`
`
`
`l6.3%. wherpaa in mum lawns,
`totalling ml);
`1mm», 1310;253:923 and lflzflffiflfi wan the ma“ abun»
`
`Jam. spexlm, in that urdar, Walling 84.5% MW {18).
`The PI from 21mm. pineal argan thus closely nae blea
`
`Wat of trout reruns with 18:ma:4:146 predmnlnam The
`130&0: 5&3 species an clmmalanstic MP1 {mm trout.
`and and brain camprlzml manly ll}3% «3f twat plum PI
`Thu. althmugh it; ill mammal mflacwt tn film 0min and it!
`very alomly mmiamd With tlw brain, the: primal in silk
`must. ‘dentlml m retina in laws of PI companion. Fur-
`
`them-Lore, no 18:1!243 wan found. in the PE of the pineal
`argan. wherwa this molacular ape-Clea mmpfims be-
`twsen 5?! and 133% of flab brain P0 (17.13).
`Mtllmgh ILM mm lipid of him mm: pinnal arias: has a.
`Imam mutant ml" wan-.3 than. that. found. in brain a:
`retina, the level is neverthelwa atlll lfigher than that. usuv
`Elly observed in Elmer nonmeruml tissues, such as. liver or
`munch E32}. Furfiwnnnm. the Efihfin—B mnlant uf lndi‘rid-
`mil phmpbmllpida la: aimilar m that; fauml in the retina
`and brain. Umqmfly. the.
`llpid pmnwmlfiwn pf the
`pineal mama cliaplays [autumn miml of mmwa llamas.
`Di-22;Bn—3 mmlmular species may be urfique tn phu-
`tomeptor membranes as them are abundant in rod
`outer segments 0f many animal apecies and have re—
`«me film been faund. albeit in mafia: nmaunm, in the
`WWW rallm M” larval barring (19). The QMWI” new
`mama at“ tlw pimal phuwmmptpm, Mulch cammpond m
`wawlike @113, m not as well dimeloped mmpm'ad to
`£35,050 of the mfinal phnmmpmr (3}, and tlm merall
`amount of mtar segment. membrane per phatmeccptur
`cell is probably mmiderably law in the pinml than. in
`film mtinu: Thin might; “plain why the. high lamla cf cli-
`mama malamlm waving whlch Malaria the: PC,
`and especially ?3 and PE, of tram: and cod retina {17.18)
`wars not. nhaemd in tha tmut pineal.
`In mnclusian, the present study demonstrates far the
`first film: that, in terms {If lipixl campasitlmn, the pineal
`mmblsss the mtina mum almly than the brain or
`Mall” timma. Phatmmpmr mambmnea cam mm M ism
`law from a pawlaulon of purified pineal calla: (34.) and
`mlynis of their mmponent 329nm may disclnm apccific
`adaptatium in lipid compositlan. This opens in