(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:20)
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1020
`
`

`

`
`
`15mm (5133 952-3247
`
`Order .# 05085310D904184553
`
`Tue Nov 16 1410510; 2094;
`
`Page 3 of 5
`NOV 16 ‘84
`93:62PM
`
`.312
`
`R.J. HENDERSON ETAL.
`
`i1'iA
`
`ga
`
`‘2
`,3.
`2i
`5%
`
`j g
`
`Fly analogy with the retina, it is probable that 22:611-3
`in a major PUPA of the lipids in the photoreceptor cells
`of the fish pineal organ. To emmine this hypothesis, we
`determined in the present study the detailed lipid
`composition of the trout pineal with particular attention
`to the PUFA content and molecular species of phospho-
`lipids. As far as we are aware, the detailed lipid compo-
`sition of the pineal organ from fish has not been re-
`ported previously and only a limited amount ofinl‘orma-
`tion is available on the lipida of the organ from
`mammal-a (20.21). Information gained from the muslysil
`of the trout pineal organ is ofbaaic importance as roan-v
`maiian pinealocytos are sooopted as being phylogeneti—
`Cally derived from the fish pineal photoreceptor oelll (2}.
`
`MATERIALS AND MEN-1005
`
`Fish and pineal organs. Rainbow trout (Omorbynchus
`myizionl of average weight 800 g were obtained from a
`commercial fish farm (Piaciwlture Bellet, Angoulemo.
`Frame) where they had been maintained under natural
`mnclitiono of water temperature and phoiijfiriod. One
`hundred fish were killed by decapitation. Pineal organs
`were removed immediately from the fish, frozen in liquid
`xfitrpgen and stored at —33'C until taken for anniyido.
`Chemicals and solvents. Phospholipaae C from Bacil-
`lus cereus was purchased fmm'Boehringer Corporation
`{London} Ltd. (Ewes, East Sussex, England). Omiyl.
`chloride and anthraoene 9-carboxylic acid were supplied
`by Aldrich Chemical Co. (Gillingham, Dorset. England).
`*1! nC-l-scv Chan-“.1 nlc n-nll binnhnmu-nln “39'“ nurnl'nnflnl‘
`from Sigma {Poole Dorset, England), and solvents of
`loghaperformance liquid chromatography (HPLC) grade
`nnnnn ”kl
`nal fun
`Dn- Lukum. FLA-":1tfllflrl‘wILn—kn-n
`"In: uuuulleu sculu Ilablsumu unuuunhma \lrnlwsuui u,
`Poeblcasbire, Scotland)
`the
`Lipid extraction and analysis. After thawing,
`pineal Grit—LEE were Wiig‘ncu Kiln ufifiiiar—Fed w i Teflon-
`in-glaas homogenizer. The organs were homogenizedin
`37 mL of chloroformlmethanol (2:1, volfvol) essentially
`ea (jaunt-ed by Christie (22-) to entree: Lipid; Solvent
`was removed under a stream of nitrogen and the result.
`ing lipid extract desiccated overnight under vacuum in a
`PAI— "Vs-151m vu
`any
`gun nus1M
`n-eulln-'n'hnad Inlan-
`A inwlxn nurl Mn‘n‘l’l+fl we": Ellie-n-
`reweighed to obtain the weight of the lipid extract which
`-2-
`an-I‘ L-
`was redissolved in chlomfomu'methanol (2:1 volfvolJ
`all.“ 5“?!er IifluflI in au—fifiifnierE 01 mhrugell £1.- —I'U \J “C'
`tween analyses
`To establish the lipid class composition, aliquots of
`lipid Hyaci- were subjefid to high-perfomnnce thin-
`Layer chromatography {HPTLCl alongside authentic
`standards using hexaneldiethyl ether-{glacial acetic acid
`
`(onion-1o hi unit an thin hmlnnlnn nnlunnt Int- Hm Annu-vunn--u—u
`
`ration ofneutral lipid classssmand methyl ocetotelpro-
`pan-Z-oll'chlorofomlfmetbanolf0.25% (wtfvoll at]. KCl
`(25:25:25:10:9, by vol) for the wparntinn of polar lipids.
`To confirm which polar lipid classes were present,
`aliquots of total lipid were also subjected to twodimon.
`alone] HPTLC. The polar lipid developing solvent sys-
`tem described previously was used for development in
`the first dimension and chlorofonn/acetone’methanoll
`acetic acid/water (1024:22221i by vol} was employed for
`
`development in the ascend dimension. Developed chro-
`matograms were visualized with copper acetate in phos—
`phoric acid (23). Lipid class composition was quanti-
`tatad by double-development HHLC coupled with scan-
`ning denaitornetry, as described elsewhere (24).
`Estimates of the relative amOunta of the plasmologen
`and the discyl forms of ethanolamina glycorophoapho-
`lipids (EGP) were obtained by acid hydrolysis of the iso-
`loted EGP in situ on an HPI'LC plate followed by chro-
`matography and quantitative phosphate staim'ng as de-
`scribed by Bell and Dick (25).
`For the analysis oi" fatty:acid composition, individual
`“Pia cusfiéfi Win—”E reparfited Dy EWI-P'dil‘l'léfh'fluml TLLI LII-l
`20 x 20 cm gloss platen coated with silioa gel G 60 (0 25
`mm thick) using the solvent system described here. The
`separated (1135338 were visufllmeo Dy spraying the chro—
`matogram with 0 1% 2',1‘—dicblomfluoreooein1n metha-
`nol containing 0.01% butylated hadroxytoluene and by
`viewing under ultraviolet light. Tfiacylglyooroio (TAGS)
`were purified by redeveloping the chromatogram in the
`reverse direction of the second development using
`1.. ..... Ll. A‘L
`“H. ”IL.-.'-1_--A..'- ‘- :J {In[two I“. A“
`llcwuauiuuljr's Guiana“ m—im awn“; uflu Lou (Ann, L!" v'us;
`after removal of the individual polar lipid clasaes. The
`fatty acids of the separated lipid classes wen: converted
`to their methyl esters on the adsorbent by arid—cot—
`alyzed transestarification (22). An aliquot of total lipid
`was also subjected to the name procedure. The resulting
`fatty acid methyl esters wen: purified by "mm and re-
`covered from the adsorbent with hexane/diefliyl other
`(1:1, volfvol).
`Fatty acid methyl esters were analyzed on a Packard
`439 gas chromatograph equipped with a fused silica cap~
`illsry column (50 m x 022 mm id.) coated with FFAP
`\ua—hu-I. 15:.“th IwJuca, usurious LHIIEHUI-Ill. Ul-
`All-mine (C: I". (1" “HAn Van-non TTnil-nll v;nflllnrr|1 Rum;
`ple application was by ummlumn injection, and hydro-
`gen was used as the carrier gas. During the course of an
`analysis, the oven temperature was pmgrarrmed to in»
`crease from 50 to 225'0. Samples were also analyzed
`using an Omegawex 250 fiised silica column (30 m x
`l)25 mm i(L fingelehem U.K. Ltd" Essex. Unitgi fing-
`dom} with the oven temperature programmed from 50 to
`2°60C. Fatty acid components were identified by refer-
`ence to a woll-chamctrmized fish oil fatty acid mixture.
`and the unsaturated nature of components was con-
`firmed by tie-analysis of samples after catalytic hydro-
`genation over .PtOz 'l‘ho %mrated components were
`quantitstad using a recording integrator linked to the
`chmmatograph.
`Analysis ofnwleculor species. A {loo-pg portion of total
`lipid was separated into the component phospholipids
`by HPTLC alongside 20 pg of a cod retina total lipid
`sander-:1 us..._imr methyl occtatrjpropanvz-oychlorw
`formhnetbanow25$ (Moll aq. KCI (25:25:25:ID:9, by
`vol) as the developing solvent. The standard spots and
`the edge of the hands. of pineal organ lipids Wen visual-
`ized by exposure to iodine vapor, and the bands of ad-
`sorbent, containing PC, PS, phosphatidylinoaitol (PI)
`and EGP, were scraped from the plate. The phospho-
`lipids ware hydrolyzed on the silica. with phospholipace
`C using a two-phase system of 1 mL diethyl ether and
`1 mL 911.11 M opium borate buffer; pH 15 at room tony
`
`
`
`I 1
`
`woos, Vol. 29, no. 5 (1994}
`
`000002
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:21)
`
`AKER877ITCOOO70478
`
`

`

`lfrom (613) 952—8247
`
`Order # 050853100904184553
`
`Tue NOV 16 14:05:01 2004
`
`Page 4 9f 5
`NOV 16 ‘84 @3382?“
`
`313
`
`”Pm COMPOSITION OF TROUT PINEAL ORGAN
`
`
`
`‘
`
`, parature under nitrogen for 2 h (26). At the end of the
`incubation period, 1,2-diradylglyoerola were extracted,
`dried down under nitrogen and finally desiccated under
`vacuum for 1 h. 9-Anthroyl chloride was prepared from
`panchromatic carbowlic acid and onlyl chloride as de—
`embed by Goto er a1. {27}. The diradylglycerols were de«
`rimmed and purified by a modification of the method of
`'I‘akanlura and Rita (28) as described elsewhere (19}.
`- he 1.0401,;- ,'-enyl-2-acyl derivatives were removed
`during the final HPI‘LB purification step. The 1,2-dia-
`cyl-S-anthmcyl-on-glycerols were separated by HPLC at
`19—20‘C on an ODS column (25 :4 0.46 cm. 5 pm particle
`size; Beckman Instruments UK. Ltd. High Wycombo.
`Bunkinghamshire, United Kingdom) using a Pye Uni-
`cam P114010 pump (Pye Unicorn Ltd... Cambridge.
`England} and two iaocrafic solvent systems, methanol}
`propan-Q—ol (4:1, volfvol) at a flow rate of 1.0 mUmin,
`and acetoniu'ile/propan-Z-ol (7:3, vola‘ml) at. a flow rate
`of 1.0 mlfmin as described by Takamura and Kito (28).
`Peaks were detected using a Waters 470 scanning fluo-
`rescence detector (Millipora UK Ltd. Edinburgh..Seot-
`land) with excitation and endeaion wavelengths of 380
`and 4.60 nm. respectively, and quantified using a Shi-
`madzu CR3A recording integrator (Annabella. Lubon.
`United Kingdom}. Peaks were identified by their mie—
`tiva retention time using 16:0f22:6n-3 as a reference
`peak. Di—docosahexaenoylglycerol (di-22z6n—3; Nu-Chek-
`. Prep, Elysian, MN) was also available for direct com—
`parison of retention times, as were a range of samples of
`known composition from previous studies (17,18,291
`Each sample was chromatographed three times in each
`solvent system and the standard deviations calculated.
`More final peak areas were calculated by subtraction,
`the standard deviations of the contributing peaks were
`added to give the final error.
`
`RESULTS
`
`The lipid content and lipid class composition of trout
`phwal organ an: presented in Table 1. Around 4.9% of
`the wet weight of the pineal organs was lipid, of which al-
`most half (47%) was in the form of TAG. PC was the
`major polar lipid present (16.5% of total lipid), followed
`
`TABLE 1
`
`
`Lipids of mm Pineal Org-n: Lipid Clue Competition“
`
`Lipid clans
`‘le Tblal lipid
`chnhewyl eaters
`8.3 e 0.3
`Tfia'J‘fiilfili
`Eli I ii
`Free atty acids
`8.4 z 0.0
`Cholesterol
`8.7 :t 0.4
`Diarylglymrnls
`1.5 = 0.1
`Elhanolamino glycorophaapholipida
`9.9 n: 0.6
`thphatidylglyoaml
`1.4 :a 0.1
`Phosphatidylinnailol
`3.2 = 0.3
`F‘hoaphaudylserine
`3.4 t 0.3
`Phoenhatidylcholine
`16.5 1 0.8
`Sphinggmyelin
`1.7 t 0.1
`“Vain“ m moans 2 SD of flame determinations.
`
`by EPG (9.9%}. No choline plasmalogeos were detected,
`whereas ethanolarnina plumalogena accounted for 11%
`of the total EPG fraction. PI and PS each accounted for
`loss than 5% of the total lipid, and both phosphatidyl-
`glycerol (PG) and apingomyelin (SM) were present at less
`than 2%. No cardiolipin or cerebmsidea were datected.
`Palmitic acid (18:0) comprised 23.7% of the fatty acids
`in the total lipids and was the most abundant fatty acid
`in the pineal organ (hole 2). The monounsabxated
`18:1n-9 and polyunsaturated 22:6n-3 accounted for 17.6
`and 12.4%, respectively. ofthe total fatty acids. Overall,
`saturated. monounsaturated and polyunsaturated fatty
`acids accounted for similar proportions of the total lipid
`fatty acids. The fatty acid composition ofTAGs (Table 2)
`was generally similar to that. of the total lipid although
`the proportion of 18:2n—6 (12.8%) was notably higher,
`and that 01’1620 lower, than in total lipids.
`
`TABLE 2
`
`Fatty Acid Composition {m} of Total Lipid and Lipid
`Classes from Truut Pineal Organ"
` Any] chain
`
`'[lrLal lipid
`TAG
`PC
`EGP
`3.9
`3.5
`0.6
`0.2
`14:0
`15:0
`0.9
`0.3
`0.3
`0.9
`15QDE‘A
`W
`.....
`-—
`9-5
`15:0
`23.?
`11.4.
`34.91
`10.7
`16:1m9
`--
`--—
`0.S
`._
`16:1n—7
`5.8
`8.0
`1.3
`0.7
`17:0
`0.6
`0.4
`0.3
`0.3
`18:0DMA
`—-
`-
`—--
`0.4
`187.1no9DMA
`—
`—
`—
`2.2
`13: ln-mlu'ix
`—
`—-
`—
`0.7
`18:0
`7.2
`4.2
`5.4
`10.2
`15:1n-9
`17.6
`18.5
`1.3.3
`10.3
`183.34
`4.0
`3.7
`2.4
`3.9
`15:2n-6
`9.3
`12.8
`1.3
`3.4
`18:311-3
`1.2
`2.0
`—
`0.3
`20:1n-9
`2.5
`3.1
`0.4
`1.4
`2.032116
`0.6
`1.2
`031
`0.9
`20131145
`0.4
`0.2
`0.3
`0.4
`20:411-6
`1.6
`0.5
`2.0
`7.2
`ifiziiowi
`——
`0.2
`—
`—
`20:4:1—3
`0.9
`1.4
`0.3
`0.6
`20:5!1-3
`3.5
`3 .9
`4.6
`4.8
`afizlnall
`1.7
`2.0
`=
`=
`22:1n-9
`0.3
`0.3
`——
`——
`22:21:35
`0.1
`0.3
`-
`~—
`22:5n-6
`—
`0.2
`0.2
`04
`22:52:45
`1. 2
`1.3
`1.3
`2.2
`22:6n-3
`12.4
`12.6
`24.6
`36.0
`M: Ln-9
`0. 7
`0.1
`—
`0.4
`Unidentified
`0.0
`1.4
`0.9
`0.7
`
`Total saturated
`'Ibtal inseam-saturated
`Tbtal P‘UFA
`'l‘dtal n-S
`Total 11-6
`main-6
`
`36.3
`32.5
`81.2
`15.2
`12.0
`1.60
`
`25.5
`35.7
`37-1
`21.9
`15.2
`1.44
`
`40.6
`23.0
`35.5
`30.8
`4.7
`6.55
`
`23.3
`[9.5
`56.2
`43.9
`12.3
`3.57
`
`‘EGP, ethanolamine glycerophoephalipida; PC. phoephatidyh
`choline: TAG, hiacylglyeamls; PITA, polyunsaturated fatty acids;
`DNA. dimauw”laconic.
`
`
`
`33;:
`
`.-
`
`‘
`
`ii
`
`3i
`
`,
`
`000003
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:22)
`
`UPI 3. Vol. 2o. no. 5 (find;
`
`AKER877ITC00070479
`
`

`

`firm (513) 952—324?
`
`Orcievr # 05085310N‘04134553
`
`Tue Nov 15 14:05:01 2004
`
`Page 5 af 8
`NOV 16 '01:: 63:62?“
`
`314
`
`TABLE 8
`
`EU. HENDERS‘DN STILL.
`
`www...x‘ewiyawlw.mwe”.».m.,
`
`in monounsaturated fatty acids and PUFA whinh mm-
`pnsad 44.1 and 31.1%, mpeallveiy. of the total fatty
`adds in min phoapholipid (Tabla 3). The levelw all 16: 13.?
`and [3:1n~7 (1?.0 and 10.2%} in PG were: higher than in
`my other lipid class, and, of all the phnsphnlipids PG
`had the highest. mutant of 18:91:06. In SM mare than
`half (58.2%} Of the fatty acids were manmxnsalumwd,
`mainly dun w the premnee (if a very high pmporticn of
`2421 (45.9%). 931% wmgfigefl 99.13 39% m" the SM fatty
`acids (Table 3).
`in small
`In all
`liyid class-es. 20:511-3 was present.
`amnunta and newer umdad 5.0% of the «Important
`fatty acids. The long chain monoenaic fatty acid
`22rln~11 observed in total
`lipid was wnnentramd in
`TAG, whats it acmunmd for ammd 2% of the fatty
`grids. Of all the lipid dame-8.. TAG also cantalnad the
`highest. levval of 14:0.
`The primpai malecular speciaa of PC, P'E= P6 and PI
`are pmnlaed in Table 4. Tire: molecular species pre-
`dmfinated in 9C. namely 161M2£n~3 and lfiflilszl,
`which marital for 33.2 and 28.5%, rwpecfiwly. of the
`total molecular awe-ties all” this phosphollplel. lXP‘UFA, dia
`what-Mod and fimmmaatummd special each coma
`prinedloosthanfi‘flwoftheumlmnndmamnmtof
`mnomafumtedeUFA spasm totalled 4.9%, wiflfin
`which [Emmfimfl was the majw component. Malecular
`spades! remaining 22:6n—3 were particularly abundant in
`PE. Di-22:6n»3 mmpriwd 13.0% uf the total, and
`warms anti gamma. were bath present at. levels of
`mare than 15%. In PS. 18:0mrfiw13 amounw fur almost
`half (43.9%} of the total malecular sparks, and
`16:032611-3 was the only ather species present at a level
`mr greater than 10%. Th: mam. abundant molecular
`ape-ciasaf PI was! l8:M0:4n-6 which amounted for 37.5%.
`Mather spacing machining 20:4nn6, [Gsw02m~6. cum
`prim 14.0% :35 the mnlwular ageuiaa at” P1 and
`mmmsw munwd for 10.8%, the highest level for a
`apecies containing 20:511-3 observed in any of that phon-
`plmlipida examined.
`
`W A
`
`lllmugh the: pineal organ i5 an adjunct of tha brain, it
`in kxmwn to have evolved From a wall-djfl'eremiawd phu»
`wreceptive organ that. in fireauenfly considered to he a
`fimctianal Uu‘rd eye in lower vertebrates (3). In fact, to
`date, the [impel-flea establishad far the pineal photore-
`mpwm have been extended to the rgtinal phobomaepwrs
`and aim: meme; (3}. The majar Menace batwmm the
`pineal and asthma relates to the: neuronal organization,
`which is simple in the pineal but complex: in the retina.
`film. pineal phuhmpwra mane mutant with second
`order neumns that send their arms to brain renters.
`Retinal phammwpmm, on the lather hand, am in contact
`with bipolar calla that, in tum. ammunicaw with gar:
`glint: cellar and nummua inmmw that am pregam-
`{hm—imam}, amacrins. mwmaxiform calla). Come»
`quently, the ratio af photoreceptor: to other memos is
`much higher in the pineal than in the retina. and the
`lipid compmaman of the pineal man can be expected to
`reflect that of film phowrewpmr mam.
`
`AKER877ITC00070480
`
`mu...“ “mama“
`Fatty Acid Composition. (M) a! Lipid Clams m Trout
`a m v-3“...
`
`Acyl chain
`14:0
`1620
`15:0
`1321:1«9
`16:11:»?
`17:0
`15:0
`18: En—‘E
`18:11»?
`lflz2n~6
`1323:!«2!
`Elma—$1
`WEn'G
`2:013:16
`20:41:45
`20:3:1-3
`20:4.n-3
`Qfizflfivg
`22:1.rv11
`22: 11143
`22:42’r5
`22:5n-fi
`22:5u~3
`”fin—3
`24:0
`Milwfi
`Unidmfifisd
`
`
`
`“mm.-wu—MW~-__.._.....u.
`
`Pl
`0.3
`01
`34.0
`«-
`a-
`(Ml
`3.8
`5.3
`1.3
`1.5
`(In!
`{1,3
`0.2
`0.3
`2?.4
`..
`9. I
`4.?
`-~
`W
`9.5
`-—
`(1.4
`7.0
`‘w
`0A
`0 4
`
`PS
`03
`0.7
`3.0
`‘w
`9.3
`0.4
`38.3
`5.5
`2.6
`1.4
`0.4
`1.2
`0.0
`-~
`1.0
`-—
`«a
`l 3
`w
`....
`3.9
`0.4
`3 0
`29 4
`-
`-...
`0.6
`
`m
`0.8
`0.3
`10.?
`m
`1?.0
`0.5
`5.4!
`15.5
`16.2
`8.8
`0.9
`1.4
`1.!
`m 0
`I 8
`-
`0 5
`k 8
`M.
`...
`Q 9
`—
`fl '3
`19 S
`m
`-»
`Ll.
`
`SM
`(1.5
`0.4
`19.2
`m
`0.5
`0.6
`9.5
`83%
`1.9
`2.2
`w
`0.4
`LB
`:0
`0.5
`-
`0.2
`0.9
`w
`0.6
`w
`—
`0.2
`2.9
`0.3
`45.9
`1.3
`
`313
`11.7
`42.5
`{9.3
`'1th saturated
`58.2
`“J
`18.6
`‘72
`Thus] mmmmalunlad
`5.9
`311
`38.2
`$3.2
`M PUFA
`4.2
`23.5
`34.1
`13.3
`M :13
`4.?
`13.6
`4.1
`20.0
`This] m6
`0.80
`1.73
`3.31!
`0.435:
`n-flmefi
`‘PG. phmphafinlylglmml; PI, phnaphuudylmuriml‘, 1P5. phm~
`phaddylsefine; 5M. sphiugumyallm; PUFA. polmaalumted fatty
`acids.
`
`Each at the polar lipid clma had a apacific fatty acid
`campoaitian. In PC, [6:0 and 22:6n—3 were. the principal
`components and together scrawled for nearly 60% 9f
`the total fatty acids present. "m this phmpholip‘id
`(Table 2}. In wntraa’t. 15:0 comprised nnly 10.7% of the
`fatty acida in BOP, whereas um travel 95 22031.3 (36%)
`mm the highwt nbaawed in. any lipid clam M a come--
`nuance, the mud mum of PUFA. in E9? (53.2%) was:
`tbs highest at an lipid clause. Only the E0? Mullen
`prom-109.6 dfmaflwl “am; by unnamgmfiallm of 1-D.
`alk-1’—enyl linked. ether chains.
`PI was unique among the lipid classes in that. 2014x143
`was a majm‘ mmponem and unwanted fur 27.4% of the
`fatty acidr, whamw 22:62:43 campfiwd may 70%
`(M13 3}. A5 a consequence, the overall ratio at" 1145 to
`:16 P'UFA in PI was the lowest of any iipié than. The
`saturated fatty acid 1820 was also a major fatty acid in
`PE. P3 was characmfimd by a high mntant 0" 18:0 and
`22:6m3, caupled with a law mutant of 1610. PG was rich
`
`UMDS. Vol. 2a m, 5 (1994)
`000004
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:23)
`
`

`

`me (5133 952-an?
`
`
`Ordar # 05085 310119041845 5 3
`
`Tue Nov 16 14:05:01 22094
`
`Faga 5 of B
`NOV 16 ”fit?
`6135329“
`
`31$
`
`LIPID WMPOSITIQN 0F TRGW WNW 085W
`
`TREK J1
`
`
`
`118 at 19.0
`{.3 1c (M)
`2.4 3 (H;
`
`0.7 x £12
`L8 :2. 0.3
`16$} 3: ‘03
`11.8 t {M
`LI 1: #111
`35“ a: $13
`%&9 a 6.2
`(51$ 5: 0,].
`‘L‘? 1: [M
`
`0»? ’1; 0.1
`w
`{3,3 a M)
`”all!
`1.3 3: D.)
`
`Li a C4,]
`
`23 t 0.!)
`M! :2: M)
`
`m
`0.3 x 0.0
`(U :f: 0.0
`
`0.2 at CU
`4i a EM?
`1113 : $3.5
`M.“ ;_ 5A3
`‘16 x: 0.52
`1113 a: 011
`7‘1 3.: 0.5
`37.5 : LC!
`{M u 0.1
`
`{3.3 : 0‘0
`‘13.? x {M}
`{M} t ELK
`41::412
`-
`
`w
`
`543' a: 6.3
`0.3 a 0421
`
`
`Mflmw 18pr M Fhmphuflplds Wm Max M flrsflm‘
`
`Ema
`P‘G
`my
`55%
`n
`DS-PUIM
`28:5?236
`3235/22.;fi
`22312113
`SammeA
`Hflf‘l'éfi
`£51W9£
`mamms
`Ifitfimflcfi
`16:3'22‘5
`msmm
`13:31:???sz
`lflfl'fifla
`18;W:5
`MDMWWHWLWA
`13:33:35
`ifiilflflfi
`mmaza
`18;?!2324
`Wfifllflfi
`Dininturuhd
`E5JW16fl
`Sazmaudfmammnmm
`1&WI3:1
`l8flfl8:1
`Dimaaumacwaicd
`W}. t 0.0
`1.2 2 8.1
`0.6 a: 0.1
`0.8 x 0.1
`18.11181
`
`-
`{3336”}
`..
`._..,
`m
`2D:1f18:1
`MJKIW WV [HUIN in?“ mi: WVflni W WERE“?! “WIT"{HEHHHK it; A a“ 11 W“ «sum Wt um M “WNW “Half Mimi} WILD“!
`bread cm the sml 993mm Mnlewhr 5W manna Mm any and; at aka-y madmm“ arm mt maimed {mm W»,
`W mmnmm Mamba: modes mm in:mm madam mm mm mnwnm flfima and 23:51:43 W: th
`phafidfichakmvs. PE Wmvhaudflmhamdmm; P! nhmgnmwmmimt. vs macaw-imam; FWA, mimnmmmmd
`fatty mm
`
`0.6 x 010
`m x 0.3
`1.6 :1: 0.1
`
`if} A: (117
`8K7 I 0.1%
`33.3 : L6
`L6 3 0‘3
`3‘1 2: (m
`1.2 :r. 0.2
`337 a: {3,2
`035 :t 0.1
`1.6 an 0.52
`
`0.5 a 111
`M} x: (1.1
`2.9 a: 1.?
`-—
`£16 a: M
`
`m9 5' 0;}
`
`2&6 3; L0
`2.5 :2: $3.1
`
`1.0 x 0.8
`13.0 a 0,3
`4.5 s 01.3
`
`38 1563.!
`4.2 I 8.1
`3-0.1 1 8L!
`.23! x: ELI
`1.51 2 EM
`8"? a £11
`9.6 t 3.3
`2.7 x 0.!
`0,6 t 0.2
`
`0.9 a 0.1
`3‘? 3': 0‘}
`151» t (M
`2.63%1
`M 2 (H
`
`0.1 1 0.0
`J
`3.2 t {M
`0.2 a: (3,1
`
`A m.
`...........
`j t-..
`The iipid content 11? the tram pineal H9% of we:
`Wfiifihw:5 air-I‘m?“ mail-"ta? hfiuwu) wuwuvfi unfit. Iflfifiu WI“
`the brain and patina (6 8 mci 3 1% respectively) of that
`name apaciea {102). Howemt, the: {we} of TAG in the lipid
`af ihc: pinEai {437%} is: mnuiégmbiy higher than tha Swain
`and 30% fauna in the lipid (If the bmm and mating, but.
`m mvenhalms draw in the: mm. The mm a! TAG in
`mm and man of trout am known M M "higher than
`in the ewe tissues {mm and (10). An influencing ram;-
`MA
`t-k».1 =_L
`.. n-..L
`may be the fast that the mm «Examined mam famed
`W11 AL n? WU“ KJHJW" Hint m nan), “Niall!“ m““E,
`have higher lipid minimum :11 Meir flea}: than than wild
`wunmmm (am Wham the lipid cim manhunt of
`new: annex fiifim Minoan Niki um‘ {Emmi fifih at
`whether high TM} Imam m a specific feature: 01F the:
`lipids; of trout neural timmes» remains to be establifihedg
`is nombie feature 0‘? the brain 01’ fish and mm in
`general is mm high pm'parmn at Bf}? m, the 3mm lipid
`( 11.2231)» In bath mfi mm mm, the: ESPM mtm in brain
`31in is afipmmawb‘ 31:1 {W}. The: 25am} 5‘? EFL? is: the
`pmeai
`Iipfid is nutabiy Ewe:
`than that of PC, 31m
`EGP/PC ratio being 116:3. This value is closer to the
`RH Dhfleflifia fur the mm» (If these twa phwphoiipiiia in
`
`the mum 12f both Mat and «ml (10). The. athancv‘iamine
`fiwmfiagea mfitfifit a?” this pimai is film-‘1 Btu-aria? than
`that of fish brain (3648% MEG?) (25) and flame: m that
`of retina (tfifi af ESP; Ml. MM. unpublisheai data).
`T‘na Wm m” cerahxmfiw in tins iipida m? 13% pineai 5m
`aim mare typing} all retina than brain. as is tha law
`amwnt of ammmmne mimmflagenn (31}. The ub-
`51mm of wrfiinifipm in significant mutants: in pineal
`Eixsidn is unusuai an mm mm chase in a common mmpm‘r
`meat 42! mitochtmdfin] membranes and has been founé in
`E‘i'pid amm‘i fmm {nah Push WEBB and Maia am;
`'I‘he total Ikpid ami component lipid dams ml” the
`pineal organ; with the: mmymon of SN! were cit-Imam
`mm by high mm“ ofW‘Fn. fievmfimims, iha room
`mm; of PUPAm the total 1mm aftmut organ £31 2%)13
`law: than this wmaponding vaiues of 41A and mm
`mmmfi far the: “brain anti mm, mpectiw’iy, 0f the
`ma memes (M3) and m wwiderahly 3%» than the.
`MFA watt-213M1436%)01“ mm} Livia (mm mi pineni (2m
`,— ”3‘...
`1:..11
`3W5 Wefiuuwn in said?" Wufi mung My; ‘wuf'w-‘w “ii-am
`chm £811qu to wall mtmblisbed magma For exam
`pi», the Mug-«main mmamfin fatty acid 22:11:41 my
`mma only in Tfififi ’I’m ammafihis {my acid, which
`
`000005
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:24)
`
`BMW mm no» M15514)
`
`AKER877ITCOOO70481
`
`

`

`,.~W“-.—..-;.a.»a".uH»
`
`.....4-.—.....____._.______.....‘....
`
`From *(6135 952~5247
`
`0rder # 05085 310DF‘0418455 3
`
`Tue Nov 113 14:05:01 2004
`
`Page 7 of 8
`NOV 1E ’Bd
`6319121901
`
`315
`
`KJ. HENDEREON 5T5);
`
`originates in calmmd zmplankmn, is a common feature
`0f fish phmghnlipids (32). The: PUFA 1812:1415 was also
`confined to TAG. It is newbie: that the mm! lipid of
`pineal gland mnmina higher levels of this fatty acid
`than brain in the rat (20}. although it. remains to be es.
`mblished whether the 18:2n-6 is specifically associated
`with TAG. The presence M high levels nf 16:0 in PC la a
`characteristic: feature of this phospholipid.
`including“
`that wxmacwd fmm Wu: brain and retina (10) and
`bewine pineal urgan (21). The EGP of neural tissues am
`known to be specifically enriched in 22:6:1-3 (31}. and
`this war: alsn a numble faamre observed in the presmt
`study of the ERG? fmm £318 trout pineal. The value fur
`22:6n~3 in E0? (36%) is alightly higher than the content
`in EGF 9mm tmut brain (Mm. but less than that of the
`retina EGF {43.3%} of“ the same species {‘10}. interest—
`ingly, the 22:6n~3 contents at? PC, PI and PS found here
`in the pineal are all intermadlate between those found
`in 121143 brain am! in Lbs retina.
`The PI a? the: pineal urgan mnformed m the well am
`mbliahed pattern in fish flames, whmby PI has a
`higher canterfi af 20;-‘m~6 than ether phmphfilipida and
`is consequently sch aracterized by a low ratiaa sf 21-3 to n»fi
`PUFA (32). The actual content. of2014n-6 in the pineal
`Pi {214% of total fatty fluids») is markedly "higher than
`that rammed {N trout brain (10.2%) and mum (14.3%)
`and for the. same biauuea of cm (4.4 and 12.5%, reaper:-
`tiveiy) {10). The results suggest that. in teams of the
`20:4n-6 content of PI the pineal exceeds retina which, in
`mm, anaemia brain, In the rat the level of 20:4&3 in the
`A, n? l:_.‘
`A-
`:vtntuuwdfi uui’fi “[6 F111“; Ulifloll "I m lSiglbfil “WK
`that found 2;} brain lipids £20) Thiam mayhem with
`film idem mailing to which phamhmmsdwtion might. an»
`fivate phosphalipaae A2 with tho: aubnequani formation
`of eimaanoida €33) An invalvement 0f eimsanoids de-
`
`nvod {mm 20:411-6 in the light—dependegt control of
`melatonin pmducuon is that-atom mfijcamd
`Commm writs the overall fatty acid mmposinmn at
`1501”, PB «unstained the: highaeat level (13.0%} of di~22:6
`malawlar amass af the: pl‘mphfilipidi magnifier}. "How
`ever, tbs valua was less thaa that observed previously in
`trout. brain (14.9%), and maulderably less than that in
`trout minim! (111.3%) (1‘73. Likewiae. 6.1423 molecular
`spacial; of PC and P8 ware. km abundant in. the pineal
`flan the Justina, wd only plum} PC had a high” (022:6
`cunhent
`than the correfiponding‘ phosplwlipicia from
`brain. The Othfl! molecular species were all 83 expected,
`nn‘ ran 113-1.“
`with 16M2:6 and 1620218 I flarninating FE 1613/2216
`18‘:“‘23.; 3110 13:mm:6 3%“ Wl'flpmiflg' U 0"le70 0‘
`PE and. 13:0’22:6 being the predominant P8 species. In
`mums; P: m m in gnaw m be mlutimly impov‘u‘
`IMP?! win ffiim“ W "ma m‘uxmfii mmmxu'
`species, While C29 PUFA species are abundant (17.18).
`In trout, retinal PI was pmdominanfly l0:W20:40-6
`1411.11”) arm Dram II'L LQiUJ'ZULDn-d (42.3%! {All}. 11119 ”5*
`
`we amclficity m the molecular spaciw mmpasltion of P!
`was confirmadm md in. which 18: 01:20411-6 was the wee
`Anmmnny “um: no :m 'man! and m ('11: '7 M"! An 1a. ~_
`mu map“at; u. .H‘m mm .w
`m. wanmm, H»
`apectiveiy) and 18:1120:«in-6 was the next must abun-
`dant (18293 In brain. 18:0&0; fin—3 and m:U20:5n-3
`__...:..-
`.1" 5L- “h?!{1¢\ ”.‘dl. -...-
`WUIUIJI IBIZU U‘V‘uf {Lia-ll tIKE 1" K 0” Hum 111ufruuflmx spuuu'ifi
`
`totalling may 16.3%, whemaa in retina 16:0/22:6,
`18.0204. 18:02:06 and 10202025 ware the mast abun-
`dant. specieg, in that order, totalling 04.5% a! PI {18).
`The Pi from trout. pineal nrgan thus closely resembles
`that of trout retina with 18:0f20mm6 predmnmam. The
`301012015043 species an charaawristic of FI from truut
`and and brain campfiscd nnly £0.39?) at’ trout pineal PI.
`Thug, althaugh it in aituamd adjavmm to the brain and is
`very cleanly ”minted with the brain, the pitwal is a!»
`must identical my ratina in harms of PI mmpoaicion. Fur—
`thermore, no 1819243 wan fennel in the PC of the pineal
`argan. whereaa this molacular species comprises be-
`:wwn a? and 3.3% ofz‘iab brain FEE (17.18}.
`Mflmugh 0% mm lipid 9! m trout pineal arm“: has a
`lower content. af man-a than Hurt found in brain or
`retina, the level is neverthelesa still Haber than that usuv
`ally observed ‘2: Elmer non~neural tissues, such as. liver or
`muacle (32). Furthermore. the 22:611-8 content mi individ—
`ual phoapbnlipma is similar m that found in me retina
`and brain. Cmmux the lipid cmwafifian 0f the
`pineal organ diaplws faatuma typical of manning tisama.
`Mafiifinfi malecuiar species may be unique in p'nu—
`‘wmephor membranes as these are abundant in rod
`outer segments of many animal apecies and have re-
`cently aimmn faumi. aiine‘n: in amnflar amounts», in the
`allwne retina of larval herring (19). The out/er Beg»
`mantra of the pineal phawmwpmm, which correapond to
`mnedike @1503, am not as well cleveloped compared to
`those of the retinal phoneme-emu: (3), and the overall
`amuunt of outer segment. membrane per phnioreccptor
`cell is pmbably cfirfiéfimlaly less in the pineal them in
`the retina. This might; uplain why the high Levels of di«
`22:05:10 mlamlm species which dwmctar‘iu the PC,
`and eepecixmy % and FE, oftmu‘t anti and neutral €17.18}
`were not ohaemd in the. trout pineal.
`In conclusion, the present study demonstrates [or the
`first time that; in terms of lipiul composition, the pineal
`mmbles the wane more tslmly than the brain or
`athar timwm Photarmpmr mambmnea am: new be iao»
`luwd AKUm a mefiljull GIfpu‘lilm‘i runner: New magi} fifid
`analysis of their component lipids may discloser specific
`adaptations in lipid composition. This opens interwting
`pampw‘cives fur the study 01' the fighifiependeni Signal
`production \by theme calla.
`
`85m
`
`‘l, Hawker. M, {1935} in Phomwiodwm “datum: and (A:
`film! (Qatari. 3.. am! Clerk, 3., m.! 9.1".
`'3'8-92, Pimmn,
`Kantian.
`i. Collin JP Valium, Y Falcm J Film. JP 311mm R. and
`A Kiel-hand R. 1198mm}: 31;"qu Cytnl 53.4451-Mfi
`a. 3“me lmmu.m ‘5»,WA. WWW].
`J.-.F. (1393) in Rhythm in M18 {Ali MA" ed) up 16749-3,
`Plenum Press, New: York.
`4. 55mm; .1». mm cm 5mm Re; 233. 5515-573
`5. Maine],
`3%..
`amt! Ekatmm, R [1933} Neumnch‘m 2.5.
`107l~107fl
`E. Hmtim, MIL Herbert, 3., Maxim”. NIL, and Wm, AC.
`(1935) it; thQMrwm, Maigmrsfa and My Ping”! {Evans-J
`13.. and Clark. 3.. am pp. $5.77, Piurmn, [Milan
`'1'. Bram. Nu Jams, 35., Randall, 0.. Thrush, MC. Swiss. 3..
`Spring“, «L. SWIM! .1, and Harlan G. (1992} in T15: Rain»
`haw Tram? (Gail, (3.3., ed.) pp. iiidfié, Eiwvimr. mmm.
`
`000006
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:25)
`
`AKER877ITC000704
`
`mnaumkrwymmpun. 2
`
`

`

`{mm 30313“) 9524247
`
`Ciréer 5‘ 05035 3:00P04134553
`
`Tue Nov 16 14:05:01 2004
`
`page a of 8
`NOV 16 '30 @3162PM
`
`317
`
`LIPID GGMPOSI'I'ION 0F MDT PINEAL 035m
`
`23,
`
`23.
`
`24.
`
`iii
`25.
`27.
`
`’
`
`Christie, va- C1982) Lipid &Wym. am mm. mm. 51mm,
`Porgfimm How. (Mam,
`me, Mail.“ Bums. 33a!” and Maud. if". (1969} J. Chm«
`may; 43, gamma
`Olefin, RE“ and Heudarsmm, RAJ. (1939} J. Exp; Mar.
`Emilia 135—197.
`Bell. MM. “flick. JR” [199-3) Lipids 28, 19422.
`Emlmnn, U. (1955) «I. Am 051 Chm. Soc, 43, W.
`(from. «L. Gum. NZ. fihnmm. Ft, Shim, M. Knmawu. 8.. Suzuki
`K, and thnm, T.
`(13483) Amalyflm Claw-mien Mia )‘47,
`3027—400.
`Tnkamura, ii. and Kim, M. {High-J" Biochem. 105?, {REP—439:
`« Hell, MN. {19189} Lipids 24‘ 585—688.
`van VIM, I, am! Kama, MS. (1980) Am. J. Elm. Nair. 51,
`12
`Entry, (‘3. H035) Frog. W Ram 24. GEMIM,
`* Hendemm ELL and Wm: DE. {1937) hag )2pr Rex. 25,
`231—33?
`r Redbuxn, Silk, and T‘Bsanuamfiforalw. H. {1360} Neuroi. New
`robin}. 49, 3—3“).
`Begay. V, Fakmn, J‘. 'l'hihauk, 0,, Ravaull. 3.?“ and Catlin. J;-
`PJ. {1992} Neumndmfinal. 4, 33?-345.
`
`5:59""?
`
`Nay“, EB». and Small, 11?. [1987) J. Pineal Res. 4. SET—am.
`Hialeah”, 345., land Mum RE. (1933} Flag. lipid. Rm 2.2,
`7&1‘3L
`Ether, ISLE“ LLI‘ad H9. 0 _, Dfl. { 29%) FILM Phyaia-I. Bmhcm. 5,
`229-239.
`ngand. RD” and Andemn‘. RE, {“33} Exp. Eye Rm 3‘7.
`159-173‘
`Rodngue: d6 'T‘urm‘ 1:13q Wan, WC“ and Ewan, NJ},
`(1991) J. Mfume“; N , 38$?m%?5,
`Namingcr, Mil. Emma W18” Lin. 11$“ gamma, L” and
`Luigi, S. @9385} Pat-s. ”9.32. AM. 3-5! UfiA 3-3, 6.9214025.
`De La Pu. M.A., and Andaman, ILE. {1932) Int-eat. Optical
`Via. Sci. 33, @91—20‘3‘5.
`Louis, E. WEegand. RD“ anal Andaman, RE. (£988) Biochtm~
`11m} 21!, 2x31441020.
`36:35am. km. Wiegand, R0,. and Andaman. RB. {1991} Exp.
`Eye RM. 52, 21%238,
`Bell, MM, and Tanker. DJ?» t" £989) BMW J. 3W. BOG-uglfi‘
`Rafi, ELY, and flick, 3,151 “WU Lipids 25, 566-573.
`. Bell. MM, and Dick, 5.3. {1993}J. Mar. Bad Ass. U452 25‘,
`679—688.
`8m, E", Ghnn'b, 5L, Crank M‘, Molina“, P; and Damn-deg LL
`[1591} 3mm Binphxu. MW 1081. Win-78.
`, mm, J”. $astm Fifi. amt Shaman HI;‘ (1969) J. Wm»
`rocMnt 15, W74? 14.
`
`.6103.
`
`[mm NW 19. 1993,, and in mvimd form March 23, 1994;,
`Envision mum March 23, mm
`
`000007
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:26)
`
`UPIDS, Vol 2% m, 5 (1994)
`
`AKER877ITC00070483
`
`