Linoleic Acid‐Derived Metabolites Constitute the Majority of Oxylipins In

UC Davis UC Davis Previously Published Works

Title Linoleic acid-derived metabolites constitute the majority of oxylipins in the rat pup brain and stimulate axonal growth in primary rat cortical neuron-glia co-cultures in a sex-dependent manner.

Permalink https://escholarship.org/uc/item/7642789z

Journal Journal of neurochemistry, 152(2)

ISSN 0022-3042

Authors Hennebelle, Marie Morgan, Rhianna K Sethi, Sunjay et al.

Publication Date 2020

DOI 10.1111/jnc.14818

Peer reviewed

eScholarship.org Powered by the California Digital Library University of California This article isprotected rights by All copyright. reserved. 10.1111/jnc.14818 differencesofversion Record. and c theVersion Please between this throughcopyediting,typesetting, the proofreading pagination and process, whichmay lead to This article undergone has for and buthas accepted been been not review publication fullpeer Phone: (+1) 530 Email: USA 95616 CA, Davis, Environmental (3162), North RMI and Avenue, Shields One Davis, Agriculture California of University of Sciences, College Technology, and Science Food of Department Dr. Y. Ameer Taha + *These au School ofVeterinary UniversityDavis, Medicine, ofCalifornia, USA CA, Sciences, University Davis, ofCalifornia, USA; CA, 1 Articletype : Original Article AMEERDR. TAHA Y (Orcid ID : 0000 Department ofFoodand Technology, ScienceAgricultureEnvironmental Collegeand of Corresponding author: Linoleic acidLinoleic

Accepted ArticleMarie Hennebelle and stimulate and in growth primary axonal neuron cortical rat [email protected] thors contributed equally work. thors tothis

- derived metabolites in constitutemajority the oxylipins of - 752

1 - * 7096 Cristina Grodzki Cristina , Rhianna K.Morgan

- 0003 - dependent mannerdependent 4611 2 , LeinPamela J. 2 - 7450) * , SunjaySethi

2 Department ofMolecular Biosciences, 2 , Ameer Y. Taha

2 , Zhichao ZhangZhichao - glia ite this article as ite this doi:

co 1 +

cultures 1 , the Hao Chen rat pup brain rat pup

in asex in 2 , Ana -

This article isprotected rights by All copyright. reserved. hydroxyoctadecadienoicacid (13 neocortex were tovehicle exposed ethanol), (0.1% respective gas chromatographychromatographyliquid and totandem coupled mass spectrometry, metabolites (oxylipins) wereone inbrains quantified from development. isknownregardinglittle the levels orrole metabolites (OXLAMs) Inadult rats, omega ABSTRACT 1. SP hydrolase; epoxide soluble sEH, acid; fatty polyunsaturated PUFA, gamma; receptor activated metabolite; acid linoleic oxidized OxLAM, chromatography acid; eicosapentaenoic EPA, acid; docosahexaenoic p450; cytochrome Abbreviations: Keywords: title:Running

Accepted extraction; phase solid E, Article

ly. Primaryly. n

fatty neuronal acids, OXLAMs, morphogenesis, oxylipins

To address this this To address

A aahdnc acid; arachidonic AA, with flame ionization detection; detection; ionization flame with - hydroxyoctadecadienoic acid - 6 l DPPC, DPPC, euro inoleic acid (LA, known toregulate signalingthe brain multiple processes in n - TAG, triacylglycerol; triacylglycerol; TAG, glia co gap, f 2 - diheptadecanoyl - HODE) attyacids various and brain within pools, their oxidized lipid - cultures

E coetrl ester; cholesteryl CE, 18:2n , (s)

or or

of LA and metabolitesof during its brain

PL, phospholipid; phospholipid; PL, prostaglandin E2 ( prostaglandin E2 derived - 6)

increases - sn serves as

TRPV1, transient receptor potential vanilloid vanilloid potential receptor transient TRPV1, - LA glycero

from postnatal day 0 LA, linoleic acid; LOX, lipoxygenase; lipoxygenase; LOX, acid; linoleic LA, ,

the OXLAM the

F, re at ai; GC acid; fatty free FFA,

- axonal growthaxonal

day female oldmale and pupsusing a precursor to oxidized LAa precursor tooxidized - 3 - phosphocholine COX, cyclooxygenase; CYP450, CYP450, cyclooxygenase; COX, PGE2 PPARγ, peroxisome PPARγ, 13 )

at -

10 - 1 male and female rat - 1,000

(DPPC); (DPPC);

. nM

However, - proliferator proliferator - I, gas FID, for 48h DHA, - This article isprotected rights by All copyright. reserved. represented already intake is breastmilk human in composition LA that knowing g/day) (~16 a the increased high towards shifts agricultural US, the development and growth normal for calories daily of 2% to 1 at required INTRODUCTION morphogenesis inthe developing precursor, and majority thedeveloping ofoxylipins in alter significantly outgrowth axonal inmale butnotfemale increased cortical neurons. outgrowthaxonal femalebutnotma in derivedLA from vs.female pupsrevealed male that sexes. 32% ~ and free fatty whereas acids, less than3% detectedtriacylglycerides, offatty phospholipids, inbrain cholesteryl acids esters to test the Accepted Article5% reportedbrain) inadultrat

neuronal of total OXLAMs. Brainof total concentrations OXLAMs.acid OXLAM notdiffer did fatty andbetwe LA and (In Linoleic ir effectsneuronal onmorphology i 20, Insull 2007, nis (Blasbalg

outgrowth 13 highlights a novelLAhighlights role and of mount of LA in the in LA of mount

acid (LA, 18:2n (LA, acid - HODE significantly increased axonal outgrowth. significantlyHODE axonal increased Separate analysescultures of

12% of total fatty acids acids fatty total of 12%

t al. et

t al. et either sex OXLAMs constituted 46to52%constituted ofmeasuredOXLAMs

01 US Dprmn o Arclue 2016) Agriculture of Department U.S. 2011, . Of 13 these, -

male 6) is an essential omega essential an is 6) 1959) food supply food . Th le cortical neurons, whereas and rat I te 1980’s the In . - is studydemonstrates A oba ad on is uig h lt 13s has 1930s late the during oils corn and soybean LA

brain female brain . (Putnam

- In both male and female pups, LAIn male andaccounted female both pups, for HODE was accounting abundant, themost forHODE

despite lowreldespite 13 from 2% of daily calories to approximately 7% 7% approximately to calories daily of 2% from - HODE in at 1, 50 and 1000nM, at 1,50and1000nM,

et al. et . , LA composition composition LA ,

iety eae t maternal to related directly

- 1982, Jenness 1979) Jenness 1982, 6 polyunsaturated fatty acid (PUFA) (PUFA) acid fatty polyunsaturated 6 differentially differentially ative that 13 OXLAMs constitute the constitute OXLAMs

abundance of their LAabundance oftheir - HODE (Hansen

significantly increased oxyl of influencing .

hs s concernin is This at 10 ua breastmilk human , providing , ipins PGE2 PGE2

et al. et 0 nM

(versus

dietary

neuronal neuronal 1958) did not did not

en 8% of of 8% 31 . In . LA - g This article isprotected rights by All copyright. reserved. hydrolaseepoxide (sEH) al. (Engels non Taha 1994, al. et (Cunnane acids fatty other al. et thebrain LA enters DHA, however, ~30% accumulates myelin and microglia (phospholipids, DHA not changed 18:3n 20:4n (AA, acid arachidonic product, desaturation 1958) calories daily

Accepted Article 2000) - enzymatic

- . 2008a) y h brain the by

3), eicosapentaenoic acid (EPA, 20:5n (EPA, acideicosapentaenoic 3), AA+DHA Contrary to common common to Contrary Prenatal

et al. et , cytochrome (CYP450) p450

significantly in the developing rat brain rat developing the in ]

. equaling 1986, Funk & Powell 1983) Powell & Funk 1986,

Upon entry, however, it it however, entry, Upon

(Porter sphingolipids develo

(Lien or

(Green possibly possibly (Sinclair & Crawford 1972) Crawford & (Sinclair

mn i caatrzd y h rpd n slcie crto of accretion selective and rapid the by characterized is pment 4 et al. et t al. et -

(Moghaddam 8 times the minimum requirements minimum the times 8 over the

et al. et al. et Sanders 1994, belief

1995) and others) others) and ovre it oiie linoleic oxidized into converted

2016b, DeMar 2016b,

past few decades past few 1999) , dietary levels of ot of levels dietary ,

(Hassam or enzymatic pathways involving cyclooxygenase (COX) (COX) cyclooxygenase involving pathways enzymatic or

(DraperHammock& 2000,Bylund et al. , which contains contains which , is wih tlzs th utilizes which , , lipoxygenase (LOX) lipoxygenase , rapidly

eurd o te auain of maturation the for required et al. et

- 1996)

3) an 3) et al.

et al. et 1984, Cunnane 1984, d docosahexaenoic acid (DHA,22:6n acid docosahexaenoic d

β .

1975) enzymes as - (Blasbalget al. 2011) - 6) and omega and 6) In contrast to contrast In oxidized oxidized

2006) her PUFAs her <2% LA of total fatty acids fatty total of LA <2%

at comparable rates[ at comparable em

. OXLAMs are synthesized are OXLAMs . establis into carbon dioxide carbon into shown in o om ope lpd structures lipid complex form to

(Noguchi

et al. et

- cd eaoie (OXLAMs) metabolites acid AA and DHA, and AA 3 alpha 3 ,

hed in hed including LA’s elongation LA’s including

1994) Figure 1 .

et a et al. et

infants ioei cd (ALA, acid linolenic ern, astrocytes, neurons, . Similar to AA and and AA to Similar reviewed l.

1998) 2002, Burger 2002, .

, very little little very

(Hansen et al. et (Hansen

recycled into into recycled

compared to to compared

or soluble or soluble - in

AA and and AA 3) through through

(Chen ,

have have LA

et -

This article isprotected rights by All copyright. reserved. acids fatty oxidized us brain rat developing the in OXLAMs of abundance 1 and 1 at neurons neur of differentiation the promote to shown was which PGE2, outgrowth axon for control positive 13 and LA of effects axonal h relevant physiologically morp neural and epithelial (Dehaut 0. reported al. 1 not but mM 0.35 2017) activation (PPARγ) Alsalem vanilloid potential receptor transient gated 2019, Hennebelle ydroxyoctadecadienoic acid (13 acid ydroxyoctadecadienoic Accepted Article0 1 to 1 oeei o cnrl ern. o et hs yohss we hypothesis, this test To neurons. central of hogenesis . In vitro In Collectively, these studies suggest that suggest studies these Collectively, rat adult in abundant are OXLAMs 0.

et al.

t al. et mM when applied to applied when mM 1 mM 1 (Akeo similar

1993) , LAand ,

03 Ruparel 2013, ,

et al. et and

et al. 0

µM findings in PC12 cells PC12 in findings .

- (Nagy

derived from LA and other PUFAs other and LA from derived like (PC12) cells. (PC12) like

in chick moto chick in

1996)

2017, Ramsden 2017, its its (Wong concentrations

hydroperoxide t al. et - . LA was LA . HODE were compared to those of of those to compared were HODE

pheochromocytoma (PC12) pheochromocytoma t al. et et al. et -

HODE), on HODE), 1998) neurons

2016) (Krug 2012)

also

They also raise also They et al.

and hippocampal neurotransmission neurotransmission hippocampal and of

were reported to reported were

exposed to lower to exposed . peripheral and central nervous central and peripheral found to increase neurite growth at growth neurite increase to found peroxisome ,

We also address also We LA to exposed LA and one of its its of one and LA

t al. et - 2016, Tahaal. 2016b) et (TRPV 1 axonal growth in primary rat cortical neurons. cortical rat primary in growth axonal

LA or its metabolites its or LA

03 Stiegler 2013,

the possibility that OLXAMs regulate the the regulate OLXAMs that possibility the - ) channe 1)

- ing proliferator activated receptor gamma gamma receptor activated proliferator (i.e. oxylipins) (i.e. growthcell epithelial retinalreduce

cells concentrations of 0.001 to 0.01 mM 0.01 to 0.001 of concentrations

ed the paucity of paucity the ed

agtd eaoois o quantify to metabolomics targeted concentrations of concentrations Rho (Kamata ot abundant most

t al. et ls etdra se cls into cells stem oectodermal -

kinase inhibitor Y inhibitor kinase regulate

(Patwardhan , where they regulate, where pain quantified

2011)

n a ppban Fatty brain. pup rat in et al. et tissue tissue the morphology of of morphology the information on the on information Hneel e al. et (Hennebelle ,

LA ranging ranging LA 0.1 and 0.3 mM, 0.3 and 0.1 2007) n AA and

(Ferdouse XAs 13 OXLAMs, h efcs of effects the

t al. et . Dehaut et Dehaut . - 27632, - derived

2009, et al. et from The The

at a - -

This article isprotected rights by All copyright. reserved. µ in reconstituted and vials µ 900 containing HCl methanolic 1.2% crushed (GC gas by confirmed was composition acid containing Upon light h 12 a and °C) 22 (approx. temperature with housed individually were Animals were not pre Committee Use and Care Animal Institutional Animals EXPERIMENTALPROCEDURES differencesto test for inOXLAM sex compositionandresponse pools lipid (FFA) acidfatty free and triacylglycerol (CE),(TAG) als were oxylipins to precursors acid

AcceptedL. Article

- FID) purchased L arrival - The overall study design is shown in shown is design study overall The All

registered. of the top hexane layer was mixed with 450 µ 450 with mixed was layer hexane top the of

with a pestle a with

nlss as analysis 1 experimental 8

6 at UC Davis UC at

g/kg protein, 6 protein, g/kg from Charles River Laboratory (Hollister, CA, USA) CA, (Hollister, Laboratory River Charles from

and mortar, weighed to ~100 mg (n=4) and transesterified in 3.6 mL 3.6 in transesterified and (n=4) mg ~100 to weighed mortar, and previously described described previously 100 rcdrs ee prvd y h Uiest o Clfri Davis California of University the by approved were procedures , the dams were placed placed were dams the , µ 2

L hexane L g/kg fat, g/kg 400 µ

quantified o Ltoluene -

chromatography coupled to flame ionization detection ionization flame to coupled chromatography 589 prior to GC to prior corncob - dark cyclew dark

(Taha

g/kg carbohydrates, g/kg (IACUC Protocol (IACUC Figure 2. Figure .

Water and hexane (1 mL each) were added and mLeach) added (1 were hexane and Water

n ri popoii P) coetrl ester cholesteryl (PL), phospholipid brain in bedding.

on the on t al. et - FID analysis FID L

ith food ith

Timed p Timed 2018 Teklad global 18% protein 18% global Teklad 2018 water, centrifuged, transferred to GC to transferred centrifuged, water, 2016a) nml wr kp udr constant under kept were Animals .

. Number

. In brief, a few pellets were were pellets few a brief, In . All e All and water provided ad libitum. adwaterprovided and and regnant . The

xperiments were xperiments 5 16 3

injection volume was volume injection

20541

g/kg ash. g/kg days post days Sprague ) . The study was study The .

- Dietary fatty fatty Dietary - Dawley rats rats Dawley conception designed

diet diet

of of

1 .

This article isprotected rights by All copyright. reserved. analysis 1.25 in reconstituted g x test original the to chloroform mL 3 adding by extracted layer chloroform were which homogenates, m Kimble Sigma b 0.6% containing methanol µL 850 in min 1 for homogenizer frozen mother) acidFatty described below d or analysis lipidomic to subjected al. isoflurane) (e.g. methods common others because scissors sterile using L

Accepted Article

2018) for 10 for ehnl olwd y 1.125 by followed methanol - Aldrich, St Aldrich, pups female four and male Four were pups female and Male

. nitrogen. liquid in glass

were euthanized on postnatal day 1, and their brain excised excised brain their and 1, day postnatal on euthanized were .

min. min. uhnsa a promd ewe 8 m o 2 noon. 12 to am 8 between performed was Euthanasia concentration - tube The bottom chloroform layer was pooled with the first one, dried one, first the with pooled was layer chloroform bottom The .

containing total lipids was transferred to a new test new a to transferred was lipids total containing - s Louis, MO, USA MO, Louis,

containing 3 containing

following cold following

m L s in various lipid various classes s in

chloroform otxd n cnrfgd at centrifuged and vortexed rzn ris ee w were brains Frozen

L m ) - . euthanized

setos f otcl tissue cortical of issections induced anesthesia induced L chloroform. chloroform. The brain h brain The :

ehnl (2 methanol 0.1M KCl in water water in KCl 0.1M arbitrarily

t it o o post on or birth at

omogenates were transferred to transferred were omogenates in pup brains pup in were found to alter synaptic formation synaptic alter to found were The brain homogenates were rinsed with rinsed were homogenates brain The

: selected 1 eighed and homogenized using a bead bead a using homogenized and eighed v:v , . -

tube, vortexi tube, This method of anesthesia was chosen chosen was anesthesia of method This ) and stored stored and ) 4 x g x 646

from 2 2 from n poe wt te et of rest the with pooled and

utylated hydroxytoluene utylated

fo The - neuron r aa dy 1 day natal

dams (2 per sex from each each from sex per (2 dams - ng and centrifuging and ng o 10 for tube. Total lipids were re were lipids Total tube.

brains were excised and and excised were brains and immediately flash flash immediately and at

- -

80°C 80°C la co glia min. min. y decapitation by under nitrogen under

100mmx13cm ni further until h b The - cultures as as cultures

(BHT; (Xu at ottom

646 the the 0.7

et - ,

This article isprotected rights by All copyright. reserved. of µL hundred and vortexed were Samples sample. each 4 for temperature room heat and vortexed were Samples sample. 2010) Fukubayashi triheptanoin tothe TAG fraction. fraction,20 CE to the of DPPC µg Sixty CEs. and TAGs PLs, diheptadecanoyl for standards esterified and FFAs for standards test a to transferred were standards authentication CE and TAG FFA, PL, to corresponding bands Lipid methanol. in solution dichlorofluorescein 0.02% with plates the spraying after light ultraviolet u revealed bands lipid the and hood fume the under minutes few a for dried were plates The ~1 to migrate to allowed was front solvent The system. solvent wer classes Lipid bands. lipid dipalmitoyl USA) of MA, Billerica, Milipore, chloroform: (EMD plates glass on plated were extract lipid brain of µL hundred Two (TLC). chromatography

Accepted Article moisture. Samples were transesterified in methanolic acid as previously described described previously as acid methanolic in transesterified were Samples TAGs FFAs, (PLs, classes Lipid - methanol sn - glycero uhni sadrs ( standards Authentic

- the sn - . Three mL methanol and 600 µL 600 and methanol mL Three . glycero - hexan ue otiig 0 µ tlee n approp and toluene µL 400 containing tube (2: - 3 - 5 1 - phosphocholine

, v:v , min, 1 min, e -

3 top µg of unesterifiedhepta ofµg - e separated using a h a using separated e phosphocholine )

and heat and

layer

hexane followed by 1 by followed hexane amtc cd cholesteryl acid, palmitic otiig at ai mty etr were esters methyl acid fatty containing - wr rn n eaae ae t ietf te different the identify to lanes separate on run were ) activated d t 0C o 60 for 90°C at ed the phases were phases the

(DPPC) were added to the PL fraction, 30 µg of µg 30 fraction, PL the to added were (DPPC) n Cs wr sprtd sn ti layer thin using separated were CEs) and epta overnight at 80 at overnight decanoic acid to the FFA fraction and 50 µg µg 50 and fraction FFA the to aciddecanoic 8% HCl in methanol were added to each each to added were methanol in HCl 8% ne:ethyl ether:acetic acid (60:40: acid ether:acetic ne:ethyl

allowed to separate for 15 for separate to allowed

m i. fe cooling After min. L - 2 cm before the top of the plate. the of top the before cm 2

pre deionized °C °C it aons f unesterified of amounts riate - amtt, rplii, 1,2 tripalmitin, palmitate, - washed washed under

water were added to to added were water vacuum to vacuum ih 200 with

h smls at samples the L iia gel silica TLC transferred Ihhr & (Ichihara scraped

min. min. rid them rid 3 , v:v:v ,

Nine nder nder 1,2 and and mL

to to - - )

antioxidant mix (0.2 mg/mL BHT, ethylenediaminetetraacetic acid (EDTA) and and (EDTA) This article isprotected rights by All copyright. reserved. acid ethylenediaminetetraacetic BHT, mg/mL (0.2 mix antioxidant USA homogenizer bead a homogenizedusing described previously nitrogen. liquid selected in brains concentration Oxylipin pup areas s totheinternal com by determined were concentrations acid Fatty time. retention on based µ rat flow a at maintained was gasand carrier the was Helium min. 36.5 was time run total The min. 13 for 240°C at held lastly and 5°C/min at 240°C to raised 185°C, to 10°C/min by increased min, 2 for 80°C at held was It 80°C. was temperature temperature detector and injector The U.S.A.). Calif., Clara, Santa Technologies, Agilent thickness; film μm 0.25 diameter, inner Sh hexane. samples g 15,871 micro l per sample. A set of 29 fatty acid methyl ester standards was used to identify each fatty acid acid fatty each identify to used was standards ester methyl acid fatty 29 of set A sample. per l

Accepted ArticleDB a with equipped U.S.A.) CT, elton, ) f uge tubes uge in 200 µL methanol containing 0.1% acetic acid and 0.1% BHT spiked with 10 µL µL 10 with spiked BHT 0.1% and acid acetic 0.1% containing methanol µL 200 in Six brainsSix S

Samples from one litter. one from mls were amples 50 into reconstituted were o 2 for

min. min.

containing were stored nseiid o Unesterified were tandard area.tandard

h tp hexane top The Hneel e al et (Hennebelle nlzd n Pri Emr lrs 0 GC 500 Clarus Elmer Perkin a on analyzed collected from postnatal day

The brains, which weighed approximately 200 mg, were flash were mg, 200 approximately weighed which brains, The 450 at

µL - yiis ee xrce b sld hs etato (P) as (SPE) extraction phase solid by extracted were xylipins 80°C 80°C

deionized µL was

layer until

hexane and FFA samples were reconstituted into 10 into reconstituted were samples FFA and hexane 2017) . (

Bullet BlenderStorm Bullet - FFAP fused silica capillary column (30 m × 0.25 mm 0.25 × m (30 column capillary silica fused FFAP 4° ad 0°, epciey Th respectively. 300°C, and 240°C

theywereanalyzedweek) (withinGC a by was water. . Briefly, the entire brain from each pup was was pup each from brain entire the Briefly, .

vprtd ne ntoe. L T PL, nitrogen. under evaporated e of 1.3 mL/min. The injection volume was 2 2 was volume injection The mL/min. 1.3 of e

1 rat pups (3malesfemales rat and 3 1 The t The

ubes were vortexed and centrifuged at centrifuged and vortexed were ubes , Next Advance, Next , - FID

ytm Pri Elmer, (Perkin system paring Troy, NY 12180, NY Troy, iiil oven initial e

AG and and AG of GC peak GC of , - arbitrarily - FID frozen in frozen .

CE CE µL

This article isprotected rights by All copyright. reserved. and min 26.1 and 26 between 35% to decreased min, 26 and min 24.6 between 99% at held min, 24.6 and 24.5 between 99% to increased min, 24.5 to 24 between 85% at held was B Solvent to min, 10 and 4 between B The 60% to min, 4 and 3 between acid. 48% to min, 3 and 0 between 40% to 35% from acetic increased gradient 0.1% with (80:15) acetonitrile:methanol was B phase mobile and water T . USA) CA, Clara, Santa Technologies, (Agilent Technology Stream Jet via (ESI) ionization electrospray negative with MS/MS QqQ 6460 a to coupled and USA), CA, Clara, Santa Technologies, Agilent µm; 1.8 size; Particle mm; C1 Plus ZORBAXEclipsea with equipped system(UHPLC) Sigma, Burlington,USA) at and 20min 15,871g centrifuging0˚C. for MA, at s The methanol. µL 100 in reconstituted and nitrogen under dried acetate, ethyl mL 1.5 by followed methanol mL 0.5 with eluted were Oxylipins psi). 15 (around vacuum under min 20 for dried S of volumes 2 with washed were Samples water. ultrapure in acid acetic methanol pre USA) CA, Milford, Corporation, Waters and 15,871 at min 10 d4 d11 in (TPP) triphenylphosphine

Accepted Article- TXB2, – poured 11(12) amples were amples S mls ee nlzd n 19 Ifnt ultra Infinity 1290 a on analyzed were amples ,

and pre and d6

- onto Oasis HLB cartridge SPE columns (3 columns SPE cartridge HLB Oasis onto EpETrE, d11 EpETrE, - 20 - HETE and d8 and HETE - x then then conditioned with 2 volumes of SPE buffer containing 5% methanol and 0.1% 0.1% and methanol 5% containing buffer SPE of volumes 2 with conditioned g at 0˚C. The 200 µL supernatant supernatant µL 200 The 0˚C. at g filtered using Ultrafree using filtered - 14,15 70% between 10 and 20 min and to 85% between 20 and 24 min. min. 24 and 20 between 85% to and min 20 and 10 between 70%

: methanol:water 1:1 - - 5 DiHETrE, d4 DiHETrE, - HETE

in methanol. Homogenized samples were centrifuged centrifuged were samples Homogenized methanol. in he mobile phase A was 0.1% acetic acid in ultrapure ultrapure in acid acetic 0.1% was A phase mobile he - rinsed with one volume ethyl acetate and 2 volumes 2 andacetate ethylvolume one with rinsed - - 6 MC cent MC - keto ad 0 L urgt mx otiig 2 containing mix surrogate µL 10 and ) - PGF1α, d4 PGF1α, was

rifugal Filter Devices (0.1 µm; Millipore Millipore µm; (0.1 Devices Filter rifugal cc, 60 mg sorbent, 30 µm particle size; particle µm 30 sorbent, mg 60 cc,

high mixed with 1800 µL ultrapure water ultrapure µL 1800 with mixed 8 column (ID: 2.1 mm; Length:mm; 2.1 150 (ID:8 column - pressure - 9 - HODE, d4 HODE, - iud chromatography liquid E ufr n then and buffer PE

- LTB4, d4 LTB4, - PGE2,

µM

This article isprotected rights by All copyright. reserved. 1 and Scientific) Fisher Thermo (Gibco, serum 2% with tissue cortical (Sigma DNase µg/mL Scientific Fisher cort dissected The 7.55). ( removed. rapidly were pooledtogether females. for anogenital by determined female neuron Cortical aftercorrecting extraction thesurrogate losses with for possible 1 Table target to used was monitoring reaction multiple Dynamic µL. 10 was injection of volume The min. 28 to 27.3 from mL/min 0.3 and min 27.3 to 24.6 from mL/min 0.35 min, 24.6 and 3 unti 35% at held AcceptedGIBCO Article xlpn uig h peusr n pout o prmtr peetd in presented parameters ion product and precursor the using oxylipins The neuronPrimarycortical rat pups pups rat . , Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1 with supplemented USA) MA, Waltham, Scientific, Fisher Thermo , Oxylipins with a signal to noise ratio above 3 were quantified against a standard curve standard a against quantified were 3 above ratio noise to signal a with Oxylipins B27

pups were pups Neocortices from a from Neocortices

was rinsed with rinsed was ( hro ihr Scientific Fisher Thermo - l 28 min. The flow rate was 0.3 mL/min from 0 to 3 min, 0.25 mL/min between mL/min 0.25 min, 3 to 0 from mL/min 0.3 was rate flow The min. 28 l as previously described described previously as cnann 23 gm ppi (otigo, aeod N, S) n 95 and USA) NJ, Lakewood, (Worthington, papain mg/mL 2.3 containing ) glia co primary Neocortical tissues Neocortical from onelitter from - euthanized Aldrich, St Aldrich, ices distance and confirmed by the presence of testes for males or ovaries or males for testes of presence the by confirmed and distance

w ere - Neurobasal Plus Neurobasal ll males were pooled together pooled were males ll gliaco - cultures:

- by decapitation by incubated at 37 at incubated Louis, MO, USA). The papain solution was removed and removed was solution papain The USA). MO, Louis, .

were - cultures were preparedfromwerecultures (Wayman , ltMX Tem Fse Scientific) Fisher (Thermo GlutaMAX ),

dissected in ice in dissected

M HEPES. The tissue was then triturated then was tissue The HEPES. M media ( media

°C for 23 min in Hibernate A ( A Hibernate in min 23 for °C cold after

et al. et Thermo Fisher Scientific Fisher Thermo

2006, Sethi 2006, - - cold Hanks’ Balanced Salt Solution Salt Balanced Hanks’ cold induced anesthesia, induced , while neocortices from neocortices while ,

standard.

3 -

5 et al. et postnatal day1 postnatal

2017)

Supplementary

and the and ) supplemented supplemented ) M HEPES (pH HEPES M Gibco, Thermo Thermo Gibco, . Pup s Pup ,

quantif 10% all male and and male ir females ex was was ex

. Cel . brains horse horse y the 7 ls ls 2 This article isprotected rights by All copyright. reserved. AcceptedSigma (BSA, albumin serum bovine 5% in blocked were cells Permeabilized min. 5 for 0. with permeabilized and min 5 for PBS with times (Sigma paraformaldehyde ofaxonalQuantification growth describedabove transesterification following LA of purity confirmed analysis solutions stock 2011) al. et Stiegler (Sigma 27632 cultures control final varying 1:1000 Article at diluted first were Chemical) (Cayman PGE2 or Treatments B27 plating, were Cells Aldrich). pre USA) NJ, Vienland, (BellCo, coverslips Cellometer a using counted were

a nd GlutaMAX fe 4 h xoue o LA to exposure h 48 After USA), LA Elysian, MN, (NuChek, utr ei a rpae ih Neur with replaced was media culture

concentrations of concentrations - lrc) a ue a a oiie oto fr xn outgrowth axon for control positive a as used was Aldrich)

( s niae i representative in indicated as ee rae wt vhce ehnl 110 dlto) Rho dilution). 1:1000 (ethanol; vehicle with treated were Supplementary FigureSupplementary 1C iety no cultures into directly . . U

loe t stl ad attach and settle to allowed H PLC - Aldrich - MS/MS analysis confirmed the purity of the 13 the of purity the confirmed analysis MS/MS LA, 13 LA,

i phosphate in ) n pae a a ocnrto o 3,0 cells/cm 33,000 of concentration a at plated and ,

- HODE, or PGE or HODE, dissolved Clue wr icbtd o 4 h 48 for incubated were Cultures . 13

13 - HODE ). - HODE (Cayman Chemical, Ann Arbor, MI, USA), MI, Arbor, USA), (CaymanHODE Ann Chemical,

- otd wi coated bsl Plus obasal Supple -

ufrd aie PS for (PBS) saline buffered

as 1000X stocks 1000X as

t 37 at r PGE2 or 5 % Triton X Triton % - etr Fgr 1A Figure mentary

2 at 2

th °C °C

aa mdasplmne ih 2% with supplemented media basal 0 µg/mL 500 1, 10, 50, 100, 500 and 1000 nM; nM; 1000 and 500 100, 50, 10, 1, ne 5 CO 5% under clue wr fxd ih 4% with fixed were cultures , ih .% HCl 1.2% with -

100 (Sigma 100 in absolute ethanol and then and ethanol absolute in poly - 2

iae niio Y inhibitor kinase Thr . - 2013, al. et (Krug n h peec of presence the in

- 4 HODE L - and and mn rne 3 rinsed min, 5 Aldrich

- n methanol in yie (Sigma lysine ee hours post post hours ee B

2 and PGE2 PGE2 and .

- ) in PBS in ) n glass on Aldrich) Aldrich) GC - FID as as - - This article isprotected rights by All copyright. reserved. distributed. normally were data lipid brain the of majority the and data length the using normality for tested were data data were analysis. included inthe study. exploratory an considered be should this Hence, brain. pup rat in data lipidomic quantitative of lack the to due determined not was assays lipid co glia neuro The brain. from lipids extracting before pup the of sex the determine to had experimenter the since Statistical (Molecular Dev of analysis image Devices) Molecular (ImageXpress, system imaging content high immunostained of captured images were neurons and Scientific) Fisher Thermo (Invitrogen, DAPI with Reagent Antifade Gold Prolong Invitrogen in mounted were Slides PBS. in BSA 5% in 1:1000 diluted antibody secondary C PBS in BSA 5% in 1/1000 diluted USA) MA, Billerica, (Millipore, for PBS in

Accepted Article with temperature room at h 1 for incubated and PBS with times 3 washed were ultures - Statistical analysis was performed using GraphPad Prism 7.04 (La Jolla, CA, USA). CA, Jolla, (La 7.04 Prism GraphPad using performed was analysis Statistical d The culture n and data and 1 h 1 - glia ata are expressed as as expressed are ata . Cells were then incubated overnight at 4 at overnight incubated then were Cells .

was determined based on historical data historical on based determined was ices, version 5.3.0.5). ices, version co - axonal culture assays were performed in a blinded manner. blinded a in performed were assays culture , analysis

fluorescein

rm sprt dissection separate 4 from

outgrowth

the ot anti goat

mean ± mean utilized

Shapiro - mouse IgG (Invitrogen, Thermo Fisher Scientific) Scientific) Fisher Thermo (Invitrogen, IgG mouse SD

a cell scoring journal in MetaXpress software software MetaXpress in journal scoring cell a .

The lipidomic assays were not blinded to sex, to blinded not were assays lipidomic The -

Wilk Wilk in an unbiased fashion using an automated automated an using fashion unbiased an in

°C (Sethi et al. 2017) al. et (Sethi test o outlier test was conducted was test outlier o

with the primary antibody anti antibodyprimary the with . The test revealed that the axonal axonal the that revealed test The .

(Dragunow 2008) (Dragunow to selectively label axons. axons. label selectively to

Sample size Sample ; sample size for the the for size sample ; Data .

that for neuron for A , thus, , utomated are - Tau1 Tau1

not All all -

This article isprotected rights by All copyright. reserved. reported by Envigo s 3.6% (16:0), acid palmitic 12.5% the with consistent was composition acid fatty measured The ALA. 6.7% stearic 3.1% (16:0), acid palmitic 13.3% Dietary fatty acid RESULTS tes Y control concentration determine one parametric a using (LA concentraitons various attreatment a by analyzed and effects pups. female and male even cases, inmost parame the in out pointed are distributed normally

Acceptedt Article . , Results 13 was acids, fatty total of percent as expressed diet, the of composition acid fatty The t unpaired An - HODE - 27632 was determined to be significant compared to vehicle control vehicle to compared significant be to determined was 27632

wereat considered significant tearic acid (18:0), 21.4% oleic acid (18:1n acid oleic 21.4% (18:0), acid tearic , and PGE2 , composition

( https://www.envigo.com/resources/data - test was used to probe for probe to used was test The axonal length data were normalized to vehicle to account for plate plate for account to vehicle to normalized were data length axonal The - -

a AOA ih ot o Dnets utpe oprsn et to test comparison multiple Dunnett’s hoc post with ANOVA way pcfc effects specific tw ). tric statistics were used toanalyzetric statistics were thedata. used o For subsequent analyses, data for each sex were analyzed were separately each for sex data subsequentanalyses, For - way

analysis of variance (ANOVA) to test for main effects of sex of effects main for test to (ANOVA) variance of analysis or interactions betweeninteractions or

acid (18:0), 20.4% oleic acid (18:1, n (18:1, acid oleic 20.4% (18:0), acid .

Axonal length in cultures treated with the positive positive the with treated cultures in length Axonal p <0.05. al or table differences in oxylipin concentrations between concentrations oxylipin in differences

iue eed. ic te itiuin was distribution the Since legends. figure

- 9), 55.3% LA and 5.3% ALA values values ALA 5.3% and LA 55.3% 9), sex and treatment and sex - sheets/2018

- datasheet

for each compound compound eachfor - 9), 55.6% LA and LA 55.6% 9),

using unpaired using - 0915.pdf ) .

t - ,

This article isprotected rights by All copyright. reserved. because post limits which high to subjected not were study this in rats the that noted be should (Hennebelle as Protein) 18% Global Teklad (2018 diet same the fed 2 in data brain previous our to pups female and male in DHA o Brain concentration between and female male pups. to 18 only but 1, and 0 days postnatal at pups of brains o Brain pools. percent the PL, within composition CElipid and FFA, TAG There study previous 3 and 1% in reported are pools fatty acidBrain concentrations -

Acceptedabove ratio noise Article

ee o infcn sx ifrne i ay f h dtce fty acid fatty detected the of any in differences sex significant no were Figure 3 Figure 72 the Of B xylipin concentrations xylipin their head size size head their rain fatty acid acid fatty rain % of total fatty acids, respectively. T respectively. acids, fatty total of %

t al. et percent distribution that

contrasts the percent the contrasts -

otm hne i ban ii metabolism lipid brain in changes mortem unesterified 2019)

on L constitute LA found 3 Table 1 Table . As shown in shown As was concentrations and percent composition within PL, FFA, TAG and CE CE and TAG FFA, PL, within composition percent and concentrations .

uniaie ifrne ae hw in shown are differences Quantitative

too small relative to relative small too . LA was only detected in brain PLs PLs brain in detected only was LA .

xlpn maue b U by measured oxylipins

Table 2 Table distrib d

~2 ution of ution

, none of the 18 oxylipins differed significantly in significantly differed oxylipins 18 the of none , % of brain total lipids lipids total brain of % he low LA brain composition is consistent with a with consistent is composition brain LA low he available microwave available were oxylipins derived from LA, AA, EPA and EPA AA, LA, from derived oxylipins

quantifiable because they because quantifiable weanlings and up to 2 months of age of months 2 to up and weanlings H PLC - month old Long Evans male rats male Evans Long old month - [ SM, 9 ee eetd in detected were 29 MS/MS, eiwd in reviewed

Supplementary Table Table Supplementary and CEs, where it constituted constituted it where CEs, and - energy microwave fixation, microwave energy Cnae Ce 1992) Chen & (Cunnane

adaptor fittings. adaptor

ocnrtos or concentrations Mrh 2010) (Murphy had a signal a had

The 2 . data

] It - . ,

This article isprotected rights by All copyright. reserved. brains. pup female and male in OXLAM abundant most the be to it revealed 13 nM. co glia Effect ofLA, brain oxylipins ( ( 10% ofoxylipins total quantifiable whereas and 20 13 tot of 52% and in shown As groups. age ( (Hennebelle et al.2019) were 83 Table Supplementary

Accepted Article- %) followed by DHA by followed %) HODE ,

therefore - - To test whether LA whether test To rats adult in oxylipins main The omega and ALA brains pup female and male than oxylipins more times ~6.5 had brains male Adult 24% cultures were exposed to exposed were cultures -

OE a coe fr hs mrhmti suis eas or iioi analysis lipidomic our because studies morphometric these for chosen was HODE AA , 9 of total OXLAMs of total OXLAMs - -

EPA , HODE 13 al detected oxylipins in female in oxylipins detected al ,

- Figure 3 oprd o non to compared HODE HODE -

and 12(13) and n DHA and . - 2 and PGE2 and 6 dihomo 6

; - ), and the percent distribution was remarkably different betwe different remarkably was distribution percent the and ), derived metabolites ( metabolites derived Figure 3 Figure third piethird chart ,

13 in both sexes, respectively. sexes, in both - - - HODE eie mtblts represented metabolites derived EpOME individual

- - in both sexes in both irradiated gamma

on (first and second pie charts), pie second and (first

, or PGE2 or , axonal axonal length ).

were the most abundant, representing abundant, most the were e te ae it ee the were diet same the fed

- linolenic acid metabolites were not detected not were metabolites acid linolenic

compound

dl rat adult an 9 , respectively ( d male pup brains pup male d %)

altered neuronal morphology, neuronal altered . OXLAMs only accounted for accounted only OXLAMs .

s brains from the Hennebelle et al. study study al. et Hennebelle the from brains

at concentrations ranging from 0 from ranging concentrations at

Figure 3 appro OXLAMs accounted for 46% 46% for accounted OXLAMs , respectively. Among respectively. ,

ximately ). AA

- derived metabolites metabolites derived

cortical 1 18%, 23% and and 23% 18%, G2 a not was PGE2 - 32

7% of adult of 7% % , 18 ,

en these these en in pups, pups, in st neuron

- them - 1000 1000 25 % - , This article isprotected rights by All copyright. reserved. Acceptedexperiments the of results Figure Supplementary unpaired t (P< vehicle to compared length axonal increased reports previous with female cortical or neurons growthon axonal of male ( nM 100 at neurons cortical male in length axonal enhanced o effect significant no had HODE ( nM 1000 and 50, 1, at neurons cortical female in length axonal enhanced LA lengthsignificantlyaxonal the sexes.but cortical effect male nosignificant in on had neurons of effects significant revealed test comparison multiple Dunnett’s ArticleLAand p=0.0059). = (F (6, 3.206; 118) 145)=25.93; (F(1, HODE 142)=10.95; (F(1, LA both in sex of effect significant a also was There length. axonal on p<0.0001) 143)=7.053; (F(6, PGE2 and p=0.0254), 145)=2.489; (F(6, HODE proliferation and differentiation used was it but brain, pup in detected h eut o ec niiuldseto o LA for dissection individual each of results The one by cultures female and male of analyses Independent Two - test as shown in test as shownin - , highlighting the way ANOVA revealed a sig a revealed ANOVA way

pooled analysis although analysis pooled Ku e a. 03 Sige e al. et Stiegler 2013, al. et (Krug s

2, 3 and 4 respectively 4 and 3 2, p Figure < 0.0001) < importance of performing multiple (versusdissections.importance ofperforming multiple individual)

(Wong et al.2016) 4A, 4B and 4 4Band 4A, xnl egh n eaecria nuos u significantly but neurons, cortical female in length axonal n -

because treated neurons, and a significant interaction between sex sex between interaction significant a and neurons, treated some

nificant effect of LA (F(6, 142)=10.95; p=0.0277), 13 p=0.0277), 142)=10.95; (F(6, LA of effect nificant

differences differences

C previous study reported that it induced neuronal induced it that reported study previous ). . .

.5 f old ae n fml dt by data female and male pooled of 0.05

Overall, the Overall, in the independent analysis

were observed in in observed were 13 2011) - 4B HODE ). PGE2 had no significant effect significant no had PGE2 ). Rho , data

treatment that varied between varied that treatment - a AOA ih ot hoc post with ANOVA way , and PGE2 and ,

- are iae niio Y inhibitor kinase

in agreement with the with agreement in

4A individual p 001) n 13 and 0.0012) = ). Conversely, 13 Conversely, ).

( are provided in in provided are 4C ).

Consistent Consistent dissection - 27632 - - -

This article isprotected rights by All copyright. reserved. six on performed study previous ( metabolites 2016b) al. et Taha 2019, 7% in oxylipins of life more concentrates rats older that to due diet in values reported our to compared lower more) of the support 13 and neurons, cortical 13 neuron of length axonal oxylipins measured DISCUSSION

Accepted Article- sex were growth axonal on HODE OXLAM composition reported in adult male rats male adult in reported composition OXLAM ( Supplementary Table Table Supplementary (Cunnane &(Cunnane1992) Chen differences in strain or age of the animals used in the two studies two the in used animals the of age or strain in differences hn h dt wr nraie t ttl xlpn, XAs ae p 46 up made OXLAMs oxylipins, total to normalized were data the When Quantitatively s we study, present the In

its oxidized metabolites. oxidized its overall male and female pup brains pup female and male 83

ol accumulate would ) olwd y DHA by followed %) PUFA precursors PUFA

yohss ht LA that hypothesis n ~1 in

ie po/ brain) pmol/g (i.e. - glia co glia - . OE nraig xnl ugot i ml neurons. male in outgrowth axonal increasing HODE - The main oxylipins in adult rats adult in oxylipins main The a od a brain, rat old day 2 ) . -

cultures obtained from obtained cultures

- (Hennebelle et al. 2019) al. et (Hennebelle month male Fisher male month

more oxylipins oxylipins more howed to oxylipins (e.g. AA and and AA (e.g. oxylipins to - - specific, with LA increasing axonal outgrowth in female in outgrowth axonal increasing LA with specific, derive

, regulate

, which is approximately is which brains ht OXL that total mtblts ( metabolites d n that and

brain brain s of

over time as the brain increases in size and and size in increases brain the as time over brain - 344 (CDD) rats fed a 10.5% energy LA diet LA energy 10.5% a fed rats (CDD) 344 60 M constitute AMs

- postnatal rat pups rat postnatal (Ferdouse et al. 2019, Hennebelle 2019, al. et (Ferdouse oxylipin 64 LA

. development

Differences in concentrations in Differences

day

and on a similar diet were diet similar a on 9 %)

DHA) DHA) old adult male rats male adult old

concentrations

LA Hneel e a. 2019) al. et (Hennebelle 6 -

10 - eie 13 derived h mjrt ( majority the during the first few weeks few first the during

directly and via one (or (or one via and directly . times higher than the the than higher times However, i However, .

The effects of LA and and LA of effects The

were - HODE hs results These fed a similar similar a fed

t is t 46 AA 6.5 - expected - 52% - 2 of 52%

may be be may derived derived altered

times times et al. et A . )

5 of -

This article isprotected rights by All copyright. reserved. Acceptedand be should investigated. further developm during metabolism oxylipin brain in differences sex of implications that suggests contain to found were in reported been have rather convertedphospholipids than metabolites. oxylipin into retained. is brain the entering LA that suggests analyzed L brain Articleweeks 8 for OXLAMs) with (enriched oil corn oxidized unaff were but levels, LA dietary with increased were concentrations OXLAM rats. developing in metabolism OXLAM and LA brain on information brain the entering might appear selective tobe LA from 5% only and DHA from 22% AA, from derived (comparable study), inthis tothediet used

e u to due be A to relative OXLAMs of accumulation preferential The Sex did not affect pup brain oxylipin concentrations. Sex differences in differences Sex concentrations. oxylipin brain pup affect not did Sex ), while its metabolites constituted approximately half of the measured oxylipins, oxylipins, measured the of half approximately constituted metabolites its while ),

Other PUFAs such as AA and DHA appear to be preferentially retained in brain brain in retained preferentially be to appear DHA and AA as such PUFAs Other was the least abundant fatty acid acid fatty abundant least the was e dfeecs i differences sex hi slcie noprto fo blood from incorporation selective their no XAs laig o XA accumulation) OXLAM to (leading OXLAMs into lyretained inthe the same strain same the 5 mr oyiis oprd o females to compared oxylipins more 25% ban xlpn concentrations oxylipin brain n

preferentially brain of developingbrain compared toadults. rats of of rats of reported that69% ofbrai reported relative to others to relative

(Sprague

metabolized into OX into metabolized - (Ramsden Dawley) at 9 week of age of week 9 at Dawley)

(Taha et al. 2016b) al. et (Taha

r rapid or (<3%

likely other

et al. et composition n unesterifiedoxylipinswere Fros e a. 2019) al. et (Ferdouse enzymatic enzymatic

mre ae i life. in later emerge

xlpn i pp brains pup in oxylipins 2018) LAMs instead of being being of instead LAMs

n dl mice, adult In . . The fact that pup pup that fact The . ent are not known known not are ent . There is limited limited is There

ected by feeding by ected in all lipid pools lipid all in Thus, OXLAMs OXLAMs Thus, unvr f LA of turnover brain brain

, when males males when , oxy . lipins lipins brain brain

This The

This article isprotected rights by All copyright. reserved. Accepted backgroundsimilar on ourassays, vehicle. including effect anyexperiments, 13 across used wasmedia same the since but assessed, 13 of effects the limit that at efficacy exposed concentration relationship neurons. cortical male in only 2007, Dehaut etal. 1993) calculation this in for accounted not therefore and known not is studies these PC12 Article are concentrations 0 applying after media culture the LA µM ~3.5 contained which B27, 2007) al. et Kamata 1993, al. et (Dehaut motoneuronschick µM 1 neurons cortical male not but female lengthsin axonal sex was effect this but growth, axonal altered morphology cellular

; cells Figure 4A Figure Similarly, that studies previous with Consistent

o environmental to

higher doses is not known, but known, not is doses higher

r hc motoneurons chick or versus a linear a versus - effect ) 13 .

likely to LA at LA to

N relationship has been been has relationship - HODE significantly increased axonal outgrowth, but only at 100 nM and nM 100 at only but outgrowth, axonal increased significantly HODE Kmt e a. 07 Dhu e a. 1993) al. et Dehaut 2007, al. et (Kamata eurite outgrowth eurite

. - ihn h 1 the within

HODE.

concentration chemicals 1

and h efc o 13 of effect The atog te opsto o th of composition the although , 1 Notably, levels of 13 of levels Notably, 0 (Chen

- (Wayman - M was shown to increase to shown was 300 00 M A rne bten 3.5 between ranged LA, nM 1000 .

Notably, o Notably, effect relationship ( relationship effect (Dehaut et al. 1993) al. et (Dehaut previously reported for similar neuron similar for reported previously

et al. et µM it could be due to regulatory feedback mechanisms mechanisms feedback regulatory to due be could

hwd infcn efcs f A on LA of effects significant showed specific as evidenced by signifi by evidenced as specific

ag son to shown range

2008b) - t al. et OE eebe a U a resembled HODE ur

at 1, 50 and 1000 nM (i.e. 0.001, 0.05 and and 0.05 0.001, (i.e. nM 1000 and 50 1, at culture media was supplemented with 2% with supplemented was media culture

- . Hence, the actual the Hence, . 02 Yang 2012, been not have used media the in HODE

Figure 4B Figure , and PC12 cells to 10 to cells PC12 and , following exposure of exposure following , stimulate unesterified unesterified cl clue ei ue in used media culture cell e

t al. et - ). ). HODE ther HODE - shaped shaped An inverted An

in outgrowth neurite LA concentration of of concentration LA 2014) - LA significantly significantly LA 4.5 µM. These These µM. 4.5 cantly increased increased cantly -

glia co glia Kmt e al. et (Kamata dose . - e will have ahave will e The loss of of loss The 3 00 µ 00 embryonic embryonic

U - - response neuronal cultures cultures - shaped shaped M

This article isprotected rights by All copyright. reserved. Acceptedenergy less or 2% of needs nutritional than higher but intake of levels human current to underlying their 13 and LA shown been not has HODE G2A) neuron within HODE would allow ustot high to Article subjected pups GC the because concentrations oxylipin quantify accurately to ability our limiting thus configurations, microwave current with 2017) al. et Hennebelle 2019, prevent to needed fixation microwave oxylipins other and 1.04 of density than the 13 the as The sex the to linked mechanisms that is study this of limitation A The on axonal outgrowth axonal on

receptor for various free fatty acids and oxylipins, but selectivity towards LA or 13 or LA towards selectivity but oxylipins, and acids fatty free various for receptor - - maternal suggestive suggestive HODE HODE HODE HODE - la are glia – - sex FID method used to measure it lacked sensitivity. Measuring unesterified LA in LA unesterified Measuring sensitivity. lacked it measure to used method FID 1.05 g/mL 1.05 g/mL est physiologically invitro. relevant doses

- concentration are likely overestimates because the pups were not subjected to high to subjected not were pups the because overestimates likely are specific specific in dietary LA level in the present study provided 10% energy 10% provided study present the in level LA dietary

o known not - brain at this age. this at nry irwv irdain with irradiation microwave energy - HODE concentration of 10 of concentration HODE (DiResta (Lahvic effects on neuronal morphogenesis.effectsneuronal on were not explored. This is because receptors for both compounds both for receptors because is This explored. not were . Microwave . ol hl determine help could

of 18.2 nM 18.2 of . Recent studies have characterized GPR132 GPR132 characterized have studies Recent . (Blasbalg et al. 2011, U.S. Department of Agriculture 2016) Agriculture of Department U.S. 2011, al. et (Blasbalg

et al. et Unest

et al. et h efcs of effects the

2018, Obinata 2018, erified (free) LA was not detected in pup brain likely brain pup in detected not was LA (free) erified -

fixation is difficult to implement in 0 in implement to difficult is fixation 1990)

observed in male pup brain pup male in observed ) . However, the 0 post

nM in neuron in nM in both rats and humans and rats both in et al. et

oeua pathways molecular - mortem

estv mtos uh as such methods sensitive

brain brain 2005)

concentrations . ischemia Identifying the receptor for receptor the Identifying - glia co glia - fet o L ad 13 and LA of effects

( based on a rat brain brain rat a on based - (Hennebelle et al. al. et (Hennebelle cultures is cultures and mechanisms mechanisms and - , which is close is which , as kon as known (also

1 day old pups pups old day 1 (Hansen et al. al. et (Hansen

of 13 of

- - n vivo in GCMS HODE energy energy higher higher - - ,

This article isprotected rights by All copyright. reserved. -- reviewedbefore manuscript the submission. co All draft. first the wrote AYT and MH analysis. statistical the performed and results MH, Contributions: Interest: of Conflicts concentrations explore should studies brain, development during brain in CONCLUSION 13 and LA both that showing findings neurodevelopment our of light in particularly further scores infants acids) fatty of (>9.7% composition 2017) association Choque 1958,

Accepted Human subjects Article RM, RM, . Other . promote at 15 at (Bernard In conclusion, this study this conclusion, In understand S H, Z n AG efre te xeiet. MH experiments. the performed ACG and ZZ HC, SS, between year

tudies in relation to axonal axonal

t al. et

AYT, MH, AYT,

s of age age of s al. et

in asex -- the

matern

reported an inverse association between association inverse an reported

2015) None todeclare.None

outgrowth in primary cortical neurons in a sex a in neurons cortical primary in outgrowth 05 Hla 1958) Holman 2015,

oe f leig aenl dietar maternal altering of role the effects of varying varying of effects the (Lassek & Gaulin 2014) Gaulin & (Lassek - specific manner.

al LA intake and mental and psychomotor development psychomotor and mental and intake LA al , and that that and , , neurodevelopment RM, RM, verbal IQ at 5 to 6 years of ageyears of 6 to 5 IQat verbal demonstrates SS and PL conceived the study and designed the experiments. experiments. the designed and study the conceived PL and SS and both LA and and LA both

reduced motor and cognitive scores in 2 to 3 3 to 2 in scores cognitive and motor reduced

. pdmooia suis ae on an found have studies Epidemiological that

aenl itr LA dietary maternal .

Collectively, t Collectively, OXLAMs make up th up make OXLAMs 13 - HODE, the most abundant OXLAM in rat in OXLAM abundant most the HODE,

L content LA y (Bernard maternal breast milk LA percent percent LA milk breast maternal hese studies highlight studies hese , -

RM RM dependent manner dependent et al. et on e n neu on

n ZZ and majority of oxylipins oxylipins of majority

pup 2017) - HODE regulate regulate HODE

brain OXLAM OXLAM brain rodevelopment, rodevelopment, , and , compiled the the compiled (Kim cognitive cognitive a

year old year -

. Future Future . need to need authors authors inverse

et al. et

This article isprotected rights by All copyright. reserved. the in or report thedecision paper tosubmit for publication. the of writing the in data, of interpretation and analysis, collection, design, publica the in mentioned services or products commercial any of purchase the endorse not do USEPA and NICHD NIEHS, the USEPA; or NICHD NIEHS, the of views official the represent necessarily not do work this of contents The of R number Agency Protection Environmental[grant States United number [grant Development Human Institu National Shriver Kennedy Eunice the HC], and SS to ES007059 ES011269, P01 ES023513, P30 numbers [grant Health Environmental of Institute (project the by supported was work This Acknowledgement: orEditorial isaReview unless it guidelines." wereconducted experiments "All => ifYes,insert asnecessary). sentence acomplete toform (edit phrasing " reason: following forthe notfollowed were guidelines statement: "ARRIVE => if Yes beenfollowed: have ARRIVE guidelines the and subjects, all for achieved was consent experiments wereapproved the by local ethics committee."included is in the Methods "Informed info the that ensure please yes, if => achiev approval ðics consent If yes:Informed subjects: Involves human

Acceptedwhich MondyFellowship, ScienceNell in Graduate Women the Article

it is Reviewa or Editorial, skip complete sentence => if No, includea

#1008787) , UC Davis College of Agriculture and Environmental Sciences, Environmental and Agriculture of College Davis UC The authors thanks Shiva Emami for her help with the statistical analysis. statistical the with help her for Emami Shiva thanks authors The

USDA 5 HD079125 U54

National Institute of Food and Agriculture, and Food of Institute National

tion. The sponsors were not involved in the study study the in involved not were sponsors The tion.

and

ed: in compliance with the ARRIVE R21

D 83543201]. MH is a recipientais MH 83543201]. D - HD095391 also funded part of this study. this of partfunded also

te of Child Health and and Health Child of te - 01A1

) and by the the by and ) the National National the Hatch/Taha Hatch/Taha

and T32

This article isprotected rights by All copyright. reserved. in Changes (2011) R. R. Rawlings, and F. S. Majchrzak, E., C. Ramsden, R., J. Hibbeln, L., T. Blasbalg, Bernard, J. Armand, Peyre,H., Y., Garcia, M., C.,Forhan,Agostini, Charles, A.,De M., A.M. and Heude,B. (2015) B. Heude, and A. M. Charles, M., Agostini, De A., Forhan, C., Garcia, M., Armand, Y., J. Bernard, and V. Chapman, A., D. Barrett, A., Bennett, S., M. Chan, H., P. Arya, P., Millns, A., Wong, M., Alsalem, acid linoleic of effects Comparative (1996) S. Okisaka, and H. Yorifuji, T., Kanda, T., Hiramitsu, K., Akeo, Dagger p<0.0001. at controlpositive (Y **** p<0.01; at ** p<0.05, at significance Y a in differences determine to used was concentration one determine to test hoc a post comparison multiple using separately analyzed were sex each for data Data dissections). independent (LA) acid prostaglandin linoleic A.) of Figure Teklad Global 18% Protein; chart)were 2. Table from derived as pups female and male in oxylipins brain Figure Neuro (PND) The birth. gave they until facility animal controlled light 2 Figure metabolites. Figure FIGURE LEGEND:

Accepted27632 Article - glia were also isolated from pup brains and exposed to LA,glia to exposed also from isolated were and 13 pupbrains 0 or 1 or 0 Am JClin N omega of consumption Intelligence Child and Colostrum in Quotie Levels Acid Fatty Polyunsaturated Breastfeeding, (2017) cohort.EDEN 3 and 2 at cognition child and colostrum in levels acid linoleic between association The peripheral in Pharmacol, role their and processing nociceptive to 9 ligands TRPV1 endogenous the of contribution The (2013) A. D. Kendall, cells in vitro. epithelial pigment retinal bovine of morphology and growth on hydroperoxide acid linoleic and 4 3 1 : . .

. vs vehicle control. control. vehicle vs

Flowchart of the study designed. Pregnant rats were maintained in a temperature and temperature a in maintained were rats Pregnant designed. study the of Flowchart Brain oxylipin Brain Axonal length of primary rat cortical neurons incubated with different concentrations concentrations different with incubated neurons cortical rat primary of length Axonal

obtained from 2 from obtained an nyai ptwy ivle i te ytei o oiie lnli acid linoleic oxidized of synthesis the in involved pathways enzymatic Main

ntat Age 5 2 (PGE2) E2 and their brains excised. Brains were subjected subjected were Brains excised. brains their and utr,

168, - 27632) 27632) Current eye research, Pediatr Res,

93,

1961 -

6 6 Years. 950

distribution in pups and adult rats adult and pups in distribution . Data are presented as mean ± SD (n = = (n SD ± mean as presented are Data . andfor vehicle males with treated 13 - - 1974. - (Hennebelleet al. month old Long Evans male rats fed the same diet as the pups (2018 pups the as diet same the fedrats male Long old Evans month 962. - , Results were considered significant at p<0.05. at significant considered were Results 3 and omega and 3

77, 13 B.) were J Pediatr,

829

nlzd y t by analyzed - -

835. 15, xonal length xonal

183, hydroxyoctadecadienoic - REFE 6 fatty acids in the United States during the 20th century. 20th the during States United the in acids fatty 6

467

43 - 2019) RENCES 476. -

50 50 e43.

). in cultures treated with the p the with treated cultures in

- ANOVA way

pups were de were pups - a AN way

nlmaoy an mechanisms. pain inflammatory (n=3 pups (n=3 - specific effects. specific to - HODE. fatty acid or oxylipin analysis. oxylipin or acid fatty

OVA OVA

Data for male adults (3 adults male for Data cd (13 acid ( - + HODE or PGE2. HODE . rfet p00 between p=0.06 reflects )

capitated on postnatal day day postnatal on capitated o sbeun analyses, subsequent For

per sex per

olwd by followed 7

- Asterisk (*) denotes denotes (*) Asterisk 9 - -

HODE) HODE and 13 and HODE An unpaired t unpaired An wells ) . Distribution of Distribution ositive control ositive

, and C.) C.) and ,

from from

Dunnett’s Dunnett’s

y in the in y - HODE rd - four

r J Br test test pie pie

This article isprotected rights by All copyright. reserved. AcceptedBazinet,Hammock,R. S.,B. D., S. K. Lee,J., Yang,Y., Otoki, P., A.Kitson, Hennebelle,H., Metherel, M., A. radioactive fed orally of incorporation The (1975) A. M. Crawford, and J. A. Sinclair, G., A. Hassam, inacids fatty Essential(1958) F. H. Wiese, and J. D. Adam, N., A. Boelsche, E., Haggard, M. E., A. Hansen, Glozman,Green, P., S.,Kamensky, B.and (1999) E. Yavin, Developmental changesin prostaglandinby linoleicacidsynthase endoperoxide Metabolismof (1983) W. S. Powell, and D. C. Funk, I Profile Oxylipin Brain The (2019) M. H. Aukema, and T. Winter, S., Leng, A., Ferdouse, Nijkamp, and H. Willems, F., Engels, linoleic microsomal liver human a as CYP2C9 of Identification (2000) D. B. Hammock, and J. A. Draper, Dragunow, M. (2008)High tissue brain of Measurement (1990) E. Arbit, and H. J. Galicich, F., Ali, N., Lau, J., Lee, R., G. DiResta, Brain (2006) P. R. Bazinet, and I. S. Rapoport, M., J. Bell, L., Chang, K., Ma, J., H. Lee, Jr., C., J. DeMar, Pouplard T., Miltaud, I., Bertrand, Article F., Dehaut, (1994) A. M. Crawford, and A. R. Iles, K., Craig, S., Brookes, D., J. Bell, C., S. Williams, C., S. Cunnane, Cunnane,S. C.and Chen,Z. (1992) Y. Quantitative changesin long acid linoleic Dietary (2015) P. Legrand, and P. Guesnet, B., Delplanque, D., Catheline, B., Choque, Re NS21: (2008b) W. J. Hell, and A. B. Barres, J., Milbrandt, F., J. Chang, B., Stevens, C., Y. Chen, Bazin and K. S. Orr, T., J. Green, T., C. Chen, Bylund,Analysis H.Oliw, E. J.,(1998) J. Ericsson, cytochrome andof P450 metabolites arachidonicof and Burger,F., Marks, Krieg, Furstenberger, P., Positional F.and G.(2000) of brain of effects hypercapnia/ischemia: following concentrations oxylipin Brain (2019) Y. A. Taha, and P. 417 gamma infantnutrit Res, acid. docosahexaenoic of accumulation prenatal preferential The acids. fatty and lipids from adult and fetal blood vessels. 54, n Dietary by Modulation Lett, non under macrophages alveolar pig guinea by acid hydroxylinoleic acid epoxygenase. usingdensity pycnometry. phospholipi brain in adult rats. arachidonate the of source negligible a is acid linoleic of elongation embr and cells PC12 of length motoneurons. neurite the increase acids fatty polyunsaturated 2429 acidsandfatty choleste 13C labeled uniformly of Utilization developmentpostnatal inrats. intake value, study inrats. alpha of presence the in requirements and mod acid uptake and turnover. 55 linoleicliquid acids by chromatography oxygenation catalysed by mouse(12S) -

68. 67 -

420. 40, - 209, 2436. -

80. -

linolenic acid and linoleic acid into the liver and brain lipids brain and liver the into acid linoleic and acid linolenic 960 dissection and dissection time.

ified supplement B27 neuronalfor cultures.

249

Biochim Biophys Acta,

- ion. III. Clinical manifestations of linoleicacid deficiency. 966. - 253. Neurosci Lett,

Arch BiochemBiophys, contentanalysis inneuroscience. rol accumulating intheneonatal ratbrain. - 6 and n and 6 ProstaglandinsLeukot Essent Fatty Acids, The British journal ofnutrition, Acta Neurochir Suppl (Wien),

161, The American journal o

- 1761,

3 Polyunsaturated Fatty Acids in Male and Female Rats. Female and Male in Acids FattyPolyunsaturated 3

133 Biochim BiophysActa, . . 18) Cyclooxygenase (1986) P. F. - et, R. P. (2008a) Regulation of brain polyunsaturated fatty polyunsaturated brain of Regulation (2008a) P. R. et, oynauae fty cd i h snhss f long of synthesis the in acids fatty polyunsaturated - - 136. -

lipoxygenase isoenzymes. - 1050 linolenic acid are lower than the historica the than lower are acid linolenic J Lipid Res, mass spectrometrymass ion with trap MS.

376,

- - 1059. ateax A ad anal, . 19) n (1993) M. Maingault, and A. Barthelaix,

199

60, 205. Nat Rev Neurosci, J Neurosci Meth,

f physiology, 671

51,

754, - 113, - chain fatty acids during and fetal early 682.

34 -

- 57 and stereo 1056 36.

- Biochem J, 71.

Journal ofneurochemistry,

- 262,

aaye frain f 9 of formation catalyzed - - 79, 1068. stimulated conditions. stimulated J Nutr,

of suckling rats. suckling of 171,

R14

- 779 selectivity fatty acid of

91. 239

66,

- 348 Pt 2, rat brain membrane 19. - Anal Biochem, 788.

565 247.

l 2 % of energy of % 2 l

s Resistant to Resistant s -

576. oi chick yonic

329 Lipids, -

defined - 335. J Lipid J Lipids, - chain ds of ds

FEBS 265,

10, 62,

- 6 -

This article isprotected rights by All copyright. reserved. Acceptedan W. P. DeVoe, E., S. Carlson, C., J. Putnam, 9 of unsaturated of oxidation radical free of Mechanisms (1995) A. K. Mills, and E. Identification S. Caldwell, A., N. Porter, (2005) T. Izumi, and in TRPV1 of Activation (2009) M. K. K. Hargreaves, and N. A. Akopian, E., P. Scotland, M., Tatei, A. Patwardhan, S., Nakane, T., Hattori, H., Obinata, of specificity The (2002) E. Niki, and H. Kuhn, A., Nakamura, J., Hamahara, H., Yamashita, N., Noguchi, G., J. Alvarez, P., Tontonoz, L., Nagy, (2010) J. E. Murphy, Moghaddam, M., Motoba, K., Borhan, B., Pinot,F. and Hammock,B. (1996)D. Novel metabolicpathways or arachidonic dietary maternal of Effect (1994) F. C. Kuhlman, and J. R. Yuhas, G., F. Boyle, L., E. Lien, oppo have milk human in acids docosahexaenoic and Linoleic (2014) J. S. Gaulin, and D. W. Lassek, J. Lahvic, Articlea of Evaluation (2013) M. Leist, and D. Merhof, F., Schonenberger, F., Matt, V., N. Balmer, K., A. Krug, of intake maternal between Association (2017) N. Chang, and H. E. Ha, Y., Kim, E., Lee, H., Kim, H., Kim, in outgrowth neurite of Induction (2007) E. Gohda, and Y. Kawamoto, A., Tai, H., Shiraga, Y., Kamata, II. milk. human Jenness,R. (1979) The composition of human milk. of acids fatty The (1959) Jr. H., E. Ahrens, and T. James, J., Hirsch, gas Jr., W., for Insull, esters methyl acid fatty of dieta maternal milk: Human (2007) M. Preparation S. Innis, (2010) Y. Fukubayashi, and K. Ichihara, Holman,T. R. (1958)Essential fatty acids. to response the in participates acid Linoleic (2017) al. et H. A. Metherel, Z., Zhang, M., Hennebelle, at ais n h fty cd opsto o eyhoye hshtdlhln and phosphatidylcholine erythrocyte of composition acid fatty the on phosphatidylethanolamine in human infants. acids fatty lipids. NatlProc Acad U Sci A, S contributes metabolites acid linoleic oxidized by cord spinal the coupled receptor G2A. fatty free oxidized other and acid hydroxyoctadecadienoic Chem, lipoxygena macrophagegene expression through ligand activation of PPARgamma. head ProstaglandinsOther Lipid Mediat, requires eicosanoids linoleicfor and arachidonic acid metabolism. linoleic acid Essent FattyAcids, countries. 28 of sample a performancein cognitivewith relationshipstest the receptor GPR132. 87, neurotoxicants.developmental for screen specific as assay growth neurite human results theof MOCE n PC12 cells by the medium fats. dietary of exchange Invest, and balance caloric of manipulation by produced Alterations 404. chromatography. 4342. neurotransmission. regulate that metabolites oxidized through injury brain ischemic

- L., Ammerman, M., Li, P. et al. (2018) Specific oxylipins enhance vertebrate hematopoiesis via hematopoiesis vertebrate enhance oxylipins Specific (2018) al. et P. Li, M., Ammerman, L., 6 to n to 6

2215

Lipids,

383, 38, - - 3 fatty acid ratio during pregnancy and infant neurodevelopment at 6 months of age: of months 6 at neurodevelopment infant and pregnancy during ratio acid fatty 3 2231.

se 443

619 on raton pup fatty acidprofiles.

- Brain fixation for analysis of brain lipid brain of analysis for fixation Brain 30, catalyzed lipid peroxidation and the effects of radical of effects the and peroxidationlipidcatalyzed -

450. - 626.

277 J Lipid Res,

91,

H cohortstudy.

- 290. Proc Natl Acad U Sci A, S

195 J Biol Chem,

106, -

chain fatty acid octanoi - 201.

51,

- 18820 oue mcoae raito: n itrcl perspective. historical an irradiation: microwave focused

635 Nutr Rev, hn H ad vn, . . 19) xdzd D regulates LDL Oxidized (1998) M. R. Evans, and H. Chen,

91, 280, Nutr J, - - 18824. 640. d Barness, L. A. (1982) The effect of variations in dietary in variations of effect The (1982) A. L. Barness, d

63 ry lipids and infant development. infant and lipids ry

40676 -

Lipids, 67.

16,

Semin Perinatol, 16,

Biochim BiophysActa,

Am JClin Nutr,

23.

- 115, 33 40683.

29, - c acid.c 35.

9252 53 - mediators of signal transduction and brain and transduction signal of mediators

- 59. Neuroscience, - 9257.

36, cd a lgns f h G protein G the of ligands as acids

225

106 to inflammatory hyperalgesia. inflammatory to - 239. -

scavengingantioxidants. 1290, - 114.

146,

Cell,

Proc Nutr Soc, Nutr Proc

327 ProstaglandinsLeukot

1073

93, - 339.

229 - 1081.

Arch Toxicol, Arch - 240. Sci Rep, Sci

66,

- liquid J

397 Biol Clin site

7 - - - ,

This article isprotected rights by All copyright. reserved. AcceptedKania D., Yang, Early (2018) D. C. Mintz, and P. Zhang, Y., Fang, C., Chang, Y., Wang, M., Xu, P., R. Mathena, J., Xu, Rai E., Ahmad, N., Ussyshkin, T., C. Wong, Pes D., Bruun, V., Ledoux, A., Lesiak, D., D. Bose, D., Yang, A., G. Wayman, Mean Beverages: V. Derkach, and F., W. Grant, T., Saneyoshi, D., Food Marks, S., Impey, A., G. Wayman, from Intakes Nutrient (2016) S. R. A. Agriculture, of Department U.S. E. C. Ramsden, and D. B. Hammock, I., S. Rapoport, D., Zamora, J., Yang, M., Hennebelle, Y., A. Taha, acid docosahexaenoic brain rat in changes Threshold (2016a) M. Chen, and L. Chang, Y., A. Taha, Articlechemical of Assessment (2011) M. Leist, and F. Matt, K., A. Krug, V., N. Stiegler, Crawford, and J. A. Sinclair, Sethi, S.,Keil,K. Chen, H.,P., Hayakawa, K., inhibitor, P450 acid linoleic in cytochromerich diet maternal a of influence The (1984) J. D. Naismith, The and M. Mistry, A., T. Sanders, (2012) M. K. Hargreaves, and P. Chen, D., Green, S., Ruparel, Majchrzak A., Ringel, E., C. Ramsden, S Schuster, M., Hennebelle, E., C. Ramsden, mechanisms. receptor ryanodine via neurons hippocampal rat cultured in connectivity neuronal P (2014) J. P. Lein, SynapseFormation Actionsvia the on mTOR Pathway. Gene to Exposure Developmental Neurosci Res, expression. gene target Wnt regulates and differentiation and proliferation neural promotes Health Perspect, PCB CREB Activity Amounts Consumed perIndividual, byGenderand Age. increasing with concentrations levels of dietary linoleicacid. oxylipin cortex cerebral and plasma rat of Regulation (2016b) ProstaglandinsLeukot Essent Fatty Acids, reductions graded following concentration and incorporation Sci, neurit human the developingbrain. rat Dendritic Growth in Primary Rat Neurons. 3,3' of Detection brainon and retinal docosahexaenoicacid in the rat. Molecular Pain, keto 12 pro mice. Acta MolCell Biol Lipids, in aldehydes and oxylipins, acids, fatty brain on products peroxidation .

- 121, -

conazole, inhibits oxidized linoleic acid metabolite acid linoleic oxidized inhibits conazole, 5 rmts edii got va yndn receptor ryanodine via growth dendritic promotes 95 and anti and - dependent transcription of Wnt - - Korwel, I., Ghogha, A., Chen, H., Stamou, M., Bose, D. D., Pessah, I. N., Lehmler, H. J. andJ. H. Lehmler, N., I. Pessah, D., D. Bose, M.,Stamou, H., Chen, A., Ghogha, I., Korwel, eedn dnrtc roiain eitd y CaM by mediated arborization dendritic dependent

73 - 87. -

Toxicol Sci, 94, high in imaging cell live multiparametric by outgrowth e nociceptive lipid autacoids: Implications for idiopathic pain syndromes? pain idiopathic for Implications autacoids: lipid nociceptive

120,

. - 759

ihooihnl n ua Mtra Pam ad t Efcs n xnl and Axonal on Effects Its and Plasma Maternal Human in Dichlorobiphenyl B 3 arpeetvl atr mrhmti ad ucinl aaees of parameters functional and morphometric alters atropselectively 136 CB

M. A. (1972) The accumulation of arachidonate and docosahexaenoate in docosahexaenoate and arachidonate of accumulation The (1972) A. M.

- 997 775.

1863, 138, Journal ofneurochemistry, - 1002.

- ProstaglandinsLeukot Essent

379 Hong, S. F. e F. S. Hong,

1206

- - 392. Bhogal, R., Li, H. and Crawford, D. A. (2016) Prostaglandin E Prostaglandin (2016) A. D. Crawford, and H. Li, R., Bhogal, a Aetei Aet i Piay ern utr Disrupts Culture Neuron Primary in Agents Anesthetic ral Li, Li, Lin,X., Y., Lehmler,H. J., Puschner, J.B. and (2017) P. Lein, . et al. (2018) Effects of diets enriched in linoleic acid and its and acid linoleic in enriched diets of Effects (2018) al. et . - 1213. - 2. 2.

Neuron,

105, Toxicol Sci,

t al. (2016) Dietary linoleic acid linoleic Dietary (2016) al. t

- 50, 34.

19,

The British journal ofnutrition, 897

158, IntJ Mol Sci, -

NHANES 2013 1753 mediated peripheral inflammatory pain. inflammatory peripheral mediated - 909.

401 Fatty Acids - - 1758. -

- iae atvto ad enhanced and activation I kinase eedn mechanisms. dependent 411. n itr alpha dietary in sah, I. N. and Lein, P. J. (2012)J. P. Lein, and N. I. sah,

. and Soderling, T. R. (2006) R. T. Soderling, and - .

2014 - density cultures. density - induced impairment of impairment induced - induced alterations in alterations induced .

ici Biophys Biochim - ioei acid. linolenic

51, - dependent Mol Pain, Mol

57 Environ Toxicol - 66.

2 J

This article isprotected rights by All copyright. reserved. Accepted PHOSPHOLIPIDS TRIACYLGLYCEROLS Article CHOLESTERYL FATTYFREE ACIDS of male and female pups. rat fractions (PL) phospholipid and (TAG) triacylglycerol (CE), ester cholesteryl (FFA), acid fatty 1. Table

Myristoleicacid Stearic acid Palmiticacid Arachidonic acid Dih Gondoic Acid Linoleic Acid Vaccenicacid Oleic Acid Palmitoleicacid Stearic acid Palmiticacid Myristoleicacid Myristicacid Total Arachidonic acid Palmitoleicacid Myristoleicacid Stearic acid Palmiticacid Total fatty acids Linolenic Acid Arachidonic acid Dih Linoleic Acid Gondoic Acid Palmitoleicacid Myristoleicacid Stearic acid Palmiticacid Myristicacid Total fatty acids Arachidonic acid Oleic Acid Palmitoleicacid Fatty acid concentration (nmol/g) and (nmol/g) concentration acid Fatty omo omo fatty acids ESTERS - - gamma gamma

- -

18:1 n 18:1 n - - - -

------18:0 18:0 18:0 18:0 - -

- 18:2 n - 18:2 n 16:0 16:0 16:0 16:0 14:0 14:0

20:1 n 20:1 n 20:1 18:1 n 18:3 n 18:3

------linolenicacid linolenicacid

- - - - 16:1 n 16:1 n 16:1 n 16:1 n 14:1 n 14:1 n 14:1 n 14:1 n

- - 20:4 n 20:4 n 20:4 n 20:4 n 9 9

- - 6 6 - - - - 9 9 7

- - - -

7 7 7 7 - - - - 5 5 5 5 6 6 6 6

- - 20:3 n 20:3 n - - 6 6

15718.4 2206.8 4886.7 1477.9 5524.4 7047.0 1240.1 1153.9 3031.7

composition (% of total fatty acids) in brain free brain in acids) fatty total of (% composition 280.9 812.3 222.7 266.4 476.5 424.6 219.0 725.7 187.9 129.6 451.5 675.9 399.1 273.8 268.5 129.4 427.9 216.0 529.7 215.6 228.7 336.0 Mean Mean 94.6 54.4 97.7 92.0 Malepups rat

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

SD %fatty acids Mean 48.6 742.5 59.8 785.7 71.8 322.1 514.9 91.7 45.1 370.3 883.6 37.4 14.4 79.7 254.2 1011.6 155.0 1240.7 3083.3 73.0 189.0 273.9 55.0 281.8 70.9 16.7 355.5 461.5 66.2 88.9 342.6 19.3 66.2 90.4 91.7

10% 25% 7% 5% 10% 19% 21% 11% 34% 11% 0 0 1% 3% 13% 2% 17% 37% 1% 3% 5% 23% 25% 12% 35% 3% 7% 18% 3% 7% 8% 11% .4 .3

% %

15714.9 1177.6 4865.2 1468.0 5518.8 7285.8 1187.7 2863.5 267.4 813.7 183.7 168.5 347.9 167.5 327.1 187.1 138.4 440.6 675.0 344.5 846.3 117.3 244.5 128.3 291.3 134.4 198.9 471.9 227.7 231.8 255.0 92.4 74.3 65.0 79.0 Female ratpups

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

20.7 577.3 29.1 83.4 9.4 12.3 21.8 35.9 12.7 15.5 225.1 16.3 20.2 41.5 30.4 159.9 40.7 359.8 497.2 45.4 70.3 75.3 17.1 22.0 23.6 10.0 35.1 1024.0 93.9 113.5 364.2 28.8 110.2 97.5 13.1 SD%fatty acids

11% 0 0 1% 3% 13% 2% 17% 37% 1% 3% 8% 14% 29% 15% 34% 4% 7% 15% 3% 8% 9% 10% 11% 27% 7% 8% 6% 14% 30% 14% 28% .4 .3

% %

This article isprotected rights by All copyright. reserved. linoleic esters, myristic, female in and acid phospholipids. vaccenic cholesteryl and phospholipids male male in acid stearic triacylglycerols, in male in acid palmitic acid esters, cholesteryl gondoic acids, and fatty free myristoleic male in acid palmitic, oleic and palmitoleic were These distributed. normally not were pups female and male between ( observed were pools lipid 4 the of any in composition percent t unpaired an by analyzed were (nmol concentration mean are Values p

Accepted Article>0.05).

Total fatty acids Docosahexaenoic Acid (DHA) Docosapentaenoic acid (DPA)

Shapiro - Wilk test showed that most data were normally distributed. A few fatty acids fatty few A distributed. normally were data most that showed test Wilk

- - test. test. -

22:6 n 22:5 n /g) o infcn dfeecs n at ai cnetain or concentration acid fatty in differences significant No

- ± SD and mean % composition of n=4 pups per pups n=4 of composition % mean and SD ± - 3 6

42671.8 3432.9 1500.4

± ± ±

7736.4 565.0 271.3

8% 4%

42828.0 3450.4 1551.7 cd n female in acid

± ± ±

1401.8 130.6 58.0 s . Data Data .

8% 4%

This article isprotected rights by All copyright. reserved. Accepted DiHETE, prostaglandin; PG, acid; dihydroxyeicosatrienoic DiHETrE, dihydroxyeicosatetraenoic acid; EpDPE,epoxydo acid; acid; epoxyeicosatrienoic EpETrE, acid; hydroxyeicosatetraenoic HETE, acid; trihydroxyoctadecenoic epoxyoctadeenoic EpOME, Abbreviations: 14(15) distributed, normally t (Unpaired female and male between S ± mean as expressed are Data Total oxylipin DHA EPA Article AA LA Table - - derived metabolitesderived derived metabolitesderived - - derived metabolites derived derived metabolitesderived - 2 EpETrE infemales. EpETrE . Oxylipin Oxylipin 19(20) 10(11) 1 15 14,15 14(15) 8(9) 5(6) 12 5 9,12,13 9,10,13 12,13 9,10 12(13) 9 13 9 7,18 - - - HETE oxo HODE - - - s deoxy HETE HODE

- - OE hdoycaeaini ai; oxo acid; hydroxyoctadecadienoic HODE, - detected EpETrE EpETrE DiHOME - - - - DiHETE DiHETrE DiHOME - - - - ODE concentration EpDPE EpDPE EpET EpOME - - TriHOME TriHOME

PGJ2

except

o 9 for

D (n=3 D s

- (pmol/g) male theof female pups. brain in and OE 12(13) HODE, - test; DiHOME, dihydroxyoctadecenoic acid; TriHOME, TriHOME, acid; dihydroxyoctadecenoic DiHOME,

pups per sex per pups 10.2 17.5 12.7 98.7 21.4 2.8 1.7 2.8 3.7 4.5 0.4 2.2 4.4 0.9 3.3 6.7 2.0 1.0 0.6 p <0.05).

Male ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

cosapentaenoic acid.

- 0. 0. 0. 1.4 0. 0. 1.0 2.7 0. 5.7 3.2 14.5 10.9 33.2 3.0 0. 6.2 0. 0. pM, 9,10,13 EpOME, Shapiro ) 9 4 2 5 2 4 6 3 3 . No significant difference significant No .

- Wilk test Wilk - D, oxo ODE, 103.4 11.3 15.0 25.5 - 3.0 1.7 2.6 4.7 4.8 0.5 3.4 5.4 1.1 2.3 8.7 9.0 2.4 1.0 0.8

rHM, 12 TriHOME,

showed that the data were were data the that showed Female

- octadecadienoic acid; acid; octadecadienoic ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0. 0. 0. 1.3 1.5 0. 2.6 2 0.0 9 1.3 10.2 3.3 22.7 1.0 0. 4.1 0. 0.

.4 .5 s 8 5 7 3 4 3 2

were

- HETE and and HETE

observed observed This article isprotected rights by All copyright. reserved. Accepted Articlemetabolites. Figure 1 .

an nyai ptwy ivle i te ytei o oiie lnli acid linoleic oxidized of synthesis the in involved pathways enzymatic Main

This article isprotected rights by All copyright. reserved. Accepted Article Figure 2: Flowchart study of the design

This article isprotected rights by All copyright. reserved. Accepted Article Figure 3 . Distribution of oxy Distribution lipins

in malepup and female

brainadult male rat and brain.

This article isprotected rights by All copyright. reserved. Accepted Article(PGE2) ( acid Figure LA) . 4

Aoa lnt o cria nuos incub neurons cortical of length Axonal . ,

) 13 B) -

yrxotdcdeoc cd (13 acid hydroxyoctadecadienoic td ih ifrn dss f ) linoleic A) of doses different with ated

- HODE) ad ) prostaglandin C) and ,

- E2