Developing intestine is injured during absorption of oleic acid but not its ethyl ester

Developing intestine is injured during

absorption of oleic acid but not its ethyl ester.

O R Velasquez, … , P Tso, K D Crissinger

J Clin Invest. 1994;93(2):479-485. https://doi.org/10.1172/JCI116996.

Although lipids are essential nutrients in the mammalian diet, we have shown that fatty acids are injurious to epithelial cells of developing piglet intestine during luminal perfusion. Furthermore, the intestine of young animals sustains greater injury than that of older piglets. In an effort to understand the mechanism for this developmental injury, we investigated whether changes in the chemical configuration of oleic acid would alter this damage. Mucosal permeability, as quantitated by the plasma-to-lumen clearance of 51chromium EDTA, was evaluated during luminal perfusion with oleic acid as compared with its ethyl (ethyl oleate) and glyceryl (glycerol-1-mono-oleate) esters, solubilized with taurocholic acid, in jejunum of 1-d-, 3-d-, 2-wk-, and 1-mo-old piglets. 51Chromium EDTA clearance

increased significantly during oleic acid and glycerol-1-mono-oleate perfusion, but did not increase during perfusion with ethyl oleate or saline. This result was not secondary to failure of absorption of ethyl oleate, as [14C]oleic acid and ethyl [1-14C]oleate were absorbed to a similar extent. Furthermore, developing intestine was able to remove the ethyl group and then re-esterify the fatty acid to form triacyglycerol. These studies indicate that oleic acid-induced mucosal injury can be abolished when the carboxylic group of the fatty acid is esterified with an ethyl, but not a glycerol, group. Since the ethyl ester is also absorbed and metabolized similarly to the free fatty acid, this […]

Research Article

Developing

Intestine Is

Injured during Absorption

of Oleic

Acid but Not Its

Ethyl

Ester

Otto R. Velasquez,* AllenR. Place,' Patrick Tso,* and Karen D. Crissinger**

Departments of *Physiology and tPediatrics, Louisiana State Universityv Medical Center, Shreveport, Louisiana 71130; and §Center of Marine Biotechnology, UniversityofMaryland, Baltimore, Maryland 21202

Abstract

Althoughlipidsareessential nutrientsinthemammalian diet,

wehave shown thatfattyacidsareinjurioustoepithelialcellsof developing piglet intestine duringluminal perfusion.

Further-more, theintestine of young animals sustains greater injury thanthat of older piglets. Inanefforttounderstand the mecha-nism for this developmental injury, we investigated whether changes in thechemical configuration of oleic acid wouldalter

this damage. Mucosal permeability, as quantitated by the

plasma-to-lumen clearance of

"1chromium

EDTA, was evalu-atedduring luminal perfusionwitholeicacid as compared with its ethyl (ethyl oleate) and glyceryl(glycerol-1-mono-oleate)

esters, solubilized with taurocholic acid, in jejunum of 1-d-, 3-d-, 2-wk-, and 1-mo-old piglets.

5"Chromium

EDTA

clear-ance increased significantly during oleic acid and glycerol-l-mono-oleate perfusion, but did not increase during perfusion

with ethyl oleate or saline. This result was not secondary to

failureof absorption of ethyloleate, as

_1[4Cqoleic

acidandethyl

11-'4Cioleate

were absorbedtoasimilarextent.

Furthermore,

developing intestinewas able to remove the ethyl group and then re-esterify the fatty acid to form triacylglycerol. These

studiesindicate thatoleic acid-induced mucosalinjurycan be

abolishedwhen thecarboxylicgroupofthefattyacidis esteri-fied with an ethyl,but not a glycerol, group. Sincetheethyl ester is also absorbed and metabolized similarly to the free

fatty acid, this may providea means ofsupplying long-chain fatty acidstodeveloping intestine without causing mucosal dam-age.(J. Clin.Invest. 1994.93:479-485.)Keywords:

necrotiz-ingenterocolitis* mucosalpermeability, fatty acids-lipid

ab-sorption*infant nutrition

Introduction

Numerousinvivoandinvitrostudieshavedemonstratedthe

cytotoxiceffects offatty acidson intestinal cells ofadult

ani-mals ( 1-4). Perfusion or incubation of intestinal tissue with

fatty acids solubilized with bile saltsproduces significantcell

damagethatisnotseen withbilesalts alone (2, 3). This

lipid-Portions of this workwerepresentedat the meetingofthe American

Gas-troenterological Association,May 10-13, 1992, SanFrancisco,CA.

Addresscorrespondenceto Dr. Karen D.Crissinger,Departmentof Pediatrics, Louisiana State University Medical Center, P.O. Box

33932,Shreveport,LA71130.

Receivedfor publication6May1993and in revisedform 26 August 1993.

inducedinjury increasesproportionallywith theconcentration (2) and with the carbon chainlength(3)of thefattyacid.

In developing piglet intestine we have demonstrated that mucosalinjuryinduced bydietaryfattyacids is alsodependent

onthefattyacidconcentration andcarbon chainlength(5, 6).

Ingeneral,medium-chain fatty acids

(MCFA)'

appeartobe lessinjurioustothe intestinal mucosaofdeveloping animals comparedtolong-chain fattyacids(LCFA)atconcentrations

thatmaybepresentpostprandiallyin theintestinal lumen. We havealso shown that thesensitivityof the intestinalmucosa to

theinjuriouseffects offattyacidschangesas afunction of age

suchthat theintestineof1-d-oldpigletsismorepronetoinjury

comparedto thatof1-mo-oldanimals (5).

Inaddition to thelowcytotoxicityofmedium-chain fatty

acids in intestinal mucosaofdeveloping animals, MCFAare

digestedandabsorbedefficientlyfrom thegastrointestinaltract

withoutthe needforbile saltsolubilization, providingareadily availablesourceofenergyfor the newborn (7). This is

particu-larly important in the neonatal period, when pancreatic and

biliaryfunctionsareimmature(8,9). However,dietaryMCFA

cannot betransformed into essential LCFA inthebody(10).

Mammalslack theenzymes totransform MCFA into LCFA of

the n-6and n-_{3series,whicharenecessaryfor thesynthesis} of complex structurallipidsinbiological membranes andare precursorsfor eicosanoidcompounds ( 11 ). The LCFA

consid-eredessential inhumannutritionarethe18-carbonchainn-6 linoleic acid (18:2, n - _{6) and its} derivatives, in particular arachidonic acid(20:4,n -_{6); and the}n - 3fatty acids lino-lenic acid ( 18:3, n-_{3) and itslongerchain,more}

polyunsatu-ratedderivatives,eicosapentaenoic acid (20:5,n- 3) and

do-cosahexaenoic acid (22:6,n- 3),ashave beenidentified(10,

12).Therefore,thesefatty acidsmust be providedintheinfant diet for normal growthanddevelopment.

Previous work from our laboratory has shown thatoleic

acid,acommondietary long-chain fatty acid, induces increases

in mucosalpermeability indevelopingpiglet intestine. These

changesinpermeabilityareassociated withmucosalinjury ob-servedwithlightand electron microscopy(5). Moreover, we

demonstrated that theoleic acid-induced injury is similarto thatobserved with the essential fatty acids linoleic and lino-lenicacids(6).

There arestudies thatsuggest that thecytotoxicityof

long-chainfattyacids maybe relatedtothe presenceofafree

car-boxylgroup in thelipidmolecule. Zhu et al. have shown that

esterificationof oleic acidandlinoleicacid with a methyl group

significantly decreases thecytotoxic effect of these LCFA on

1. Abbreviations used in this paper: CE, cholesterol ester; DG, diglycer-ide; EO,ethyl oleate;LCFA, longchainfattyacids; MCFA, medium

chain fatty acids; MG, monoglyceride; n, omega; PL,phospholipid; PUFA,polyunsaturated fatty acids;TG,triacylglycerol.

Lipids and MucosalInjury inDevelopingIntestine 479 J.Clin.Invest.

©The AmericanSociety for Clinical Investigation,Inc.

0021-9738/94/02/0479/07 $2.00

Ehrlich ascitescarcinoma cells in mice ( 13 ). Theyconcluded thatpart of the cytotoxicity of LCFA can be attributed to the existence of a free carboxylic group. Similarly, Raz and Livne demonstrated that methyl esters of oleic, linoleic, and linolenic acids do not show the hemolytic effect observed with increasing concentrations oftheunesterified (free) fatty acids( 14). Inthis study we investigated whether the injurious effects of oleic acid on intestinal mucosa of developing pigletsare influenced by changesinthe chemical configuration of the carboxylic group of thefatty acid. Our objective was to gain a better understand-ingof the pathophysiology of lipid-induced injury in develop-ing intestine. In the course of this investigation we observed that ethyl oleate was harmless to the intestinal mucosa. This intriguing observation raised the question of whether the intes-tinal absorption and metabolism of ethyl oleatewas compara-ble tothose of the free fatty acid. This question was addressed by studying absorption of ['4C]oleic acid compared to ethyl [1-'4C]oleate. The results of this study provide insight into the mechanisms of mucosal injury induced by dietary fatty acids and suggest that ethyl estersofLCFA may be an alternative means of supplying these important nutrients to immature in-testine.

Methods

Intestinal permeability studies

Animalpreparation. 20 Hampshire/Yorkshire piglets of either sex wererandomly selected among groups of -d-old, never-nursed ( 1-7 h of age; 1.51±0.08 kg;n=5);3-d-old,fasted for 18 h(2.2±0.12kg;n

=5); 2-wk-old,fasted for24 h(4.27±0.42kg;n=5);and1-mo-old,

fastedfor24 h(7.38±0.63 kg;n=_5).After intramuscularinjection of ketamine hydrochloride (20mg/kg) andxylazine (2 mg/kg),the ani-malswereanesthetizedwithpentobarbital sodium (15mg/kg)viaan earvein. Maintenancedosesofpentobarbital (5 mg/kg)weregiven intravenouslyasneededduringtheexperiment.

Immediately following anesthetic induction,the animalswere arti-ficiallyventilated viaatracheostomywithanintermediateventilator (HarvardApparatus, SouthNatick, MA)at atidalvolume and

respira-toryrate tomaintainnormalarterialblood gases andpH(pH/blood

gasanalyzer; Instrumentation Laboratory,Lexington,MA).

Polyethyl-enecannulas wereinserted into the right externaljugular vein, for

administration ofpentobarbitalandfluid forhydration,and into the

left carotidartery tomonitorsystemicarterial pressure (physiologic

recorder; Grass InstrumentCo.,Quincy, MA)andtowithdraw blood samples. Body temperaturewasmaintainedat-370Cwithaheating

pad andaninfraredheating lamp.

The abdomenwasopened throughamidline incisionand therenal vessels were ligated to preventexcretion of5"Cr-labeledEDTA(New England Nuclear,Boston,MA),whichwasinjectedlater. Four 10-cm loops ofjejunumwereisolated and cannulatedatbothproximaland

distalends with polyethylene tubing, after which the intestinal and

abdominalcontentswereirrigatedwith normal saline and covered with plastic wraptopreventevaporativewaterloss.

Preparationsoflipid solutions. 5-mMsolutionsof oleicacid,

glyc-erol 1-mono-oleate, andethyloleatewereprepared. Thelipidswere

solubilized with 10mMtaurocholicacid(sodiumsalt)andemulsified

withanultrasonichomogenizerfor 10 min(Labsonic 2000; Ultrasonic

Power, Freeport, IL). All solutionswereiso-osmolar(- 300mosM),

thepHwasadjustedto - 7.4with 1 NNaOH,andtheywere

main-tained inawaterbathat370C.All the chemicalcompoundswere ob-tained fromSigmaChemical Co.(St. Louis, MO)atthehighestpurity

available.

Experimentalprotocol. After surgery, thecannulated loopswere

perfusedwithwarm normal saline viaaGilson minipulsperistaltic

pumpatarateof 1 ml/mintoachieveaconstantflow.5"Cr-labeled

EDTAwastheninjected intravenously, such that therewere atleast

25,000 cpm/ml (100-150MCi/kg). 10 min were allowed for tissue

equilibrationof the labeled EDTA. After theequilibrationperiod, the

four intestinalloops were initially perfused with normal saline during 20 min (control period). After this control period, the different fatty acid solutions in each experimental group were perfused simulta-neously in different loops for 20min(lipid period). Subsequently, the

perfusatewaschanged to normal saline for 60 additionalmin (recovery period). Luminal perfusatefrom the effluent cannula (4 ml) was col-lected at 10-min intervals during the experiment to monitor5 Cr-la-beledEDTAclearance. 1 mlofblood(centrifugedtoobtain 0.5 ml of plasma) was removed every 20 min to monitor plasma5"Cr-labeled

EDTA radioactivity. The blood was replaced with an equivalent vol-umeof6% Dextran 70 (SigmaChemical Co.). 5"Cr-labeled EDTA

activity in plasmaand in 4-mlaliquots of perfusatefor each 10min,

was measured in aCompuGammaspectrometer (model 1282; LKB Instruments, Inc.,Gaithersburg,MD).

At the end of theexperiment,the animal was killed with an over-doseof pentobarbital (60 mg/kg), and each loop of intestine was re-moved, rinsed with normal saline, and weighed.

Calculation of5Cr-labeledEDTAclearance.Theplasma-to-lumen

clearanceof5"Cr-labeledEDTAwas calculated as follows: Clearance= cpmDXprX 100

cpmplX_wt

where clearance is given in ml /min per 100 g,

cpmP

is cpm per ml of

perfusate,pr is theperfusionrate,_cpmp,iscpm per mlofplasma, and wt is weight of the intestinal segment in grams.

Dataanalysis.Allvaluesfor5'Cr-labeledEDTA clearance are ex-pressed asmean±SEM.Athree-factorANOVA, followed by Duncan multiple range post hoc testing if the ANOVA revealeddifferences

amonggroups, was used to evaluate differences among age groups, fattyacid solutions, and experimental periods (SAS Institute Inc., Cary, NC). DifferenceswereconsideredsignificantatP<0.05.

Absorption studies

Animal preparation.21piglets of eithersexweredividedinto groupsof

l-d-old,never nursed(n= _14),and2-wk-old, fasted for24 h(n=7)

subjects.Theseanimalswereanesthetized and handledasdescribedin theprevious section,with theexceptionthat the renalpedicleswereleft

intact.

Preparation oflipidsolutions. Solutions of oleic acid and ethyl

oleate in normalsalineat aconcentration of5 mMwerepreparedand labeledwith[1-

_'4CIoleic

acid(20nCi/micromol),andethyl [1-

14C]-oleate(20nCi/micromol), respectively. 10 mMtaurocholic acid

(so-diumsalt)wasadded and thesolutionswereemulsified for10minwith

anultrasonichomogenizer.ThepHwasadjustedto - 7.4with 1 N

NaOH.

[1-14C]Oleic

acid was obtainedfrom New England Nuclear Re-search Products(Boston, MA) and hadapurity of99%. Theethyl

[1-14C]oleate wassynthesized using p-toluenesulfonic acid-catalyzed esterification (15).Theethyl [1-_'4C]oleatewaspurified by chromatog-raphy ontapered preparativesilica Gplates (AnaltechInc., Newark, DE)using hexane/diethylether(85:15, vol/vol),and detectedby

ra-diometricscanningafterscrapingthesilica andelutingwith hexane

containing2% diethylether. Fromradiometricscanningof the TLC plateusingaVanguard (model 2001;Digital DiagnosticCorp.,

Ham-den,CT), theradiopuritywas98.7%.

Experimental protocol. Afteremulsification,reference samplesof

thelipidsolutions from the top,middle,and bottomofthecontainer

were obtained. Radioactivity was measured in these samples to ensure ahomogeneous distribution of thelabelingmolecule. Thenavolume adequate to fill the intestinal loops withoutcausing distention (2mlin

l-d-old,and5 ml in2-wk-old)wasplaced into differentloopsfor I h. 12to16 loops perlipidsolution per age group were used forstatistical analysis.Inseven1-d-old piglets,the solutionswereleftin theloopsfor up to 3 hand loopsweresequentially excised everyhtodetermine progressive absorptionand intestinal metabolism of thelipid.

excised andplacedover ice.Theremaining volume inside the loopwas

collected inseparateconicaltesttubes. Eachloopwasthen rinsed thor-oughly with three washes ofacold solution of 10 mM taurocholic acid and the washeswerecollected andcombined in separate conical tubes. All thevolumes were recorded. The samplesweremixed witha vortex

shaker andanaliquot(50ju)from eachsamplewastaken for radioac-tivity determination. Lipid absorption was determined as follows:

absorptiveindex=

100% ofinfusate dpm-% of total dpm recovered in lumen. Lipids in the effusate collected from ethyl [1-'4C]oleate-infused

loops were extracted by the method of Blankenhorn and Ahrens ( 16) andwereanalyzedby TLC. The intestinal segmentswerethenopened longitudinally, the mucosaseparatedviabluntdissection,andplaced

in separate Erlenmeyerflasks containing50 mlof

chloroform/metha-nol2:1 for lipidextraction(17).After24h,analiquot(0.5ml)from each flaskwastaken and blown down undernitrogenforradioactivity

determination. Lipid transport was calculated as follows: transport index=

absorptive index-%of total dpm recovered in mucosa _x

₁₀₀

absorptiveindex

Lipids from thejejunal mucosa ofl-d-old piglets extracted with chloroform/methanol 2: 1, were also used to determine the distribution ofradioactivityindifferent lipid classes as analyzed by TLC.

Radioactivity determination.Thesampleswere mixedthoroughly

with 10 ml ofanaqueous miscible scintillationcocktail (Poly-Fluor;

Packard Instrument, Downers Grove, IL) and then counted for 10 min in a liquid scintillation spectrophotometer (model 1209 Rackbeta;

LKB Instruments,Inc.).

TLCanalysis.The TLCanalysiswasperformed usingSilica Gel G plates (20 x 20 cm), which had been activatedat 120'Cfor 30 min. Thesolvent system usedwascomposed of petroleum ether/anhydrous diethylether/glacialacetic acid (105:21:0.6, vol/vol/vol).Thelipids

werevisualizedafterstaining with iodine vapor and wereidentifiedby

co-migrating lipid standards (18). Theappropriate lipid spots were

thenscraped intoscintillationvials,and10 ml of scintillation cocktail was added forradioactivity determination. TLC plates used for photog-raphywerestained with 25%phosphomolybdicacid in methanol.

Dataanalysis. Allvaluesareexpressedasmeans±SEM. A three-factorANOVA, followed by Duncanmultiple-rangepost hoctesting (SAS Institute,Inc., Cary, NC)wasusedtoevaluatedifferences among agegroups,lipid solutions,andtime.Differenceswereconsidered

sig-nificant atP <0.05.

Results

Permeability studies

Perfusion of developing piglet jejunum with 5 mMoleicacid

produced significant increasesin mucosalpermeabilityin all agegroups(Fig. 1 A). Thisresponse was moreattenuated in

1-mo-oldpiglets compared toyoungeranimals. Mucosal

per-meability returned to nearbaselinevaluesafter I hof luminal

perfusionwith normal saline such thatatthe endof the

experi-mentthesevalueswere notsignificantly differentthancontrol

atany age.

A similar response was observed in developing intestine afterperfusion withglycerol 1-mono-oleate(Fig. 1 B).

Clear-ancevalues also returnedto nearcontrol valuesatthe end of the recoveryperiod. Incontrast, after luminalperfusionwith

ethyloleatenosignificant alterations ofEDTAclearancewere observedatanyageduringanytimeperiod (Fig. I C).

When thepermeabilityvalues obtained after perfusion with the3lipidswerecompared in 1 -d-oldand 1-mo-oldanimals, it

A

a

o 2.00

C

E 1.50

E w

o 1.00

z

.4 4c

UW 0.50

- 0

U

.4

o3 0.00

Lu

B

0 0

C

E E

E Lu 0

z

4

cc

4c

: Lu

0 4

a

LU

= 1-DAY =i1 3-DAY = 2-WEEK = 1-MONTH

_ OLEICACID

F

F-CONTROL LIPID

T

...I....

L

F:::::.A

RECOVERY

_ 1-DAY = 3-DAY = 2-WEEK = 1-MONTH

2.00

1.50

1.00

0.50

0.00

1-MONNO-OLEATE

+*

*

CONTROL LIPID RECOVERY

C _ 1-DAY =li 3-DAY = 2-WEEK [lI-MONTH

0 0

0E

-iE

z

4 4

qc

Lu

-1

0 4c 0

Lu

2.00 r

ETHYLOLEATE

1.50 F

1.00 V

0.50 h

0.00

CONTROL LIPID RECOVERY

Figure 1. Jejunal5'Cr-labeledEDTAclearance(mean±SEM)after luminalperfusionwith 20 min of normal saline(CONTROL), fol-lowedby 20 min of 5-mM solutions of either oleicacid, glycerol 1-mono-oleate,orethyl oleate, each solubilized with 10mMtaurocholic acid (LIPID), followed by 60 min of normal saline(RECOVERY).

Values were obtained at the end of eachexperimentalperiod in 1 -d-, 3-d-, 2-wk-, and 1-mo-oldpiglets(n= _{5 animals per age group).}

*in-dicatesP<0.05 vs.control withinalipidsolution. +indicatesP <0.05vs. I-mo-oldanimals withinanexperimentaltimeperiod.

wasclearthat onlyoleic acidand glycerol 1-mono-oleate

pro-ducedsignificantincreases in mucosalpermeability in bothage groups.Moreover, the mucosal injury observed after perfusion

with oleicacid and glycerol l-mono-oleate in 1-d-old piglets

wassignificantly higher than that observed in

1-mo-old

ani-mals(Fig.2).

Absorption studies

Ethyl oleate is more hydrophobicthan oleic acid orglycerol

1_{-mono-oleate. Under the experimental conditions used in this}

studyit is possible that the reducedeffects ofethyl oleate on

a

a

0

V-I-.

a

LU

2.00 r

1..50 [

1 00

1.00 F

*

_ OLEIC ACID

L=i MONO-OLEATE

ETHYLOLEATE

0.50 F

o.oo L

1 DAY

I-F

0

0

B

-J

I-0

I-. 1 MONTH

Figure 2.Comparison of EDTA clearance (mean±SEM) in jejunal loopsluminallyperfused for 20 min with 5 mM oleic acid, glycerol

1-mono-oleate, orethyl oleate, eachsolubilized with 10 mM tauro-cholic acid inI-d-old and 1-mo-oldanimals (n=5animals per age group). *indicatesP <0.05 vs. ethyl oleate within an age group.

+indicatesP <0.05 vs. 1-mo-oldwithin a lipid solution group.

75 [

50 [

25 F

0

- MGPL

'' _DG

FFA

TG

[IIICE

EO

INFUSATE EFFUSATE

Figure 4. Distribution of'4Cradioactivity (mean±SEM) in the lipid extractedfromthe ethyl [ 1-4C]oleatesolution placed into the lumen andfrom the effusate collected after 1 h inl-d-oldpiglets (n=3 ani-malswith 4intestinal segments per animal).

lization and,therefore, reduced mucosalcontactand absorp-tion of the ethylestercomparedtothefreefatty acid.To deter-mine whether the physicochemical characteristics of ethyl oleatewereresponsiblefor its attenuatedeffect,wecompared the intestinalabsorptionof ethyl [ 4C]oleate and oleic acid in

l-d-old and 2-wk-old animals.

Fig. 3 showstheabsorptionrateof ethyl[ 1- 'C]oleate and

[1-

"C

]oleic acid in developing piglet intestine. Approximately

75% of the ethyl

[1-"C]

oleate wasabsorbed from the lumen after 1 handnodifferencewasobservedas afunctionofage.

TLC analysis of the effusate collected further supported the high absorptive capability ofdeveloping intestine (Fig. 4). Most of the radioactive lipid infused was absorbed and only

- _{3% of thelipidpresentin theeffusatewasin theethylester}

form. However, it couldnotbe determinedfrom the TLC

anal-ysis if ethyl [1-_4C]oleatewastaken upintact or was

hydro-lyzed before uptake by the intestinal mucosa.Aslightly higher

uptake of

[1-"'C]oleic

acid than ethyl [1-'4C]oleatewas ob-served inbothage groups.This differencewasstatistically

sig-nificant for the 1-d-old piglets (Fig. 3).

Toascertain theproportionoflipidthat hadactually been

transportedoutof the intestinalmucosaafter 1h, theamount

ofradioactivelipidpresentin themucosawasdeterminedand thetransportindex for eachlipidwascalculated(Fig. 5).

Ap-proximately

65%of the total

[1-

"'C]

oleic acidplacedintothe

lumenwastransported from the intestinalmucosa atthe endof thefirsth.Thispercentage wassimilar in bothage groups.The proportion ofethyl [1--

"C]

oleate transported was similarto

that of [1-'4C]oleic acid in 2-wk-old animals. Interestingly,a

significantlyloweramountof ethyl [I-"C]oleatecomparedto [ 1-

"'C

]oleic acidwastransportedoutofthemucosaafter1hin l-d-oldpiglets.Thissuggestedthatalthoughasignificant

pro-portion ofethyl oleatewasabsorbedfrom the lumen in l-d-old

jejunum, there wasadecreased abilityto metabolize and/or

transporttheabsorbedethylester outof the intestinalmucosa atthisage.

To investigate whether this reduced capability to handle ethyl oleate improved with time, the radioactivelipids were

placed intoseparatejejunalloopsofl-d-oldpiglets for3 h.Fig.

6 shows that theabsorption of ethyl[1-

_4C]

oleatewas

essen-tially complete by the second infusion h. Atthistime, uptake

was close to 100% and the difference in absorption rate

be-100

0-x

x

_ ETHYL OLEATE

z

I-OLEATE o

0.

Co

z

I-75

50

25

- ETHYL OLEATE

OLEATE

0

I DAY 2 WEEK

Figure3.Percentageof totallipidabsorbed(mean±SEM)after

placementofethyl [1-'4C]oleateor[1-14C]oleic acid injejunal loops

for1h inI-d-old and 2-wk-oldpiglets (n=7animalsper agegroup).

*indicatesP<0.05vs.ethyloleatewithinanagegroup.

I DAY 2 WEEK

Figure5.Lipidtransportindex(mean±SEM) expressedas%of total

lipid (ethyl [1-'4CIoleateor[1-"'C]oleicacid) placedforI h into

jejunal loopsof l-d-old and 2-wk-oldpiglets (n =7animals per age

group).*indicatesP<0.05vs.ethyloleatewithinanagegroup.

100

01%

x 75

w

Lm

z

50

Co

_so

m

0

1 2 3 4 5

100

1-0

x 75

z

'>

50

r

a.

o 25

co m

0

40

CE

EO

ETHYL OLEATE

OLEIC ACID

1 H 2H 3H

Figure 6. Absorptionrate(mean±SEM) ofethyl [1-14C]oleateor

[1-'4C]oleicacid afterplacement into thejejunum for 1, 2,or3 hin l-d-oldpiglets (n=7animals). *indicatesP<0.05vs.ethyloleate within a timeperiod.

tweenethyl[1-'4C] oleate and [1-14C]oleic acidpreviously ob-served was abolished. When the transport index was deter-mined, aprogressive increase intransportwithtime for both lipidswas observed (Fig. 7). Theproportion of [1-'4C]oleic acid transportedwassignificantlyhigherthan theethyl

[1-

'4C]-oleate transportedduringthefirst 2 hof the experiment. No

statistically significant differences between the percentage of either lipid transportedoutof themucosa wasobservedbythe third h. Atthis time, the proportion of transportedethyl

[1-14C]oleatewassignificantly higherthan the valueobtainedat 1 h.

The mucosalradioactivelipidwasseparated intodifferent lipidclasses byTLC after1hof lipid infusion.Asshown inFig.

8, ethyl oleate separated well from triacylglycerols and free fatty acids. The quantitative analysis of this distribution

showed that afterluminal placement of eitherethyl [1-

"'C]-oleateor[1-14C]oleicacid,mostoftheradioactivitywas

recov-ered in thetriacylglycerol(TG)fraction (Fig.9). Onlyasmall

percentage (< 3%) of the mucosal lipid waspresent asethyl oleate,suggestingthat theintestinalmucosaof1-d-oldpiglets

was able toeffectivelycleave theethyl group and re-esterify the

resultantlipid into triacylglycerol.

TG

FA DG MG+PL

Figure 8.TLCof thelipidextractedfromjejunalmucosaof l-d-old

piglets 1 hafter luminal placementof eitherethyl [1-"'C ]oleate

(col-umn2)or[I-"'C]oleicacid(column5).Columns 1,3,and 4are

standardsfor ethyl oleate, neutrallipid,and oleicacid, respectively.

Discussion

FFA arehighly toxic molecules capableofkillingordamaging

cells in a variety of tissuesincluding red blood cells(3, 14),

enterocytes( 1-6), severaltumorcell lines (19, 20),and heart muscle cells(21 ).Althoughthemechanism(s) offatty

acid-as-sociated injury is unknown, alterationsinthe chemical configu-ration of thehydrophobicgroupofFFA mayleadtochangesin

theircytotoxic effects.We and others have shown that the inju-rious effectsofFFA on intestinal cellsaredependent on the length and,therefore, thehydrophobicity ofthecarbon chain

(3,6). The degree of saturation of thehydrophobicgroup does notappear toplay animportant role in determining the toxic effect of fatty acids (6). Information regardingthe effect of changes in the configuration of the hydrophilicgroup onthe toxic potential ofFFAissparse. Afew studies have evaluatedin

vitrothe cellinjury inducedby methyl estersoflong-chain fatty acids(LCFA),including oleicacid,in red blood cells ( 14) and

tumorcells (13).Theseexperimentsshowed that the toxic ef-fects of LCFA were abolished after esterification with the methyl group, leading the investigators to conclude that the

cytotoxicityof LCFAwasdependenton thepresenceofafree carboxylicgroup(13).

100

0-x 75

Lu

0

z

- 5

0 CL)

z

25

0

1 H 2H 3H

100

1 75 ETHYL OLEATE O

m OLEIC ACID _a

24

50

-I

O 25

0

I--0

Figure7.Lipid transport index(mean±SEM) expressed as % of total

lipid(ethyl [I-"C]oleateor[1-'4C]oleicacid) placed for 1, 2, or 3

hinto jejunal loops ofl-d-oldpiglets (n= _{7animals). *indicates P}

<0.05vs.ethyl oleate within a timeperiod.

MG PL

DG

FFA

TG

CE

EO

ETHYL OLEATE OLEIC ACID

Figure9.Distribution of mucosal '4Cradioactivity (mean±SEM) in differentlipidclasses in l-d-oldpiglets (n=7animals) after

place-mentof ethyl [ 1-14C]oleateor[1-14C]oleic acid in jejunal loops for 1h.

LipidsandMucosal InjuryinDevelopingIntestine 483

we

Theobjectiveofthe present study was to evaluate whether

modifications ofthe carboxylic moiety by the formation of esterscould alter the mucosal injury previouslyobserved(5)in

developing piglets after perfusion with oleic acid.Bothglycerol

1-mono-oleate and ethyl oleate lack a free carboxylic group,

and themajor difference betweenthese twocompoundsrelates tothepolarity of theester. Theglycerol moiety oftheglycerol 1-mono-oleate with two hydroxyl groups is more polar than

oleic acid withasingle hydroxyl group. Oleicacid is in turn much more polar than the ethyl ester as observed with TLC

(Fig.9). Due to the reduced polarity of the ethyl oleate

mole-cule, its abilityto behaveas anamphiphilemay beimpaired, thus lowering its cytotoxicity. Amphiphilic molecules, which

possess detergent-like characteristics, may interact with cell

membranes andcausealterations ranging fromsubtlechanges

inpermeabilitytomembrane lysis, accordingto the

concentra-tion of the detergent (22). This direct effect mayexplainthe

rapidity ofthefatty acid-induced injuryobserved in thisand

otherstudies. Theconcentrationof oleic acid used in this study

(5 mM)consistentlyproduced a20-fold increase in mucosal

permeability (Fig. I), and has been shown to be associated with cell membrane disruption and cell deathobserved with light and electron microscopy (5).Themechanism of this

am-phiphile-induced alteration incellmembranes isunknown but may involve alterations in membrane fluidityand/or

mem-brane breakdown andsolubilizationintomixed micelles (23).

Analternative explanation forthereducedmucosalinjury observedafterperfusion with ethyl oleate is suggested by the behavior of thislipidinwater. Inthis study, sodium taurocho-latewasusedto solubilize thelipidsinwater, andunderour

experimental conditions(pH 7.4,T370C)oleic acid and glyc-erol 1-mono-oleate were adequately solubilized. In contrast,

ethyl oleate formed a turbid solution. These observations correlatedwithpreviousreportsbyHofmann (24)that micel-lar solubilization ofsolutes with bile salts appearsto be

in-fluenced by thepolarity ofthecompoundsuch thatnonpolar

moleculesaresolubilized lessefficientlyinanaqueous

environ-mentthanpolarones.This suggests thatthereduced effect of ethyl oleatewassecondarytothereducedsolubilityofthislipid

in water, leading to reduced micellar solubilization, reduced mucosalcontact, andtherefore, reduced mucosal injury.

Pre-viousstudiesinvitro in red blood cells and Caco-2 cells have shown that stearic acid (C 18:0)has asignificantlylowerlytic potential at 37°C compared to other 18-carbon mono- and

polyunsaturated fattyacids(3).Thiswasattributedtothe

insol-ubilityof thefattyacidsecondarytogelformationatthis

tem-perature,which is below itscriticaltemperature. Toascertain

whether thebehavior of ethyloleate inwater was

responsible

for the lack ofinjuriouseffectsonintestinalmucosa,we

evalu-ated theethyloleate-mucosa interactionby determining the

absorptionofthislipidandcomparingit withthe

absorption

of oleicacid.In2-wk-oldanimals - 70%ofthelipidinfusedwas

absorbed after 1 h and no statistically significant differences

wereobservedbetween theuptake of oleic acidandethyloleate

(Fig. 3).In 1-d-oldpigletstheoverallabsorptionratefor both

lipidswasalsohigh

('-

80%),but theuptakeof oleic acidwas

significantly higherthan the uptake ofethyl oleate (Fig. 3).

This differencewasabolished with time(Fig.6).Theseresults

suggest that mechanisms other than lack of interaction be-tweentheethylesterandintestinalmucosamay beresponsible

forthe reducedeffect ofethyloleate.

Toourknowledge, this isthe first reportonthe

absorption

of aLCFA and its ethyl ester in developingintestine. Important findings have been described in numerous studies, however, in

which the absorption of long-chainpolyunsaturatedfatty acids

(PUFA) in different chemical forms (including FFA, triacyl-glycerol, and arginine salt) has been compared to the

absorp-tion of ethyl esters of PUFA in adult rats (25) and humans

(26-29). In general, it has beenobserved that the absorption of

ethyl esters of PUFA is less efficient compared to the other chemical forms, especially FFA. However, it has also been

shown that the ability to absorb ethyl esters significantly

im-proves with exposure time (25). The different absorption of ethyl esters of LCFA may be the result of poor hydrolysis by

pancreaticlipase (30). It has been suggested that the resistance

to hydrolysis could result from the ethyl component of the molecule (26). Studies by Sarda and Desnuelle have reported a lower hydrolysis of ethyl oleatecompared to the hydrolysis of

triolein (31 ). Furthermore, it has beenshown that the

hydroly-sis of ethyl oleate is significantly lower than thehydrolysisof thehomologous methyl oleate (32). Despite thereduced

hydro-lysis of ethyl esters of LCFA, this and other studies (33) have reported uptake rates higher than 90%, suggesting that the

ab-sorptionof the ethyl ester is probably very effective and may takeplace without prior enzymatic hydrolysis. Once inside the

enterocyte, it appears thatan intracellular esteraseexists that hydrolyzes the ethyl ester, allowing the incorporation of the liberated fatty acid into de novo synthesizedtriacylglycerolsin thecell (Fig. 9). Studies in adult animals have alsoreported the

possible intracellular metabolism of ethyl esters of PUFA. In ratsreceiving eicosapentaenoic acid and docosahexaenoic acid ethyl esters, no ethyl esters were found either in 4-h lymph samples or in blood (34).

An interesting observation in our study relates to the re-duced ability of l-d-old intestine to transport the absorbed ethyl oleate(Fig. 5). In rats, the ability of small intestine to transport lipidinto lymph can be determined by cannulation of a major intestinal lymph duct (35). This allows the calcula-tion of the lymphatic transport index (36). However, in devel-oping piglets lymphatic cannulation is technically very difficult andeven though we have been able to cannulate a lymphatic andobtain lymph for a short period of time in 2-wk-old piglets (data not shown),thisprocedure isimpracticalin 1 -d-olds due to the size and the fragilityofthe vessels. Another problem is the difficulty in ensuring the complete recovery of intestinal lymph from a certain segment of the gut. Therefore, in our model, transport index was calculated by subtracting the

pro-portion ofradioactive lipid present in the mucosa from the

proportionof lipidabsorbed fromthelumenand dividing the result by the absorptive index. This provides lipid transport values that are normalized for the variability in uptake rate that may be observed in differentexperimentalanimals. Our results showed thatin 1-d-oldanimals more ethyl oleate was present in the intestinal mucosaafter1 h compared tooleicacid,

indi-cating a more efficient transport of oleic acid.This phenome-non was not observed in 2-wk-olds (Fig. 5). The reduced abil-ity of

1-day-old

intestine to transport the absorbed ethyl oleate wasnot due to the inability to incorporate the lipid into triacyl-glycerol, because only a very small amount of ethyl oleate was found in the mucosa at the end of 1 h (Fig. 9).

ofoleic acid in the mucosaand wouldgive ahigh transport

index value. Another possibility is that oleic acid, after causing mucosal epithelial disruption,mayreach the interstitium and

the circulation through a paracellular route, reducing the

amountofthelipid detected in themucosa.Thisspeculationis

notsupportedby thefact that oleic acid produces similar

mu-cosal injury in 2-wk-old piglets (Fig. 1) and in thisage group no

difference in the amountof oleic acid andethyl oleate

trans-ported has been found(Fig.5).Regardless of the mechanism, it is obviousthattheinitialdelayinthetransportof ethyloleate

improves significantly with time (Fig. 7). Overall, these results indicatethatethyloleate is efficiently absorbed, metabolized, andtransportedoutofthemucosaindevelopingintestine.

Takentogether, the results ofourstudymayhave

impor-tantclinicalimplicationsindevelopingintestine. Immaturity

ordisruption ofthedevelopingmucosal barriermayresult in

clinical diseasestates towhich the newborn issusceptible,such

as necrotizingenterocolitis, toxigenicdiarrhea, andintestinal

allergy. We haveshown in this studyandothers(5, 6) that bile acid-solubilized dietary fatty acids, especially the long-chain

unsaturated species, in a concentration that may be found

postprandially (after pancreatic enzymedigestionof

triglycer-ides in infant formula)areabletodisrupttheintestinal

muco-salbarrier.Becauselong-chain fattyacidsareessential for

nor-malgrowth and development andmustbeprovidedin thediet,

it is importantto findthe means tosupply fatty acidsto the newbornwith minimal alterations of the mucosalbarrier,thus

decreasingthehazard of intestinal pathology. Ourstudy

indi-cates that esterification oftheinjurious LCFA oleate with a

nonpolar ethylgroupabolishes itscytotoxiceffects in this

exper-imentalsetting,usingdevelopingpigletintestine. In addition,

ethyl oleate iswellabsorbedand metabolized, indicatingthat

ethylestersofLCFAmaybeusefulas asourceofessentialfatty

acidstothenewbornwithoutconcomitantly increasingtherisk of mucosalinjury.Althoughwehavefoundnosignificant dif-ferences in injuryinduced by oleic acid solubilized in bilevs.

taurocholic acid (data not shown), investigation of the re-sponse to a mixed nutrient meal that has been digested and

solubilized bypancreaticenzymes andbilein anintact develop-inggastrointestinaltractis crucial before applyingthese

experi-mentalfindingstotheclinicalsituation.

Acknowledgments

This workwassupported by grants DK-43785 and DK-32288 from the

National Institutes ofHealth.

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