INT ESTINAL ABSORPTION:'ST UD IES OF ABSORPTI.ON OF CERT.AIN NAT URAL LIPIDS

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INT ESTINAL ABSORPTION:'ST UD IES OF ABSORPTI.ON OF CERT.AIN NAT URAL LIPIDS

AND LIPOPHILIC XENOBIOTICS

by

@ Jenn ifer Lynn' Hall, B.S e. (Hans.) /

A th esis subm itted ;;;'hl: School of Gra duate Studi es in partial fulfillm ent of the

requirements for the degree of Master of Science

. ..

Toxico!ogy

Memorial

University

of Newfoundland November--l98'1

/

St. John's

---

Newfoundland

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Pe'rmlsBibn

has

been gr.anted to the Nl\tional Library..gf Canada to microfilm this the sis llnd to lend.oc sell cople a of thepfilm.

",0-" .

t:

^s

,ut:~rs'~C~';:~9hto~~eer~ .

pUblication rights, and

neither the theeil!ll nbr extensive extracts from it may be printed orotherwise repJ'od~ced without his/her· writtenpermission.

\. ~1

ISBN

/

L' autorlsation a.l!tl!accord's

a

Ill. BibUotheqltenaUonale du Canada de .icro-Ulmer ce t te th~seet de preter au de vendre des exemplaires,d u

film. •

L'~ute'ur

^{(tLtulaire du droit}

d'auteur) 88 r'serve "f e e autre,s drafts de publiclIt1.on, J

:~t/aait:he~: ~~'lldee_'cton~: \

doivent.etre Impriml!8 ou aq.trelllent repro~lts sans son autorhl!l.t~_'cr1te. .

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ABSTRACT

The upta keofnutrientlipidsby theabsorptivecell ofthesmallintestinal

'! . • 1"-

mucosa :".i:~f1uenced by;,a Dumber of factors, Somelipophilic xenobiotic

compourfd's' ~re

^{alsoabsorbedbythe}

enterocyt~

in conj unctionwith the""trieot lipid.Theinteracti~thatoccursbetweenthesecompounds and.nutrientlipidsis oftoxicologicalan?physiolcgieal interest Tlie.chylomicronapp~arall'Cetime{the intervalbel w~en',ill.troduction of HC-oleicacid and the {irstappear~nceof HC. labcled>ehylomicrcns in thora cic duct lymph) is an effect ivetool tn

. ·.":l :" . > ' ,

monitorinlfch~ngesinthehandling ofthefirstlipidmoleculesbeingprocessed,by

.

^':. \

~e.enterocyte;'>The chylomicronappearancetime incoltt.ro lanimalsish.ighly·

reproducible'and agreesw"ell

wit~

previously

pUbl~hed

work. -

" ' , -

J:,~Polyeyelicarom~tichydrocarbons suchas7,12'dimethYlbeDz[al a~thracene ( (DMBA) areld!~wncarcinogensan_ adilyabsorbed.Iromthega;str~intesti~al. tract,Due

to

.tbelrlipophilicnature,

a.

substantialp~~~onorthe~b~~~~_ _"_ "

xenob~oticcanbetransport edi'blymph ehylomicrona.Ofthe PARs'tested,only

DMB~.was found to exert any effecton thechylomicron appear anceti~e

rrsulting'inan~ppe;rancetimeo~ 13~08

±

,2.10 minutes.as compa redto'4contro l

. .

value of10.81

±

1.03 minutes (p

.<

0.001). ~heseresults indicatetha~the inclusionof DMBA,in anutrientlipid test mealsigni!kautlyret ards the normal .processorupta keand/o r processing ofnut rientlipidswith inthe ea terocyte.

Exposur.eto~Ojl.molDMBA severalhour~prior.tctestmealadministr ation,I'

stlll·resuiteda delayto'chylomicronappearancetime Tnglycertde 4

nt6

analysts forthree hoursfollowing test mealadministrat ionindicatesetransient eHect,of

9holesterol had noeffe<;t on-rhe chylomicron appearance time but. the

.---- 'hydrophob ic surfactant Pluronie L-8.1'delayed'the the appeara nce.otthe

radlclabal.{16,60

±

0.77 minutes,p 4:0,01\. This resultsupports thereport attribut ing hypclipidemieactivitytothiscompcpnd.

.:- I..

f

J:

.1ii

.DM'BAsinee'tbetota ltriglyceride eutputs inboth controlandDMBA*tre atedrats

were notsi~niricantlydif~eren t. ~ AI

. ~ " . 11

SUbse~uent

studies used the

chylomic~W

appea rance time

~ec4,nf(ji'Ie-to

^, determ ine the-ceffects of other li'pophilic.compouede on lipid

~ ~~rPtioV

chylomicro~appear~ncerlme.tecbni que.Threegroupsof.rats~ereused~orthis st udy.Grou pAreceived aI~id^{emulsiontestmeal}cotitainingl*moD~leill~^{its " .} sourceofmonoglyceride.Group Breceived1'1.tes,tmealcontaining2-mo'noolein an d.i-oupCreceivedates.t'mealwit hout any

monOgl;~L.JllfsenL...T~

.chylomicronappearancetimes forthese.three groups didnotdiffer significantly.

.Thisindicat es thatthee-glycerc pbosphete pathway iscapableor hand lingthe Ilrat lew molecules orlipid as efticientl yas the mohoglyceridepathwaydespitethe

r-

nigherenergycostre9uired todoso.

Theerticiencyorthe e-glyeerophosphatepa th waycompared with the

~

monoglycerldepathway

'0'

triglycerideresynthesis was examined usi ng

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. .

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.. ..

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[KE Y WORDS!

An'tbracene t.a-Bensenthrecene Benzo[l\jpyrene Choleste rol

ChY'pn;aicronAppeara nceTime 7,12.0imet hylb t nzanlhracene hydr ophobicsurfactant ....~ PluronieL-81

PolycyclicAromaticHydrocarbons

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~.,p_/'

AqkNOWLEDG~MENTS

; _{, .}

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1deeplyappreciatetheguidan.~e

ani

encouragementof mys~perviS~r:·Dr.

.-.fA.B·arr owman .His constantenthusiasmandendless supply of-ideas'made rny

.

.

^,~

research ,very enjoyable. My thank sare extended to. the members,or my.

superviSor!"committ ee,Dr.J.C.Orr"and Dr. "N.'tamPbell for

t~eir.

^valuable

assistance.

(. ^.

.Man ythanksareex~en dedtoJim RossandAnisurRahm anforpat ientl y te.achingme the_ne~ssar~':I~15oratorytechniquesandtoGarryChem eoko_rld~

LoriCollins tor alwayslending

.

.;.'ahelpinghand.

" V .

-) gratefullyacknowledge Iinanctal'suppo rt provided bythe School of GraduateStud iesand

tli~~u lty

^of

r-.1edicin~.,

. . .

Finally',1 \vculdliketothank David,myparent san

if!

my~rother.,Richa:rd .I

for theirconsta ntloveandsupport.

I

i

I

TABL'E OF CONTENTS

I

1.INT R Op UCT IO N . '

_.L1.Bac(~oundinformat ion

1.1.1. ,

Lipid digestion andabsorption

1.1.2.Factors and disease states tliat influence chylomicron appea rance time

1.1.3. Polycyclic aromatic hydr ocar bon s 1.1.4.Metab olismof polycycli c arom atic hydro car bon s 1.1.5 .PlutonicL-81

1.2;Object ives

2. MATERIAL S ANDMET HO DS 2.1.Animals .

2.2. Surge ry 2.2.1.Anaest hesia 2.2.2.Surgery • 2.2.3.Duodenumcannulation 2.2.4.Thor aciclymphduct cannulatio n 2.2.5:.Closure

2.2.6.Post-operativehousing 2.2.7.Sacr irice ,/' . 2.3.Testmealsandlymph collectio ns

2.3.1.Test MealComposit ions(Ta ble2.1) 2.:3.1.1. Contro ltestmeal

2.3.1.2. Polycyclic aroma tichydr ocarbon test. meal 2.3.1.3.3H·D.MBA

2.3.1.4.Choleste rol 2.3.1.5.Plut onicL-81

2.3 .1.6.a-Glycerophosp ha tepa thw ay efficiency 2.3.2.TestmeaI administration

2.3 .2.1.PluronicL-al '_

~,3)~

^Lymph

s~m~le ~ollectipn.

.. 2.3,4.Liquidscintillationco unting ~

2,.4.Experiment alprocedur es

I )

2.4,1.Chy lomicro nappear ancetime determin a tion . . 2.4.2.Erreet orPAHsonchylomicro nappearancetime 2.4,3.Dose effectorDMBA

\ .

'13 15 16,

, '1

20, 21 21 21 22 22 22

2' 2'

26 26 26 26 26, 2S 2S .28 28 32 32 33 33 34 34

\34 34

\

I \

chylomicron 34 vtt

~A.4:.yErrect 01~h~r hydrophobic xenobiotlca

appear an cetime I

2.4.5.Enzy meindueflonexperiment ... 35

2.4.6.Triglyceride Flux arter administration of a test meal ' " 35

co nta iningDMBA (\

2.4.7.Chylomicronapl)Iil''"' 'tim. in rats given atest moat 36'"

withoutmcncgly eerideorwith2-moDoglyceride

2.4.8.Triglyceride nux arter administratio noratest mealwithout 36

monoglyceride . ").

3.

RES~~~s~atistics

^I

;~

3.1. Effectorpolycyclicaromatichydrocarbonson lipid absorptionand 38 handling

3.1.1.Chylomicron appea ra ncetimes (Table_3-1) ,38 3.1.1.1.Contro lchylomicronappeareneetime .3&

3.1.1.2.Polycycli carom at ichedr ccarbcn s (Ta ble 3-1,Figure 40

. 3-1)

3.1.1.3.3H.DMBA 40

3.1.1.4.LOw doseDMBA 40

3.1.2.Triglyceride nux 40

3.2.

~i~~~t Eo~z~~hee;~~~~t:6;h:~:ri~:~tbi~tieS

⁰⁰lipid absorpt ionand,

Jl

~1~~1~ghYIOmicron 'appeara~ce

lim; s (Table 3-2) 54

\ . 3.2.1.1.Cholesterol 54

3.2.1.2.PluronicIrSI '. 54

3.3.Alpha-glycerophosphatepathwayefficien cy 54

3.3.1.Chylomicronappearancetime . 54

3.3.1.~.Monoglycerideabsent 54

3.3.1.2.z-Monccletn 62

\ 3.3.2.Triglyceride[lux 62

• 3.3.2.1.Monoglycerideabsent 62

3.3.2.2.2-Monoolein 67

t.DiSCUSSION ' .At>

4.1.Chylomicronappea ra ncetime .•.~.::.:. J;o 1.2.Influenceorpolycyclicaromatichydrocarbons ohlipidabsorpt ion 71 '1.3.

~:~~~s~e~~~omicron

appearancetime '.'

~~

4.4.Pluronic1..-81 . . 76

4.5.AJpha-glycerophosphatepath wayerricicncy ~8

SUMMA RY 81

.:

REF ERE N CES.

viii

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Table2-1:

Tab le 2·2:

Table3-=1:

.Tab le3-2:

LIST OF TABLES

Composit ionoftestmealscont aining polycyclicaromatic 27 hydroca rbons.

'Compositionof testmea lscontaininglipidsand Plurcnic 31

~81. "

ChylomicronAppearanceTimes after administrationof 3{1 test meals cont ainingpqps'cyc!ic aromati chydrocar bons ChylomicronAppearance Time after administ rationof 5.')

various testmeals. ~

rI':' ^{".; '}

, .

.,/.

- ..

;

. .... LIST OF FlGURES'

Plguee3- 12:

\ J

. .

FigureI-Iv Factorsaffecting lipid absorptionin theente rceyte. 10 Figure2-1: Placem e nt

or

the duodenum and thoracic lymph duct 23

cannulae. ••

F'lgur e2-2 : Polycyclic'aromatic hydrocarbonsusedin this study.. '20 Figure3-1: Ch~lom'ic~onappear ancetimein animalsgfvena control 41

orPMitest meal.. .

Figure 3-2: ChYIOmiCro~apearence

ume

in animalsgivena eoutrol 43 :

··-erDMBAt eal. ' ., :-

Figur e-3-3: .Cbylcmicro appearance timeandappearanceof DMBA 45- ./

inanima lsgivenaDMBA test meal.

Figure3-4: g~~icronappeara~cetimeiiianima lsJiv~n ~ mM 47

"FIgure3-6:.

;;~:~~s~:i:t~onr~~x~ e:::~ol:r-~:;A ;e~~r~e~~.rio~ ~er

^'40

Flgur.3-6.·C~lomicron appearance tim,'" polyeychc aromatic: " .t". llJIIrocar bon bydroxylase inducedanimals. '

Fig'utea~7: C.lomicro~ ^{appearance jtme in animals given a 56} cholesterol-sup plemente dtest meal • 'F igure3-8: 'Chylomicron appearance timein animalsgivfln a test 58

meal containingPluronicI.r81.~ - FI~ure3-9: Cbylbm icronappearancetime "in.animals given'a test 60

" meal without monoglyceride.

Figure3~ 1 0; Chylomicronappearancetime'inanimals'givena test 63...

mealcontaining1·or z-moeeclein. .

Figure3-11: Triglyceride n'ux over a·one

h~

period -en er

"6!f

~~:;;~s:~~t;on f~~xa t :te;ne~l w~~eou~~~';l0~I:r~:~.d\rtU ~

^.-

administri~ion.of ate~tmealcontaining2-mo~ooleitl.. ~

I F·

DMBA

'AP

. <~~~

~I ^1,2-BA

'pm PAR CAP

;,

'E

J:ABP TIlFA

\'LOL

' ER

VI

xt

ABBREVIATIONS

7.12-Dimet.h y lben[al &nt-braUD ' Benzo[a]pyrene

Ant.hrac ene i.2-Beu,t &DtbraceDe countepermi~ut.

polycyclicarom~t-1!'hydroc arbon ch11 olllicro ~appeara nce time

8tan d a rderror

ofthhean

htty .d. " 10.,.; P'i ) D

trig lyceride fattyacid

very10'11'densit y'lip~~roteill'8

smootl:!.endoplasmicreticulUD vfeccu e ho t-ropi c

Chapter 1 I NTRODUCTION

1.1.Backgroundinformation

1.1.1.Lip id dlge srlcnand absorpt ion

. .

ThelipidcomponentoftheNorth American dietco nlri~u tesaprrp ximately 43%of energyderived.fromthetotaldiet.Thi s lipidco mponent~com posed of triglycerid es, cholesterol and phospholipids (Guthrie , 1983 ) with dietar y triglyceri de accountingfo r over gO%of total lip id int'ak e.Dueto theimpo~tance~ of this dibtarycomponen t,themechanism of triglyceride digest ionand absorption

hasbeenin ~l'nsivelystudied,

The pathwayfrom in~estiollofdiet ary triglycerid etoabsorptioncan be dividedinto sixgener alste ps:i)int ralu m inaldigestionii) mice ll a rsolu bilizat io n iii]permeatio n from tbefumentothecelliv]intrace llular reesterificationvv]

chylomicro nformation and vi) transportofthechylom icr~nfromthecelltothe circulation(Shia u,1087 ).

TheIIltir~lateaim oftrigly ceride digestionisto pro d uce compounds thatate morehydr ophilic thantheparen t nonswellinglipid (Sh iau,1987 ).Thedigestion processbegins inthemouthwherebodytemper atureliquifiesmanyfatssuchas

. .

'"

butterand margar inean dmechanicalaction break sdow nlergelipidpar t icles: a processwhichservesto increas e the surface area.of'the, lipid increasing its

• bloavailability fordigest iveenzy mes. Lipolyticenzymes hydro lyse.triglyceride resultin g inthe produc t ionof,diglyceridt's,monoglyceri des,fa tt y acids and ....

glycerol. Tbislipase activitybegins wit h lingual lipas esecret edfrom serous glands

.

(vonEbne r's glands ) onthedorsalaspect ofthet~I;g.ue.

^{. .}

TheoptimalpHof 4of thisenzym eallowsit:0conti nuehydrolysisin. theacidicgastricmediu m- .This enzyme racksPositionalspecificityyetit prefe;entia llyatta ck s tbe medium

.

"

chaintriglycerides.Once expose d-t~biles3.lt~,th~act.ionoflinguallipaseis __inhibited(Liao,HamoshandHam os h,1984}.

, ,

Lipoly.tic.a ctivityin the sto machis largely dueto.gastriclipase'whose activity·bears elcseresem blance tothat~fthe lingual'lipase_.Because oftb,is similarity theexistenceofthesetwo enzymesasseparate ent itieshast '"

questioned.Approximat ely 10.30% oftriglycerideishydrolysedinthe stomae to formpartiala('ylgJycerolsand free fattyacids (Liao, HamoshandHamosll.I83). -;

In tralum inaldigestion

Thegreate stproportion oftriglyceride digestionoccurs in thesmall intest ine

.

atthe oil-water interface.Here.pancreaticlipase,secretedby the acinarcellsof thepancreas,hydr olyses triglycerideat theIand3positio ns withapH.optimum ofapproximat ely'S(Borgst ro~,Hl6-4)_lnIQ6-j",\Borgstromdemon s t ratedlbat the presenceor~lesa ltsintheintes tine resultsinthe-rt>dud ionorth e pH optim um for this enzymefroms.to6 whichcortespo ndswit h thepHoft~e^{smallinte stine} Themech anismby which this shirtoccursis unclea r.It has been postu lated that thebilesaltmayinducecha ngesintheconformationofthelipase.

,~.;/"

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The presenceofpanc r eaticlip asein tbesmallintestin eisDot sufficie n t for lip olysis to occurat theprev ailing pHandbilesaltconcen trations,Thepancreas

~"cre.'.cta rofactor,tolipa".',e', intbeformof procolipa.se.Proteo lyticenzymesbreak down"

thi~'c'o~pound

^to

th;~active

^colipase

~bicb bint'l~

^{totbe lipase}

i~

^a1:1ratio.

This'bindingisfacilitale~,bythe presenceof bilesalts. Ithasbeen post ulate d th a t'

th:.c;liP~e

may,anch orthe

l~~e

^a,t

t~e

^{oil-wa ter}

in~errace

^"inthe presence .I'

~~

^{bileslllt;.•}

( ~o~s;~om .aQ~,.oo~rr,

^IQ751·

T,h~ 1iP(lS~

^{clln',then'act at the}

. Int errace bringing~lksa: tmicellesdoseto th:siteofhP~IY~IStoremov e the •~ lipolyticproductslrom th e_Inter face.If not \.ll1lt0ved,

t~e

pres ence of these productsacc~mulatingat the oil:wa terin ter~aceres ultsin inhibit.ion of further hyQroly\ is.

Mleellaesoluhlllaaelon

''':-:-

Micellarsolub ilizat io noflipidprod uctsmust. occu r prior to-'absor ption"

,P rim~ry",bilesaltssecreted"by theliv erareconvertedtosecon darybile,saltsin the i,nt~~,nal-tumen due to bacterialbiotrinsforma t ion, At ~eri tlcalmicellar concentration,bilesalt mon omersformaggr('gatl's called simplemicelles.When lipolyticproducts are incorporatedintothesemicell es. mixedmice llesare for med [Careyand Small,' 1070),This process increases the water solubilit yofthelipids"

by a factorofapproxi mate ly 1000.

. . ^.

Theevents orlipid digestion,asdescribed above, canbe seen by light microscopyas afourphase seriesco nsisting or anoil phase.calciumsoap,viscous isotropic (VI) phase 3o.d mi; ellar ph~e[Patton , VetterI Ha~o~b,Borgs~roin,.

" Lindstrom andCa rey, 1085) "Theoilphaseconsists"oftriglyc eride and~iglyceride existingas a spheric al formin wat er.Lipiddigestio n by,pancreati c'lipase canb~

r ·

,

ob"servedatthe oil-waterinterrace.'The first.product

p te,

the calciumsoa pis seen:lS;~~ghbrittl e shellsurrou ndingthe

on

droplet.Isola tion andanalysisof thisshellrevealsit s

cornp~sitjon

^{to be}

o~er

^00%

~ty

acid and ca lcium.The viscous isot ropic phase follo ws altercalciumsoap Icrmation ceases. The compo'siti~^{ofthe Vl}phase is unknownsinceit hasnol yetbeenisola ted.F~om the fact thatmi~turesof prctou ated fattyacids and~,\ogly c eri d t'5exh ibit ch¥ acterist ics virtuallyidentiealtot9t.orthe\-1phas~itis suggestedthat these twocom~onentsmake\ipabrge propodiocofthethis phase.Thefinalphaseis the micellar phasewherebile saltsdisperse theVI phaseintodis cretewat er

~

.

solubledisks,ormixedmicelles: Smallparticles

or

tbisphase areno t visibleqy light

m'""c~py b ~t

^a'siae

~,"um

^{":, ,,}between.thi

Ph~~d t~;

^VIPh"·'.

.Anoildropletremnant isleft over after digestionoccurs.Thecomposition ofthis remnantmaybe.concentratedpolarconstituen tscftheinitialdroplet.It is, ,belie vedtha tthed~opletdoe s notconsistofnonpola r~il?idasthese compounds

movefreelyinto theVI P?asethro~ghahydr o carbon continuum.

Palton's stud iesIn1981resulted in the proposal of a hydrocarbon cont inuum intriglyceride which remainsintactduringdigestio n

o r

triglycerideto mcnog jycendeend fntt y 'old.Thepresence 01sucha,pa""lcontinuumallo·w. .\

(racelipidsto

now

^{th roughinto theproductphase:Pattonobserved}tbis.flnet hod 01tr-ansport witb both

~;';ot."'

^andthe

aromatic

hydjccarbon

p"y l.~

^-.

. 'I ,

Per meation!'rom,t helumentothecell - '

Once

digestio~

^{occurs, th}e' mixed micellescontainingtbelipoly ticproducts ate presente dtothebrushborderofthe ente rocyteofthe smallintesti ne.Two welldefinedbarriers slow this.movement.Thefirst istheunsnrr edwaterla ye r

.<

f

that lies-adja c enttothemicrovillous membrane ofthecelio

thisaq ueousenvironmentisIa cihntedby thesemixedlipid:bil;~altmicelles;The .second barrieris tilelipidmem brane.Micelles are tbo bgbt toreleasethe lipolyt ic .productsat th eceltme~brane.Thedif~~sionof theseli~j4Sa~roS!' th'Jmembr~ne isdependen tonthe individual permeabilityco efficientandconc entrat io nofeach

.

. . . .\~

ofthe produc ts.Long-chamfattyaCI~swhichare highl y hydrophobiccrossata faste rratetha nthoseofa shor terchainlength (Sallee,1919).

Bijrsaltsare rea bsorbedfrom theintest ine partl y by

,

pass ivediffus ion inthe smallintestine and partly byactiv,e tr ansport inthedistalileum.They'returnto theliver viathepor t al vein.About iO%of the intes t inal bile'salts escape'this cnterohepaticcirculationand is excrete din~efeces.

I . (

In the cellmemb rane,the lipolyt ic productsassociate

prererenti~lIy

^withthe

lipid matrix.A methodof transport is required tomo ve the lifolyticprodu\cts

to

the smoothendoplasmic reti c ul um withinthe cell. In1912, OcknerIManDing,

..,

Poppe nbausenandHoisolated. prot ei nwithamolecu larweight of12,000from thecytosolof thtent erccyte.This pro te inhasa high affinity rOf

l

fatt,y:~cidsandis refer redtoasthefat ty acid bindingprotein{FABP}(Ocknerand Manning,lQ74).

Areasorthe small

lDtc~.jDe~

^whe re fat absoqi tl0 I\..occ u rshaveJlghcon centrati o ns ofth isprotein TbeFABP

"er"'~"~lIY

^"me, unsatur ated

lolg _{,h,~ r.tt~}

^acids

andItISbelie vedthe t.reeete r triceuouofthefatt y acidsrequir esFABPtranspo rt

as a

"""I

^{stepID,h"andMa}nning, 1074)

I

I

glycerophosphate pathwa y.

Reesterification oflipelytlc products totriglyceri deoccurs inthe smooth endoplas micreticulum.Thisresynt hes is processoccu r;; through

..

a ser ies

.

^'of bioebern ical pathways."Cenera tly.fat ty acids ate<aerivete d toacyl Cox

(',M·-.. .. ' ~

•derivatives -in the preseo;eofCo.A.AT?(derived hom glucosemet abolism} and M,;++ by theenzymeacyl eoAsynthe tase.Triglycer ideresynth esiscan then follow oneof twodistinctpathways:oilthe1J1o~Oglycerid epathwayor ii) theo-

\ 1JoI • ~

.:.l.- .:.-'~

In tbe mo no.glyceride~at hway, z-moncgfv cerlde derived frolTl"")d ietary triglyceridedigesti on is esterified with a.ctivatedfa~tyacidstoformadi¢ceride.. \' '''

Q . ". Esterificationwit h anot her Iatty acid&resu ltsintriglyceride format Ion.Bo tb

o r>

theses~eps teq(jr~the presencej an acyl

cs«

ac~l!.raDsferase.Th i~is..thejnelc r pathwayoftriglycerideresynthesis (Shiau,1987).

It)

" When-f-monogjycetideisint roduced intothe intcsrin a!lume n,'itsult imate .:fa t e depends I~gely^{on the}fat ty acid' moiety involved. l-mcnopalmit in is

reesterifirtthrough tbe monogly cerldepath~ ay(as .described above.)tof<;Jnn triglyce;id ewhere asr-monoclemfollows a differentroute, thee-glyc erophos ph ate pa thwaydescribedjb~o,w.. Inadd ition ,themethodbywhichthet-monoglyce;ide is presente dtothe enteroeyteis

im~rtant

ⁱⁿdet ermining,the mechanismby whichth e 1-Isomerishandledbytheabsorptive cell.

Luminalhydr olysiscan resultintheproductionoft- monoglyceride.Alevel

. '

of6%,t-m~nooleiI!hasbeenrep ortedfrom bydrolysis"oftriolein(Matts onand

Volpen bein.\HJ64).This mon..?glyceridfisproducedasaresultor.2.mo~oglyceride isomerizalio~el. m?oooleintravelsto theintest inal mucosawhereit is ,hydrolysedfurtherto glycerolandfat tyacid.TLese arethenusedin triglycerid e

·

resynt hesis. The ~tilization of l- rnoacglyceride as a. substrat e..ror'the

., ~

monoglycer:de pathway oftriglycerideresynthesisisdisputed bysomeeesearchejs (Mat tso.n andVolpenh ein.Ig64)and supportedbyot hers(Brown andJohnsto n, lQ64a.;BrownandJohnston,Hl64bj

In the absence of monoglyceride,triglyceride i,s.resyntaesleed by'.

~he

Q.gly cerophosp~a.tepa thway(Shi\u, Popper, R~ed , Umstet ter,Cap ueti ~nd/

Levine,lQS5). Hexosemetabol ism resultsi~theforma t ionof a-glycerg phosphate' whichwhen combinedwitht}Yo molecules~facylCoAbecomes phosphatidicacid.

L-.,.ph~phatidatephosphohydro'lase hydeolysssthe..,phosphatidic aci to a

diglyce ride.(S~iau..HI87). Asubseque n tacylat ionrea ction results intriglycer ide forma tion.

Chylomicro n f<fm atio?

The newly formed triglyceride~ropletthen acquiresphospholipidin the SER.This par t icle, is transportedtothe Golgiapparat uswhe rea glycoprotein '.,coatisadded. Thes~rfaceofthjfullyformedchylomicron is composedofa momila yerof

phospboli~ids,

apoproteina,free iObolester ol anda

sma~1

amountor triglyceride.Thecorecontainsthe majorityor tnglyceeldesandcholes te rol esters. anda~outone thirdofthe total free cholest erolinthechylomic ron. The .mecha nism bywhichthispacka gin,g occursand the regulatory.~ecban ~smswhich governitsasse mblyare genera llyunkno wn.Thepresenceofapopro teln s ,however, is essen tialforthisprocesstooccur.

(

^.

. .

~ T.aDOPO: t'or tbe

'b"'~ml

^{..a outof8t heee11.}

Onceform;d,the chylomicronsarepicka ge<!.into secreto ryresicleswhich tU t titoan~fusewiththebasolateral~rtioDoftlieeuterocytecellmembran.p~

Theehylomieeoasl,rtthe neaocy tosedinto theintercellularspac e. They tbenpass

":"~. ,..~., .'-..

throughgaps in the basementmembrane ,erossthe lam ina propriaandenterthe la rtu ls, adist aneeof approximately SO..m (G..ranger, 1981).Travelof the 'chylomic ronsthrough thelam inaprop riais pr~bably by passive dirrusion, facilitate dbymat rixhydration(Tso,Barrowm a nendGranger ,1086).Thusthe flu idmovementthatoccursconcurrent .ly withchylomicron tra nsportmayassisl the mov ementofthesere'lll.tivelyb'ulky~articl.~lIthroug htheinterstitiu m and greatly decreasethe chylomicron,transittime(Tso and Balint,IQS6l. Tso,Pitts

~ ,

.. andGranger(HISS) repor1 thatthe chylomicron..tra nsittimeis.dependenton- thenowrateoflymphwbe,lymphno wis

<

40I',Ljmin. At non abovetb~.lI, thereisaminimum transittime ofradiclabeledfaUya.C:idsbetw een theintestio'al .lumenandtheint estinal lymphaticwhichisoftheorder

\

ofIo-ISminutes(1.50et

ai,1~6 ) .Prov id ~thehydration of theint~rs titiumill heldrela.tiyelyconstant, cbaDte! inthetimerequired Lad~tec tthese chylomicrons intbe intestin al lym pharicscan beusedto indica techa ngesin lipid absorptionaedhandlingby theenterocyte.

1.1.2.Factorsand disease statestha t Inrtu e n eeehylomle ron app e arance tIlll e

N:

mentioned previously,the ceylcmieronappearanc~time{CAP)'ie.the time intervalbetweeninrroduction ofaradiolebeledratty acid in theduodenum andtheappearanceofthelabelin lym ph chylomicra,can be alteredbya number

offa ctors {Figure U).

.

The rate offat absorption fromthe lumen,lymph(ormat ion;.lymph nowrate and int cr stitinl matrixhydration all appea rtohavesacute effectson the chylomicron

"

,

app earance time in thelymph (Tooet a],1086;Tso and Balint"1086 ;Tso,Pit ts,• and~ranger,1085 ;Tso,BL~h,BalintandRodgers,1982).

Variousclinicalconditionshevc beenid~ntifieathat resultin'changesin chy lomicro nappearancetime whichint~rnresults inchronicabnormalhandling ofdietary lipids. Some ofthese mal absorpti on syndromesarcdescribed below (F ig ure 1-1.). Asmentioned 'preJio llsly,apoprote insareanessentialelerqent in chy lomicron forma tion.Inhibition ofprot einsynthesis markedly.effects the"

chylomicro ntrall~portof triglycerides intothe lymph(Glickman, Kir~chand Isselbach~19i2).Abetalipopr~teine_mia,an inb.ornmetabolicabnor~ alityresults in adeficiency'inapolipopeotein B(a p0 !31synt hesis. T? isclass ofapo prot eins are requiredin the earlystages ofchylomic ron for mati on.Morphologicai exa mination ofthe eatercejteeofaffli;ied- patient srevealslarge unbound lipid droplets within

•thecell. Chylomicro nsare notformedandtherefore a malabsorption synd romeis obse rved(Malloy andKane,IgS2j.:

.

^{~ (}

Zincdeficiencyhasbeenlinkedtoimpairment of intestinal triglyceride

Figu re1· 1 : FactorsafTec:tlnglipidabso rptio n In theenter-ccyte, Theleft sideofthefigur eshows the

struc t u resinvolvedin

4(:' dietary lipid absorption. "'\

...

\ .

/

^'

/

=

\V~. ~i

Abetalpoprotenemia (obsfol"lC'ofogosl Al"'derson 's Disease ldforfoel InI'nalel'lylo m OCf'On asurrbly?l

, _ __ ZincDetciercy

BasementMembrane • ('MIllohanofprot " "Sy" I,,"1S')J•

.

. ... . ^{~ \}

^..

^{~ ,}

Intestinal (.c:x=:.~.~~ Intersti tialMwrlxHydration

1.YlI'llrotic V essel - - . - , • r ; • • • • ^{LY'T'llh Row}

. e::.-:- . ... ~

Bindng Proteins

GdJ i

Apparatus

Monoglyeendes FaltyAcids

~ ~

Smooth EndoplasmicReticulum

SecretoryVesCies· lllf.llf~

. ,

Rough Enc:bplasmlcReticulum

{

...

I'

trans port possibly throughinhibitionofintest1oallipoprottill syn thesis.A decreas e in nettriglycE'!ide tt,aosportbas beenreportedbyKoo,d

T~d

^{(lQ771..illeats.} reda zincdelicien#diet.EI~roDmicrograph sortheintestinalmucosao,rthese rats showaccumulatio n01large. lipiddroplets..Thesedroplets areDolmembran e bound andtendto ecelesee. Markedr~uct ioDin tbegranularendoplasmic

. . . ( ), r.G" . . d T

reticul um-andaDIDactlveappe~rlllg olglappa ratusIs/observe. h.edr.o pl elsare unable to~nter^thein tercellularspaceandtherefore

ao

not ap pearIDthelymp h. Instudieswhere asu belieieallevelofzinc

d.efieie~5

^{induce dinrats,marked}

mor p hological altera t ionsin intesti n alnascen tehylomicrcns

aJ~

^decreasesin

apoproteinCandEnreobserved{KoD, Henders on.Algi la~andNorvell,HI8!)).

,.

. . ..

Anderson'sdise as e orchylomicfu n' rttentiondiseaseitarecessivelyinherited rond itio D,OI'lipid mal absorption.Patientswith thisdise ase exh ibitnoinereesein pl asma triglycerlde levels afterbeinggiyen anoralfatload. Ultr-ast rucrural

e~aminalion

^{ortheen tercejtesofthes:;alientsalterratloadingrevealI.large}

~

.

numberoffatparticl es nsicula.tingtbeendoplas micreticulum.fnaddit ion,these ,chylomiaonsized piu ticlesare clustered withi nthedilatedvesi cles 01theGoig i rene.faltransportcu r.crtheeaterocyreapP!~rsto be impa ired[Hoy,Lev y, Green, Sniderman,Le tarte,Buts.Orq uin,Bro c hu,Weber,Morin.Ma rceland Deckclbaum, 19S71. Thedefectin faltransport i'll' thisdisease is notyet"

underst oodbut itispost ulatedtheteither thefinalchylo mteronas~mblyorthe, emey toaispathway oft~ecmp letedcb ylomicronisarflil~..tt!d.

In add itio n to clinical cond itio nsan d physical parameters, various

.

senobioticshavebeen Ic uadto .rrtttlipid abs orption.Includedare tbe Pluronic

.,:,.

13

,',

(

)

polyolswhich are a group ofhydrophohicsurf actants and colchicinewhichisa drugusedin treat mentofacutegout,

1,.1.3.Polycyclicaromatic hydr oca r bo n '

Polycyclicaromatichydroca rbo ns(PAHs)are animportan tclassqfchen»c al. compoundsman)' ofwhichha ve been found tobe po tentcarcinogens.Th;se compoundsare compo sedof thr eeormorefused benz e ne ringsandaregenerally forme) Mare s ultof inc: mPle tecom bustion ofcarbonaceousmaterials

{ B1u~~r, ·

10;6). Researchinto thepossibleadv~~seeffec tsof these ecm po unds onhu~an health hasbe encar riedoutsince'dib~n z(a,h )anthrll:cenewas recognizedas a

carcinoge~

^{in the}early partofthiscentury.

As

ofIgt S.over'100PAHshavebeen ident ified

( Lo

and Sandi, Hit s).Earlys;ud ies

demonstr~ted

the carcinoge nic

effe~t

of>top ical

applicali~n

^{of'c'oaltars on mouse skin andlater the'compounds}

re-sponsibleforthiscarcinogeniceUect wese sho wntobelo ng totheclassofPAMs .

,

-

^~,

Initially, research'roncentratedonoccupa t ionalexposuretoth is class'of compo undslargely as a resultoftheproposalforanenvironmen tal basisfor mc

t , '

^{by Pottin177?}

^Pot~

^{concludedthat,t he high}

incidenc~ ~f

^{scro talcancer}

III

C~'~neY

swe eps was ares ultof longtermexposu re to ca rbonsoot.Laterit becameappare nt that

hU~lan

^{exposureca n}

oc~ur

^{fromanumberofothe:}

so~rces,

Onesuc hrout e ofexposureisviaPAHcon.tamintit~.foodslUUs,PAHsappearsin a'wide varietyorrood typesfrom a.v~rietycreo ureee. Commonly,food.

I ' ,

contamination by these com pounds isa result of.environmentalpollution deposit edonleafyve getab lesOrIromsmoke-curedorcharbroiled meats,Heati ng ofoilsand othercookingproceduresalsoresultillPAHfor mation(Gray,and

Mbr~-on,

1981).

/

14

PAHs sue hasbeeaclsjp yreoefor medas aresultofindustr ialemissio ns have beendetect ed inlett uce,kale,spinach,leeks andtomatoesin5igniriean ~qua ntit ies [LcandSand i.1978).Fortu nately,rinsing ofthesefoodswit hwat erremo ves

,

muchoftheeontamination.PAHcontamin ation"·has also been reported in marg arines,butt er andvegetableoils producedfro,illcontaminated plant raw materials[Hcdia,Pyysaln andwiekstrom.1986 )

ThebulkofPAHconta minationoffoo d

resu~s

^fromcuring

~cert ai n

for msofcooking suc has grillingoverchar coal{Loand Sandi,1978 ).Sinceth e PAHs aregenerallylipo p hilic ,associa t ionwiththefatcomponents ofmealand

I (ish.iscommon.

TheamOulIct'ofPAH deposition onsmo ked foodsdepen dslargely onthe

wood deposition temperature (Gilbert and Knowles, 1975 ). Beca use the penetrat ingability ofthesecompoundsisrela tivelylow, surfac" futsof meats cont ai nhigherconcentrationsof these compounds than the inner cuts(Gilbert andKnow}es,1975)

Charbroiledmeatand fishalsocontainsignificantlevels of PAHs.Here,the fat contentofthefoodandthe dista ncefromthehea t sourceare keyfactors.

PAHconta mination i9.this caseisduetopyroly sis offat.During thecharbroiling process quantit iesoffat driponthecoals.Pyrolysis ofthisfatoccursandIorms polycyclicaromatichydrocarb ons.These are thenvolat izedand deposite d onthe meat(Lijinskyand Sh ubik,H:l65).

15

1.1.4. Metabo lism or poly e)Ccllearom !lUr;hy dro e e j-bone

Asoutlinedintheprevioussection,the gastrointestinaltr-aetis a keyroute forhuman exposure to polycyclicaromatichydrocarbons.Itis believedthattrace amounts oflipid-soluaqlecom poundscanreadilydissolve in dietaryfat and then ran betransferr ed to mixedlipid-bilesalt, micelles (LaberandBarrowman,HlSJ ).

~ ,

Suchbeingthecase,lipophilic PAl-Is may be passivelycarried intothe en terocyt e

throughthe hydrocarbonco nt inuum postulatedtoexist in the small intest ina l lumen (juringfatdigl'St io~and absorption{Patton.(181),

Onceabsorbedinto the ent erocyte. PAHs~aytravel oneof tworoutesint o the generalcircu latio n.Lindstrom,BarrowmanandBorgstrum(HI87)showedtha t acertain port ionof7,12·d imethylbenz[ajanthracene (DMBA) absorbed.iro mthe smallintestineappears unmetabolized in the chylom icronfraction ofthe lym ph.

Therelativeimpor t ance ofthisroute wasdetermine dbyLeber andBarrowma n

,

.

(i{l87). From these studiesitWl\Sfoundthat the lymphaticroute

contrib~tes

^2.0%

ofthetotalrecoverableDf\..lB Ahom~thlymph and bile combined.Once.inthe systemic circulation, it is not.known whet her Dr-,mA remain s with the chylomicron remnan t afterexposuretolipoproteinlipasl\,activ ity in pesipheral tissues.Irresp ective of ttis,DMBAislargelyavailabl e'formet abolismby the liver s:

followedbybiliaryexcretion(Lindstromet al., HI87 ).

Theport alvenous bloodroute forDMBA transpo rt from tbe enterocyte appears l(J beamoreimporta nt pathw ay~hanthelymp ha tic rout e.10 theearly 1960'sPAH metab olismintheeut erccytewasdescr ibed(Wat tenbe rg,Leongand ( "

Stra nd,1962).The enterocytepossessesenzymeactivitythatoxidizesPAHssuch

v.

as benzoialpyre ne (BaP)to hydroxy l derivativesand subsequently qui nones.

Inductionofthis,enzymeactivityoccursfro m priorexpos u reofthecells toPAIls oroth erin ducers (Wattenberg,l{li l), In addition,th eglutat hione-S-tra~srer:l..Se enzy mes are inducedin the smallintestine after benzotajpyrene ad min istr a tion .and the distributionofth is enzyme parallels wit h thatofthebenzo(a )pyrt>oe h~rox yla.s esystem(CliftonandKaplowitz,11l:j7). Oncemetabolized,thePAH rrel~bolit('~,^probablyin a conj ugatedfor m, are tra nspor t ed to theJive~'l.^the portalvein(Bock,Claus bruchandWinne:IOiO)

Inthe liVft,PAlls undergo PhaseJ"(us u allyox id a t io ns) and PhaseII (conjugations)met a bolism ,the end result being theconvers ionofthelipoph ilic' parent compounds towa t ersolu blespecies v.:hichand can be excrete dinbile .(D ipple ,1083 ;Digiov an niand Jucha u,1980), WhileP,AHsarebiolo picall yra t her .iner tcompounds,so meoftheir metabolites'arehigh lyrea ct ive electro philic substances which are the ultimate carcinogenic produ cts of this class of cempounds (Grover,HI86).

1.1.5.Pluronle1..-81

Anothergro upofcompo undswhich appearassociate d with thelipidport ion

•,of the die t are the hy dr o p hobic sur factantsofwhich the Pluron.ic polyol detergents aremem bers. Com pounds in this grouphave beenusedasem ulsifiers

,

^{"'\ ' .}

in food s,such as milkshakes,and,insmalldoses,they arewell toleratedendfr~e from toxic effects.

The hydrophobic component.

0\

^th;s..:.su bst a nces..aries between these

admi'n~tration st~dies

^{(T50.Bishopand}

~odg~rs, 1~8l).

11

absorptio n.In 1077, Bcehenek and Rodgers showedthatrats given aliptd'test meal containing asmall quantity ofthePlutonic compo undsshowed~.decr~

in lipid absorplion proporhonal to the hyaropbob id ty of the Plut on ic .adminetered.III animals giveD these detergents withahydrophobiccom.pont olof

atleast

sacc..

inhib ition oflipid absorptionwas obse rved.Det ergentscoolai ning lessthan6ifOb~drophobicitydidnotia terjerewithlipid absorptionand,in~me rases,even enhancedabsorp tio n

One

or

the pluronie compou nds,Plut onicL-81,whichis composl'd'of polyoxyet hyJeneandpolyoxYP,ropylenecopolymersandhasa molecularweigbt.2t'].'. 2750,and hasabydecphoblccompo nentof 00%.Tro tBalin t and Rod gers,(10M ), has shown thatthiscompo~nd^{depresseslymphatic}transpo~t^oftrigl~erid, f~tty.

acid (TGFAIandC'holesterolin rats given1 fhro.n!CdO:e.(3-4wks ]

~r

^{L.81:.ln:.:··}

ad dition,the same

i nhibitio~

^of lipid transpor t'can beobserved"hi.acute •

. :,- , " ,:

j-,~

.The

'tftec6.~nism

^{involvedintb·is}

e~t

^Onlipid

·absor~tion

^basbeen reeenn y-

.inves:i gated.

JIJ~IIY: im~~irment ~; di~estion

^{andabsqrptibO}

~r

^{th.; IjpidIntbe}

. '. . . .. .4 . '..

^,1 '.'#' .:"

..jll:testihallumen 'wassuggestedasthemec~~ism_b;"'-w.hicbPl~ ro~.:c.^t:8}.ex~tl.S its

ernCt.-l~chenek.

^{andRodgers,10771..}

;.~ 'th~l ~as b~'ed

^{on invitrostudies.}

' , ' , • U '

by Green,Heald, Baggaley,Hindley and Mor gl.ll,(1076).,ItwaSlatersbcwc that in cases·

wb~ere

^{IIlargedose'ofplutonic L-SI}

. 'w f g;ven,-cli~ er(t~ ~~as

^as

~O',"I~,d,

^'bot,when

, m ;" ~o'"

^were,'dlniOi" " ; d,this did

r' ^~,.

-, Instead.'~i~.Luve~ normally r~om the lumen in~o tbe enterocyte -:~_ut

~C'.('Umulllleswilhin the small intesti nal mucosa(Brunelle,Bochenek,~brabam.

t,

' (:

".

18

Kim andRodg ers,197Q).Itisthe re foreappare ntthat Plur cni eL-SIdoesDot

interrer~

^{with precessesin} the int":inal lumen,...hieh in turn,

iDdil'lt~

^&

int raC'E'lIular"event.

Inordertoinv~tig"lethis,electr onmieroseepic studiesYi~{'employed.

, ' " I

. These.studiesrevealed accumulationof lipid inthyeeteroeyte.OnlyVt>rylow-

~ d ensii~v

lipoprcteie-sieedperue leswere-iranSPofted int ot'oe

ly~ph

^{by tM}

Pl.1i;on i; L-81exposed

~eils.

These studiesshowtbat tbere,

j~

a signitiC'anl

l . . jnhib!~ion. ,J(

intracellulartransp6rt..2!.chylomicron-sizedparticles andaresult ing .blockageoi chylomicron secretionby theenrerocyt e(Tsoet aL,IgBl)

The"lackof

~nhibition ^at

VLDL produ ction suggests tbatseparate pathways exi!!! forehylc micronand VLOLaSsemblyandtransportin thesma lli~I~linal m~eO$aor therat,Ekc~useo!this prererentialinhibi tion~yPluroni cI:r81,this compo undisbeingusedu&tool to invesligatechylom icron andVLOLassemb ly andseereuc e.

"-

Evidencerromothersources supportsthe 5uggt"Slionoratwopathway syst em.IIWMsh~wDthat apalmitateinfusioncausesamarkedincrease inVLOL Iran sportwhereas oleateandlinoleetedidnot. Oleate andIinoleate,c~ost'll^an increased chylomicronout put (Ocknee,Hugb es and lsselbacher,(969).More

~u pportingevidencecamewhen itwas shown that \be ratty acidcompositionof

. .

theVLDL andchylcmicrons differ.Furth er,Mah.ley ,Denn ett,Morre,Gray, T~hwaiteand Lequire (IQ11)showed microgr aphs or separatepopulations-or'

"VtDLandchylomicronsin Golgivesicles, \

\

r '

.'I

(.

, .

. ^.

.. Basedonthis ('video,ee,Tso,Drake,

Bla rk

^andSebeein (HI84)hypothes ised that theintestinallipidtransportwouldnolbe alteredifa

~fUSCd

^that..

st imulated \-1,D[, production

i~

conj unct ionwitbPluj-onieI.r81.It.basbeenshown thatphosphaud ylcholine stimulates VLDL product ionin humans and has litt.le eHectOilchylomicron produ cti on[Bell andGrundy,lQSO). Suchbeing the case Tso and coworkersJIQS4j ~howe~ that the concomitant infusionof egg phospbotid ylcholio eand PluronicL-81 result ed in nochange in transpo rtoflipid>

to the lymph ascomparedtopbcepbatidylchohn einfusionalone.

Itisthereforeapparentthatifchylcmicrou assemblyand"sec: et ionare alteredby this ccmpcubd ,the chylomicronappearancetime.should be lengthened withtheadministrati~nor Pluronic L-81ina lipid emulsiontest meal.

"

\

20

1.2.Object ives

1.To determinethe effect , ifany,of7,12-dimethylben~lalanthraceDe, benio( a )pyrcne,anthr acene, benz anth racene, and cholesterol on chylomicron appearancetime,IfaneffectisObs{'T.ved,todetermine the mecha nismbywhich thecompo undsexerttheeffect.

J

2.Toassesstheefficiency ofthe o-glyeerophosphate'pathwayintriglyceride resynth esis intheabsence ofmonoglycetideinthe diet

._'"'1- '"

3.Todetermine theeffect. if any.of a Pluronic poly~ (L.8l) on chy[o:ni~ ronappearancetime.

<,

.\

"

Ohapter 2

MATERIALS AND METHODS'

2.1.Animals

MaleSprague- Dawley rat sweighing between2i 5-3ipg werepurchasedfrom Canad ianHybridFarms(Nova Scotia, Cana da ) and were main t ain edunder standa rd ligh tand tempe rst urecondit ions(12 hourphotoperi od ,74•Fiand 40%

humidity}. The rats werehoused, inShoe-Boxcages; plasticrect angularcages withwir~bar.deta c,ha ble covers (3 rats / cag e)withls awduslbedding.Free access

t..?rood IPturina Rat Chow,RalstonPurina Compa ny) and tap'w~ter ^was perm itted.

2.2.Su rgery

2.2. 1. Anaesthesia

Diethvlct her vapour used

"

to indu ce and maintain-anaesth es ia.

Anaesthc1'iawasind ucedbyplacingtheratin a glass jarconta iningeth er-soa ked

. ,

cottonswa bs.Thejarwas covered withahea vyplexiglaslid., Theratwas remove d once anesthesi a"Y33achieved (1:2 minut es ].Anaesthe siawasma intai ned

~Ilringsurge:ybymeans .of a maskconsisti ngora 150mL beak erconta inini ethersoakedgau ze.The mask wasrem ovedor replacedasrequir edwithcare beingtake " tomo nitortheratforsig nsof respiratorydistres s.

'pyloru s (F igure2.1),'

22 2;2.2.Sur ge ry

Therat

w?S

placedin a dorsa l recumbent positionwith eachlimbsecured.

Th E' abdo men was sha vedwith anelec tric shave r and a left sub-cost alincisionwas madein the abdomen.Bleed ingfromtheincision line was usua llyminimalbutif necess ary,hemosta-iswas.achievedby applying gen tle-pressur ewit h gauze.The incisionwas heldope n by aweiueoerret r actor

2.2 .3.Duode n um can nu la ti on

Poly ethy lene tubing (pEno.Clay,Adams'i !=1.2i mm:rl?= o.86 10m)

"measuri n g45em in iengthwas introdu cedinto .theduod enumthrough a small

r

punctu reontheventralsurfaceofthestomachapproximate ly'Iemfrom'the .

/ '

^,

The tube-waspassed inJotheduodenumfo rapproxi mately2ern.and wassecured by apurse-stri n g,sut ur eof4-0silk,The tubingwas exte riorizedthrougha stab wo u nd in- theright flankoftheabdomen.Approxima telyone mLof O.Qr:;;NaGI sali ne wasinfusedthroughthecannulainto(he duod enu mtoens urefree passa ge of mal crial intothe duodenum andtoch eck forleakagearoundthepurse-string suture,

2.2.4. T'hoeacl eIYI:?Ph due tcann ul a tion

Thora cic lymphduct e;nnulationwasperformed based onthemet hod of Bo llman,Cain and Grind ley(lg·18 ). Theintestinalorgans.were reflect edtothe righ t andcovered witha warm sa line-soakedga!lze ."Approxima t ely2emof the thoracicI)'mphductwas'dissectedfrom the aortaandthesurro u ndingtissueby blu ntdissection.A stra ndof4· 0silkwas passed aro und thevessel'asfarcrania l

. '

Figure2·1:

Placemen~ or

the duod enumand th.Ol~dclymphdod cann ulae.

.. _~4

..

-s E .

~

. ^'

-as possible and tiedtooccludethevessel. A nickwesmadein the lymph-duet

25

I

•

caudal to the ligat ure and heparin ized'polyerbyleee tubing m....lSuring

'a pproximately"Semin lengthfPE 00,ClayAdams,OD= 1.27

1m;

ID=O.86

mm] was insertedand heldin placebya droporcyanoacrylate glue

~Kr'azy

^Glue)

(figure2.1).Thetubi:llN&S~riorizedihrougbastabwound in tiieabdominal wall.Gentlesuctionf.nthecaoDula ohenh~lpedto establish aconsta ntlym ph' Ilcw.

. :

~J09Ure

^{- ,:}

The abdominal?rganswere returned tothp ir properposit ions The abdominalmusclelayenwere closed using4-0silk in asirtpleinterruptedsuture patternand the skinwas closed with 7.5 mmMichaelsurglcal"c1ips.

2.2. 6.Post-oper ative'housing

Theratwas placediiia Bollman-ty perestraintcageimmed iatelyafter' .surgery,priorto"recoveryfrom anaest hesia.Thisprovidedsu ffiei~ntandhuman e

immobilization topreventac~esstorbecannulae.Aphysiologicalsalin~solution (0.9%NaC l1ront aining 5%glucose was infusedintotheduodenumcannulaat a rate or2.-1mL/hrror the 2thrrecoveryper lod.Food andwa~were~b:ld.

Therestrainedratswe!e keptat aeonstantt~mflerll.t U reor74•Fwitha12hour photoperiod.

I

l

Rats thatdid not exhibit9.

~nstant

^lymph

f1o~

^onthefirst post.op'erative·

'> •

2.2.7.Sacrifice

26

. . '

day wereremov edfromthe study AJItats weresacr ificedby anoverdose

o r

sodiump<,nloharbital[Euthanyl).

. ^.

2.3.Te st!meals an dIympncollecti o ns

.~ .

2.3.1.Tes tMealCom p osi.ti o ns(Table2.1) 2.3.1. 1.Controltestmeal

A lipidemulsion testmeal wasintrodu ced intotile duode num via the duoden al cannula.The testmealconsist edof 40mMoleic acid(Siftma)la belled with15 mCi/molll.14

q

oleic acid (Amersharnl,20 m.\lI-monccl ein~"d28.ftmM•

. . ----

Z~dium tl}u ro('~olate in 0.5

mL

phosphate-buffered solut ion. Solven. were

. .

evaporated off todry nessunderII.light streamornitrogengas.The phosphate-

• l"

bufferedsolutionwas composed of

e.rs

mMNa.zHP 0₄;16.5 mMNaH2PO~;ll~

mM NaCIand 5~MKCI(pH6.4). Emulsificationoftho.:testmealwas achieved byvortexin g and by10 minute sonicationin aColePar mer sonicat ingbath.The temperatureofthe

._,

testmealremained below34•C afte rsonication

^..

.An aliquot of~stmeal wasremo'Vedfor scintillatio nco unting

2.3.1. 2.Poly cy c lic aro ma tichydr ocarbo,"!testmea l

Allpolycyclicaroma tichydrocarbons were of thehighestpur ity available andwereused wit houtfurtherpurification. The test meal described in sect ion 2.3.1.1. was

su pple~ented

^{with 1Orru\1\ of.}

eit~er

henzb(a)pyr@ne, 1,2·

benzanthracene, 'oran thra~ne(Sigma ChemicalsCbmpany).The PAllswere- ' dissolvedin anorga nicsolventpriortobeingadded to thetestmeal.As above,'.

!

\

' /

( \

^"

~

. ~- ~

I . ~

"<

:i'

' /

27

~ ~ ~

:Ou

I.

~ ! ..

^:!!'O

^{" .}

re o

-~

~ r~ h

:Oi!i

~ ^~

~ ~~

^~

:e

:!:C;

;a ' ~ ~

~ ~ · ~~ i ~~

:Ou

J

~ _~·Gi

~ - ~

:!:·o

- .

28

thesolvents wereevapot~odrynessunder.IL gentle strea mofnit rogen'.The structuresofthecompo undsusedi~thisst udyIl~SbPWDin Figure2.2 2.3.1.3.3H_DMBA

Todetermine the appeara: ce ofthefirst~MBAmolec ulesintbelymph,10 mM DMBA(Sigm a )labelledwith6Cijmol3

n:m•

1BA(Amers ham)was combined

{

withtheles t meal described in sectio n2.3.1.1. The oleicacidlabelwas increase d to15'0inCijmolfor thisexperiment.Thetestmealwasma de intoanemulsion and admin iste r ~d vjathe duodenalcannula. An aliquot was remov ed for scintillatio n counting .

2.3 .1. 4.Ch olesterol

10 mMcholesterol(QQ+%pure,SigmaChem icalCompany)was add edto thetestmenldescribedin section 3.3.1.1.

rs-:

^testmealwas made into an emu lsionandadminist eredas abolusdose viathe duodenumcannula . 2.3 .1.5.PluronicL-lh

The hydrophobicsurfac tant,PlutonicIrSl(HASf' Wy andott e,Wyand ott e, MI.),wasadded tothe testmealdescr ibed insection.2.3.1.1.at aconce nt ra tio nof 0.25mgJmL.the test mealwas thenl'nlU lsifi~dand analiquotwas take nfor scintillationcounti ng.

2.3.1 .8. I>- G lycero p hosp h ate pathwayefficien cy

two dlfferen t test meals were used to test the efficien cy of the e-glycerop hosphate pathway . Atestffi\ealwaspre paredas

o f med

insection .2,3.1.1.exceptthat 2·monoolein wasusedin place of t-moncclein. Anothertest meat-w~^{pfepar_:dasdescr ibedinsection2,3.1.1.}except that the'!-.ffionoolein wasomitte dfr~m^theprepuratjon le-avingthetest mealwit hnomon oglyccrid e

(

Figure 2·2: Pt'l)'C'yel "icaromatichydrocarbons

usedin this st u dy• < )

'.i 'i '.

..

.. , .. \ .

. .J

. . .. . :: ;. . '~ ~

30

. ''? '.' \'

';' .

1.2-BENZANTHRA CE':NE

\ 1

~. . "1

r

I

.J 7.12-DIMETHYLBENZANT~A~EN~ '

^r

~ . ' . . ~

BE~ZOla)py~ENE 0 .

\

'r

. '

. ~

. , J - '

JI ~

....

!~ f\

, .

~ ^I ~ S ' ¹

l~ J~' . ~~'

.. _{o '}

~

g

~ ^~ ~ ^~~

! ^{1 .\ ~ ~ i 'i~ ~}

^~o

^~

^.;

a

" ^~ _{~! ~ ~ ~} ^~

^~u

^{i '" " ~ ii}

_-=

~

~ ~] ~~ _~

i§ . ,

_~o

o~

. ~

\ ^~

]

~ _~ l ^\. 1 ~ ^{~ . e ~} _~ ^{!~ ."} - ^I ! ^t

~.

~ ~~ , ^f

.~

~

^~-

~

_i'!

^a '\;

^{-~ .;~}

l ^"

"

-15 _~ f

_,3^~

~ ! ^:li _~ f ^!~ _~~

'I

^':!:'O

I i

:,;'

.

~

~

N

~ :t. e~

~ ! ^{~.~ 51 .! ~,} M " ~

.---...' _{. ;.~}

32

Theoleicacidlabelwas increasedto150mei/m olfor this exp eriment.Allaliquot or scin t illationcounting.

ealadminist ratio n

D,tswereper formed00theIir stPjt-OPE.' rativ:day.Attim",ZNO, the glucosein fusionwas brieflyinterruptedandthe test mea!w~administere das apulse through theduodenumcan nu la. Testmeals were adminis t ered directly intotbeduoden~m^{to avoid}vari~tio ns^j'ngMf'ricerpptying. 0.5

m C

physiological saline wasusedt~^c'learthecann~la^{thereb yensu r ingtb;testmeal hadbeen} de.liv ered

into~uOdenum.

^{The rafwas immedi:telYrei urned to the 5%}

glucos e-salin e

~inr\lsion

^{at arateof 2.4mL/br.}

2.3 .2. 1.Plu ronlc L-81

Testme aladmi nistrationwas ca rr iedout asabo eawith oneexce ptio n. Two 'bours priorto the testmealad mjnistrat~nthe 5% glucose-sali nesolution infusi on wasre placedwith a 5% glucose-sali nesolutio nconta iningeith er28.5 mM sod iu m tauroebclate inthecontrolanimalsOf28.5mM sodiu mtaurocholateand0.25 mg/m L Plur oni c L-81intheerpeei mentalan imals.Theeatsremain ed on this modifiedinfus ion overthe ent ireexper imentalperiod.

Forthisset ofexperiments, ther; teoflerusic oofthe~%glucose-saline solut io ns[includingthemodifiedsolut ions) was.16~/h r,

(

I ·'·

2.3.3.Lymph&. m ple eollectlo n

Lymph

~as

collected inpreweigbed,test

tUb~. T o a~oid d~tting.

^0.008

^ln

^g

hepa rinwss added to each tub" priorto weiw;hing .Lymph wa,S collect ed cont inuouslyover the uperirnentat iootimeatdesignate<! in tervals.'Lymph

sam pleswere eclleetedinaD'automatictract ioll collector(LKB 7000Ultu.rac)

.thusrl."Su~gin few"r disturba nces fortherats.

I

Testlubes contai ninglymph werereweighedtodetermin elymphweight.

t:sing1\.'spl'cificdensityof1.00,thelymphvolu me was_cal~latedtobe eq uivalent tothelymph~l.'igh tper unit time(Barrowman,IQ66l.

2.3.4. Liquidlu:ln t lll at ioncounting

lOOpLof.lympb wascombined with10mLofliquidscintilla tioncock tailfor direct scintillation countingbyaBeek man LS 8100scin~il~ncoun t er.Each

.

samplewascou nt~,r0r10 min utes or tothe2%two sigma error.

The lymph sampleswere analysed"todete rmine.the proportionofoleic acid andOro-lEAin the chylomicron Iracticn oftbe lymph.·Toisolatethechylomicro n lrect ion. pooled lymph was diluted 1:1 with O.Q% NaCl solution.

Ultrac entriruga.tionat30,000rp mfor30minutes separated the mixtu re i.nooan opaque supern atant containing thecbylomicraanda c1ear)n£ranatantphase.The

'opaquephasewas separatedan d 100,..Lof theclearinfranata n t wastakenf~r

scintillationcounting. By compa.ringthecpmof.the whole lympha.nd tba t orthe infranat ant, the percentage orlabel inthe chylomicron fraction can be

J

34 2.0(.Experimentalprocedures

2....1.Cb110mlcronappearance timedeter mlaat lo n

Todetermieethecbylom icr·)11 appearan celimerbeco~trol

iest

mealwas administered viatbe duodenu mcaDDula. Priorto espeeimeetenoc.lymph

sampleswere~olll'ttedat 2minut~intervalsfor a period of 8 mill,After test meal administration.lymph sampleswereeollee ted tor30minu tes at 2minute intervals,and at30min uteintervalstorthenext150minutes.

2....2.ElTect.of PAHson chylomicronappearancetim e e

In anima ls givena.PAH testmeal,Iym pbsan.les werecollectedat 2 minutesintervals

r or 6~ minut.es ~llowed

^{.bY 30minutesinterval}eolleet icnsfor thenextIZOmio•.

2....3.Doseeffectof DMBA

The

t~t

^meal

descJb~

^{in section2.4,2 .above}containing D!l.lBA

\li..,

modified'Ouch thattheconcentrationofD~ffiAgivl'Uwasonetenthofthe original.

. . .... . .

meal,S~mpleswere .collectedasaboveandtbechylomicronappe arancetimewas determ ined,

2."-.4.ElTeet

or

otherh)'dr ophobic·xen obi oUe.on ehylomkron appea.r aneetime

In ratsgiven acholesterol-supplemented testmeal,lymph sam pleswere inkenat2min u te interva lsfer60minu tesandat.3Dminuteinte r valsCorthenext, 120 rninutes.

. .

In'ratsgiven the.P\'uM)D icLo8t

test~eal.

^{thoraciclym phsampleswere}

takenat 2minu te

interv~~'lo;

^{60minu tes, '}

,

^.,~'

~.', "

35

2.4. 6. En zy meIndu ctio nexperi ment

Tworandomly chosen groups ofrats wereused in this experiment.Cro u p A was given

20~A

^disWlved in 0.5mL etbanoi'as a bolus ·dose approximate ly5 ho urspriortotest mealadministration.GroupB wasgiven0.5 mLethanolasa.bdlusdose atthe same timeastbeprevio usgroup.Alterthe Drvm~^tes\meal was administered tobotJ1groups,lympoqsamples were collected at 2 minut ei~tervalsforone~urand\t30minuteinter valsforthenext 2 hours Aliquotsweretakenfor scin tillationcount ing. Animalswerereturn edtothe5%

glucosesalin e infusion immed iately after administration of botbthein d uction dose and testmeal.

2.4.6.Trig lyceri deFluxafteradmi nis t ration ofateet mealcon t aining DMBA

In thisexperiment the totaltriglycer ide nuxovera 3 hourperiod was determined in rats giveneit hera cont rol tcstmealoronecontainin g DMBA;0.5 ml,ofthecontrolor Dl\1BA testmeal was administered as a bolusdose.·L y mph sam p les were collectedat15m:nuteinten:al s oyerthe 3 bourper iod..Two15 min u tecollect ions were taken priorto administrationofthe dose.

The triglycerid ein pmol/Lw~quantiriedby aIN-test usingan enzym atic process on aHitaohi705AutomaticAn'\i yzer (e.v.,3%)as describedbelow.

triglyeeridee

+

3HzO~glycerol

+

3ReOOH

\

)

3.

Il;l:ycerol+~TPglycerolkinD~eglycerol.J.pho sp hate,+ ADP

ADP+phospho enolpy ruv a tepyr~v<l'eki~<I~1!pyruva t e+ATP

py ruv ate

+

NADH+

n+ !::!!!!

lactat.e+-NA D+

Triglyceride concen t rations were co n verted to totallr iglyceride perunit timeend theflux everthe 3 hourperiodwascalcul~ted.

2.4.1.Ch y lomic ro n appearancetim ein ratagive n8.tes tmea)with out monoglyeer-Ideor with2-~onoglyceride

The chylomicr on appea rancetime was determinedforTa '.sgiven thetest meal witho ut monog lyceri.1!or with 2-monoglyceride descr ibedIIIsection 2.3,1.5.

Lym phsamples weretakenat2 minuteinterv alsfor one hourand at30 minute interva ls forthe next2bou~s.

'2.4.8.Triglycer id e nuxaft eradministrat ion ofates t meal wit hout

mo no glyee ri qe

.'

-"----

n,.

efficiencyby. whichthee-glycerc phosphate pathw ay ca

•

n resynth esize .triglyceridecanbe furtherdeterminedby

eXamin~

^the

triglycerid~

^{flux overa3}

hourperiod . For thisexpe riment,'t hreegro upsof ratswererandomlychosen.

\

:'t: ·.

37 ^.~

\

\

GroupA receivedacont rol test meal. ,describedinsect ion2.3.1.1.

and~uP B

r~eiveda testme alwithou t.monoglyceritle asdescri bedin section2.3.1.5. Group Cr: ceived a test mealcon taining2. m~mogl)"ceride .Lymph samp leswer~taken a.t 4minute interva lsIoronehourand~t30minuteinterva lsfor thenext2hours an danaly sedlortr iglycer ide on'the Hitachi705Automati c Analyzer.

2.4.9 ,Statistics

Allchylomicron"appearancetimes ar eexpressedasmean

±

standar derror of themean(SE)

Ch y lo micronappea rancetim esare compa re dusingStudent's T test ata.

sjguificnncelevel

or

p

<

0.01 uJess other wisenoted.

38

Chapter '3 ) J RESULTS

Cannul a Dead Space Tim.=cannula~

averagelymphflo. per min

\

.- j

. .

3.1.Effect of polyc yclicaroma t ic hydrocarbons jn lipid ab s or pti o n and han4ling

3.1.i.

cfomb~'

^appeaeanee",;,ea (Ta ble 3- 1)

3.LI.l _Ce nt r e!.•

h11~m"ro

^{• •}

~p

^{. .ra nee}

Um~

Inorder to deter minethe true app eara nce time oftheradiolabelin the

lymph,itwas nec-essaryto cor rectlor thetb~r a('iclymp h duct cannu ladead spare

TbI G:",,,.",I,t 'd

^{hom thelymphnowandcannulavolumeasshown;0 ' \}

the followingform/ulae,

,

This{eldspace timewasthen su btractedfromthe observed

ap~e·aran('e·

timetode terminethe trueappea rancetime;

The chylomicronappeara ncetime as measuredbY'

I~C

^{appearance in the}

thoraciclymphductwas10.81

±

1.03 minutes as showninTable

?-l.

Th isvalue

~presentsthe~on trol tPpearancetime.

, "

r: _r

39

§

~ ~ 'Q 0 ^'Q

fI

^{~ ~}

'e-

Il

~ t

~

~

⁰

f"

r~:::::

. ^..

^{E e}

.

^E

1; ^~+I

5 -

⁰

^::; ~ ^{g; .;}

~ ^ill

^{~. ~.}

~ ^~l l

^{-~ ,~}

⁹

^{ee ,}

^-, - ^{- , ., ~ ., ; l(j -,}

.,;;

.:;:

i

^~u.

~ ~

~ i .

~ r

lj

I ^j

~

^u

s

^~~ ^~

~ ^e ~

i ^~

~ ~ ^{! ; ~}

^{~ ~~}

s . ~

~

^§ . f .

'" ^!,(

^~~

! i ^~

^{Q 0}

!

^;;

^.:; ~

^{~ ~}

--..

..1

-- ,

-Ii_

I

40

~.1. 1.2.Pol YC1clicaromati~hyd rocarbons(T able 3· 1,Figure 3- 1)· The chy lomicronappearancetim esin those rats givenatest mealcontain ing eitheranthracene,1,2- henzanthracene,01'"ben zo(a)pyrene

~re

^{'showninTable.3-1%}

Nonecrtbesevaluesdiffersign ificantlyfromthatofthecontrol.

3.1. 1.3.3H_DMBA

Uponadd itionoftheDMS..\.tothe test'rneal,the chylo micronap pearanc e timewas23.08

±

2.10minutes (Figu r e3-2).

• The ap pearanc e time of DMBA inthelymph(20.4

±

1.98minutes)coincides withthe"chyl o micron appearan cetim e(Figure3-3)..

I -

Thechylomicronappearance timein tbis experimen t was signitieaurly differen tfromtbechylo micronappeg ranca tim eofthecontrolgroup 3.1.1.4.LoW'doseDM BA

When theeouc e nrrattonorDl\1BAadd edtothetestmealwasreduced to onete nththe concentration insection3.1.1.3.,nochangewasobservedinthe chylo micronappeara nce timeascom p a redtothecontrol(Figure3--4).Here,the chylo mic ron appearance time was1l.28

±

O.Sg minute s.

3.1.~.Trig ly ee rldenux

Thethre ebourtriglyceride(luxinanima.lsgivenacontroltestmeal orone containing D1\1BAis shown inFigure3-5.

... •

Thebaselinetriglycerideoutput wascalculate d and subtract ed fromthe total output to determine'the triglyceride output-due to the test mea l Multivariate an;lysis (AJ'iOYA)waS used to com pa re the two groups.NC'

'.',

' .

"

Figure3-1: Chltomleron AppearanceTimein

• ~ a!lim ,lagiv ena control . or PAlltestmeal :acbbarrepresen tstbemean

t±

SEj.

./

18

16 11.

12

VJ UJ

10

~ z 8

.. . ~ ~ ⁶

I.

2

\ 0

"

~ .

r+ n-

, r+

~

CONTROL A 1.2-BA . B(olP

n=8 n=8 n=8 n=8