Intestinal apoproteins during fat absorption

Intestinal apoproteins during fat absorption.

G Schonfeld, … , E Bell, D H Alpers

J Clin Invest.

1978;

61(6)

:1539-1550.

https://doi.org/10.1172/JCI109074

.

To compare the roles of apolipoprotein (Apo) A-I, B, and E (or arginine-rich apoprotein,

ARP) in the intracellular production of intestinal chylomicrons (and/or VLDL), these

apoproteins were localized in rat intestinal mucosa by the light microscope method of

indirect immunofluorescence. In addition, tissue levels of ApoA-I and ApoB were measured

during fat absorption by radioimmunoassay. Antisera were produced using ApoA-I isolated

from rat plasma high density lipoprotein, and ApoB and ARP from plasma VLDL by column

chromatography. The apoproteins yielded single bands on polyacrylamide disc gel

electrophoresis in urea and in sodium dodecyl sulfate. Anti-apoprotein antisera were

produced in rabbits. These antisera appeared to be monospecific on double-antibody

immunoprecipitation of 125I-labeled apoproteins. In fasted animals granular staining of

ApoA-I was noted in the supranuclear (Golgi) regions of epithelial cells in the top third of the

villus. At 30 min, when fat droplets were seen in the supranuclear cytoplasm of the cells

along the top two-thirds of the villus, intense ApoA-I staining surrounded droplets in the

cytoplasm. At later times when epithelial cells and lamina propria both contained fat

droplets, bright ApoA-I stain surrounded many droplets in the supranuclear cytoplasm of

cells and in the lamina propria. Over the same period of time, tissue levels of ApoA-I rose

10-fold. The distribution and time-course of ApoB staining was nearly identical with that […]

Research Article

Find the latest version:

Intestinal Apoproteins during Fat Absorption

GUSTAV SCHONFELD, ELLIOTT BELL, and DAVID H. ALPERS, Lipid Research Center

(mtid Departments of Preventitie Mledicinie,

Medicine,

and

Pathologry,

WVa.sh

ington

University/

Schtool of

Medicine,

St. Lonis, Missotnri 63110

A B

S

TRA

C

T To

com)1pare

the roles

of

apolipoproteini

(Apo)

A-I, B,

anid

E

(or

argininie-rich

apoprotein,

ARP)

in

the initracellular

prodtuctioin

of

intestinial

chylomi-crons

(and/or V7LDL), these

apoproteins xvere

localized

in

rat

intestiiail miucosa

1y

the light microscope method

of'

indirect

imimutiniofluorescenice.

In

addition,

tisstue

levels of ApoA-I

aindl ApoB were measuired duiring f:at

absorption b) radioinmmunoas sa'.

Antisera were

produced uising ApoA-I isolated froim

rat

plasma high

density

lipoprotein,

and ApoB

and

ARP

from

plasmcla VLDL

bv coltunii

chromatography. The

apoproteins

yielded

sinigle banids

on1 polyacrvlamide

disc

gel electrophoresis

in

turea and

in

sodiumin

dodecyl

stulf'ate. Anti-apoproteini

antisera were

prodtuced

in

rab-bits. These

anitisera

appeared

to

be

moniospecific

on

double-antibody immiiuntoprecipitation

of

'251-labeled

apoproteins.

In

fasted animiials granular staining of'

ApoA-I

wvas noted

in

the

supraniuclear

(Golgi)

regionis

of

epithelial cells

in

the

top

third of the villus.

At 30

mmin,

when f:at

droplets

were seen in

the

supranuclear

cytoplasm

of

the cells

along

the top two-thirds of' the

villus,

intenise

ApoA-I

staining

surrouniided

droplets

in

the

cytoplasml.

At

later times

when epithelial cells and

lamina propria both containied f'at droplets, bright

ApoA-I stain

sturrouinded maniy

droplets

in

the

supra-nticlear

cytoplasm of cells and

in

the

laminca

propria. Over

the

samiie period of time,

tisstue

levels of ApoA-I

rose

10-fold. The

distribution

anid time-coturse

of

ApoB

staininlg

wvas nearly identical xvith that of ApoA-I.

Con-comitantly, tisstue

ApoB levels doubled.

By

contrast,

in

ftastinig

rat

intestinie, staininlg

of

ARP

wvas sparse,

ptunctate, andl

confined

to

the

lowver

(qtuarter

of

the

vil-lts. After fat

feedinig,

stained droplets were

seeni

only

in

the

laiminla

propria

near the base of' the villus

even

thouigh abundant

ARP

wvas found

in

cells along

most

of this

length of the villus.

Stain was

never

seeni

to

surrouniid

any

droplets inside cells.

Thuis,

ApoA-I

ancd

ApoB

appeared

to

participate

in

the

intracelltular

assemiiply

of' lipoproteins

in

gut,

whereas

ARP

did not,

althouigh

ARP was

found

wvithinl

mutcosal

cells.

Reccived for publication 14

jily

1977 and in revised

form

16Jat1n

aril 1978.

Liver

and

intestine

differed

in

their

stainable

con-tents of ApoA-I and

ARP.

Wlhereas

intestine stainied

heavily for ApoA-I and lightlyr for ARP, liver stained

heavily for ARP and lightly

for ApoA-I. Both organs

stainied

for

ApoB. These findings stuggest

that there ma'

be

some

(quantitative "specialization"

of the two

organis which secrete lipoproteins.

INTRODUCTION

Dietary fat

is

absorbed

across the

intestinal epithelial

cell

of

mammllals

via

clhvlomiiicronis and

very lov densitv

lipoproteins (V'LDL)' (1).

Several

apoproteins

have

been identified

in

chylomicrons isolated from the

in-testinial lvmph of mani and

rat

(2-4).

Amnonig these

are:

(a) ApoA-I (5), which

is

the major apoprotei

n

(Apo)

of

plasmiia high density lipoprotein (6, 7) and

is an

ac-tivator of

lecithiin

cholesterol acyltrainsferase (8, 9);

(b)

ApoB

(4), the major protein

of

plasma low

density

lipo-protein

(LDL)

and

a

major

protein

of'

ApoVLDL (6,

7),

which

appears to

be

essenitial

for lipoprotein foirmatio

n iin

the

liver and guit (10, 11); (c) arginine-rich protein

(ARP or ApoE) (12)

which,

in

rat, is

found

in

all

plasma

lipoproteins (6, 7) aind levels of which appear

to in-crease in

VLDL

and

LDL in

response

to

intakes of

high f:at, high cholesterol diets

in

several animal

spe-cies

(13-15); and

(d)

ApoC, which

consists

of

three

proteinis

that

moduilate

the activities of'

lipoprotein

li-pase

(16, 17) and of

lecithin

cholesterol acyltransferase

(9).

It is

suspected that intestinal

VLDL

have similar

apoproteins, but more studies

are

needed.

These apoproteins have been identified by

electro-phoretic and

immuinologic

techniiqes

in rat

lipopro-teins

(.5-7, 12, 18,

19).

The

electrophoretic

patterns

of ch lomicron apoproteins isolated from intestinal

lyZmph greatly

resemble

those of

analogouis

particles

isolated from the

Golgi apparatus of

intestinal

mucosa

(20).

In

addition,

while

this work was _{in progress,}

Glickman et al. (21, 22), have localized ApoA-I

anid

I_{Abbrcriatiotis nsedin thiispapcr: Apo, apoprotein; ARP,}

arginine-rich apoprotein;LDL,low.density

lipoproteini;

SDS,

socliuil

dodecvl

sulfate;

V'LDL,

very low density lipo-protein1.

ApoB in isolated gut epithelial cells of the rat and

dem-onstrated that

apoprotein

localization

is

altered during

fat absorption.

ARP

and

ApoC

have

not

been similarly

studied. These findings

suggest

that

at

least

the

ApoA-I

and ApoB of lymph lipoproteins

are

ac(uired before

these

particles exit from the gut epithelial cell.

During fat absorption, radioactive amino acids are

incorporated into several moieties

of

lymphatic

lipo-proteins (22-24). Although the electrophoretic

meth-ods used

in

the

elegant earlier studies do

not

allow

for

une(fuivocal

identification

of

each individual

apo-protein, recent findings (22) do suggest that ApoA-I

and

some of

the

other

apoproteins ac(uired

during

chy-lomicron and/or

VLDL

production

are

newly

synthe-sized.

We wished to compare the involvemiient of three

apo-proteins, ApoA-I,

ApoB,

anid

ARP, in

the

formation of

intracellular lipid packaged

for export. The

light

micrio-scope

method of indirect

immunofluoreseenee,

applied

to

frozeni sections of rat small

intestine,

comnbined with

tissue meassurements

of'ApoA-I

and ApoB have allowed

us to follow the behaviors

of apoproteins in

the

intesti-nal

epithelial cell and along the length

of the villus

during f;at absorption, and

to

distinguish between the

roles of' ApoA-I and ApoB on the one handl, aind the

role of

ARP

on

the other. Part of' this work has been

presented (25).

METHODS

Male Wistar rats weighing 250-300 g were miiainitainied on Purina Rat Chow (Ralston

Purinia

Co., St.

Louiis,

Mo.)

andl

tap water ad lib. After overnight f;asting, the animlals were

giveni 1.5 ml of corn oil by gastric tube withouit aniesthesia. At time intervals thereafter the anlimals wvere anesthetizedl with ether andsamples ofproximaljejunutmlil were taken for staining with oil red0and for indirectimmu-nofltuoreseence assay. Bloodsamples werealsotaken from the inferior vena cava foranalysisoflipids.Plasmiiatriglyceride and cholesterol levels were measuredby the methodology of the Lipid Re-search Clinies (26).

Specific

proteinis

were detected in tissues by anl indirect immun(ofltuoreseenceassayonfrozenisection substrate.0.5-cim lengthsof intestine taken from animals uinder etheraniesthesia were opened longitudinally, rinsed in ice cold phosphate-bufferedsalinie(0.05 MNaCl,0.04 Msodiumphosphatebuif fer,pH 7.2, isotoniie), (uick-frozen in li(quidnitrogen-cooled Freon (EI. dii Pont deNemours &Co.,

Wilminigton,

Del.)at

-158XC, and stored in screw-cap vials at -70(C uintil used. 4-,um thick frozensectioniswereeut from the tisstue blocks in

amicrotomiie-cryostat; blocks were orientedsothatcross

sec-tions of the intestine were providled. Sections were placed

on microseope slides and air-dried for20 min at room tem-perature. Sections were theni fixed in methanol for4 min at

-20°C, thenacetone for2mIi at -20°C, andagainiair-dried (2 1). Theremiiainiderof theindirectiimmunofluoreseenieeassav was performed as previously dlescribed (27). Rabbitaniti-rat

apoproteini

anitisera (see belov) and the fluoreseeini-conijul-gatecd IgG fraction of' goat anti-rabbit IgG (heavy and light chain specific; Meloy Laboratories Inc., Springfield, Va.) wereboth

used

atdilutions of 1: 100. Diltutions wvere freshly prepared inphosphate-blufferedsaline.

Controls conisisted of'substrate ineubated on glass

micro-scope slides as follows: (a) with buffer tlone, i.e. without

eitherrabbit anti-ratapoproteiniantiserulmorfluioreseein-goat anti-rabbit IgG conjugate;(b) with conjulgate alone;(c) with nonimmune rabbit serum (at same dilutionas immune sera) andconjugate; (d) with immunesera directed against irrele-vantanitigens(e.g.humiicancellmembranes)and conjugate; and (e) with specific anti-rat apoprotein antisera absorbed with theappropriateapoproteinantigens. Adjacent4-gmthick

sec-tion-s were stained1 with hemllatoxylini and eosin, and oil red

0,au(I examiiinedtoassist inthe identification of tissuie struic-tturesand to follow the progress off;at absorption.

Twoorthree sectionisateach time point and for each apo-proteiii were

independently

examinied

by two different ob-servers. The entire area of eachcrosssectioni ofintestiniewas

suirveye(l.

About 60% of the

gradinigs

(0-5) were identical between the observers. Thegrading difteredby 1rankorless

over95%ofthe time. Where differences were observed the m11eanii ranikinigwastise(d.

The anitisera uise(dintheimmuniitiiiofltuorescenec stutdieswere

obtained by usinig antigens isolated as follows: ApoA-I vas isolated

froml

rat

plasmna

high density lipoprotein (d

1.070-1.19)by colullln

chromiiatographlv

as previously describe(d (6, 28). This material

yielded

a single band on sodiumii dodecyl stulf'ate (SDS) polyacrylanide gel electrophoresis (28). ARP

was isolated bycolumnclhromiiatographlyfrom ratVLDL

uisinig

the imiethod ofKoga etal. (6) initially,anid laterthe methlod of Marshi (29). In miiost inistaniees a single pass throuighl the SephadexG-200(6)orthe Bio-GelA 1.5(29)coluimn(Bio-Rad Laboratories,

Richmiionid,

Calif.)yieldedasinglebandon SDS

polvacrylamide gel electrophoresis (Fig. 1),however,in

somlle

prep)arations

fromii thebSeplhadlexG-20()coltumnn a

smlllal,

miiore

ral)idlyv migratinig

bandwasnoted.Inthelatterinstaniees,ARP wasreelromatographedontheSepha(lexG-200 column

yield-ing

atsinigle bandonSD)Sgelelectroplhoresiswith ani apparent

olectular

weight

of 35,000 (30).Twvoanitigeniie

preparationls

were ise( to

produice

anti-ApoB aniitisera. First, LDL was

isolateld

fromiirat

plasllna

betweenthe(lensitiesof 1.025-1.050 ib two

centriftogation

s

ait eatch (lensity.

These

prepIarationis

)r'medl

only

onie

precipitin

linle

with

ainlti-rat

serumiii on

in-mnoelectrophloresis

and

immunllvilodliffutision.

LDLwasutsed(as

immuniiiiiiogeni directly.Trhe anitisera obtained arecalle(d "aniti-LDL" anitisera (31). In addition,ApoB was isolate(d fromii rat

VLDL by

coluimni

clhromiiatographly on Sephadex G-200 (6). The

malterial

inthe ApoB-containing

peak

(peakI)(6)did niot

enter SDS oruirea gels (Fig. 1) (32, 33) (wlhereas (I 1.025-1.050 LDL(lid

conitiai siiaill aimiouints

of material wiih

mi-grate(d

inthe

argininle-r-icih

anid

ApoC

regions

oftheSDS

gels).

The antiseraproduicedwith ApoBarecalledl "anti-ApoB"

aliti-sera.Anitisera were

p)rodliced

in rabbits (28).

The specificities ofantiserawereevaltaitedby

double-aniti-body immintioprecipitationi ofvarious test, radiolabeled apo-proteins (28). We used either rabbit anti-rat apoprotein aniti-seraandagoatanti-rabbitIgGantiserumn,ortherespectiveIgG fractions isolatedl

froml

the rabbit anid goat anitisera by (NH4)2SO4 precipitation followed by DEAE chromatography (34).To asstire

miaximiuimiii

precipitationi

ofrabbit IgG by goat

antibodies,

rabbitIgGwasiodinatedwith1251and lactoperoxi-dlase (28,35)and purified

by

column

chronmatography

(1.5x30

cmcolttimn,0.05M barbital buffer, 1 mMEDTApH 8.6). 1251-RabbitIgG was

adlde(d

as atracer torabbitantisera or to rabbit IgG

preparations

(- 150,000

cpmpi/mnl),

andincreasing

amiiounlits

of goatanti-rabbitIgG

anitiserum-

orgoat IgG were

addled

tothe

trateer-containiing

rabbit

aniti-rat

antibody

preparations.

Tubes contained 150,ulof

0.05

Mbarbital, 1

mlM

EDTA, pH 8.6,3% bovineserumii albumini (bovine serumilalbuininbarbital), 100() ofrabbit

antiserumil

(final dilution 1:1,000- 1: 16,000) orrabbit IgC

(final conenltrationi

1.5-50

/g/ml, e(quivalenit

to

do0

.0..

₄

A B C D

FIGURE 1 Apoproteinsof high density lipoproteins ApoA-I,

ARP, and ApoB in SDS gel electrophoresis (A-D, respec-tively).

dilutionof 1:10,000 to 1:200oforiginal antiserum), and 100 ,ul ofgoatanti-rabbit IgGantiserum(dilution1:20-1:100)orgoat IgG (70-120

,ug/ml).

Incubations were for 16 h at 4'C. Tubes were centrifuged, precipitates were

"\vashed"

by

resuspeni-sion and recentrifugation in barbital buffer at 4'C, and precipitates were counted in a Packard Autogamma spec-trometer(PackardInstrument Co. Inc., Downers Grove,Ill.). Inthe rabbitIgG/goat IgG system mass ratios l1/20 (rabbit/ goat) yielded maximum precipitation oflabel. In the rabbit antiserum/goat antiserumoptimalratios\vere s 1/50(vol/vol). To assessthe specificities of the rabbit anti-rat apoprotein antisera, assay tubes contained 250 ,ul of bovine sertum albumin-barbital (0.05 Mbarbital pH 8.6, 3% bovine

seru-m

albumin), 100,ul of rabbit antiserum orrabbit IgG

(diltution

1:1,000- 1:4,000 or 4.6

,tg/ml)

and100,ulof the appropriate 1251_ labeledapoprotein (- 15,000 cpm). ApoA-Iwaslabeled vith lactoperoxidase (28, 35). LDL, ApoB, and ARP werelabeled with chloramine-T (36) as modified by us (37). Incuibations with firstantibodywere carried out for2daysat4'C. Goat

anti-rabbit IgG antiserum or goatIgG (50 ,Au) was thenadded in

optimalconcentrations andincubation wascontinued for

an-other 16 h at 4'C. Tubes were

centrifuged

and counted as

described above. "Nonspecific"tubes were includedineach assay.Inthesetubes,appropriate

amouints

ofnonimmune rab-bit serum or nonimmune rabrab-bitIgGweresubstituted for im-mune sera orIgG. Results areexpressedasBo x 100/T, where Bo=theprecipitated counts (minus the counts in the "non-specific" tubes)dividedbyT, the total countsadded(minus the countsinthe "nonspecific" tubes).

Tissue contents of ApoA-I (28) and ApoB (31, 37) were assessedby radioimmunoassay.Mucosal scrapings ofproximal jejunum werehomogenizedinthree volumes of 0.05M bar-bital pH 8.6, 1% TritonX-100andcentrifugedat105,000 g for 30min. Afloating fat cake, a clear midzone"supernate",and a pellet were obtained. Known amounts of'251-LDL and 1251_

ApoA-I wvere placed in separate aliquots of homogenates before centrifugation. More than 95% of each label was re-coveredinthe"supernate"aftercentrifugation.Inthe assays, dilutions of supernates prepared in the assay buffer(bovine serumalbumin-barbital0.1% Triton X-100)displaced 25IApo/I and 1251-LDL in parallel with the appropriate standards. Coefficients of'variation in these assaysaveraged 10%.Thus, the ApoA-I and ApoB contents of' tissuescotuldbe measured withprecision. Results are given as nanograms of apoprotein permilligram of'homogenate protein (38).

RESULTS

Anitisera.

Each

of the

anti-ApoA-I antisera

bound

large

proportions

of the added

1251-ApoA-I

(Tables

Iand

II). Antiserum

R 150-1

and the

IgG

isolated

from it

appeared

toprecipitate

'251-ApoA-I almost exclusively.

R 150-1 was

chosen

for the immunofluoreseence

ex-periments.

Anti-ARP

antibodies bouind 1251-ARP, but

not 1251_

ApoA-I

(Tables

I

and II). However, they did bind large

proportions of

'251-LDL. On

the other hand,

'251-ApoB

vas

bound only minimally,

stuggesting

that

the

anti-ARP antisera

(and

IgG preparations) were

binding

to

'25I-ARP

and

not

the '251-ApoB determinants of

1251_

LDL. Antiserum

R 178-3

vas used

in

the

immunofluo-reseence experiments.

Anti-LDL

antisera contained antibodies which were

able

to

bind both

'251-LDL

and

1251-ApoB and small

TABLE I

Specificities of

Rabbit Anti-Rat

Apoprotein

Antisera

12"I-Labeledantigenprecipitated Antisera 251-ApoA-1 25I-ARP "251-LDL 2'1-ApoB

Anti-ApoA-I

R149-1 86-88 22 12-16

R 150-1 70-85 <5 5-7

R 186-2 85-87 16 11-16

Anti-ARP

R178-2, -3 <5 53-73 47-51 7-11

R 179-1 10 49-55 73-77

Anti-LDL

R134-1, -2, -3 <5 6-23 74-88 41-47

R135-1, -2, -3 <5 20-23 76-81 Anti-ApoB

R176-1, -2, -3, -4 <5 3-9 73-87 65-69 R177-1, -2, -3, -4 <5 4-8 77-83

Specificities were determinedby double-antibody immuno-precipitations using whole antisera from rabbits and goats. Results of two to three experiments are given as ranges of Box 100/T. Final dilutions ofrabbit anti-rat antisera were 1:2,000-1:4,000. Precipitations (percent of added) in the presence of nonimmune rabbit sera (instead of rabbit anti-sera) were as follows:

'251-ApoA-I,

<3%; 1251-ARP, 8%;

25I-ApoB, 9%;

'25I-LDL,

<3%.

TABLE IL

Specificities ofIgG FractionsofRabbit Anti-Rat

ApoproteinAntisera

'25I-Labeledantigenprecipitated IgGfractioni 2'l-ApoA-I 125I-ARP 1251-LDL 2'1-ApoB

Anti-ApoA-I

R 150-1 77-88 <1 3-7 <1

Anti-ARP

R178-3 <1-3 51-67 30-43 <5

Anti-LDL

R134-1 2-3 8-10 80-83 38-42

Anti-ApoB

R 176-1 <1-3 2-3 76-77 63-67

Specificities were determined by double

immunoprecipita-tion using IgG preparations isolated from rabbit and goat antisera. Results of two to four immunoprecipitations are given as ranges of values for Box 100/T. Precipitations of added labels (percent added) when a pool of nonimmune rabbitIgGwassubstituted for specific immune IgG were as follows: 125I-ApoA-I, 1-3%; 1251-ARP, 4-7%; 1251-LDL,4-7%; and 1251-ApoB, 4-5%. Rabbit and goat IgG final

concentra-tions were 4.6and93.1,ug/ml, respectively.

proportions of 1251-ARP but not

'251-ApoA-1.

The

anti-ApoB antisera

had similar

specificities

foir 1251-LDL and 1251-ApoB but they bound virtually no 1251-ARP or 1251_

Apo-I.

R 134-1 and R 176-1 were used.

Apoproteint

localizatiotn.

After the

intragastric

ad-ministration of corn oil, plasma

triglyceride

levels

in-creased

from

25-30to - 250

mg/dl

over

the

6

h

of' the

experiment, whereas cholesterol levels stayed

rela-tively

constant

between

60

and

70

mg/dl

(two

to

three

rats per time

point). Oil red

0 staining

of the

small

intestines

of

these

animals

revealed the following

(not

shown):

in

fasting

rats

virtually

no

staininig

was

de-tected either

in

the

epithelial cells,

or in

the

laminia

propria.

At

30

min,

many

fat

droplets

were seen

par-ticularly

in

the

apical

portions of' epithelial cells,

in-volving

cells

in

the

top

two-thirds

of the villus.

A

slight

amounit

of' staining

was seen at the bases of cells

and

in

the lamina propria.

At 3-6 h both the

apical and

basal

regions

of

epithelial

cells and

the lamina

propria

along the

entire

length

of the villus were

engorged

with fat droplets

(Table

1II).

In

the

f;asted

state,

ApoA-I

granular

fluorescence

was

present

in

the

supranuclear

regions of epithelial cells

in

the

upper

three-fourths

of the villus (Fig.

2A

and

Table

III).

At 30 min,

the

intensity

of staining of' the

apical cytoplasm

increased and fluorescence

sur-rounded droplets

in

both the supranuclear and

inifra-nuclear regions

of

cells

in

the

top

two-thirds of the

vil-lus

(Fig.

2B),

whereas, granular staining appeared

in

cells down

to

the bottom of' the

villus. The lamina

pro-TABLE III

Specific Staining of Apolipoproteins in

Jejunum

duringFatAbsorption

Intensity of'staini

Lamiiinapropria

Periph-Epithelialcellcytoplasm eral

inter- Lacteal

Staini

Timne

Difluse Golgi Grains stitiulm lunmen

h

Oil redO 0 0 0

0.5 2 2

1 4 4

2 5 5

3 4 4

4 5 5

5 2 2

6 2 2

ApoA-I

ApoB 0 0 1 1 2 2 4 4 4 3 4 3 2 5 2 2

0 0 2 0 0 0

0.5 1 4* 0 1* 2*

1 1 4* 0 1* 4*

2 1 5* 0 3* 4*

3 3* 5* 0 3* 4*

4 3* 4* 0 3* 4*

5 3* 4* 0 3* 4*

6 3* 4* 0 3* 4*

0 ₁ 2 0 4 4

0.5 1 3* 0 4* 4*

1 1 3* 0 4* 4*

2 1 3* 0 4* 4*

3 2* 3* 0 4* 4*

4 2* 2* 0 4* 4*

5 2* 2* 0 4* 4*

6 2* 2* 0 4* 4*

ARP 0 0 0 1(1/8) 0 0

0.5 0 0 4(1/4) 0 0

1 0 0 4(1/4) 0 0

2 0 0 4(1/4) 2* 0

3 0 0 4(2/4) 2* 0*

4 0 0 4(3/4) 2* 2*

5 0 0 4(1/4) 2* 2*

6 0 0 4(1/4) 2* 2*

This table

summarizes

the

mean

gradings from three

sep-arate

experiments. ApoA-I,

ApoB, and ARP were stained

and

analyzed

by

indirect

immunofluoreseence

as described in

Methods. Intensity was graded from 0 _{to 5. Diffuse stain}

refers to

smilooth

staining throughout the apical

cytoplasmii

of cells in the upper

three-fourths

of the villus. Golgi corre-sponds with

graniular

staining in areas of supranuclear

cytoplasmii

of cells in the upper three-fourths of the villus.

Grains

refersto punctate staining

throughout

the cytoplasm of cells in the crypts. The

fractioni

in parentheses indicates theproportionate

distance

from base of crypttotip of villus

in which cells contained staining. The asterisk refers to

sectiot)s in

which

stainl

clearly

sturrounided

f:at droplets.

FIGURE 2 Immuniofluoreseent localization of ApoA-1 near the tip of a jejunal villus.

ApoA-I

imm1iunofluorescence in gran1ules is

apparenlt

in the supranuclear region1s of

epithelial

cells in the f;asted rat (A). At30min(B),ApoA-I stainini;g surrounds fatdroplets primarily inthe sipra-nuIiclearregions ofepithelialcells. Dropletsare alsoseen1intheinlfranuclearI

region;s

ofepithelial cells. Nonspecificstaining can beseeni inthelamin9apropria.1,lumeln; n1, n1ucleus;c,cytoplasm;

11),

lamin11a

propria.

Antiserunm

R 150-

1,

x

_1,250.

pria

also

demiionistrated

some

specific fluoreseeniee

sur-rounidinig

fat

droplets (not showni).

At 3-6

h

(not

showni),

there

were

maniy

more

bright droplets

in

both

the

apical

and basal portions of' epithelial cells and

inthe

lamina

propi-ia,

eveni

in

cells and laminia

propria at

the base

of the villus.

Over

the same

period

of time,

ApoA-I

conl-tents of

jejunial

mucosal

scrapinigs

rose

from

70to 414

ng/mng proteini,

whereas plasma levels of ApoA-I

in-creased

insigniificanitly

by less than 1.5-fold (Table

IV).

The

f;asted

state

anid timlle

sequenIee

of

immiiiiuniofluo-reseeniee

for ApoB was

nearly

idenitical

to

that

seen with

ApoA-I (Fig. 3

and

Table III).

At 30

mnii

fluoreseenee

surrounlded

f:at droplets

in the

supranuclear

portionis

of'

the cells

(Fig.

3B). The degree of

fluoreseenice

was

not

so

intenise

as

with

ApoA-I

(Table III).

Tissue ApoB

levels

rose

only

approximiiately

twofold whereas

plasmiia

levels

remiiainied conistanit

(Table IV).

The

behavior of'

ARP was

(quite differenit.

In the

fasted

state,

there

was

nio imimiiluinofluioreseeniee

in

cells

niear

the villus

tip (Fig. 4A).

Punctate

imimluniofluores-cence

due

to ARP was

conifinied

to

epithelial

cells at

the base

of

tlhe villuts anid

in the

subvillus

glanids (nlot

TABLE IV

Apoprotein Levels in RatJejuuinuz

dtirinig

Fat

Absorption

Jejuniumii Plasmiia

Timiie ApoA-1 ApoB ApoA-1 ApoB

/I

0 70 117 22 32

0.5 73 177 17 22

1 110 237 27 29

2 170 224 31 18

3 312 199 28 22

4 414 302 27 19

5 533 278 29 22

6 592 257 26 18

Plasinas anid homiiogenates of miuicosal scrapinigs (105,000-g supernates) were

anialyze(l

for their apoprotein contenits by

radioimilmunioassay.

Tissue resultsare giveI as nanogramiis of apoprotein perimiilligramii homiiogeniateproteiin. Plasma values are

milligramiis

per deciliter. Each time point represents a

single aniimal. Siimilardatafromii0to 4 h were obtained from aniotlherset of animals.

I

FIGURE 3 limmunofluorescent localization of ApoB at the tip ofajejunal villus. In the fasted rat (A), ApoBis seenasa

graiiular

stainprimarily inthe

supra'iuclear

regionsofepithelial cells. There is nonspecific

sthining

of the

apical suirface

of the epithelial cells as wvell as the lamina propria in these sections. At 30

mim

(B),

ApoB

staining

is seen

surrounding droplets

primarily

inthe

suprimuclear cytoplhsi

Is.abels arethe same as in Fig. 2.

Antiseru-m

R 176-1, x1,650.

shown).

Even at 30

miin

(Fig. 4B), ARP

stainiing

wvas

still

punc'tate

and

confined to the cells

alonig

the lower

foOurth of'the

villtus (Table

III

and(

Fig. .5A).

There wvas

nlo staininig arouind the intracellular f'at

droplets

nior in

cells

at

the

vilhis

tip.

At :3-6i

h, punctate

staining

in-volved

more of the villu.s lengthl (Fig. 15B) being seen

in

cells over as

miiuch

as

the lower

tlhree-fouirths of' tie

villus.

Hovever, eveln at that time

wvhen

cells were

en-gorged wvith

f;at

anid ApoA-I anid

ApoB fluorescence

arotiind

fat

droplets

was

initen.se,

there wkas still no ARP

staining

arouind

the intracellular

(droplets.

Specific ARP

staining

was

seen arouinid f'at drol)lets

in

onily the lauiina

propria (Fig.

4C).

The

iiteinsity of fluorescence after

fat

feeding

was

comparable

to

thiait

seeni

fOr

ApoA-I

and ApoB

(Table

III).

In all

conitrol

preparationis,

nio

granutlar staininiig

in

cells,

or

staininlg

or

droplets

\within

cells

orin

the

lamn-ina propria was

notedl

(Fig. 6).

A

variable

amiiouint

of

staiiniing

of'

the apical

surface of inmieosal

(cel

s

anld of tlle

interstitial

structuire

of the

laniina

p)ropria

was

seen.

The

outline of fat

droplets

never deilloilstrated

in

inunlllofli-orescence

wvhen

norm1lal

rabbit

serumi11

was u

se(l (Fig.

6B). These

apoproteins

were similarly

identified with

the above

techniiques

in

ftastinig

rait

duodnium

audi

ileum,

but not

in

stoiiiach

or

colon.

The

immunmofluorescenit localization

of'

apoproteinls

in

liver

dlifferedc

fi'oni

that

fOuIInd

in

thec initestine.

Whereas ApoA-I

staininlg

in

the

intestine

was

bright,

in

liver the

stain

was

only

moderate

in

intensity (Fig.

7A).

Carbohydrate

fee(dinlg,

wvhi(h

iimo'reases

\VLD)L

se-cretion

bN

liver, did

not

alter the staining of ApoA-I.

ApoB

and(I

ARP

were both

stained brightly in the

hepa-tocyte,

particularly near the bile canalicular portions

of

the cell (Fig. 7B, C).

D)IS

CUSS ION

Unequivocal

i(lentification of'

individual apoproteins

in

tissuies

re(quires that antisera be monospecific. It is

lhelpf'ul

to this

encd that

immunizinig

antigens

be

pure,

and

it is

essenitial that the

specificities of' the antisera

obtained be

adeqtuately

characterized. The antigens

uised

here

were

homogeneous

in two or three

electro-phoreti'

systems

(Fig.

1).

Similar results have

beeni

reiportedl

by others (6, 7, 17, 18, 39). The

specificities

of

the antisera

were

tested

by

double-antibody

im-iunoprecipitations

(Tables

I

and II),

and

in

the

im-munofluiorescenlce

assay before and after

absorption by

appropriate

apoproteins. By both of these criteria

spec-ificity

appeared

to

be

adequate. The adequacy of the

immulnofluiorescence

assay itself was tested

by

includ-ing several

commonly

accepted controls. Thus, the

spec-ificitv of the

localizations appears

to

be established.

Chylomicron and

VLDL

formation

in

the

intestinal

epithelial cell

dturing

fat

absorption

is a

complex

proc-ess

re(quiring:

(a) the esterification of

lipids, (b)

the

svnthesis

of

newv

proteinis,

(c) the assembly of lipids

and

apoproteins,

and

(d)

the

secretion

of the

assembled

product

(1).

On

electron

microscopy

(40-42),

within

minutes of

the

in-gestion

of' a

fat-containing meal, lipid

FIGURE4 I mUntofluorescenit localization of ARP withini cells. No specific fluoresceince is seen atthe villustip either

inthef:aste(dstate(A) or at30m.in30minafteradiiiiinistratioll ofcorin oil

_p)llnctate

fluiorescenice is seeni in the cytoplasm ofmucosal cellsatthevillus-crypt

jtinctioni

(B). At30mim

anid

thereafter many flat droplets are seeni in cells (niot showin), lbut nonie are staine(l. By 4 h after corn oil a fexw

(Iroplets

(d)inthelaminiiapropria arestaine(d with anti-ARP (c). Note

againl nionspecific staininig inthe laminiaii propria. Labels are

the

samile

as in Fig. 2.Antiserumil R 178-3, x975.

0L

F~IGURE.5 Immunofluorescent localization of' ARP

alonig

the villus. At 30 mmn after corni oil initraceliuilar

granuiles

specific fo(r ARP are seeni in the muitcosal cells of' the crypt and along the lower one-fourth of the tot villus-crypt height (A). B'y 4h, graniiies are seeni in cells fulrthcruip the

villuis (B). The area illustrated corresponids to about

one-fouirth of' the enitire villus-crvpt height (average 10 cells).

Cellsatthe villus tip w~ereneve'r seenitostaini fu(r ARP. Even at 4h, nio fiat dIroplets (d) were seeni to be suirroundi(ed by specific- stainiing. Nonspeciftic staininig of'the laminia propria

can- be seeni. Labels are the samne as in Fig. 2. Antiserumn

R 178-3, x875.

globuiles

are

seeni

beneath

the

bruish

border

miemibrane.

These

miigrate

toward the

suipranucilear

region

of'

the

cell

accumuitlatinig

in the various

portionis

of'

the

Golgi

apparatuis, inceluding

the secretory

vesicle.

Lipopro-teinis

are

extruided fromi

the latter

strutuetres

inito the

lamiina

pr-opria,

where

they

finid

theirway

inito the

lac-teals. It is

niot kniowni

whether the two

lipoproteins

are distinct classes

uinder

independent

conitrol,

or

mierely

two

enids

ofabroad

density

continuumin.

Indeed,

ithas

niot

been

possible

to

distinguiish

the

intracellular

produictioni

of

chylomicrons anid

VLDL

fromi

each other.

Therefore, ouir

finidings

miay

be

applicable

to one -or

both

of'thie lipoprotein

classes

unider discuissioni.

ApoA-I

anid

ApoB

were

clearly

demionstrable in in-testinal muitcosa

by

radioimmiunoassay

andinthe

Golgi

regioni

of' the gut

epithelial

cells

by

immuinofluores-cencee,

even in the fasted state when VLDL secretion

proceeds at a

low\

rate and

chylomicron formation

is

minimiial

(43).

Obviously

evenl

this

low

level of'

lipo-proteini productioni

is

sufficient

to

maintaini detectable

pools

of'apoproteins

within the cell. Fat f'eeding rapidly

altered the picture.

Initially,

fluorescenit

staining of'

the

cell

increased and intracellular fat

(droplets

were

stur-roundcled

by

apoproteiins.

Later, stai

ne(d

droplets were

seenl

throughout

the width and height of' the villus.

There

wvere concomitanit

rises in

tisstue

levels

of'ApoA-I

and ApoB which

indlicate

that the increases in

fluores-cence were

indeedl due

to

increased

tissue cWontents

rather

than

to

changes

in

the

immultlnoreactivities

of

the

apoproteinis.

These

findings suiggest

that: (a)

ApoA-I

and ApoB were

ac(lqtired

by

lipoproteinis beffore

they entered

the

Golgi

regions and

(b)

that

lipoprotein

lipidls

were

accompanied

by

ApoA-I andcl

ApoB as

they

were secreted

from

the cells. The rises in

tisstue levels

are also

compatilble with

the notion

that both

apop)o-teins

were newlv synthetizecl dtlring f;at

absorption,

althouigh

the

magnittu(le

of

the

chaniges;

may

represenit

both synthesis

and(

recycling

of apoprotein

s

f0rom11

plasmiia. Incorporationi

of'

radioactive

amiiino

acids

inlto

ApoA-I

(22, 44)

anid(

what may be ApoB (23)

provi(le

futrther

proof'

for

apoprotein synthesis

by

intestinial

cells.

The

contrasting

behaviors of

ApoA-I and

ApoB

on

the

one

hand

and

Arp on the other are striking. Little

ARP

stainiing

was

seeni

in

the

intestinial

epithelial cells

of

f:astinig

animals,

anid

what there was of it

seemied

to

be

confined

to

the

lower parts

of'

the villi and to the

glands. No

stain

was

ever seen to

sturround

droplets

inlside

of

cells

evenl

at the height

of' chylomicron

fur-mationi.

This

was so

despite the f'act that the degree

of ARP

fluiorescenice

wvithin

cells was significant after

fat

feeding and

exceeded

that of ApoB. Although ARP

levels

in

tissue

canniiot

be

measured

at this time, it

is

reasonlable

to

asstume

that

fluorescence and

tissuie

levels

woould

be

correlated,

as

in

the case of

ApoA-I

andl

ApoB

(Tables

III

and IV). Droplets stained with

ARP

were seen

only

in

the

lamina

propria.

Clearly

the

roles of ApoA-I and ApoB differ

fromii

that

of'

ARP.

ApoA-I and ApoB

seem

to be intimately

connected

with fat

absorption and lipoprotein

prodcuc-tion, and the

intestine

may

contribute importantly

to

the

circulating

pools

of these

apoproteins (21, 23).

ARP

may

not be involved

in

chylomicron

and

VLDL

forma-tion

under

these conditions although staining

within

mucosal

cells increases with fat

feeding.

The

stain

seeni

in

the lamina propria may be

due

to ARP

secreted

separately from the intestinal cell,

or from ARP

se-creted

by

liver

and found

in

intestinal

lymphatics.

We

found ApoA-I fluorescence

in

abundance

in

the

intestinal epithelial cell, but little

ApoA-I

staininig

in

hepatocytes. Recently Felker

et

al. (45),

reported

that the rat

liver

secretes

approximately

10times as

much

ARPas

ApoA-I.

The

intestine stains

heavily

for

ApoA-I

FIGURE 6 Lack of staining of riat intestinal 1iticosaII cell

cytoplasimi by nlonlilllmllmIne ralbbit serumlll anid fltoresc'ein-lab)eled goat anti-rab)b)it IgG. SectionIs atre takeni iiear the

tips ofjejtinial xillifromi afitsted rat(A), and 30 mini (B), and

from the ery7pt regioni (C) at 30 min after adminiistratioIn of

corn oil. Some stainiing of the api)ial surEace ot the

epi-thelial cells and lainina propria was lIoted, butrio granlules

were seen in the cytoplasimi nlor was staininlg f'Ound arouInllC fat droplets. Labels werethe samileas inl Fig. 2. x975.

4

.4

a

nd only very lightly

for ARP.

Glickmian

etal.

(4, 22),

found

the

predominiant

apoproteini of

chylomicrons

to

be

ApoA-I, whereas ARP was present but miiuch

less

abunidant. Similar findings

have beeni

reported by

Fainaru et

al.

(5, 12).

These

data

support

the

idea that

under the

condlitionis studied,

the

gut

secretes

ApoA-I

preferentially,

whereas the liver

preferentially

secretes ARP.

The

findings

of Rooke and Skiimmer

(44)

that

both

tisstues are

capal)le

of

synthesizing ApoA-I

suiggest that

the differenices between gut anid

liver

may

be

(Iuianitita-tive

rather thani (jtualitative.

ACKNOWLE DGMENTS

The technical

assistaniee

of Alberta Bailey, Robert Rox,anid

Roger Urbaniis greatlyappreciatedl. We wouild like to thank

)r.A.R. Brownforassistinlginthelipidfeeding experimienits. This investigation was supporte(d by the following granits anndcontract from the DHEW: Contract N01-H\-72-2916-L, Lipid Researeh Clinics Program andlPHSgrantHL-15427-05, Nationsal Heart,Luing, Blood Institute(Dr. Schonfeld); Grant

GM-21096, Nationsal Institute of General Medical Sciences and Grant Ca-15556, National CancerInstitute(Dr. Bell);aind

GrantsAM-14038and ANI-05280, National Institute of

Arthri-tis, Metabolism and DigestiveDiseases (Dr. Alpers).

REFERENCES

1. Gangl,A., aindR. K.Ockner. 1975. Intestinal metabolism of lipids aindl lipoproteinis.

Gastroetnterologyj.

68:

167-186.

2. Alaupovic, P., R. H.Furmiiani, WV. H. Falor, MI. L. Suilliva n,

S. L. WValraven,aindA.C.Olsoni. 1968.Isolationand chalar-acterizationi of huimiian chyle chylomiiicrons and I

lipopro-teinis. Antn.N. Y.Acad. Sci. 149: 791-807.

3.

Kostnier,

G., aindA. Holasek. 1972.

Clharacterizationi

aind( (Itlantitation of the

apolipoproteinis froml humtiain.ll

chyle chylomicrons.

Bioc/lwemtistrml.

11: 1217-1223.

4.

Glickmiian,

R.NI., andK.Kirsel. 1973. Lymphchylomicron formationi dturingthe inhibition of'protein synthesis. .1. Clitn. lIncest. 52:2910-2920.

5. Faintaru, NI.,R. J. Havel,aindl T. E. Felker. 1976. Radio-imnmunoassayofapolipoprotein A-I of rat

serumii. Bioc/hi

im.

Biophys.

Acta.446: 56-68.

6. Koga, S., L. Bolis, and A. MI. Scainti. 1971. Isolation and characterizationof'subunlitpolypeptidesf'romiapoproteius of rat serumiii

lipoproteini.

Biochimin.

Biophyts.

Acta. 236: 416-430.

7. Bersot,T.P., W. V. Brown, R. 1.Levy, H. G.

Windmuleller,

D. S.

Fredricksoni,

and V. S.LeQ(uire. 1970.Further

clhar-acterizationiof the

apolipoproteinis

of rat

plasmiia

lipopro-teiins.

Bioch-lemlistry

. 9:3427-3433.

8. Fielding, C. J., V. G. Shore, aind P. E. Fielding. 1972. Aprotein cofactor of'lecithin: cholesterol

acyltranisf'erase.

Biocheml.

Biophyjs.

Res.

Communti.

46: 1493-1498.

9. Souitar,A. K., C. WX'. Garner, H. N. Baker,

J.

T. Sparrow,

R. L.Jacksoni, A.NI. Gotto, andL. C. Smith. 1975.Et'f'ect ofthe humlan plasmaitapolipoproteins and phosphatidyl-cholinieacyl donioron theactivityoflecithin:cholesterol acyltransferase.

Biochlemistry.

14: 3057-3064.

10. Fredrickson, D. S., A. NI. Gotto, and R. I. Levy. 1972. Famnilial lipoprotein dleficiency. In The Metabolic Basis

of'Inhlerite(d Disease. J. B. Stanbury, J. B. Wtyngaarden,

and 1D.S. Fredricksoni, edlitors. McGraw-Hill Book Com-panv, New York. 3rdedition. 493-530.

11. Lees, R. S., and E. H. Ahrens, Jr. 1969. Fat transport in abetalipoproteinemia: the effects of repeated infusions of beta-lipoprotein-rich serum. N. Engl. J. Med. 280:

1261-1266.

12. Fainaru, NI., R. J. Havel, and K. Imaizumi. 1977. Radio-imnmunoassayof arginine-richapolipoprotein of rat serum. Biochimn. Biophu s. Acta. 490: 144-155.

13. Bersot, T. P., R. NV.Niahley, M.S. Brown, and J. L. Gold-steini. 1976.Interaction of'swinelipoproteins with the low density lipoprotein receptor in human

fibroblasts.J.

Biol. Chlemol. 251: 2395-2398.

14. Shore,V.G., B. Shore,andR.G. Hart. 1974. Changesin

apolipoproteinis

an(l propertiesof rabbitverylowdensity lipoproteinsoninductionof cholesteremia. Biochemistry.

13: 1579-1584.

15. Rodriguez, J., G. C. Ghiselli, D. Torreggiani, and C. R.

Sirtori. 1976. Verylowdensity lipoproteins in normal and cholesterol-fed rabbits:lipid and proteincompositionand metabolism.Athterosclerosis. 23: 73-83.

16. Herbert,P. N., R. NI.Krauss, H.G. WVindmueller,andR. S. Shtulman.1973.Purificationiof' the rat apoproteinactivator of'

lipoprotein

lipase. Circulatiotn. 48: 112. (Abstr.) 17. Havel, R. J., C. J.Fieldinig, T. Olivecrona, V. G. Shore, P.

E.Fieldiing,and T'.Egelrucl. 1973. Cofactor activity of pro-tein

_comp)onents

ofhuman very low density lipoprotein withhydrolysisof'triglycerides by lipoprotein lipase from di'fferenit sources. Biochemtistry. 12: 1828-1833. 18. Gidez,L.I., J. B. Swaney, and S.Mlurname.1977.Analysis

of' rat

serumil

apolipoproteins by isoelectric focusing. I. Stuidies of 'the middle molecular weight subunits.J. Lipid Res. 18: 59-68.

19. Swaney,

J.

B.,and( I.1Gidez. 1977.Analysis of rat serum

a1polipoproteins

by isoelectricfocusing. II. Studies of the low

Imlolecutlar

weightstubuinits.J. Lipid Res. 18:69-76. 20. Windmmmeller,II. G., F. T.Lindgren, W. J. Lossow, and R. 1.Levy.1970.On thenatureof circulating lipoproteins of imitestinal origin in the rat.Biochiim. Biophys.Acta. 202: 507-516.

21. Glickmimani,R. NI., J.Khorana,and A. Kilgore. 1976. Local-izatioIn ofapolipoprotein B in intestinal epithelial cells. Science (Wash. D. C.). 193: 1254-1255.

22. Glickmiian,R. NI.,and P.11.R.Green. 1977. Theintestine as a source of'apolipoprotein

A,.

Proc. Natl. Acad. Sci.

U.S.A. 74:2569-2573.

23.

Nindmilueller,

H.G., P. N.flerbert, and R.I. Levy. 1973.

Biosynthesis

oflymnphand

plasmiia

lipoproteinapoproteins by isolated

perfuse(d

ratliver and intestine. J. LipidRes. 14:215-223.

FIGURE 7

Immltinofluioreseenit localizattiomi

ofApoA-1 (A), ApoB (B),

ani(I

AlI' (C)in livertaken froma14-hfastedrat.

Identical

restiltswereobtainedat varioustimesafter

initragastric

adminiistra-tion ofcorn oil. The anitisera usecd were R 150-1, R 176-1, alld R 178-1, respectively. ApoA-I stainl in hepatocytes is faii t amld granuliiar, wvhereas ApoB and ARP staining is prominienit and grantular (n, ltclells; c, ecytoplasmi). NIluch of the stain is

conceniitrated

niear the bile canialicumlar regions of the cells(b).Bright,difl'tusestaininigofsinlusoids (.s)isseenlinall sections. Nonspecific staininlg with normlal rabbit serrumn (I)) is fhint. No granulalr fluorescence is

seenl. x1,250.

24. Kessler, J. I., J. Stein, D. Dannacker, and P. Narcessian. 1970. Biosynthesis of low density lipoprotein by cell-free preparations of' rat intestinal

mucosa.J.

Biol. Chem. 245: 5281-5288.

25. Schonfeld, G., A. R. Brown, C. E. Bell, Jr., and D. H. Alpers. 1977. Apo(Lipo) protein A-I production by rat small intestine. Gastroenterology. 72: 1127. (Abstr.) 26. Lipid and Lipoprotein Analysis, Lipid Research Clinic

Manual of'Laboratory Operation. 1: May 1974. National Institutes ofHealth Publication 75-628. Bethesda, Md. 27. Bell, C. E., Jr., S. Seetharam, and R. C. McDaniel. 1976.

Endodermally-derived and neural crest-derived differ-entiation antigens expressed bya human lutngtuimor. J. Immunol. 116: 1236-1243.

28. Schonfeld, G., M. S. Frick, andA. P.Bailey. 1976. Meas-urement of apolipoprotein A-I in rat high density lipoprotein and in rat plasma by radioimmtunoassay.J. LipidRes. 17:25- 30.

29. Marsh, J. B. 1976. Apoproteins of'the lipoproteins in a non-recirculatingperfusateof'rat

liver.J.

Lipid Res. 17:85-90.

30. Shapiro, A. L., andJ.V.Maizel, Jr.1969.Molecuilarweight estimation of'polypepticles by SDS-polyacrylamide gel electrophoresis: fuirtherdata concerningresolvingpower andgeneral considerations. Anial.Biocherm.29:505-5.14. 31. Eaton, R. P., and D. M. Kipnis. 1969.Radioimmtunoassav ofbeta lipoprotein-protein in rat sertum. J. Clin. Invest. 48: 1387-1396.

32. Davis, B.

J.

1964. Disc electrophoresis. II. Method and application to hulman sertum proteins. Ann. N. Y. Acad. Sci. 1121: 404-427.

33. Schonf'eld, G.,S.W. Weidman, J. L. WA'itztumnl, an(d R. MI. Bowen. 1976. Alterations in levels and interrelations of plasma apolipoproteins induiced by diet. Metab. Clin. Exp. 25:261-275.

34. Williams, C. A., and M. W. Chase. 1967. In Methods in Immuinology and Immunochemistry. Academic Press, Inc., New York. 1: 307-335.

35. Marchalonis, J. J. 1969. An enzymic method for the trace iodination ofimmunoglobulins and other proteins. Bio-chem.J. 113:299-305.

36. Huinter, W. M., and F. C. Greenwood. 1962. Preparation of iodine-131labelledhumangrowthhormone of high spe-cific activity. Nature(Lond.). 194: 495-496.

37. Schonfeld, G. R. S. Lee, P. K. George, and B. Pfleger. 1974. Assay oftotal plasma apolipoprotein B concentra-tion inhuman

subjects.J.

Clin. Invest. 53: 1458-1467.

38. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Proteinmeasturementwith thefolin phenol

reagent.J.

Biol. Chem. 193: 265-275.

39. Swaney, J. B.,H. Reese,and H. A. Eder. 1974. Polypep-tide composition of rat high density lipoprotein: charac-terizationbySDS-gel electrophoresis. Biochem.

Biophi,s.

Res. Commun. 59: 513-519.

40. Palay, S. L., and L.

J.

Karlin. 1950. An electron micro-scopicstudy of the intestinalvilluis.II. The pathway of fat

absorption.J.

Biophys. Biochem.Cytol. 5: 373-383. 41. Cardell, R. R., Jr., S. Badenhausen, and K. R. Porter. 1967.

Intestinal triglyceride absorption in the rat. Anelectron microscopical

stuidy.J.

Cell Biol. 34: 123-155.

42. Sabesin,S. M.,and S. Frase. 1977. Electron microscopic

stuidies

oftheassembly,intracellullartransport,and secre-tion of chylomicrons by rat intestine. J. Lipid Re.s. 18: 496-511.

43. Ockner, R. K., F. B.Htughes, and K.

J.

Isselbacher. 1969. Very low density lipoproteins in intestinal lymph. Origin, composition,and roleinlipid transport in the fasting state. 1.

Clinl.

Invest.48: 2079-2088.

44. Rooke,

J.

A., and E. R. Skinner. 1976. The

biosynthesis

ofratsertum apolipoproteins by liver and intestinal mu-cosa. Biochem.Soc. Trans. 4: 1144-1145.

45. Felker, T. E., M.

Fainarui,

R. L.Hamilton,and R.

J.

Havel. 1977. Secretion of the arginine-rich andA-I apolipopro-teins by the isolated

perfuised

rat liver.]. LipidRe.s. 18: 465 -473.