Proc. Natl. Acad. Sci. USAVol. 75, No. 7, pp. 3459-43463, July 1978Medical Sciences

Secretion of lipoprotein-X by perfused livers of rats with cholestasis(plasma lipoproteins/apolipoproteins/bile/electron microscopy)

TUNDE E. FELKER*, ROBERT L. HAMILTON*t, AND RICHARD J. HAVEL*f* Cardiovascular Research Institute, Departments of tAnatomy and *Medicine, University of California, San Francisco, California 94143

Communicated by John A. Clements, May 8,1978

ABSTRACT The major abnormal plasma lipoprotein ofcholestasis (LP-X) was isolated from bFood plasma and fromperfusates of isolated livers of rats with biliary obstruction. Inboth cases LP-X was composed mainly of about equimolar partsof phospholipids and unesterified cholesterol; the small proteincomponent was primarily the arginine-rich apolipoprotein. Byelectron microscopy, LP-X appeared as a unilamellar liposome(690 A mean diameter, range 400-1000 A) with the trilaminarstaining image typical of phospholipid bilayers.

Extensive block staining of cholestatic livers for 48 hr withwarmed uranyl acetate (370) permitted the visualization ofvesicles indistinguishable from LP-X within hepatic paren-chyma. These trilaminar-staining vesicles occurred predomi-nantly within bile canaliculi. They also were seen in nearbycytoplasmic vacuoles or invaginations between hepatocytes andin the space of Disse. Similar vesicles were not seen in the en-doplasmic reticulum or Golgi cisternae. These observations raisethe possibility that the vesicles are formed within bile canaliculiand are transported from the canaliculi to the space of Dissewithin pinocytotic vacuoles.

Cholestasis may be defined as the interruption of bile flow fromthe biliary passages of the liver with consequent appearanceof biliary constituents in blood plasma. In humans, prolongedobstruction to bile flow causes a progressive and often equimolarrise in plasma unesterified cholesterol and phospholipid (1-4).This unique hyperlipidemia results largely from the accumu-lation in plasma of a major abnormal lipoprotein that is sepa-rated with normal low density lipoproteins (LDL) by ultra-centrifugation. This abnormal lipoprotein, which differs greatlyin lipid and protein composition from normal LDL, is com-monly called LP-X (3, 5). Most normal plasma lipoproteins arepseudomicellar (6) with a core of relatively nonpolar triglyc-erides and cholesteryl esters covered by a monomolecularsurface of phospholipids, unesterified cholesterol, and apo-proteins, whereas LP-X is largely a unilamellar liposome ofabout 400-700 A diameter (4, 7-9). It probably exists in vivoas a bilayer vesicle of equimolar phospholipids and unesterifiedcholesterol containing small amounts of plasma proteins (mainlyalbumin) in its internal aqueous compartment together withsome apolipoproteins adsorbed on its surface (4, 6, 8, 9). Severalother abnormal plasma lipoproteins have also been reportedin patients with cholestasis (8, 10-13).The origin of LP-X is unknown, although it is generally as-

sumed to be produced by regurgitation of bile components intothe blood (for review, see ref. 14). Recent studies have shownthat LP-X occurs in the plasma of animals after surgical inter-ruption of the common bile duct (15-18). This reports showsthat LP-X and other abnormal lipoproteins accumulate inperfusates of isolated livers from rats with biliary obstruction.Our electron microscopic observations provide a clear dem-onstration of LP-X-like vesicles within bile canaliculi.§

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked"advertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

3459

MATERIALS AND METHODSLiver Perfusion. Male Sprague-Dawley rats (350450 g) had

free access to food and water. Cholestasis was produced underether anesthesia by excising 2 mm of double ligated commonbile duct. In sham-operated controls the peritoneal cavity wasopened and the bile duct was manipulated. After operation,animals were injected intraperitoneally with 0.3 ml of penicillinG (500,000 units/ml, Squibb & Sons, Inc.,-Princeton, NJ) andintramuscularly with 0.2 ml of streptomycin sulfate (0.4 g/ml,Pfizer Laboratories, New York, NY). Forty-two hours laterisolated livers of rats from sham-operated controls and thosewith cholestasis were perfused for 6 hr in a recirculating systemby techniques described elsewhere (19-21). The perfusionmedium, which was pumped at a rate of 1 ml min-1 g-1 ofliver, was composed of three parts of Krebs-Ringer bicarbonatebuffer, pH 7.4, containing 1.5 mg of glucose per ml and 1 partof thrice-washed rat erythrocytes. The bile duct was cannulatedto permit bile to flow during perfusion of livers.from unoper-ated or sham-operated control rats.

Isolation of Lipoproteins. Plasma from control animals andthose with cholestasis was collected into chilled citrate solution(19). Lipoproteins from plasma and perfusates were isolatedby sequential ultracentrifugation at 40 in a 40.3 rotor (BeckmanInstruments, Inc., Palo Alto, CA) (21). Lipoprotein fractionswere recentrifuged and dialyzed for at least 24 hr at 40 against0.9% NaCl/0.01% NaN3/0.04% EDTA (wt/vol) at pH 7.0. LDLfrom cholestatic animals (2-4 mg of protein) was applied to a1.2 X 90 cm column of 4% agarose gel (Bio-Gel A-15m, Bio-RadLaboratories, Richmond, CA). Fifty 2.0-ml fractions wereeluted with 0.2 M NaCl/1 mM EDTA/0.02% NaN3 at pH 7.0.Absorbance at 280 nm and protein content of each fraction weremeasured.Chemical Analyses. Protein was determined by a modified

procedure of Lowry with bovine serum albumin as standard(22). Lipid composition of lipoproteins was determined bystandard methods (23). Polyacrylamide gel electrophoresis wasperformed in sodium dodecyl sulfate (NaDodSO4) by a modi-fication of the procedure of Weber and Osborn (24). Lipopro-teins (5-30 gg of protein) were denatured at 900 for 3 min with5% mercaptoethanol and 1% NaDodSO4. Gel electrophoresiswas carried out at 2 mA per gel for 19 hr. Gels were stained withCoomassie brilliant blue in a solution of ethanol/acetic acid/water (45:10:45 vol/vol) and destained in 10% acetic acid.Apolipoproteins were identified by comparison of electro-phoretic mobility with purified rat apolipoproteins (25-27) andby immunodiffusion (28) against monospecific antisera.

Morphological Studies. Whole plasma and lipoprotein

Abbreviations: LP-X, major abnormal lipoprotein of cholestasis; VLDL,LDL, and HDL, very low density, low density, and high density li-poproteins, respectively; NaDodSO4, sodium dodecyl sulfate.§ A preliminary report of this study was presented at the annualmeeting of the American Society for Cell Biology in November 1977(J. Cell Biol. 75, 375A).

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FIG. 1. (Upper) Negativelystained preparation of wholeplasma from a rat with cholestasisshows LP-X, which appear as

flattened vesicles that often formrouleaux. (Lower) Negativelystained preparations show lipo-proteins isolated from perfusate ofa cholestatic liver. (Left) ParentLDL fraction (1.015 < d < 1.075g/ml) before gel filtration. (Mid-dle) LP-X from the first peakeluted from a column of4% agarose

gel. (Right) Round particles (250 Adiameter) with a more electron-lucent "core" recovered from thesecond peak. (X129,600.)

fractions were examined by electron microscopy after negativetaining with 2% potassium phosphotungstate, pH 6.5 (4). LP-X

was prepared for study in thin sections (20). Livers of plasmadonors and perfused livers of rats with or without cholestasiswere flushed via the portal vein with 25 ml of Krebs-Ringerbicarbonate buffer followed by 30 ml of fixative containing 1%paraformaldehyde and 2% glutaraldehyde (29) in a 0.1 Mphosphate buffer, pH 7.4.

Tissue was postfixed in buffered 2% OS04 (30) at 30 for 18-48hr. To visualize lipid bilayers, liver cubes were extensivelystained en bloc for at least 48 hr in 2% aqueous uranyl acetateat 370 (20). Tissue was dehydrated in acetone and embeddedin Epon (31). Thin sections were stained with uranyl acetateand lead citrate (32) and photographed in a Siemens 101 elec-tron microscope at 80 kV with a 60-Aim aperture.

RESULTSThe amount of protein in the LDL fraction (1.015 < d < 1.075g/ml) of perfusates obtained after perfusion of cholestatic liversfor 6 hr was about double that of sham-operated control rats.Concomitantly, recovery of protein in very low density lipo-protein (VLDL) from perfusates of cholestatic livers was re-

duced about 90%. Recovery of protein in high density lipo-protein (HDL) was unchanged. The amount of VLDL andLDL protein recoveredy varied widely although both sham-operated and cholestatic rats lost comparable weight (15-30 g)after surgery.

Characterization of LP-X in Plasma and Perfusates. Wholeplasma of rats with cholestasis contained particles that oftenappeared in rouleaux in preparations negatively stained withphosphotungstate (Fig. 1 upper). These structures closely re-

semble LP-X described in plasma of human patients withcholestasis (4).LDL fractions isolated from perfusates of cholestatic livers

contained a mixture of particles. Many appeared in negativelystained preparations as large flattened structures in rouleaux,whereas others appeared as smaller spherical particles with a

more electron-lucent "core" (Fig. 1 lower left). These differentparticles were separated into two major peaks by gel chroma-tography. The elution patterns of LDL fractions from plasma

(Fig. 2 mIddle) or liver perfusates (Fig. 2 bottom) of rats withcholestasis were closely similar. The first peak, eluted in the voidvolume, contained very little protein but caused substantial lightscattering, whereas the second peak was protein rich with lesslight scattering. The void volume peak contained the largerLP-X particles, which appeared as flattened vesicles (Fig. 1lower middle). The middle portion of the second peak (Fig.1 lower right) contained the smaller spherical particles witha more electron-lucent "core." When the smaller amount(0.4-0.6 mg of protein) of perfusate LDL of sham-operated ratswas subjected to gel chromatography, a somewhat differentelution pattern was found (Fig. 2 top). There was insufficientmaterial for further characterization.The composition of LP-X purified by gel chromatography

(Table 1) was the same for plasma and liver perfusate. It con-tained very little protein (3.3-5.5% by weight) and, like LP-Xfrom human plasma (3-5, 8, 13), was composed mainly of al-most equimolar phospholipids and unesterified cholesterol.Lipoprotein eluted in the middle portion of the second peakfrom plasma, and perfusates contained much more protein andnonpolar lipids (Table 1).

Polyacrylamide gel electrophoresis in NaDodSO4 showedthat the parent LDL fractions from perfusate and plasmacontained proteins with the mobility of B-apolipoprotein, al-

Table 1. Composition of eluates from columns of 4% agarose gel(% by weight)

LP-X* Second peaktPlasma Perfusate Plasma Perfusate(n =3) (n =4) (n = 2) (n = 2)

Cholesteryl esters 2.4 ± 0.7 1.3 ± 0.8 12 15Triglycerides 2.8 + 0.9 3.9 + 1.6 19 22Unesterified

cholesterol 28.2 ± 0.6 24.8 ± 1.5 12 8Phospholipids 62.7 0.6 65.5 3.5 34 28Proteins 3.8 0.5 4.7 0.8 27 27

* Mean ± SD. LP-X represents the main part of the void volumepeak.

t Mean. Fractions pooled from the middle of the second peak.

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Proc. Natl. Acad. Sci. USA 75 (1978) 3461

VOA280 mg/ml

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FIG. 2. Elution patterns of LDL fractions (1.015 < d < 1.075g/ml) from columns of4% agarose gel. (-) Absorbance; (---- -) proteinconcentration. LDL fractions were isolated from: (Top) liver perfusateof sham-operated rat; (Middle) plasma of rat with cholestasis; (Bot-tom) liver perfusate of rat with cholestasis. Top and Middle are fromthe same column; Bottom is from a different column. Vo = void vol-ume.

bumin, arginine-rich apolipoprotein, and C-apolipoproteins,(Fig. 3). LP-X appeared to contain Arg-rich apolipoprotein asits major apolipoprotein, with small amounts of albumin andC-apolipoproteins. The particles from the second peak appearedto contain mainly B-apolipoprotein. LP-X gave a strong reactionof identity with Arg-rich apolipoprotein upon immunodiffusionagainst antibody to Arg-rich apolipoprotein but not antibodyto B-apolipoprotein, whereas lipoprotein in the second peakreacted with antibody to B-apolipoprotein. In preliminaryexperiments in which [3H]lysine was added to the perfusate ofcholestatic livers, 3H was associated mainly with Arg-richapolipoprotein in purified LP-X and with the B-apolipoproteinin fractions from the second peak.

Electron Microscopic Observations of Liver. Low-mag-nification electron micrographs of perfused and nonperfusedlivers from rats with cholestasis showed many vesicular struc-tures between adjacent hepatocytes (Fig. 4 left). Highermagnifications indicated that these trilaminar staining vesicleswere contained mainly within the bile canaliculus (Fig. 4right). The vesicles were closely similar in staining propertiesand size to images in thin section of LP-X isolated from plasmaby centrifugation (Fig. 5 right). These vesicular structures werenot seen in bile canaliculi of nonperfused or perfused livers ofunoperated or sham-operated control rats. LP-X-like vesicular

B - --m- mAlbumin _

la-IV _.

ARP--- _ _ _

A-I

FIG. 3. NaDodSO4/polyacrylamide gel electropherograms of

perfusate lipoproteins from cholestatic liver. From left: perfusate

HDL for the identification of apoproteins, (30 ,tg of protein); parent

LDL fraction of perfusate (28, ug of protein); top fraction of void

volume from 4% agarose gel column (LP-X) (8 gg of protein); middle

portion of the second peak from gel column (9 Mg of protein). B, B-

apolipoprotein; A-TV, A-TV apolipoprotein; ARP, Arg-rich apolipo-

protein; A-I, A-I apolipoprotein; and C, C-apolipoproteins.

structures with bilayer staining properties were often seen incytoplasmic vacuoles or invaginations near similar particlesfilling a bile canaliculus (Fig. 5 left). These vesicular structureswere most abundant in bile canaliculi, but were also foundbetween hepatocytes and in the space of Disse (Fig. 6). Nonewas evident in Golgi vacuoles or in cisternae of endoplasmicreticulum. The vesicles within bile canaliculi appeared to berandomly scattered within the hepatic parenchyma. No obviousmorphological landmarks were associated with their presenceor absence.

Similar appearing vesicles were present within multivesicularbodies (Fig. 5 left), but those vesicular structures were seenin all sections of rat liver without the requirement for extensivestaining with the warmed uranyl acetate that was necessary toclearly demonstrate the LP-X-like vesicles of cholestasis.

Perfused livers from unoperated and sham-operated controlrats contained many secretory vesicles filled with nascentVLDL within Golgi areas, whereas perfused or nonperfusedcholestatic livers invariably appeared to contain very limitedamounts of secretory vesicles in the Golgi areas. Moreover, theGolgi cisternae of cholestatic livers usually appeared empty andcontained few nascent VLDL particles, especially after per-fusion.

DISCUSSIONOur findings appear to establish an hepatic origin of LP-X inrats. The lipid composition and morphologic characteristics ofLP-X obtained from perfusates of liver or from plasma of ratswith cholestasis were indistinguishable. Although the apoli-poproteins of LP-X from those two sources appeared similaron NaDodSO4 polyacrylamide gels and upon immunodiffusion,quantitative analyses remain to be performed. The LP-X par-ticles from perfusates of cholestatic rat livers also closely re-semble LP-X isolated from plasma of human subjects withbiliary obstruction (4, 5, 9), suggesting a similar origin in thetwo species. The apparently higher proportion of Arg-richapolipoprotein relative to albumin and C apoproteins in ratLP-X as compared to human LP-X (12) may be related to theproportionately higher content of Arg-rich apolipoprotein innormal rat plasma lipoproteins.

Although the mechanism of origin in LP-X within the liverremains obscure, our morphologic observations provide cluesabout the pathway by which LP-X reaches the blood stream.Bilayer vesicles with the same size and trilaminar stainingproperties as LP-X isolated from plasma or perfusates occurredpredominantly within bile canaliculi. Indistinguishable vesiclesalso appeared within adjacent cytoplasmic vacuoles or invag-

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3462 Medical Sciences: Felker et al.

inations of the hepatocyte cell surface, between adjacent livercells, and in the space of Disse. However, such vesicles were notfound within the cisternae of the smooth or rough endoplasmicreticulum or within cisternae of the Golgi system. These ob-servations suggest that the LP-X-like vesicles may be formedextracellularly, perhaps within bile canaliculi. We observedapparently intact tight junctions associated with canaliculi filledwith LP-X-likce vesicles, even those adjacent to the occasionalcells in which the cytoplasm was destroyed by necrosis. The

Proc. Nati. Acad. Sci. USA 75 (1978)

>\a-s+ FIG. 4. (Left) Low-magnifi->

. - Ott.::~cation image of a perfused rat liver

+r ~ Ark. Y'sui with cholestasis shows a typicalJ ,61 ,56 t!#%? change caused by biliary obstruc-; fat tb ~~tion: the presence of material that. .! t U; J ;4 'tappears vesicular within a bile

(1j7^j ,; # * a canalicular region. (X5184.)Svi 7^trv I'- At*1 (Right) Higher magnification of'Fi. # * vvffl these areas illustrates more clearlytn;.w no 7 that the material consists largely of>i~~ ~~__APJe i esicles with trilaminar staining.tpF~e7 e @ Otis

Junctional complexes (arrow) and; lurninal microvilli identify this

-> Z E ~~~~area containing the vesicles as a_ _ ~~~~~~~bilecanaliculus. (X43,200.)

frequent observation of LP-X-like vesicles within cytoplasmiccompartments or invaginations of the cell surface (Fig. 5 left)suggests a possible pinocytotic route of transport around tightjunctions of the bile canaliculus to the spaces of Disse.

Other investigators (15, 33) probably did not observe tri-laminar staining vesicles of LP-X size within liver during cho-lestasis because of the difficulties of staining such phospholipidbilayers. We had learned previously that in order to visualizethe trilaminar staining pattern of bilayer vesicles it is essential

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FIG. 5. (Left) Bile canaliculararea of a perfused liver with cho-lestasis. The bile canaliculus isidentified by the tight junction(arrows) and luminal microvilli.The lumen of the canaliculus isfilled with bilayer vesicles of simi-lar size. Indistinguishable vesiclesare also evident within mem-brane-bound vacuoles (V) near thebile canaliculus. A multivesicularbody (MV) is also present (thisstructure is seen without the spe-cial staining procedure required todemonstrate the vesicular struc-tures within the bile canaliculusand cytoplasmic vacuoles). (Right)Thin section of the plasma LDLfraction from a rat with cholestasisillustrates the characteristic tri-laminar staining image of the lipidbilayer of LP-X. The range(400-1000 A) and mean diameterof these particles (690 A) are thesame as those vesicles within thebile canaliculus (range 4001000 A;mean 646 A) shown at Left.(x43,200.)

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Proc. Natl. Acad. Sci. USA 75 (1978) 3463

FIG. 6. Vesicles of the samesize and staining properties asLP-X appear within the space ofDisse in this perfused liver withcholestasis. (X12,960.)

to incubate them in warmed (370) uranyl acetate for at least48 hr after OS04 fixation (see Materials and Methods and ref.20). Because some multivesicular bodies were always seen innormal livers stained by routine techniques, they probably arenot directly related to the LP-X-like vesicles described here.

Cholestasis altered other aspects of hepatic lipoprotein me-tabolism that await further investigation. In addition to LP-X,cholestatic livers produced increased amounts of another aty-pical lipoprotein in the LDL fraction. This particle evidentlyis pseudomicellar because of its high content of triglyceridesand cholesteryl esters (Table 1). It is not a typical LDL becauseits content of triglycerides exceeds that of cholesteryl esters, andits image in negatively stained preparations frequently showsa more electron-lucent "core" that is not evident in similarimages of normal rat or human LDL.1 The lipid compositionand predominance of B-apolipoprotein suggest that this particlemay resemble a newly described abnormal LDL (called LP-Y)(13) in humans with cholestasis.

Although the recovery of HDL protein was similar in per-fusates of unoperated controls, sham-operated controls, andcholestatic livers, HDL from cholestatic livers contained morenascent discoidal particles (20, 21) and proportionately lesscholesteryl esters. In these respects they resemble the lamellardiscoidal HDL in humans with biliary obstruction (8, 10,12).

In sharp contrast to the increased LDL fraction of perfusatesof cholestatic livers, VLDL was greatly reduced. Livers ofsham-operated rats secreted even more VLDL than unoperatedcontrols, indicating that the surgical stress could not accountfor this large difference. The electron microscopic observationsare consistent with the chemical data: we observed an obviousdiminution in the number of Golgi secretory vesicles thatcontained nascent VLDL particles in cholestatic livers.

The excellent technical assistance of Carlene Chang, Agnes Frank,Alyce Green, and Julie Liaw is greatly appreciated. We thank Dr. L.S. S. Guo for immunochemical analyses. T.E.F. is a postdoctoral re-search fellow of the Bay Area Heart Association. This research wassupported by Arteriosclerosis Specialized Center of Research GrantHL-14237 from the U.S. Public Health Service.

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