Erythrocyte echinocytosis in liver disease Role of abnormal plasma high density lipoproteins

Erythrocyte echinocytosis in liver disease. Role

of abnormal plasma high density lipoproteins.

J S Owen, … , H Isenberg, W B Gratzer

J Clin Invest.

1985;

76(6)

:2275-2285.

https://doi.org/10.1172/JCI112237

.

Echinocytes were frequently found in patients with liver disease when their blood was

examined in wet films, but rarely detected in dried, stained smears. When normal

erythrocytes (discocytes) were incubated with physiologic concentrations of the abnormal

high density lipoproteins (HDL) from some jaundiced patients, echinocytosis developed

within seconds. Other plasma fractions were not echinocytogenic. There was a close

correlation between the number of echinocytes found in vivo and the ability of the

corresponding HDL to induce discocyte-echinocyte transformation. On incubation with

normal HDL, echinocytes generated in vitro rapidly reverted to a normal shape, and

echinocytes from patients showed a similar trend. Echinocytosis occurred without change in

membrane cholesterol content, as did its reversal, and was not caused by membrane

uptake of lysolecithin or bile acids. Abnormal, echinocytogenic HDL showed saturable

binding to approximately 5,000 sites per normal erythrocyte with an association constant of

10(8) M-1. Nonechinocytogenic patient HDL and normal HDL showed only nonsaturable

binding. Several minor components of electrophoretically separated erythrocyte membrane

proteins bound the abnormal HDL; pretreatment of the cells with trypsin or pronase reduced

or eliminated binding. Echinocytosis by abnormal HDL required receptor occupancy, rather

than transfer of constituents to or from the membrane, because cells reversibly prefixed in

the discoid shape by wheat germ agglutinin, and then exposed to abnormal HDL, did not

become echinocytes when […]

Research Article

Find the latest version:

Erythrocyte Echinocytosis

in Liver Disease

Role ofAbnormal Plasma High Density Lipoproteins

JamesS. Owen, David J.C.Brown, David S. Harry, andNeil Mcintyre

AcademicDepartmentofMedicine,RoyalFreeHospital School of Medicine(University ofLondon), London NW32PF Gilbert H. Beaven,HarveyIsenberg, andWalterB.Gratzer

Department of Biophysics and MedicalResearch Council Cell Biophysics Unit,King's College (University ofLondon),

London WC2R2LS,England

Abstract

Echinocytes were frequently found in patients with liver disease when their blood was examined in wet films, but rarely detected in dried, stained smears.Whennormal erythrocytes (discocytes) were incubated with physiologic concentrations of the abnormal high density lipoproteins (HDL) from some jaundiced patients, echinocytosis developed within seconds. Other plasma fractions werenotechinocytogenic. There was a close correlation between the number ofechinocytes found invivo and the ability of the corresponding HDL to induce discocyte-echinocyte transfor-mation. On incubation with normal HDL, echinocytes generated

invitro rapidly reverted toanormalshape, and echinocytes from patients showed a similar trend. Echinocytosis occurred without changeinmembranecholesterol content, as did its reversal, and was not caused by membrane uptake of lysolecithin or bile acids. Abnormal, echinocytogenic HDL showed saturable binding to -5,000 sites per normal erythrocyte with an association constant of108

M-'.

Nonechinocytogenicpatient HDL and normalHDL

showed only nonsaturable binding. Several minor components ofelectrophoretically separated erythrocyte membrane proteins bound the abnormalHDL;pretreatment of the cells withtrypsin

orpronase reduced oreliminatedbinding.Echinocytosis by

ab-normalHDLrequired receptor occupancy, ratherthantransfer of constituentsto orfrom the membrane, because cellsreversibly prefixedinthe discoidshape bywheatgermagglutinin,andthen exposed toabnormal HDL, didnot becomeechinocytes when

the HDL andlectinweresuccessively removed. Binding didnot cause dephosphorylation of spectrin. We conclude that the

echinocytes of liverdisease are generated from discocytes by

abnormal HDL, andweinfer thattheshape changeis mediated by cell-surface receptors for abnormal HDL molecules.

Introduction

The initialassessmentof erythrocyte morphologyinliver disease is usually based onexamination ofdried, stained blood films.

Portions of this studywerepresentedattheMedical Research Society meeting in London (1981.Clin. Sci.

(Lond.].

60:2P)andatthe

Inter-nationalAssociation fortheStudy of the Liver meeting in HongKong

(1982. Hepatology. 2:150).

Address reprint requeststo Dr. Owen, Academic Department of

Medicine,RoyalFredHospital School of Medicine(Universityof

Lon-don),Rowland HillStreet, LondonNW32PF, England.

Receivedfor publication27April 1983 andinrevisedform24 June 1985.

Unfortunately, drying may cause shape artefacts to which wet preparations and scanning electron microscopy(SEM)'are not subject. "Target" cells are common in stained smears from jaundiced patients, but result from distortion because they are

codocytic(bell-shaped) in wet films. In 1968, Grahn and col-leagues (I) found spiculated erythrocytes in wet blood films from

patientswith liver disease; they were not evident in dried, stained specimens. This observation has largely been disregarded. They

pointedoutthat these cells (whichweshall call

echinocytes-seeMethods)werealso present in "spur cellanemia,"ahemolytic

disorderseenoccasionally in severe alcoholic liver disease and characterized by the presence of acanthocytes2 (1-4). Acantho-cytes are bizarre, irregularly spiculated cells recognizable in stained smears, aswellasinwetfilms.

Grahnetal.(1)foundthat theserumfrom bloodcontaining

echinocytes causedechinocytosisofnormalerythrocytes;serum

from blood that contained acanthocytes also induced formation

ofechinocytes, butnotof acanthocytes. They therefore argued thatan additional factoracting in vivo mightbe necessary to

produce acanthocytes and suggested that the spleen might be involved. They were unable to identify the serum fraction(s)

responsiblefor theformationofechinocytes.

HuiandHarmony (5) produced echinocytes byincubating

normal erythrocytes with isolated normal lowdensity

lipopro-teins (LDL). The active constituentwasapparently

apolipopro-tein B(apoB), inasmuch ascyclohexanedione blockage of the arginineresiduesof apoB reducedechinocytogenicpotency(6).

Thehigh densitylipoprotein(HDL)of patientswith liver disease is often enriched in apoE, which competes with the apoB of normalLDLfor "apoB,E-receptors"onthesurface ofnucleated cells(7).Wehavethereforeinvestigatedwhether abnormalHDL

fromjaundiced patients, like normal LDL, wouldcause

echino-cytosis. We found echinocytes in patients with liver

disease;

morphologicallysimilar cellswereformedrapidlywhen normal erythrocytes were incubated with abnormal HDL from these

patients. Echinocytosisdidnotresult from additionorextraction

of membrane components; theimportant factor seemed tobe

reversible,saturablebindingof abnormal HDL

particles

tothe erythrocyte surface.

Methods

Patients. HDLwere isolated from 18 inpatients withliver disease of varying severity (Table I). Diagnostic methods included liver biopsy,

1. Abbreviations usedinthis paper: apo, apolipoprotein; LPDS,

lipo-protein-deficientserum;SEM,scanningelectron microscopy.

2. Theseirregularyspiculatederythrocyteshavealso beentermed"spur

cells"and"spur-shapedcells"(2,3)or"burrcells"(1);we

_prefer

touse the nomenclature of BrecherandBessis(9),asdescribed in Methods, andcallthem"acanthocytes."

J.Clin. Invest.

©TheAmericanSociety for Clinical Investigation,Inc.

0021-9738/85/12/2275/11 $1.00

Q%ON 0 " 0 O "1 000 o000w1 0 0 ,Q

) Oen en, O t-- O

zzzz 0O O

Z Z Z Z Z CmkN 0% 0 t 0l "Ft 'I

____t" _oo00o00

00o O tO 0 - O

0

kn 0,0 ON -0O OCIA O 000

00 0 ^ 0 N 0 eC 00ON_q -N - 0 - N

-ON IDN t 00 -0 (ON-s 0

- O" O M ON

%000 \0 "o N -0 (N -0 00) C0N .0 e 0

eQ0(N0000 _...(n e

r-~ Ne en (N (N (N e^ (N (N e^

Cr

-CrC

- N Cr tN- "o t N 0 000 (N t N t a-M - d 20 0 Rt - N n N n (N C

(N 0 %o Rt -N C -

--ON

o>-00--N>o-£-e~ -- - _-e , - d

C,~~~ ~ ~ ~~~~~~~C

0 CNMCrCo 0%00Se'Ce o(008 o0Cr0%N

(N CDC e~ t'- S-(ON C tn e r

Et°.E2! 2 : E Dt 6 t6U 6

0V

V-C

.0. 20.

~~ 00.

.0

~0'_~~ 0 . ₀

0o0 . 00 .0.O .0

2~~'~20 2 ~~ 2

_2a

U

0 C .0 Cu

'u<', 0

<

.5

tn tn tN e

" ew Nn (nN

N

(N 00 00

n

0 (N m 0I

m Cfvn (N 00 m o N ^ o eC (N m eo^ '

R)imXSU~~~~~x3 =2 cvt[; 2z*0iX s 6 2t L

0

ai =.0

0

a

.a

,

a=

* r.

FF

06

Q

_.

.Q

za

_Z

O

:z c

t.

a,

.0 _

.0%

0',

E-1

'A

.m

.~

°,

_

o=

( '

'0_c o

-._

2 _

=

2-o *,

cd C.) Cu 0

00

CZ_ r

D

C.-, x

'x 3

0 0 0

.Ctc

Cd-^ =>

~ _

CuU

CZ~

0.0

Cd

0. .

- _) 00

0F _ C..

C.o C.. Cu

u 3;0

0 0.= e Z ° _ o

b.

cC .2°-=

-; 0.l

_C);

2276 Owenetal.

I

2

.-1.

-DI0

.I

cholangiography, and/orsurgery.Five patientshadobstructive jaundice

(three intrahepatic, twoextrahepatic): thirteen had parenchymal liver

diseaseand twoofthem (M.F. and M.T.), both with hemolyticanemia, showed acanthocytes in dried blood smears. In the last 13patients

in-vestigated, erythrocytes were examined in wet films and by SEM. Re-versibility of echinocytosis was studied in 30 additional patients, mostly

alcoholiccirrhotics, selected because>10%of theircells wereechinocytes.

HDL were isolatedfrom three patients (K.N.,M.J.,and S.J.) with

abe-talipoproteinemia,andtheirerythrocytemorphologywasstudiedbySEM.

ThesepatientslackapoB-containinglipoproteins and their clinical details have beenreportedpreviously(8).Normalsubjectswerehealthy medical and laboratorystaff.

Reagents. Human serumalbumin (<0.005% fatty acids)and allother

materialswerefromSigmaChemicalCo., Poole, England, except Hanks'

balancedsaltsolution(FlowLaboratories Ltd.,Irvine, Scotland),

Cab-O-Siland Mercksilicagel (BDH ChemicalsLtd., Poole, England), and pronase(Calbiochem-Behring Corp.,SanDiego,CA).

Lipoproteins. Very low density lipoproteins(VLDL, d< 1.006 g/ ml), LDL (1.019-1.063), HDL (1.063-1.21) and lipoprotein-deficient

serum(LPDS, d> 1.25)wereisolated by differential ultracentrifugation

and washed oncewithbuffer ofthe appropriate density(7). The lipo-proteins were usedwithin a day or twoof isolationand were passed through a

0.2-mtm

filter before additionto erythrocytesuspensions;

tech-niquesusedforcompositional analysis, labeling with 125I,andchemical modificationhavebeen describedpreviously(7).

Erythrocyte isolation, morphology, and analysis. Erythrocytes were separatedbycentrifugationat40C ofvenousbloodanticoagulated with

10U/mlheparin. Theywere washed threetimes withHanks'solution, broughtto ahematocritof'-50%andfixed for 30min at0C by adding

anequal volume of 1% glutaraldehyde in Hanks' solution. Morphology

was examined by light microscopy of wet films and frequently by SEM

(Philips501). When whole blood was fixed, erythrocyte shape appeared

identicaltothat in washedpreparations.

UseofSEM hasallowedreclassification of erythrocyte shape anda betterinsight intotherelationshipbetweenspiculated forms (9).Inliver disease four"spiculated"erythrocyteshapescanberecognized in addition

tonormalbiconcave discs(discocytes).These arediscswith protuberances around theedge(echinocytes I), flat cells with spiculesoverthe surface (echinocytesII),sphericalorellipsoidalcellswithalargenumber of spic-ules evenly distributedoverthe surface (echinocytes III),andcells with

abulbousbody and a small numberof irregularlyspacedspicules,

gen-erally with knobbly, club-shaped ends (acanthocytes) (Fig. 1). These shapesmaygenerallybedistinguishedbylight microscopy ofwetfilms,

Figure1.Scanningelectronmicrograph oferythrocytesfromapatient

(M.F.,TableI)with alcoholic cirrhosisand associated hemolytic

ane-mia.Examples of acanthocytes (AC),and type I(E-I),type II(E-II),

and type III(E-III) echinocytesarelabeled. (Inset) Discocytes froma

normalsubject. Calibrationbar= 10Aim.

but we suggest that the identification of acanthocytes should rest on SEM. Using light microscopytheproportion of echinocytes IIand III

in a cell population was determined by counting 200 cells in three to four fields; the observer was unaware of the source of material.

Erythrocyte and lipoprotein lipids were extracted with isopropanol-chloroformandchloroform-methanol, respectively, and the concentra-tionsof individual phospholipids measured as described previously (10,

I1). Total bile acids were determined by the technique of Barnes and Chitranukroh (12).

General incubationprocedures.Washederythrocytes (usually 10 !d containing 6X 109 cells/ml of Hanks' solution) were incubated with4

vol oflipoprotein or reagent. Lipoprotein concentrations are expressed

asmilligram of protein per milliliter, andallconcentrations of lipoproteins

orreagents refer to the final incubation mixture. Incubationswerestopped by fixation with I volof1%glutaraldehyde or, if addition ofasecond reagent was required, by diluting with 50 vol of Hanks' solution, followed by immediatecentrifugation and a secondwash.Further detailsaregiven in legends to our tables and figures. Washed erythrocytes were enriched incholesterol using cholesterol-rich phosphatidylcholine dispersions (13). Binding studies. Washed erythrocytes (25 glof a 10%suspension) were added to 250zdofvarying concentrations of1251-labeledlipoprotein with 0-40mg/mlhumanserum albumin and incubated for 40 min at room temperature with periodic agitation. Cells were separated from unbound lipoprotein, by brief centrifugation of the suspension (200JA)

throughabarrier (50

_jil)

of dibutylphthalate, andbound1251 was mea-sured.Determinationsweregenerally made in quadruplicateandblanks (without erythrocytes) were subtracted; the correction was 10%.

Toidentify binding components, membrane proteins were first frac-tionated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electro-phoresis (14). The protein zones were electrophoretically transferred to

anitrocellulose membraneandnonspecific binding sites blocked by ag-itationfor 24 hin 10 mMTris-HCI, 150mMNaCI, 1 mMEDTA, 2 mM sodium azide, pH 7.5, containing 25 mg/ml bovine serum albumin ( 15). The bufferwasthenreplaced by '25I-labeled patientHDL at

0.1-1.0mgof protein/ml in the same buffer. After2hof gentle agitationat

room temperature the nitrocellulose membrane was rinsed, dried, and leftincontact withpreflashed Kodak X5 film at -70°C for upto 4h. Duplicate lanes of the original SDS gel were stained with Coomassie

blue.

Washed erythrocytes (10% hematocrit) were subjected to proteolysis by incubating for 1 hat37°C with 0.5 mg/ml of trypsin, chymotrypsin,

orpronase in 5 mM sodiumphosphate, 150mMNaCl,pH 7.4. The cellswerewashed, membraneswereprepared, and theirprotein

com-positionswereanalyzed bySDS-gel electrophoresisin0.1MTris-Bicine

N,N-bis(2-hydroxyethyl)glycine,pH 8.1 (15).The"dot-blot"technique was used to establish whether binding of patient HDLstill occurred; membraneproteinwas spottedontowashed nitrocellulose, incubated with radioiodinated HDL and theamountof binding measured.

Other studies. For measurement ofphosphorylationchanges inthe

spectrin of the membrane cytoskeleton, erythrocytes were prelabeled

with 32P (16) and incubated with lipoproteins. Cells were removed at intervals and their membraneproteinswereseparated byelectrophoresis

( 14)andstained withCoomassie Brilliant Blue G. Relativeconcentrations

ofspectrinwereestimated by dye elution with 70% (vol/vol) formic acid and the 32Pcontentby Cerenkov counting.

Erythrocytes(107/ml)weretreatedwith wheat germagglutinin (20

,ug/ml),

incubated withabnormal HDL orotherechinocyticagents for

I minat37°C,andwashed threetimes; bound lectinwasdisplaced by

additionof 20 mMN-acetylglucosamine (17). Erythrocyteswerealso treated with the oxidant diamidetointroducedisulphidebonds

predom-inantly between spectrin chains(18).Cross-linkswereremovedby ad-ditionof 15mMdithiothreitol.

Results

echino-cytesIIand/or III inwetfilms (mean 47%,range 10-98%). Fig. 2 A and Bexemplify the morphologic variation at the lower (14%) and upper (83%) ends of the range. Discocytesand

echi-nocytes I, II, and III could be seeninmostpreparations: spec-imens with the highest degree of morphologic abnormality

con-tainedmainlytypeIIechinocytesand those with the least mainly type I. No acanthocytes were seen in this group of 30 patients. Twoofthe 18 patients (M.T. and M.F., Table I), from whose blood HDL wasisolated, were alcoholic cirrhotics with a

he-molytic anemia and "spur cells." Acanthocytes were evident bySEM(Fig. I andFig. 2 C)butcomprised only4-6% ofthe total erythrocyte population;mostcellswereechinocytes II or

III.Erythrocytes in the other 16patients showed variable echi-nocytosis but no acanthocytes inwetfilmsorbySEM; in only two (A.S. and S.F.) were spiculated cells reported on routine

examination of dried,stained smears. Light microscopyof wet

films allowed discrimination of the various types of echinocytes (Fig. 3 A) and acanthocytes(Fig.3C).LikeGrahn et al.(1), we found almostnotypeII orIIIechinocytesin dried, stained films (Fig. 3 B), but acanthocytes were readily recognized (Fig. 3 D).

In all three patients withabetalipoproteinemia small pro-portions of acanthocytes were present, but most cells were

echinocytes IIandIII(Fig. 2Dand E).

Effects

ofliver diseaseHDLonnormalerythrocytes.Normal erythrocyteswereincubated for I minat370C withHDL from

jaundicedpatients ataconcentration similartothat in plasma

(500 ,ug/ml) (19, 20); echinocytes I, II, or III appeared in the

suspension (Table I, Fig. 4). With some patient HDL, only

B

I

echinocytes I and II were formed (Fig. 4 A); with HDL from others(A.S., M.T., and J.R., Table I) complete conversion to type II orIIIechinocytes occurred (Fig. 4 B). The ability ofa

patient's HDLtotransform normalerythrocytes correlated with the proportion of echinocytes in the patient's blood (r= 0.92, P<0.001;TableI). Less than 1% of normal cells became

echi-nocyteswhenincubated with HDL from normal subjects. Echi-nocytosis occurred within the time requiredtostopthe reaction (>5 s);nofurther changeswere seenwith incubation forup to 4h. The degreeof echinocytosiswasdependenton HDL

con-centration butindependent oftemperature,giving thesameresult

at0°C andat 37°C aftera 1-min incubation (Fig. 5).

Severalpatients' VLDL, LDL, HDL, and LPDSwereisolated and incubated with normal erythrocytes for Iminat37°C.Only

the HDLand LPDS fractions were echinocytogenic at

concen-trations equaltothose in the original plasma. LPDSwasmuch less effectivethan HDLbutmaystill have contained lipoprotein

components, derived artefactually by ultracentrifugation (21), becauseit became inactive ontreatmentwithCab-O-Sil. VLDL, present at lowconcentration in severe parenchymalliver disease (20), becameechinocytogenic only when concentrated above1

mg/ml. PatientLDL was inactive except with longer incubations (>1 h) and higher concentrations (>2 mg/ml). Suspending the washed cells in normal plasma or normal LPDS before addition

of abnormal HDL causedonlyminor reduction inthenumber

ofechinocytes produced. When active patientHDL was incu-bated overnight at 4°C with normal LPDS and recovered by untracentrifugation, it retained its echinocytogenic ability;

nor-Figure2.Scanningelectronmicrographsof

erythrocytesfrompatientswith liver diseaseor abetalipoproteinemia,and of cholesterol-en-riched normalerythrocytes.(AandB)Examples

of the various types ofechinocytesseenin liver ~~~

~disease.Theacanthocytes,of liver disease(M.T.,

TableI)andabetalipoproteinemia,(K.N.and ~~ 4~ S.J.,Reference8)areillustrated in C, D, and E,

respectively. (F) Erythrocytesenriched in

choles-terolbyincubation in vitro witha cholesterol-richphospholipid dispersion (cholesterol/phos-pholipidmolar ratio= 1.38comparedwith 0.85

incontm]rolcels.0

iolk

J v

_{LJ-1_.i}

Add6

'@

_Cow.it

by

"Op

a ~

~~

CHa

J024w

-.

Figure 3.Light microscopyof wet filmsand dried stained blood smearsfrom patientswith liver disease. (A) Echinocytes are clearly

vis-iblewhen wet films are examined from patient P.K. (TableI)but are

mal HDLdid notacquire shape-transforming properties after

incubation with patientLPDS.

Reversibility of the discocyte-echinocyte transformation. Echinocytes formed inseconds retained their abnormal shape

onprolongedincubation(upto 4h) withthepatient HDL, and alsoshowed little reversalafter washing and subsequent rein-cubation in Hanks' solution for up to 8 h. When they were

incubated with normalHDL therewas completereversalto

nor-mal erythrocyte morphology within seconds (Fig. 6). Normal LPDSinducedonlypartial reversaleven athighconcentrations.

Reversibility ofechinocytesfoundin liver disease. Normal

HDL(8mg/ml) wasaddedtosuspensions of erythrocytesfrom

rarely seenin driedsmears(B). Inapatient with "spur cell"anemia (M.T., TableI) acanthocytescan bedistinguished fromechinocytesin

wet films (C) and are also recognized in driedfilms (D).

30 patients with liver disease, whose blood contained at least 10% echinocytes II and III. In each case there was a rapid re-duction in the percentage of type II and IIIechinocytes(Fig. 6 andFig. 7). Complete reversal was rarely achieved, possibly be-cause the cells had adapted to theirnew morphology during

prolongedexposure totheechinocyticagentand reversedonly slowly when the agent was removed (22). Excess normal HDL alsoameliorated the echinocytosisof cells from the two patients

with spurcellanemia but the proportion of acanthocytes was

unchanged,suggesting that acanthocytosiswasirreversible.

Binding studies.Lipoproteinbinding by erythrocytes reached

equilibrium within 30 min, but the small number of sites per

Figure4.Ability ofHDLfrom patients with liver diseasetotransformnormalerythrocyte into echinocytes. Normal, washed

erythrocytes (6X 109cells/mlHanks'buffer)wereincubated for 1 min at

370C

withfreshly isolatedHDL(final

concentra-tion 500jgofprotein/ml) from patients S.F. (A)andA.S. (B). v!p --

ro-V.S.. 0

4

_i15

4

.-

%Al

v

_a

61

.

9

sb^.I

4 c

f4l04;

-01 -.rl A.

r4f

w

(nb

Mi

0

4

rp

la

1oo

1-,

0)4

*-,

0-cn

CU

C-,

LL

be-

75-50

251

.00

-/- - A

-

-0' I;_.-- - - --

-0 25 50 15 100

Echinocytogenic Agent (arbitrary units)

Figure5.Transformation of normal

erythrocytes

to

_echinocytes

at

00C

and370C. Normal,washed

erythrocytes (6

X

₁₀₉

_cells/mlHanks'

buffer)

wereincubatedfor 1 minateither0C(open

symbols)

or

370C

(solid symbols)

with

_increasing

concentrations of

_freshly

isolated

HDLfrom

patient

M.A-M.(o,

.;

100U= 1 mgof

protein/ml),

lyso-lecithin-enriched

plasma

(0,

.;

100U=430 nmol

lysolecithin/ml

plasma)orplasmaenriched with bile salts(a,_,; 100U=2.5jmol

totalbile

acids/ml

plasma).Thecellswerethenfixed and the percent-ageof

_echinocytes

IIand IIIdetermined inwetfilms

by

differential

counting

of 200cellsin threetofourfields

_using

_{light microscopy.}

celllimited

_precision

ofmeasurement.

_{Strongly echinocytogenic}

HDL

_(patient

_A.S.,

Table

_I)

was bound

by erythrocytes

in a

saturablemannerwhen added in

increasing

concentration

_(Fig.

8).

Saturation of sites wasachieved atan HDL concentration of

_~-40

nM

₍₁₀

_,ug/ml),

atwhich

_point

conversionto

erythrocytes

II and III had reached a

_plateau

at -85%. Labeled HDLwas

displaced by

unlabeled

HDL,

andcountswerealso

_{progressively}

lost

_by

_repeated

_washing

of the cells. These

_findings

areconsistent with a reversible

_binding

process, and

assuming independent,

noninteracting

sites,

the

_binding

curve canbe

satisfactorily

fitted

by

a

single binding

_processwithanassociationconstantof

-X 108M-1

_(inset

to

_{Fig. 8).}

There

appeared

tobe

4,000-5,000

sitespercell.Defatted albumin reducedtheamountof abnormal

HDL

bound,

buta

_large

_partwasnot

_{displaced (Fig. 9). Thus,}

despite

thefit

_{(within experimental error) by}

a

single

association

constant, theremaybemorethanone_typeof

site;

one

_population

being

specific,

the other

nonspecific

andavailablefor

competition

by

albumin. Such studies

_required

_large

amountsofHDLand

only

oneother_potentHDLwasexamined

(patient

_M.T.,

Table

I);

this also gavea

_{binding profile}

_{consistent with the presence}

of saturable sites.

By

contrastnormal

HDL,

and

patient

HDL withlittleor no

_{echinocytogenic}

_activity,

didnotappeartobind

tothesites.No saturable

binding

was

detected,

andinstead there

was acontinuous

uptake

of

lipoprotein

in themannerofa par-titionprocessoverthewide concentration rangetested

(0-6,000

nM,

data not

_shown).

Normal

LDL,

which did induce

eryth-rocyte

shape changes

on

_{prolonged incubation,}

also showed

nosaturable

_binding.

This

disagrees

with the report of Huiet

al.

_(6).

The dot-blot

technique

indicated that

_binding

of radioio-dinated

_patient

HDLcould beaffected

by proteolytic

pretreat-mentof

_erythrocytes

andpronasetreatment

_largely

eliminated

binding. However, trypsin caused only partial reduction of binding and chymotrypsinwasineffective,possibly because these

proteasesdegradeonly certain of theexternally accessible

pro-teins(23)orbecause cleavedfragments remain associated with the residue of the parent molecule(24). When labeledpatient

HDL was incubated with the electrophoretically

separated

membraneproteins several bandsweredetectedby

autoradiog-raphy. Thesewereweak, presumably because the small number of sites hadrelatively modest associationconstants,and didnot

obviouslycorrespondtomajor membraneproteinswhen stained

withCoomassie blueorwithperiodate-Schiffreagent.The pro-nase-sensitive membraneproteins which bound patient HDL

wereapproximatelyin thepositions of bands 3, 4.9 (or 5), and 6 in the Fairbanks numberingsystem(14). This heterogeneity

suggests that thereceptors areglycoproteins,manyof whichare

presentin erythrocyte membranes atlow concentrations(25),

somedifferingonly in theircarbohydrate moieties.

Spectrin andechinocytosis. Phosphorylation ofspectrin in the erythrocyte cytoskeleton was unaffected by lipoproteins

withinthe incubationtimerequired for shape changes viz.

sec-ondsfor abnormal HDL andup to Ih for LDL(Fig. 10). The

result forLDL conflictswith that of Hui and Harmony (26).

Low concentrations of diamide were added toerythrocytesto

causelimited thiol oxidation with the formation ofcross-links,

mainly between spectrin molecules (18). These cells became partiallyresistant topatient HDL;45-50%oferythrocytes treated

with diamide remained discocytic after addition of abnormal

HDLcomparedto <I0%ofuntreated cells. Theprotective effect

of diamide was reversed on reduction of the cross-links with

dithiothreitol.

Apolipoproteins andechinocytosis. The proportion ofapoE

in HDL from our 18patientswashigher(I13.2±7.3%, mean±SD, range 4.0-30.0%) than that in HDL from 10normal subjects (3.9±1.0%, range 2.6-5.4%; P < 0.001),butthere was no

sig-nificantcorrelation withechinocytogenic potency (r=0.26, P

> 0.05; calculated from Table I). Agents that prevent apoE

binding by apoB,E receptorsonnucleatedcells,e.g.,protamine (200 utg/ml)orheparin (10 mg/ml),orcyclohexanedione mod-ification ofapoliprotein arginine residues (7), did not impede

echinocytosis. Moreover, discocytes from patients with homo-zygous familial hypercholesterolaemia, which lack apoB,E

re-ceptors, were readily transformed to echinocytes. HDL from

threepatientswithabetalipoproteinemiaalsocontainedexcess

apoE (10.5±0.8%, range 9.6-11.1%)but were nonechinocyto-genicatconcentrationsupto 1.5mg/ml; presumablyother

fac-torsare moreimportantforechinocytosisinthesepatients, such

asthe increasedsphingomyelincontent(8) in the cell membrane.

Twoadditional,morebasicapolipoproteins, previously described (7), were found in HDL from nine of the patients with liver disease,but theirpresencedid not reflectechinocytogenic activity

(r

=

-0.31,

P>0.05).

Relationofchangesinerythrocyteshape to erythrocyte

cho-lesterol content. Echinocyteformation by patient HDL, as

ex-pectedfrom the rapidityof theprocess,was notaccompanied

bynetcholesteroluptake (TableII). Furthermore, the lipid

con-tentof theseechinocytes,and those ofpatients, did notchange

onincubation with normal HDL and reversion tonormal shape. PartialdelipidationofpatientHDLremoved 92%of cholesterol,

24% ofphospholipid,andessentially all ofthecholesterylester andtriglyceride,butdidnotaffectechinocytogenic potency. We

found,inagreementwithothers(I13, 27, 28), thatnormal eryth-rocytesbecamebroader and flatteronenrichment with

A

V

*m

1,

_-w

Figure 6.Reversibility of echinocytesproducedby incubation of nor-mal red cells with HDL isolated from jaundiced patients and of

echino-cytes foundinliver disease. Normal erythrocytes weretransformedto

echinocytes IIand IIIby HDLfrompatient A.S. as described in Fig. 4 (A).Afterwashing, these echinocytes were incubated for I minat

brane cholesterol byequilibrationwithcholesterol-richliposomes

(Fig. 2 F); many showed peripheral distortion as in typeI echi-nocytes. Type II echinocytes were rare, and probably resulted from prolonged incubation, in that they also appeared in

con-trols.

Amphipaths andechinocyteformation. Small amounts of amphipathic anions or other ligands causeechinocytosisby

par-titioning mainly into theouterleaflet of the membrane bilayer andexpanding its area relative to the inner leaflet (29-31). We studiedtwophysiologic amphipaths,bile salts and lysolecithin,

todetermine whether they could be the active components of abnormal HDL. Echinocytosis by bile salts orlysolecithin,and its reversal by addition of normal HDL, was rapid and the

echinocytes produced resembled by SEM thosegenerated by

patient HDL. However, HDL-inducedechinosytosiscould not

beexplained by detectable increasesoftheseamphipaths in active HDL;freshly isolated echinocytic HDL contained no more ly-solecithin than normalHDL(1-2% oftotalphospholipid)and had a low totalbile acid content (2-9 nmol/mg protein) when comparedtoinactiveHDLfrom fourpatientswithextrahepatic

obstructive jaundice (1 1-17 nmol/mg protein). Furthermore, we foundthe echinocytogenic effects of bile salts,lysolecithin

andpatientHDL todiffer. Normal plasmawasenriched with eitherlysolecithin (by incubationat37°C)orbile salts(byadding equal amounts of sodium taurocholate and sodium

glycoche-nodeoxycholate)untilit produced 65-85% typeIIandIII echin-ocytes on I minof incubation at 37°C. In contrast topatient

w_-ism

S

370CwithnormalHDL(finalconcentration 8mgofprotein/ml)to

give completereversal todiscocytes (B). Echinocytes fromapatient withalcoholiccirrhosisalsoreturned towards a normal erythrocyte

shapewhensimilarly incubatedwith normalHDL;thepercentageof echinocytesII and IIIpresentwerereduced from 14%(C)to zero(D).

HDL,far less echinocytosis occurred when the incubations were repeated at00C(Fig. 5). Echinocytesformedby exposuretobile salts revertedto discocytesafter three washes with Hanks'

so-lution; those generated bylysolecithin or by patient HDLdid

not revert. Washed echinocytes produced by lysolecithin

ac-quiredamorenormalshapeafter7-8hofincubation, presum-ably becauselysolecithin progressivelyenteredthe inner

mem-braneleaflet(31), but thoseproduced by patientHDLdidnot reverse. VLDL, LDL, HDL, and LPDS were prepared from plasma enriched withlysolecithinorbile salts and washedonce

byultracentrifugation, andtheir volumeswereadjustedtothat of the original plasma sample. Only the LPDS fractions were

foundto beechinocytogenic;the HDL produced <1% type II andIIIechinocytes.

These resultsseem to us toexcludelysolecithinandbilesalts

asthe active constituents ofechinocytogenic HDL. To assess

whetherother, sofarunrecognized,amphipaths mightbe present we(reversibly) fixed a erythrocytesuspensionin thediscocytic shapewith wheat germagglutinin (17). Aliquotswereincubated either with plasma enriched inlysolecithinorinbile salts

(pre-paredasabove) orwith abnormal HDL. In each case

echino-cytosiswasmarkedly inhibited,thenumberoftype II and III

echinocytes produced being reduced from 65-85%to 3-10%. Reagents were removedbywashingthreetimeswith 50 vol of

Hanks'buffer (without albumin)andthen the boundlectinwas

dissociated by addition ofN-acetylglucosamine. Cellsexposed

tolysolecithinbecamepredominantly echinocytic;asexpected,

100r

80

.

60

IV

G)

8

40k

20L

oL

+

Buffer

+ _Normal

IHDL

Figure 7.Reversibilityofechinocytes found in liver disease by normal HDL.Suspensionsof washed erythrocytes (6XI09 cells/ml Hanks' buffer) containing at least 10% ofechinocytesIIandIII wereobtained from 30 patientswith liver disease, mainly alcoholic cirrhotics. One portion of the cellswasincubated with normalHDL(final

concentra-tion 8 mg ofprotein/ml)for 1 minat370C and another with Hanks' buffer. The cells were thenfixed andthepercentage ofechinocytesII and IIIdetermined in wetfilms by differential counting of 200 cells in threetofour fieldsusinglight microscopy.

0.6

S0.4

0 0

C~~~~~~~~~

_0~~~~~~~~~

u0.2 -£

-IH~l~e h

0 VI0.40 S8

2

0.1 0.2

HDJ1

4

fHL~ree(OM)

Totalconcentration of HDL (nM)

Figure8. Saturablebinding of'25I-labeledliver disease HDL by

nor-malerythrocytes.Cells were incubated for 40 min at room

tempera-turewithincreasingconcentrations ofHDLfromapatient with

lu-poid hepatitis(A.S., Table

I).

Eachpoint isthemeanof four determi-nations; bars areISD. The molecularweight of the HDL was

assumed to be 0.25X 10'andthe curve ( ) is calculated for 4,500

bindingsites per erythrocyte and_K.= _I X 108MW.Inset:double

re-ciprocalplot ofl/[HDL]boundvs.

l/[HDL]f,.

°5 0.34 % 0

I \

No 0.2- °

C, 0.1'

0 10 20 30 40

Albumin (mg / ml)

Figure9.Inability of human serum albumintopreventbinding of liver disease HDL by erythrocytes. ThepatientHDLand incubation conditions were as described in Fig. 8 except that each tube contained up to40 mg of defattedhumanserum albumin per milliliter, Final concentrations ofpatientHDL wereeither31 ug ofprotein/ml (o)or

14

_,ig

ofprotein/ml(-).

the membrane of lectin-fixed cells took up theamphipathand

retained it during washing andlectin dissociation. Cellstreated with bile salts remained discocytic, because washing reverses bile salt-inducedechinocytosis (unlikethat inducedby lysolec-ithinorby patient HDL,seeabove).Cellsexposedtoabnormal

HDLremainedlargely discocytic, suggestingthat abnormal HDL does not actby adding (orremoving) lipophilicmembrane

con-stituents;it must occupy its receptors to beechinocytogenic.

Discussion

The literatureonerythrocyte shapein liver diseaseisconfused

byinconsistentterminology.The term"spur cell,"whichshould

be reservedforacanthocytes,has often been used forspiculated cells,seenin vivo orgeneratedinvitro,that are in factechinocytes (as defined by Brecher and Bessis [9]). Spurcell anemia, (i.e., with acanthocytes)is rare, but we havefrequently found echino-cytesinjaundiced patientswhen their blood was examined in

wetfilms; unlike acanthocytes, they were notusually evidenton

routine examinationofstainedsmears. Theproteinor

lipopro-Tome (min

Figure JO.Inability of liver disease HDL,or normal LDLand normal

HDL,todephosphorylatespectrin innormalerythrocytes.Erythrocyte phosphoruswaslabeledbypreincubation with32Piand then incubated for upto 1 hwith buffer (o),HDLfromapatient (M.T., Table I) with alcoholic cirrhosis(0;0.5 mg ofprotein/ml), normalHDL(n; 1.0mg of protein/ml)or normal LDL(-; 1.0mg ofprotein/ml).Membrane proteinswereseparated bySDS-polyacrylamide gel electrophoresis (14) andthe bandscorresponding tospectrin were removed. Their32p

contentwasmeasured by Cerenkovcountingandthevalues were

nor-malizedto aconstantspectrinconcentrationasestimated by dye elu-tionofthestainedbands.

2282 Owenetal.

;400 .

m11 -i

E O' 3(W

c _a a

i

0 w .

sw-4.

1.₂₀₀

I2

I

0 0.₁₀₀

t

0 .G

CL

0-Table II. Unaltered Erythrocyte Lipid Composition after HDL-induced Changes in Erythrocyte Shape

'5 Total lipidphosphorus

Erythrocyte source Cholesterol Phospholipid LL§ SM _Pi PS L PE PA

nmti/h107cells nmol/10'cells % % '5 5 % %

Normal cells 3.55 4.08 1.1 21.1 0.6 13.5 29.1 27.5 2.1

Transformedcells* 3.54 4.17 1.0 26.9 0.5 12.8 29.6 27.4 1.8

Transformed cells reversed with normalHDL* 3.68 4.10 1.1 27.8 0.5 12.9 29.4 26.3 2.0

Patientcells 5.68 4.32 0.8 18.9 0.7 11.8 43.7 22.2 1.9

Patientcellsincubated with normal

_HDL*

5.86 4.44 0.9 18.6 0.5 12.4 42.9 22.5 2.2

*Washed,normal erythrocytes (6 X 109cells/mlHanks'solution) were incubated for I minat

370C

withfreshly isolated HDL from patient M.T.

(finalconcentration 250

,gg

ofprotein/ml).All cells weretransformed to echinocytes II orIII.Afterwashing, one portion of the transformed cells wasincubated for I min at370Cwith normalHDL(8mg/ml)to give complete reversal todiscocytes. Similar results were found in a further

experiment usingHDLfrompatient A.S. 4 Washederythrocytes (6 X 109cells/mlHanks'solution) from patient A.S. were incubated for I min

at37TC withnormal HDL(8 mg/ml) toreducethepercentage of typeIIor III echinocytes (from 69% to21% of the cells). The experimentwas

repeated twiceusing erythrocytes from two additional patients (M.T. and M.F.); the same conclusions were drawn (data not shown). §

Abbrevia-tions: LL,lysolecithin;SM,sphingomyelin,PI,phosphatidylinositol;PS, phosphatidylserine; L, lecithin;PE, phosphatidylethanolamine; PA,

phos-phatidic acid.

tein fraction(s) present in liver disease plasma that is responsible forechinocyteand/oracanthocyte formationhas notpreviously

been identified (1, 32, 33) but our results establish that the

echinocytogenicagent canbeisolatedwith abnormal HDL.

Inliver disease, erythrocyte membranes tend to acquire ex-cesscholesterolfrom abnormalcirculatinglipoproteins (34, 35), butthis is unlikely to explain echinocytosis, inasmuch as spic-ulated cells have been found unaccompanied by elevation in membrane cholesterol content(32) andviceversa(36). The shape ofcells loaded with cholesterol inourexperiments,and in others (13, 27, 28), bore little resemblance to the echinocytes

ofjaun-diced patients and could not be reversed by briefincubation with normal HDL. Cholesterol enrichment was evidently un-related toechinocytosis inducedin vitro by abnormal HDL, for thelatter occurred in seconds, without increase in membrane cholesterol, and could also be induced by cholesterol-de-pleted HDL.

Two physiologic amphipaths producing echinocytes, lyso-lecithin and bile salts,wereexcludedastheactive constituents ofpatient HDL; echinocytogenesis by these agents, and the

propertiesof theresultingechinocytes, differed in several ways from those induced by abnormal HDL. Moreover,our

experi-mentswithwheat germ agglutinintendtoexcludeother, sofar

unrecognized,amphipathsasechinocyticcomponents ofpatient

HDL,evenwere there evidence oftheirexistence;in any case, thedifferentpropertiesof the

echinocytes

generatedbyknown

amphipaths renders sucha mechanism

unlikely.

Weconclude thatechinocytogenesisinvolves the

occupation

of

binding

sites

onthecellsurface byabnormal HDL. Howthiscausesthe im-balance ofarea between the membrane leaflets

implied

by echinocytosis

(22,

37-39) is uncertain, although cytoskeletal

changes (thoughnotdephosphorylationof

spectrin)

may be

in-volved, in thatpartial

cross-linking

ofspectrinmoleculesby di-amide inhibitedechinocytosis. Removalof abnormal HDLby

washing did notreverse the echinocytosis, possibly because a

slowpassiveadaptation by thecytoskeleton (22, 38,

39)

is re-quiredfor reversal in the absence of normal HDL.

Ourevidence suggests that abnormal HDLactsvia saturable receptors,numbering -5,000per cell. Because

binding

and

rapid

echinocytosisoccurredwithin thesameconcentration range, and

because otherplasma lipoproteinsthat do notshowsaturable

bindingwere inactive,it seems reasonable to conclude that

re-ceptorbinding and shape change arecausally related. The

re-ceptors involvedareunlikelytobe the 160,000-molwtapoB,E surface-receptors present in nucleated cells inasmuch asthese have not beenfound in mature red cells (40). Moreover, neither agents that preventbinding oflipoproteins by apoB,E receptors, northe use oferythrocytes from apatient withagenetic defi-ciency of apoB,E receptors on their nucleated cells, impeded

echinocytosisby abnormal HDL. Our tentative conclusionthat

the receptors areglycoproteins is basedon their susceptibility

toproteolysis and theirheterogeneity; proofwill require addi-tional experimental evidence. Their function is also obscure, although other cell types have surface receptors whichbind

li-poprotein moleculeswithoutinternalization (41). Theymay,as suggestedfor insulin receptors(42),remain fromanearlier stage oferythroidcelldevelopment.

Wehave not yetidentified the constituent(s) of abnormal

HDLrequiredfor receptorbindingandechinocytosis, although

theretention of echinocytogenic activity after partial delipidation suggestsanapolipoproteinasthe active component. If this isa

minor part of the total protein, thenthe association constant will beconsiderably higher than is apparent from the binding

curves. Liver disease HDLismarkedly heterogeneous(19, 20, 43) and ourpresent aim isto isolate a population of potent, homogeneous HDLmolecules andtoidentify theactive

com-ponent.

What is thepathophysiologic consequence oferythrocyte

shapechangesinliver disease?Erythrocytesurvival is often

di-minished inpatientswith liverdisease,butisnotusuallyamajor

clinical problem. Target cellsarethoughttohaveanormallife span(34), butacanthocytosisinliver disease isassociatedwith

marked hemolysis and predominant

splenic destruction,

al-though anemia isnot aprominentfeature of

abetalipoprotein-emiaorofcertainother

acanthocytic

disorders

(44).

Echinocytes

mightbeexpectedtohave ashortened lifeasthe

spleen

removes

many cells of abnormalshape.Powelletal.

(45)

considered that

onlyinpatientswith spurcells didmorphologicalterations lead tothe premature removal of cells

(though

their

scanning

electron

hemoglo-bin levelsin ourpatients didnotcorrelatecloselywith the

num-berofechinocytes in their blood. However, hemoglobin con-centration in liverdisease is altered bymany factors(34)and

the degree of hemolysis is bestassessedbydirect measurement. Areechinocytes the precursorsofacanthocytesassuggested byGrahn et al. (1) and Cooperetal.(46)andwhatis the

mech-anism for the conversion?Thepreponderanceofechinocytesin ourpatients with spur cell anemia and with abetalipoproteinemia is consistent with suchaprecursor-product relationship.

How-ever,webelieve the suggestion (13, 46) that,inliver disease,the

conversionoccurs inthespleenas aconsequenceof cholesterol

enrichmentmustbeviewed with caution. Evidence forasplenic

conversion isderived from asingle case(46)whereas,in other

disorders, splenectomy can itself produce acanthocytosis (47). Therelevance of cholesterolenrichmenttoacanthocytogenesis mustalso bequestioned, as inabetalipoproteinemiathe increase in cell cholesterol content is only marginal. It seems, therefore,

that inliver disease the echinocytic shape,inducedbyabnormal

HDL, may be a more critical factor to the conversion than the reductions in membrane fluidity (35) and celldeformability(13)

caused by excess cholesterol. Clearly, additional studies are

needed, both toestablish therelationship between erythrocyte survival in liver disease and cell morphologyand membrane

lipidcomposition, and to understand in greater detail factors regulating erythrocyte shape in general.

Acknowledgments

Wethank ProfessorJune Lloyd, Dr. BeatriceWonke, and Dr. Tom

Warnesfor allowingus tostudypatientsundertheir care. We are grateful toJackie Lewin and LindaBoxerinthe DepartmentofHistopathology for their help and patience in preparing erythrocytesamplesforstudy

by SEM. We thankMr. A.Chitranukrohfor the bile acid measurements. ThePhilips SEM 501waspurchasedwithagrant from the Stanley

Thomas Johnson Foundation. Dr. Beaven was a Medical Research

Council projectgrantholder.Dr.Owen thanks the WellcomeTrustfor

anInterdisciplinary Research Fellowship and the British Heart Foun-dationforaprojectgrant.

References

1.Grahn,E.P.,A. A.Dietz, S. S.Stefani,andW.J.Donnelly. 1968.

Burrcells,hemolytic anemia and cirrhosis.Am.J.Med. 45:78-87. 2.Smith, J. A., E. T. Lonergan, and K.Sterling. 1964. Spur-cell anemia: hemolytic anemia with red cells resembling acanthocytes. N.

Engl.J. Med. 271:396-398.

3.Silber,R., E.Amorosi,J.Lhowe, and H. J. Kayden. 1966. Spur shaped erythrocytes in Laennec's cirrhosis.N.Engl.J.Med. 275:639-643.

4.Douglass, C.C.,M.S.McCall,and E. P.Frenkel. 1968.The

acan-thocyte in cirrhosis withhemolyticanemia.Ann.Intern. Med. 68:390-397.

5.Hui,D.Y., andJ. A. K.Harmony. 1979.Interactionofplasma

lipoproteinswith erythrocytes.I.Alteration of erythrocyte morphology.

Biochim. Biophys.Acta.550:407-424.

6.Hui,D.Y.,J.G.Noel,and J. A. K.Harmony. 1981. Bindingof

plasmalowdensitylipoproteinsto erythrocytes.Biochim.Biophys. Acta.

664:513-526.

7.Owen,J.S.,H.Goodall, P.Mistry,D.S. Harry, R. C. Day,and

N. McIntyre. 1984. Abnormalhigh-densitylipoproteins from patients withliver disease regulate cholesterol metabolism in cultured human

skinfibroblasts. J. LipidRes.25:919-931.

8. Herbert,P.N.,A.M.Gotto, andD.S.Fredrickson. 1978. Familial lipoprotein deficiency (abetalipoproteinemia, hypobetalipoproteinemia,

andTangierdisease).InThe Metabolic Basis of Inherited Disease. J. B.

Stanbury,J. B.Wyngaarden,andD. S.Fredrickson,editors.

McGraw-Hill BookCo., Inc.,New York.544-588.

9. Brecher,G.,andM. Bessis. 1972. Presentstatusofspiculatedred

cells andtheirrelationshiptothediscocyte-echinocytetransformation: acritical review. Blood. 40:333-344.

10.Owen,J.S.,R. A. Hutton,R. C.Day, K. R.Bruckdorfer,and N.McIntyre. 1981. Plateletlipid compositionandplateletaggregation

in humanliver disease. J.LipidRes.22:423-430.

11.Owen,J.S.,V.Ramalho,J. C. M.Costa,and M. P. T. Gillett.

1979.Determination of lecithin: cholesterolacyltransferinmouseplasma

and theinfluence of mercaptoethanol andsulphydryl blockingagents

onitsactivity. Comp.Biochem.Physiol.63B:261-265.

12.Barnes,S.,and A. Chitranukroh. 1977. Asimplifiedprocedure

for the isolation of bileacids fromserum basedon abatch extraction

with thenon-ionic resin Amberlite XAD-7. Ann. Clin. Biochem. 14:

235-239.

13. Cooper,R.A.,E.C. Arner,J. S.Wiley,andS. J. Shattil. 1975.

Modification of red cell membrane structure by cholesterol-rich lipid dispersions.Amodel for theprimary spurcelldefect. J. Clin. Invest. 5 1:

3182-3192.

14.Fairbanks, G.,T. L.Steck,andD.F. H.Wallach. 1971.

Electro-phoreticanalysis ofthemajorpolypeptidesof the humanerythrocyte

membrane.Biochemistry. 10:2606-2617.

15.Kendrick-Jones, J.,E.M.Szentkiralyi,and A.G. Szent-Gyorgyi. 1976.Regulatorylightchainsinmyosins.J.Mol. Biol. 104:747-775.

16. Wolfe, L. C., and S. E. Lux. 1978. Membrane protein

phos-phorylation of intactnormal andhereditary spherocytic erythrocytes.J.

Biol. Chem. 253:3336-3342.

17. Lovrien, R. E., and R. A. Anderson. 1980. Stoichiometry of

wheat germagglutininas amorphology controlling agentand as a

mor-phology protective agent forthe human erythrocyte.J. Cell Biol. 85:

534-548.

18. Haest, C.W. M., D. Kamp, G. Plasa, and B. Deuticke. 1977.

Intra- and intermolecular cross-linking of membrane proteins inintact

erythrocytesandghosts by SH-oxidising agents. Biochim. Biophys.Acta.

469:226-230.

19. Agorastos, J., C. Fox, D. S. Harry, and N.McIntyre. 1978.

Lec-ithin-cholesterol acyltransferase and the lipoprotein abnormalities of

ob-structive jaundice. Clin. Sci. Mol. Med. 54:369-379.

20. Day,R. C., D. S. Harry, J. S.Owen,A.Y. Foo,and N. McIntyre. 1979. Lecithin-cholesterol acyltransferase and the lipoprotein

abnor-malitiesof parenchymal liver disease. Clin. Sci. 56:575-583.

21. Oram, J. F., J. J. Albers, and E. L. Bierman. 1980. Rapid regulation oftheactivity of the low density lipoproteinreceptorof culturedhuman

fibroblasts.J.Biol.Chem. 255:475-485.

22. Alhanaty, E.,and M. P.Sheetz. 1981. Control of the erythrocyte membrane shape: recovery from the effects of crenatingagents. J. Cell

Biol. 91:884-888.

23. Furthmayr,H. 1978.GlycophorinsA,B and C:afamily of

sialo-glycoproteins. Isolationand preliminary characterization oftrypsin

de-rived peptides. J.Supramol. Struct. 9:79-95.

24.Steck, T.L., J.J. Koziarz,M. K. Singh, G. Reddy, and H.Kohler. 1978. Preparationand analysis of seven major,topographically defined fragmentsofband 3, thepredominant transmembrane polypeptide of

human erythrocyte membranes. Biochemistry. 17:1216-1222.

25. Robinson, P. J., F. G. Bull, B. H. Anderton, and I. M. Roitt. 1975. Directautoradiographic visualisationinSDS-gels of lectin-binding

components of the human erythrocyte membrane. FEBS (Fed. Eur.

Biochem. Soc.)Lett. 58:330-333.

26. Hui, D.Y.,and J. A. K.Harmony. 1979. Interaction of plasma

lipoproteins with erythrocytes. II. Modulation ofmembrane-associated enzymes. Biochim. Biophys.Acta. 550:425-434.

27. Hui, S. W., C. M. Stewart, M. P. Carpenter, and T. P. Stewart. 1980. Effects of cholesterol on lipid organization in humanerythrocyte

membrane. J. Cell Biol. 85:283-291.

28. Lange, Y., H. B. Cutler, and T. L. Steck. 1980. The effect of

cholesterol and other intercalated amphipaths on the contour and stability

oftheisolatedred cell membrane. J.Bid.Chem. 255:9331-9337.

29.Conrad, M. J., and S. J. Singer. 1981. The solubility of

amphi-pathicmolecules in biological membranes and lipid bilayers and its

im-plications formembrane structure. Biochemistry. 20:808-818. 30. Lange, Y., and J. M. Slayton. 1982. Interaction of cholesterol andlysophosphatidylcholinein determining red cell shape. J. Lipid Res. 23:1121-1127.

31.Mohandas, N., A. C. Greenquist, and S. B. Shohet. 1978. Bilayer

balanceandregulation ofred cellshape changes. J.Supramol. Struct. 9: 453-458.

32. Cooper, R. A. 1969. Anemia with spur cells: a red cell defect

acquired inserum and modified in the circulation. J. Clin. Invest. 48: 1820-1831.

33.Martinez-Maldonado, M. 1968. Role of lipoproteins in the

for-mation ofspur cellanaemia.J.Clin. Pathol.21:620-625.

34.Cooper,R. A.1980. Hemolyticsyndromes and red cell membrane

abnormalities in liverdisease. Semin. Hematol. 17:103-112.

35. Owen,J.S., K. R. Bruckdorfer, R. C. Day, and N. McIntyre. 1982. Decreased erythrocyte membranefluidityandaltered lipid

com-positionin humanliver disease.J.LipidRes. 23:124-132.

36.Godin,D. V.,G.R.Gray,and J.Frohlich. 1978. Erythrocyte membranealterations in lecithin:cholesterolacyltransferase deficiency.

Scand.J.Clin. Lab.Invest. 38(Suppl. 150): 162-168.

37. Kuypers, F. A., B. Roelofsen, W. Berendsen, J. A. F.Opden Kamp,and L. L. M. van Deenen. 1984. Shape changes in human eryth-rocytesinducedby replacementofthenativephosphatidylcholinewith

species containing various fatty acids.J.Cell Bio. 99:2260-2267. 38. Lange,Y., R. A.Hadesman,and T. L.Steck. 1982. Roleofthe

reticulum inthestabilityand shape of the isolated human erythrocyte membrane. J.Cell Bio. 92:714-721.

39. Lange, Y., A. Gough, and T. L. Steck. 1982. Role of the bilayer in theshapeofthe isolated erythrocyte membrane.J. Membr. Biol.69: 113-123.

40. Goldstein, J. L., and M. S. Brown. 1983. Familial hypercholes-terolaemia.InTheMetabolic Basis of InheritedDisease. J. B.Stanbury,

J. B.Wyngaarden, D. S. Fredrickson, J. L.Goldstein,and M. S. Brown,

editors. McGraw-HillBookCo.,Inc., New York.672-712.

41.Owen, J. S., N. McIntyre, and M. P. T.Gillett. 1984. Lipoproteins,

cell membranes and cellularfunctions. Trends Biochem.Sci. 9:238-242. 42. Dons, R. F., L. M. Corash,and P. Gorden. 1981. Theinsulin

receptorisanage-dependentintegralcomponentofthe human

eryth-rocyte membrane.J. Biol. Chem. 256:2982-2987.

43. Wiedman, S. W., J. B. Ragland, and S. M. Sabesin. 1982. Plasma

lipoprotein composition inalcoholic hepatitis: accumulation of apoli-poprotein E-rich high density liapoli-poproteinandpreferentialreappearance

of"light"-HDL during partialrecovery. J.LipidRes. 23:556-569. 44.Shohet, S. B. 1974."Acanthocytogenesis"-orhow the red cell wonitsspurs. N. Engl.J.Med. 290:1316-1317.

45. Powell, L. W., J. W. Halliday,and B. R. Knowles. 1975. The

relationship ofredcellmembranelipidcontent toredcellmorphology

andsurvival in patients with liver disease.Aust. N. Z. J.Med.

5:101-107.

46.Cooper, R. A., D. B.Kimball, and J. R. Durocher. 1974. Role

ofthespleen inmembraneconditioning and hemolysis ofspur cells in

liverdisease.N.Engl.J. Med. 290:1279-1284.

47. Brecher, G.,J. E. Haley, and R. 0. Wallerstein. 1973.Spiculed erythrocytes after splenectomy. Acanthocytesornon-specific poikilocytes?

In RedCell Shape. Physiology, Pathology, Ultrastructure. M. Bessis,

R.I.Weed, and P. F.Leblond, editors.Springer-Verlag,New York.