Bile Salts and Cholesterol in the Pathogenesis

of Target Cells in Obstructive Jaundice

RcICARD A. COOPERand JAMESH. JANDL

From the Thorndike Memorial Laboratory and 2nd and 4th (Harvard) MedicalServices, Boston City Hospital, and the Department of Medicine, HarvardMedical School, Boston, Massachusetts 02118

ABSTRACT Free cholesterol is in rapid equi-librium between serum lipoproteins and red cells.The level of red cell cholesterol is influenced bybile salts, which shift the serum/cell partition offree cholesterol to the cell phase and which inhibitthe cholesterol-esterifying mechanism. During in-cubation in normal serum possessing an activecholesterol-esterifying mechanism, red cells losecholesterol and surface area and thereby becomemore spheroidal and less resistant to osmotic lysis.When exposed to serum from patients with ob-structive jaundice or to normal serum with addedbile salts, red cells accumulate cholesterol andincrease their surface area, thereby acquiring aflattened shape and an increased resistance toosmotic lysis. The described gains and losses ofred cell cholesterol and surface area do not involvemetabolic injury and occur with no significantchange in phospholipid content.

The red cells of patients with obstructive jaun-dice are flat and osmotically resistant and havean increased cholesterol: phospholipid ratio. Whentransfused into normal subjects these "targetcells" rapidly lose their osmotic resistance. Simi-larly, normal cells acquire osmotic resistance inthe circulation of patients with obstructive jaun-dice. These reversible changes in shape occur with

Dr. Cooper is a Research Fellow, Harvard MedicalSchool.

Dr. Jandl is an Associate Professor of Medicine, Har-vard Medical School.

Address requests for reprints to Dr. Richard A. Cooper,Thorndike Memorial Laboratory, Boston City Hospital,Boston, Mass. 02118.

Received for publication 9 October 1967 and in revisedform 27 November 1967.

half-times of about 9 and 24 hr, respectively, andoccur without impairing cell viability.

These studies indicate that the red cell mem-brane accumulates cholesterol in obstructive jaun-dice as a consequence of the elevated levels ofbile salts. The resulting increment in red cell sur-face area is responsible for the physical propertiesand appearance of target cells. These observationssubstantiate Murphy's findings in vitro indicatingthat cholesterol is an important determinant of redcell shape and that its content in the cell membranemay vary independently from the phospholipids.Presumably any process or disorder affecting cho-lesterol exchange in vivo is capable of criticallymodifying the shape and behavior of red cells.

INTRODUCTIONAn increased resistance of red cells to osmoticlysis is frequently encountered in patients withhepatitis (1), particularly during the obstructivephase, and is almost always observed in patientswith extrahepatic biliary tract obstruction. In ob-structive jaundice there is usually a symmetrical,and often a marked, shift in the osmotic fragilitycurve, which indicates a homogeneous populationof osmotically resistant cells. Lesser increments inosmotic resistance, complicated by cellular hetero-geneity, are encountered in patients with chronicliver disease accompanied by an obstructive com-ponent (2). The osmotic resistance of red cells inobstructive jaundice is attributable to a charac-teristic increase in the surface-to-volume ratio(3). Affected cells have a normal volume but anincreased surface area, which gives them a broad,

The Journal of Clinical Investigation Volume 47 1968 809

flat configuration. On stained smears the dryingpattern creates an appearance described by theterm target cell (4).

That osmotic resistance can be acquired bymature red cells was suggested by Berk (5) onthe basis of observations in patients with obstruc-tive jaundice who had been transfused. Later in apreliminary report Harris and Schilling (6) sub-stantiated this in a study which made use of dif-ferential agglutination techniques for separatingout normal red cells after their transfusion intojaundiced patients.

Little is known of the normal mechanisms re-sponsible for the shape of red cells or, accordingly,of the abnormality that engenders the flattenedshape of target cells. It is known that red cellsurvival is unaffected by obstructive jaundice perse (7) and that the shape-change is probablyreversible, judging from the fact that the osmoticfragility pattern reverts to normal within 2 wkafter surgical relief of biliary obstruction (5, 6)or after the subsidence of hepatitis (8). Althoughincreased osmotic resistance has not correlatedwell with serum levels of total cholesterol (9, 10)or bilirubin (8, 9, 11), a relationship to bile saltretention was suggested in 1931 by Geill (8).Attempts to produce osmotically resistant cells invitro by incubating normal red cells in the seraof patients with obstructive jaundice have beenunsuccessful (6); however, such incubations arecomplicated and opposed by the increased osmoticfragility which results from metabolic injury (12).Recently Murphy (13) incubated red cells underconditions that minimized metabolic damage.Nonetheless, when suspended in normal freshserum, red cells showed a progressive increase inosmotic fragility. This was not observed, however,in cells suspended in heated serum. The increasedosmotic fragility that developed over a period ofmany hours in serum was associated with a lossof red cell cholesterol, but not phospholipid, andwith a decline in serum free cholesterol due to itsesterification by the heat-labile serum enzyme,transesterase.

Cholesterol is confined to the membrane of thered cell (14), where it exists almost entirely inthe free (unesterified) form, comprising about30%o of the membrane lipids (15). Unlike theseveral phospholipids, which turn over or ex-change with serum lipids slowly or not at all

(16), red cell cholesterol is in a rapid exchangeequilibrium with the free cholesterol of serum,wherein it exists bound to lipoproteins (17).Murphy has provided evidence that the distribu-tion of free cholesterol in the cell membrane maybe responsible for the cell's biconcave shape (18).The conclusion that spherocytosis can be inducedin vitro by a selective depletion of cholesterol hadnot been confirmed before the present studies(19), and, indeed, various studies by others (20,21) of membrane lipids during prolonged incu-bation of blood in vitro have reported only a proc-ess of proportional or "symmetrical" loss of lipids,in which red cells lose both cholesterol and phos-pholipids in equivalent amounts.

The present studies of the pathogenesis of targetcells in obstructive jaundice were undertaken be-cause of the potentially great importance of aprocess that allows continued modification of cellmembrane composition and thereby of shape andpossibly function. Several observations suggestedthe hypothesis that target cells may represent aspecific increase in membrane cholesterol: (a) theserum levels of free cholesterol tend to be elevatedin biliary obstruction and there is at least onereport (9) that the increased osmotic resistancein red cells from jaundiced patients (with hepa-titis) is associated with an increase in red cellcholesterol; (b) transesterase activity is decreasedin the sera of patients with obstructive jaundice(22) and that of normal serum is inhibited by bilesalts (23, 24), which are characteristically ele-vated in obstructive jaundice; and (c) with re-

spect to the surface area/volume ratio, thepotential "hemolytic volume," and the osmoticresistance, target cells are essentially the oppositeof spherocytes which can be produced in vitro bycholesterol elution (13). The following investi-gations of this hypothesis have been reported inpart in preliminary form (19).

METHODSIncubation in vitro. Blood from normal subjects was

defibrinated with glass beads and the red cells werewashed three times in isotonic phosphate buffer, pH 7.4,with added glucose (Na, 167 mEq/liter; K, 20 mEq/liter;POI 120 mEq/liter; and glucose, 150 mg/100 ml) toremove serum and buffy coat. For most studies, washedred cells were resuspended in buffered serum (2 partsbuffer and 1 part serum) at a cell concentration of only2%, in order to minimize alterations in pH or substrate

810 R. A. Cooper and 1. H. Jandl

concentrations during incubation. In studies requiringdirect measurement of maximum hemolytic volume andfor measurements of serum-red cell cholesterol balance,both requiring higher cell concentrations, the washed redcells were resuspended in serum without added buffer butwith added glucose (400 mg/100 ml) at a red cell con-centration of 35%. Penicillin, 500 U/ml, and streptomycin,250 ug/ml, were added to the red cell suspensions. Incu-bations were performed in stoppered glass vials at 370Cin a Dubnoff metabolic shaker at 110 oscillations/min for24 hr (hematocrit 2%) or 12 hr (hematocrit 35%).pH levels were 7.3-7.5 at hematocrit 2% and 7.1-7.5 athematocrit 35%.

Osmotic fragility and estimated cell surface area. Fromduplicate incubations 0.2 ml of cell suspension was addedto 4.0 ml aliquots of saline in each of a series of tubescontaining a graded sequence of concentrations of NaCl.The NaCl concentrations of the solutions of oven-driedNaCl were verified by direct measurement of the Naconcentration. After the addition of red cells, each tubewas mixed immediately and centrifuged within a periodof from 2-3 min after admixture. Per cent hemolysis wasmeasured at 540 mu and the osmotic fragility so obtainedwas corrected for the effect of pH and "tonicity" (25).The spherical volume of red cells was calculated on thebasis of the volume increment at their mean osmoticfragility, according to Castle and Daland (26). This cal-culation depends on predictable changes in cell volume inhypotonic salt solutions of known tonicity. It is based onthe Boyle-van't Hoff law which considers the red cellas a perfect osmometer. The formulation of Castle andDaland is in good agreement with the more recent find-ings of Hoffman, Eden, Barr, and Bedell (27) andSavitz, Sidel, and Solomon (28). In the present study ithas been used to compare the spherical volume of a singlepopulation of red cells before and after incubation. Sincethis volume, the maximum volume attainable before he-molysis, depends on (a) the intracellular content ofosmotically active material and (b) the surface area ofthe limiting cell membrane, it serves to reflect the latteronly if there has been no change in the former. Thus itsuse requires that the red cell population measured be asingle population distributed about a mean, that thecation content be the same in all samples of red cellsmeasured, and that the red cells be exposed to hypotonicsolutions for a period of time shorter than that whichwould itself cause an appreciable change in fragility dueto cation changes. All of these conditions were met. Thesurface area of red cells after incubation relative to theirsurface area before incubation was calculated from theirspherical volumes. The error of duplicate measurementsof the osmotic fragility values from which this calcula-tion was made imparted a standard error of 0.5% to theexpression of surface area.

Red cell and serum lipids. The red cells of quadrupli-cate incubation vials were quantitatively transferred toextraction tubes and the serum was removed by threesaline washes. The cell button was twice extracted withisopropanol and chloroform (29). Cholesterol was mea-sured by the method of Zlatkis, Zak, and Boyle (30) and

lipid phosphorus by Bbttcher, Gent, and Pries' modifica-tion (31) of the Fiske-Subbarow reaction (32). Serumfree cholesterol was determined by the method of Brown,Zlatkis, Zak, and Boyle (33). Standard errors of extrac-tion and measurement combined (95% confidence limits)are: red cell cholesterol + 1.8%, red cell lipid phos-phorus ± 1.3%, and serum free cholesterol + 1.4%. Redcells were enumerated in the Coulter model B electroniccounter.

For measurement of red cell K and volume the con-tents of triplicate vials were quantitatively transferred tograduated Constable tubes, centrifuged, and the meniscuswashed with isotonic NaCl. The red cell button was lysedwith 1.0 ml of distilled water and K measured in a flamephotometer.' Diameters of red cells suspended in incuba-tion medium were made with a calibrated eyepiecemicrometer.2 Serum bile salts were determined by gas-liquid chromatography. (34).

51Cr osmotic fragility (35). 30-ml specimens of wholeblood from patients with surgically proven extrahepaticbiliary tract obstruction or normal subjects were collectedinto sterile acid citrate dextrose, after which 300 /Ac of5'Cr-labeled sodium chromate 3 was added. After 20 minat room temperature the cells were centrifuged once andresuspended in sterile saline for injection into a com-

patible recipient. At intervals thereafter survival of thelabeled cells was measured and their osmotic fragility aswell as that of the recipient's red cells was determinedas follows: 0.5 ml of oxalated whole blood was added toa series of tubes containing 2.5 ml of hypotonic sodiumchloride solutions. Per cent hemolysis of the transfusedcells was measured as per cent radioactivity released intothe supernatant. Per cent hemolysis of the recipients'own cells was measured as per cent hemoglobin released.Osmotic fragilities were corrected for tonicity of theadded blood and for pH according to Emerson, Shen,Ham, Fleming, and Castle (25).

Patients. Patients were chosen for study on the basisof having: (a) relatively stable, uncomplicated, obstruc-tive jaundice; (b) a uniform population of target cellson blood smear and a striking increase in osmotic resist-ance; and (c) no other significant hematologic abnormal-ity. All patients or subjects who underwent 51Cr-red cellsurvival studies gave their full consent after being fullyinformed of the nature of the studies and of their poten-tial risks. In two instances the red cell donors were

jaundiced; both were established in advance to be affectedby extrahepatic biliary tract obstruction, as verified bysurgical exploration and liver biopsy. The protocol fol-lowed in their study was approved by the review com-

mittee of the Clinical Center Ward of the ThorndikeMemorial Laboratory.

Reagents. Sodium taurocholate4 was a mixture of bilesalts, greater than 98% conjugated, consisting of 83%

1 Instrumentation Laboratory, Inc., Watertown, Mass.2 Bausch & Lomb Incorporated, Rochester, N. Y.3Available as Rachromate from Abbott Laboratories,

North Chicago, Ill.4Available from Sigma Chemical Co., St. Louis, Mo.

Pathogenesis of Target Cells in Jaundice 811

cholate, 16% deoxycholate, and 1% chenodeoxycholate.5Purity of other bile salts was stated by the supplier 4 tobe: glycocholate > 95%; cholate > 99%; deoxycholate> 95%.

Statistics. Slopes were calculated by the method ofleast squares. Significance of the difference betweenslopes and standard errors, expressed as 95% confidencelimits, were calculated by an analysis of variance (36).

RESULTS

Studies in vivo. Whennormal red cells labeledwith 5'Cr were transfused into a compatible nor-mal recipient, they manifested no change in os-motic fragility during the 32 day period of obser-vation. However, when cells from four normaldonors were transfused into four patients withobstructive jaundice, the osmotic fragility curvesof the labeled cells became progressively shifted

5 This analysis was kindly performed by Dr. W. G.Hardison.

toward those of the recipients. Fig. 1 depicts onesuch transfusion in which the osmotic fragilitycurve of the normal transfused cells had shiftedhalfway toward that of the resistant cells of thepatient in about 24 hr. In the converse experi-ment red cells from two patients with surgicallyproven obstructive jaundice and target cells werelabeled with 5'Cr and transfused into two normalrecipients, with similar results in each patient.In the study depicted in Fig. 2, the transfusedtarget cells rapidly lost their abnormal resistanceto osmotic lysis and acquired the osmotic charac-teristics of the recipient's normal cells.

Figure 3 summarizes the results of six separatetransfusion studies and shows the rates at whichthe mean osmotic fragility of the transfused cellsapproached that of the recipients' cells. The 51Crhalf-life of the transfused cells was greater than

DONOR: NORMAL o-oRECIPIENT: OBSTRUCTIVE JAUNDICE

I- .z

0.

LiI4

0.50 0,45 0.40 0.35 0.30 0.25

SODIUM CHLORIDE, g/100 ml

FIGURE 1 Changes in the osmotic fragility of normal red cells after transfu-sion into a patient with obstructive jaundice. Normal red cells became pro-gressively more resistant to osmotic lysis after transfusion into a patient withjaundice caused by extrahepatic obstruction. This change was halfway com-plete in about 24 hr. In this figure and in Figs. 2 and 3, each osmotic fragilitycurve of the donor cells was determined with 51Cr and is identified in terms ofthe time interval after infusion. The range of the osmotic fragility curves ofthe recipients during the entire period of study is depicted by the, stippledareas.

812 R. A. Cooper and J. H. Jandl

DONOR: OBSTRUCTIVE JAUNDICE 0-ORECIPIENT: NORMALv.

SODIUM CHLORIDE, g/1OO ml

FIGuRE 2 Changes in the osmotic fragility of red cells from a patient withobstructive jaundice after their transfusion into a normal recipient. Osmoticallyresistant cells from a patient with target cells progressed steadily toward a

normal osmotic fragility after transfusion into a normal subject.

20 days in five of the six studies; however, in one,

involving the transfusion of red cells from a jaun-diced donor to a normal recipient, the 51Cr half-life was 5 days. The unexplained shortening ofsurvival in this latter transfusion occurred at a

rate slower than the change in osmotic fragility.Therefore, in five studies, and probably in the

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0 ~~~~~~~~~~~DONORtJAUNDICIO4Fo/ /RECIPIENT: NORI

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sixth, the observed osmotic fragility changes rep-

resent a transformation of the cells transfused,rather than the selective survival of a more or lessresistant minor population of cells. The acquisi-tion of increased osmotic resistance in vivo ap-

peared to occur as a zero order function of time,50% of the change occurring in approximately 24

-o

-0 FIGURE 3 Rate of change in osmotic fragility invivo. The differences between the mean osmoticfragility of 'Cr-labeled donor red cells and theunlabeled red cells of the recipient at various

sL times after transfusion is plotted as per cent ofthe initial difference. The 'Cr half-life of the

ED transfused red cells was greater than 20 days inall but one instance, depicted by the lower of thetwo uninterrupted lines as of 6 hr, in which the

_ Cr half-life was 5 days.12 18 24 30 34

HOURSAFTER TRANSFUSION

Pathogenesis of Target Cells in Jaundice 813

z0

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hr. The process by which target cells lost theirresistance to osmotic lysis took place more rapidly,with 50%o of the transition complete in 9 hr. Nofirm conclusion as to kinetics is possible, the rateat which osmotic resistance was lost correlatingequally well with zero and first order kinetics.

Effect of normal sera on normal red cells invitro. Incubations were carried out in a mannerwhich minimized metabolic damage to the redcells. This was shown by the absence of a changein the osmotic fragility, in all experiments, and involume and K content, in initial experiments,when normal red cells were incubated for 24 hrat a cell concentration of 2% in a serum-buffer-glucose medium using normal serum previouslyheated to 560C for 30 min. Similarly there was nochange in osmotic fragility when 6% human albu-min was substituted for serum in this medium.However, when the medium contained fresh (un-heated) normal serum, normal red cells under-went an increase in osmotic fragility and becamemore spheroidal (Fig. 4) as reported by Murphy(13). This occurred without a change in volumeand thus resulted from a loss of membrane surface

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area. Associated with the loss of surface area wasboth a proportional loss of red cell cholesterol(Fig. 5) and a proportional fall in the serum con-centration of free cholesterol (Fig. 6). When thetransesterase activity of normal sera was destroyedby heating at 560C for 30 min before incubation,there was no fall in serum free cholesterol levelsduring incubation, and red cell cholesterol andsurface area remained unchanged (Fig. 6). De-spite the close correlation that existed whenchanges in red cell surface area were plottedagainst changes in serum free cholesterol concen-tration in normal sera, surface area was not af-fected by the actual concentration of cholesterolper se; thus there was no change in surface areawhen normal red cells were incubated with sevenhomologous normal sera that differed widely intheir concentrations of free cholesterol, providedthe esterifying activity had been inactivated byheating. Moreover, after incubation in fresh (un-heated) homologous sera there was a poor corre-lation between cell surface area and serum freecholesterol concentration per se; incubation ofnormal red cells in sera from patients with familial

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FIGURE 4 The effect of normal and jaundiced sera on the osmotic fragility of normal redcells during incubation at 370C for 24 hr. Whereas normal red cells became osmotically fragilein normal serum, they became osmotically resistant in serum from a patient with obstructivejaundice. The interrupted fine represents osmotic fragility before incubation. The dotted linerepresents the osmotic fragility of red cells (target cells) from the jaundiced patient.

814 R. A. Cooper and J. H. Jandl

0

* Normal Sera*A Jaundiced Sera

- 0 Normal Sera c Taurocholate,0.2 mg/ml10 Normal Sera c Taurocholate, 1.0 mg/ml

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80

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RED CELL CHOLESTEROL, PER CENT OF INITIAL

FIGURE 5 Relationship between the cholesterol content and surface area of red cellsafter incubation in various sera at 370C for 24 hr. Both are expressed as per cent of theinitial values before incubation. A direct correlation was seen under all of the conditionsexamined.

hypercholesterolemia resulted in a slight decreasein surface area, despite the fact that the free cho-lesterol concentration of these sera was more thantwice that of the autologous normal serum. How-ever, when the free cholesterol concentrations ofthese homologous sera were expressed as a per

cent of the concentration of free cholesterol exist-ing in each before incubation, i.e. as the change infree cholesterol level, there was close correlationwith red cell surface area (Fig. 6). Thus in bothnormal and hypercholesterolemic sera it appears

to be the relative decrease in serum free choles-terol during incubation, rather than its absoluteconcentration, which correlates with red cell cho-lesterol and surface area.

Effect of jaundiced sera on normal red cells invitro. In contrast to the loss of surface area andincrease in osmotic fragility regularly seen afterred cells have been incubated in fresh normalserum, incubation of normal red cells in sera frompatients with biliary tract obstruction, who were

jaundiced and had target cells, resulted invariablyin a gain of surface area and an increased resist-ance to osmotic lysis (Fig. 4). This gain in sur-

face area was associated with a proportional incre-ment in red cell cholesterol (Fig. 5). Although the

serum concentration of free cholesterol in patientswith obstructive jaundice was usually elevated,frequently to a marked degree, there was no sys-

tematic relationship between the serum concentra-tion of free cholesterol and the red cell surfacearea after incubation. Nevertheless, incubationsin jaundiced sera induced increments, rather thandecrements, in the surface area and cholesterolcontent of normal cells (Figs. 5 and 6). Moreover,the surface area of normal red cells was greaterafter incubation in all 11 of the fresh jaundicedsera studied, whereas this occurred with none ofthe nonjaundiced sera despite and overlap in thepercentage of cholesterol esterification (Fig. 6),and it occurred also with jaundiced sera heated to560C for 30 min. Therefore, the unique effect ofjaundiced sera cannot be attributed solely to sup-

pression of cholesterol esterification.Effect of bile salts on normal red cells in vitro.

Ponder (37) observed that bile salts added towashed red cells in a saline suspension cause

hemolysis, but that this does not occur in the pres-

ence of serum or albumin. Under the conditions ofthe present study taurocholate caused no hemoly-sis unless its concentration exceeded about 4.0mg/ml of serum.

Pathogenesis of Target Cells in Jaundice 815

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SERUMFREE CHOLESTEROL, PER CENT OF INITIAL

FIGURE 6 Relationship between changes in serum levels of free cholesterol and in red cellsurface areas after incubation of normal red cells in various sera at 370C for 24 hr. In sera

from normal subjects and from subjects with familial hypercholesterolemia, changes in redcell surface area correlated closely with changes in serum free cholesterol concentration, ex-

pressed as per cent of its concentration before incubation. The slope expressing this correlationwas plotted by the method of least squares. In all 11 sera from patients with obstructive jaun-dice and target cells the surface area of normal red cells after incubation was greater (regard-less of the extent of change in serum cholesterol level) than in nonjaundiced sera.

The calculated surface area of normal red cellsafter incubation for 24 hr in normal sera to whichtaurocholate had been added was greater than thatof cells after parallel incubation in the same nor-

mal serum having an equal concentration of freecholesterol but without added bile salts (Fig. 7).As in the previous incubations, the increase insurface area in cells incubated with taurocholate-serum was accompanied by a proportional increasein red cell cholesterol (Fig. 5). Results similar tothose with taurocholate were also obtained withglycocholate, cholate, or deoxycholate at equalconcentrations (by weight). However, there was

no change in surface area when red cells were in-cubated in a medium in which serum was replacedby 6%albumin and contained cholate in serial con-

centrations up to that which was hemolytic.

After incubation in normal serum containingtaurocholate, 1.0 mg/ml, normal red cells were

increased in surface area as determined from theirmaximum volume in hypotonic saline (38) andas calculated from their osmotic fragility curve

(26). They were also increased in diameter whenexamined directly in an isotonic suspension usingan optical micrometer. The mean cell diameter

standard error (95%o confidence limit) of redcells after incubation in heated normal serum was

7.47 + 0.27 JL; after incubation in heated normalserum containing taurocholate, the mean diameterwas 8.14 + 0.47MFL (P < 0.01).

Although red cells gained cholesterol when in-cubated at a cell concentration of 2% in normalserum containing taurocholate, the amount of cho-lesterol transferred from serum to cells was too

816 R. A. Cooper and J. H. Jandl

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SUPERNATANTFREE CH/OLESTEROL,PER CENT OF INI TIAL

FIGURE 7 Influence of taurocholate on red cell surfacearea after incubation of normal red cells in normal serumat 37°C for 24 hr. At each serum concentration of freecholesterol the red cell surface area is directly relatedto the concentration of taurocholate. The intercept ofeach slope differs from the others statistically (P <0.01).

small to be measurable as a decline in serum freecholesterol. Therefore, incubation conditions werealtered so as to increase the ratio of cells/serum.As metabolic injury to red cells occurs muchsooner with the more concentrated suspensions,it was necessary to terminate the incubation periodsooner (at 12 hr) and accordingly to employ ahigher level of bile salts. At a cell concentrationof 30-35%s in normal serum which had beenheated to 56°C and to which was added taurocho-late, 2,0 mg/ml, a rise in red cell cholesterol wasaccompanied by a reciprocal fall in the serum levelof free cholesterol (Table I ). The cumulativeincrease in cell cholesterol and surface area oc-curred as a linear arithmetic function of time(tj = 6 hr), there being no immediate shift in thecholesterol partition. Thus it appears that bilesalts cause an increase in red cell surface area bymediating a gradual transfer of free cholesterolfrom serum to the cell membrane. In none of theexperiments described above, regardless of thegain or loss of red cell cholesterol, was there achange in red cell phospholipid content in excessof the error of the method.

Cholesterol esterification in vitro. The esteri-fication of cholesterol in incubation mixtureswhich contained red cells at a concentration of 2%and normal serum was 200 ± 20 pg/ml of serumin 24 hr. With jaundiced sera, despite higherlevels of free cholesterol, esterification averagedonly 100 + 42 ug/ml of serum in 24 hr. Choles-terol esterification in normal serum was inhibitedby the addition of taurocholate in proportion to itsconcentration. The per cent inhibition was 8.3 +

4.6% at a taurocholate concentration of 0.2mg/ml; 28.3 ± 11.9% at 0.5 mg/ml; and 78.8 ±3.8%o at 1.0 mg/ml. The inhibition of esterificationof cholesterol in jaundiced sera or in normal seracontaining added taurocholate prevented the netloss of red cell cholesterol during incubation, thustending to maintain the membrane composition,surface area, and, accordingly, the shape of thecells. Cholesterol esterification occurred equallywell in normal serum incubated in glass or plasticvessels and in plasma anticoagulated with ethyl-enediaminetetraacetate (EDTA).

Red cell lipids and serum bile salts in obstruc-tive jaundice. In 16 normal subjects red cellcholesterol was 13.4 ± 0.4 ug/108 cells and lipidphosphorus was 1.08 + 0.04pg/108 cells. The cho-lesterol: lipid phosphorus ratio was 12.5: 1. Thesevalues fall within the range of values for red cell

TABLE IEffect of Taurocholate on the Cholesterol Partition between

Serum and Red Cells*

Content of free cholesterol

After in-After in- cubationcubation in heatedin heated serum with Change with

serum taurocholate taurocholate

pg/ml of suspensionExpt. A

Whole suspension 871 872 + 1

RBC 528 570 +42

Serum 343 302 -41

Expt. BWhole suspension 765 770 + 5

RBC 438 490 +52Serum 327 280 -47

* Normal red cells were incubated for 12 hr at 37°C at a cell concen-

tration of 30-35% in normal serum, previously heated to 56°C for 30.min, containing added glucose at a concentration of 400 mg/100 ml.Under these experimental conditions the cholesterol content of redcells and of heated serum after incubation remain identical to valuesmeasured before incubation. Sodium taurocholate was added to thisserum medium so as to achieve a final concentration of 2.0 mg/ml.

Pathogenesis of Target Cells in Jaundice 817

lipids previously reported by several observers(15). The cholesterol content of red cells from sixpatients with obstructive jaundice was 17.9 ± 4.3,ug/108 cells; lipid phosphorus was 1.26 + 0.23,ug/108 cells. The cholesterol: lipid phosphorusratio was 14.5: 1. The red cells from patients withobstructive jaundice showed an average increaseabove normal for cholesterol of +34% (P <0.001), for phosphorus of +17% (P < 0.05), andfor the cholesterol: lipid phosphorus ratio of+ 16% (P = <0.01).

The serum concentration of bile salts in fourpatients with obstructive jaundice ranged from4.8 to 19.5 /ug/ml.5

DISCUSSION

These studies demonstrate that mature red cellsare capable of gaining and losing membrane cho-lesterol as a specific and reversible process.Changes in the cholesterol content of red cellsare accompanied by proportional changes in theirsurface area and thus in their shape, appearance,and physical capacity for water and electrolytes.These associated losses or gains in the cholesterolcontent, surface area, osmotic resistance, and po-tential (hemolytic) volume of red cells do notinvolve metabolic damage or changes in actual cellvolume. The observation that cholesterol alone islost from red cells during incubation in normalserum, provided metabolic injury is avoided, con-firms previous studies by Murphy (13). That cho-lesterol loss is unaccompanied by changes in redcell phospholipids indicates that this process inviable red cells differs from the phenomena studiedby Reed and Swisher (20) and Jacob (21) inwhich proportional or "symmetrical" losses of cho-lesterol and phospholipid occurred, possibly re-flecting membrane fragmentation, in cells duringprolonged incubation and metabloic injury. Thepresent observations in vitro and in vivo substan-tiate the hypothesis that the target cell of obstruc-tive jaundice reflects a process whereby cholesterolselectively accumulates in the red cell membranes.This accumulation appears to be favored by twogeneral factors: (a) the serum level of free cho-lesterol, expressed in terms of the saturation ofits binding proteins, and (b) the serum level ofbile salts, which influence the serum/cell partitionof free cholesterol. As cholesterol accumulates onthe red cell membrane, the surface area is extended

proportionally. Since red cell volume and cationcontent do not change under the conditions ofincubation used, this increase in surface areacauses red cells to assume a flattened shape andallows a greater increment in volume during hypo-tonic swelling, rendering the cells osmotically"resistant." Target cells are the morphological ex-pression of an increased surface-to-volume ratioand can result from either an increase in surfacewhile volume remains constant, as in the presentstudy, or a decrease in volume while surface arearemains constant, as in hypertonic serum (39).

The progressive increase in osmotic resistanceof normal red cells during incubation in jaundicedsera in vitro mimics similar increases in vivoafter normal red cells are transfused into recipientswith obstructive jaundice. It is inferred, therefore,that the gain in cholesterol (but not in phospho-lipid) that accompanied the acquisition of osmoticresistance in vitro also took place as normal redcells became resistant in vivo. Supporting thisinference is the finding that the red cells from ourpatients with obstructive jaundice were dispropor-tionately high in cholesterol (+ 34%), althoughthere was a lesser increase in phospholipid(+ 16%) as well. Neerhout (40, and footnote 6),studying a diverse group of 24 patients with vary-ing degrees of cellular and obstructive liver disease,many of whomhad anemia and macrocytosis, alsorecorded a disproportionate increase in red cellcholesterol (+ 26%o) as compared with phospho-lipid (+ 12%o). A portion of the increases in thisheterogeneous group of patients was due to the"symmetrical" rise in both phospholipid and cho-lesterol due to macrocytosis, i.e. red cells increasedboth in volume and in surface area, and reticulo-cytosis. Selective, or "asymmetrical," incrementsin red cell cholesterol are expressed by the choles-terol/phospholipid phosphorus ratio: in Neerhout'sunselected series of patients with liver disease thisratio was increased on the average by 1 %o overnormal; in our patients with obstructive jaundicethe increase averaged 16%7o. The selective incre-ments in cholesterol observed in red cells in vitroand the predominance of the cholesterol increasesfound in the red cells of jaundiced patients indi-cates either that cholesterol gain is the primaryevent in obstructive jaundice, with phospholipidacquisition being a variable, secondary event, or

6 Neerhout, R. C. Personal communication.

818 R. A. Cooper and J. H. Jandl

that both moieties may tend to accumulate, choles-terol accumulating more rapidly because of its fargreater rate of turnover (see below). In eitherevent changes in cholesterol clearly do not neces-sitate equivalent changes in phospholipid.

The lack of any gain in red cell lipids in familialhypercholesterolemia, despite serum lipid levels ashigh or higher than occur in obstructive jaundice(10, 40, 41), emphasizes the importance of factorsapart from high levels of serum cholesterol per se.The present findings indicate that bile salts whenadded to normal serum in vitro affect the choles-terol levels of red cells in two distinct ways. First,as reported by others (23, 24), bile salts inhibitthe activity of the serum-esterifying system; thisin turn inhibits the depletion of free cholesterolfrom red cells during incubation (see below).Secondly, bile salts induce a shift in the redcell/serum partition for cholesterol, resultinggradually in supranormal levels of cholesterol inthe cells. The close association of the target cellphenomenon with disorders characterized by bilesalt retention provides circumstantial support forthe conclusion that the increase of cholesterol andsurface area in the patients' red cells is similarlymediated by bile salts. It should be noted that theconcentrations of bile salts required to producetarget cells in vitro (100-1000 ug/ml) exceedthose in peripheral blood from patients with ob-structive jaundice, wherein the serum levels usu-ally fall within the range of 5-100 ug/ml (34, 42).This difference does not represent a serious dis-crepancy, however, since the response of red cellsto the 12-24 hr periods of exposure to serumcontaining bile salts as observed in vitro onlypartially reflects the process in vivo, which is sus-tained for 48-72 hr. Moreover, in contrast to thesituation during incubation in vitro where transferof cholesterol from serum to red cells results inunsaturation of the serum binding sites, plasmafree cholesterol in vivo would tend to remainsaturated through equilibration with the total bodypool of free cholesterol (43-45).

The selective depletion of red cell cholesterolduring incubation in serum results from depletionof the serum free cholesterol, with which the cellsare in equilibrium, through the action of the serumenzyme, transesterase. This occurs in plasma anti-coagulated with EDTA as well as serum and inplastic as well as glass. Esterification of free cho-

lesterol causes unsaturation of the lipoproteinmoieties which bind it. The availability of theselipoprotein sites for recombination with free cho-lesterol derived from red cells was demonstratedby Murphy (13) who showed a return of serumfree cholesterol levels toward normal' when redcells were added to serum that had been depletedof free cholesterol by previous esterification. Pre-sumably it is the relative saturation of theselipoprotein-binding sites rather than the absoluteconcentration of serum-free cholesterol which de-termines the per cent saturation of red cell cho-lesterol. This view is consistent with the findingof normal or only slightly reduced levels of cho-lesterol in red cells from patients with Tangierdisease or acanthocytosis, diseases associated witha marked decrease in serum free cholesterol dueto deficiencies of a- and ,8-lipoproteins, respectively(46-49). The selective loss of cholesterol from thered cell membrane alters the cell in a mannerdirectly opposite that observed during the forma-tion of target cells: the depleted membrane is pro-portionally diminished in surface area and there-fore the cell assumes a more spheroidal shape.Both cholesterol and shape can be restored to thered cell in vitro (13, 50) and in vivo (50).

It has not definitely been established in vivowhether cholesterol esters are mainly formed fromfree cholesterol in plasma through the action ofplasma (serum) transesterase or whether they aremainly formed in the liver and incorporated thereinto lipoproteins (51-53). However, Norum (54)has recently described three siblings who lackserum transesterase activity but who retain thecapacity to esterify cholesterol in the intestine.They have elevated serum cholesterol, only smallamounts of which is esterified. Red cells from thesepatients are described by the author 7 as havingthe appearance of target cells and containing in-creased amounts of free cholesterol but normalamounts of phospholipid. Thus it appears thatdiminished activity of transesterase, as also occursin obstructive jaundice (22), promotes the accu-mulation of red cell cholesterol. Conversely, thestudies in vitro indicate a significant potential foresterification of free cholesterol and loss of redcell cholesterol in any situation in vivo whereinconditions of plasma skimming and erythroconcen-tration may exist, as in the spleen. The sphering

7 Norum, K. R. Personal communication.

Pathogenesis of Target Cells in Jaundice 819

of red cells consequent to their loss of membranecholesterol would presumably enhance splenic en-trapment, whereas the addition of surface to acell already sphered would help it to escape initialsplenic trapping. Evidence strongly supportingthese speculations has recently been obtained instudies of hereditary spherocytosis red cells whichbecame osmotically normal and evidenced greatlyimproved survival after transfusion into patientswith obstructive jaundice (50).

Equilibration of radioactive cholesterol betweenred cells and serum occurs in 4-8 hr in vitro(55-56) and in vivo (43-45). In contrast, phos-pholipid exchange with serum occurs more slowly,the rate of phosphatidyl choline turnover havingbeen estimated at 7% in 12 hr (16). The speedof change in red cell surface and cholesterol con-tent in the present study is consistent with themobility of the red cell cholesterol compartment.Increments or decrements in phospholipid contentwhich might occur over a much longer time coursecannot be excluded by these studies in vitro whichspan only 24 hr. An increase in phospholipid, pre-dominantly lecithin, has been found in the red cellsof some patients with chronic liver disease (40).It is unknown whether the gain in phospholipidobserved in such patients reflects a process analo-gous to that described here for cholesterol.

The ratio of change in the red cell surface areato the change in the red cell cholesterol in thisstudy is approximately 1: 3. This is consistentwith the calculations which suggest that cholesterolcontributes 30% of the total surface area of redcell lipids (57-59). Moreover, this implies thatred cell membrane protein, which accounts forabout 60% of the red cell ghost by weight (14),does not form a matrix which determines surfacearea, a fact also suggested by Maddy's inability tofind red cell membrane protein in the ,8-configura-tion (57). These studies do not shed further lighton Murphy's suggestion (18) that cholesterol maynot be evenly distributed throughout the red cellmembrane but rather is concentrated at the areasof greatest convexity. They do indicate, however,that the structure and permeability character ofthe red cell membrane do not depend upon a fixedunit of ratio of lipid moieties, as implied by sometheories of membrane structure. Furthermore, theyindicate that red cell size and shape can be modi-fied in vivo by altering the cholesterol composition

without adversely affecting membrane function orintegrity.

ACKNOWLEDGMENTS

The authors are grateful to Dr. P. 0. Ways for supply-ing sera from patients with hypercholesterolemia, toMrs. D. C. Bennett and Miss E. Streiff for their tech-nical assistance, and to Miss W. M. Sheldon for secre-tarial assistance.

This work was supported in part by grants from theNational Heart Institute (HE-07652), the National In-stitute of Arthritis and Metabolic Diseases (Ti-AM-5391), a Research Career Development Award (K3-HE-3943) from the National Heart Institute, and a grant(FR-76) from the Division of Research Facilities andResources, National Institutes of Health, Bethesda, Md.

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