Metabolic dependence of the critical hemolyticvolume of human erythrocytes: relationship toosmotic fragility and autohemolysis inhereditary spherocytosis and normal red cells.

R I Weed, A J Bowdler

J Clin Invest. 1966;45(7):1137-1149. https://doi.org/10.1172/JCI105420.

Research Article

Find the latest version:

http://jci.me/105420-pdf

Journal of Clinical InvestigationVol. 45, No. 7, 1966

Metabolic Dependence of the Critical Hemolytic Volume ofHuman Erythrocytes: Relationship to Osmotic Fragility

and Autohemolysis in Hereditary Spherocytosisand Normal Red Cells*

ROBERTI. WEEDt ANDANTHONYJ. BOWDLERt(From the Departments of Radiation Biology and Biophysics and of Medicine, University of

Rochester School of Medicine and Dentistry, Rochester, N. Y.)

Erythrocytes from patients with hereditaryspherocytosis (HS) undergo changes in cationand lipid content when incubated in vitro understerile conditions for 24 hours. These are ac-companied by changes in cell volume, osmoticfragility, and the critical volume at which hemoly-sis of the cell occurs. Normal red cells have beenfound to undergo similar changes but with a longertime scale, the normal taking 36 to 48 hours toachieve the degree of change found in the sphero-cyte at 24 hours.

The osmotic fragility of the erythrocyte dependson the relationship of the osmotically active intra-cellular contents of the cell, which determine thecell volume, to the area of the nondistensible mem-brane. Consequently, the shape of the cell has ap-peared important to many authors (1-4), since therelative thickness of the cell depends on the ratio ofthe volume to the available surface area of themembrane.

A further phenomenon that is characteristic ofthe HS cell is that of marked autohemolysis onin vitro incubation (5-7). The mechanism ispoorly understood, although it clearly bears a re-

* Submitted for publication September 9, 1965; acceptedMarch 22, 1966.

Supported in part by the United States Atomic Energy,Commission at the University of Rochester Atomic En-ergy Project, Rochester, N. Y.

t Recipient of U. S. Public Health Service researchgrant HE 06241-05.

Address requests for reprints to Dr. Robert I. Weed,Dept. of Radiation Biology and Biophysics, Universityof Rochester School of Medicine and Dentistry, Roch-ester, N. Y. 14620.

t Buswell Fellow in Medicine during the period of thisinvestigation; present address: University College Hos-pital Medical School, University Street, London, W.C.1,England.

lationship to changes in osmotic fragility and isknown to be inhibited by the provision of suitablemetabolic substrate during incubation.

Both osmotic fragility and autohemolysis of theerythrocytes have been shown to be increased inmost cases of hereditary spherocytosis. In 1954Selwyn and Dacie (6) presented evidence thatthe mean cell volume was actually decreased dur-ing the second 24-hour period of in vitro incuba-tion. They concluded that in vitro hemolysis wasin some way associated with a decrease in surfacearea of the cells.

Reed and Swisher (8, 9) and Prankerd (10)have demonstrated that in vitro incubation of HSerythrocytes is associated with an abnormal loss oflipid from the affected cells. Reed and Swisher(8) showed that when this lipid loss reached ap-proximately 20%o there was a net loss of potas-sium from the cells. In 1957 Bertles (11) ob-served an increased rate of influx of sodium intoHS cells. More recently, Jacob and Jandl (12)have suggested that increased sodium permeabilityis the major lesion resulting in decreased in vitroand in vivo survival of such cells. These authorshave suggested that accumulation of intracellularsodium causes swelling of HS cells during invitro incubation that is aggravated by the additionof ouabain and predisposes these cells not onlyto increased autohemolysis but perhaps also todestruction in the spleen. There is an apparentdisparity between this view and the Selwyn andDacie observation of cell shrinkage on incubationin vitro. Selwyn and Dacie suggested that HScells are predisposed to loss of membrane surfacearea (6), which would be the probable conse-quence of the decrease in cell lipids described byReed and Swisher (8, 9) and Prankerd (10).The study reported here was undertaken in an at-

1137

ROBERTI. WEEDAND ANTHONYJ. BOWDLER

tempt to reconcile the divergent views concerningthe in vitro behavior of the hereditary spherocyte.

This investigation has been directed towards thefollowing aspects of the problem: 1) the measure-

ment of the critical hemolytic volume of normaland HS cells under various conditions of incuba-tion, especially in relation to the effects of addedmetabolic substrates; 2) the relationship betweenthe critical hemolytic volume and changes in cellvolume, lipid content, and cation composition ofthe cell; 3) consideration of the relevance of thesefindings to changes in osmotic fragility and to au-

tohemolysis induced by sterile incubation in vitro;and 4) observations on the morphologic concomi-tants of the observed biochemical and physiologicalchanges.

Methods

Blood. Blood samples were obtained from ten hemato-logically normal adults and from five splenectomized pa-

tients with HS. Splenectomized patients with normalreticulocyte counts were chosen to avoid inclusion ofyoung cells in the erythrocyte populations studied. Onlydefibrinated normal and HS blood was used for 1) 24-hour incubations before osmotic fragility tests by themethod of Parpart and co-workers (13); 2) 48-hour au-

tohemolysis studies as described by Young, Izzo, Altman,and Swisher (7) ; and 3) studies of the critical hemolyticvolume of normal cells after 36 to 48 hours of incubation.Either defibrinated blood or blood collected into Na2-EDTA was used for 24-hour studies of cation content,cell volume, autohemolysis, and critical hemolytic vol-ume of both normal and HS cells. The final concentra-tion of Na2EDTA was 5 X 10' mole per L of plasmachosen to prevent coagulation but not to provide a con-

centration sufficiently in excess of plasma Cae + Mg"to produce alterations such as those described by Garbyand De Verdier (14) for red cell glycolytic rate when 5to 10 mMfree Na2EDTA was present. Studies of redcell glycolytic rates on donors used for the present stud-ies were carried out by Reed and Swisher (9) who foundno evidence of increased glycolytic rate attributable toNa2EDTA. Simon and Ways (15) have shown thatEDTA has no effect on autohemolysis of normal cells.Hoffman (16) has pointed out that EDTAhas no effecton cation transport in red cells unless present during os-

motic hemolysis, whereas Lepke and Passow (17) havemade similar observations on K+ permeability.

In addition, we carried out five measurements of eryth-rocyte Ca++ on trichloroacetic acid extracts of NaCl-washed normal cells examined fresh and after incubation.The measurements were made with a Perkin-Elmer atomicabsorption flame photometer. The results for normal redcells were as follows: fresh, defibrinated 2.3 + 1.1 (SD)X 10" mole per cell, and fresh Na2EDTA (5 mmolesper L plasma) 2.2 0.8 (SD) X 10'" mole per cell.

After 24 hours of incubation the value for defibrinatedcells was 4.8 ± 1.0 (SD) X 10-'7 mole per cell and forNa2EDTA cells, 5.4 ± 2.1 (SD) X 1Q0'7 mole per cell.Ca"+ measurements of one sample of HSblood yielded freshvalues of 3.0 X 1017 mole per cell and 3.1 X 10-'7 moleper cell for defibrinated and EDTA cells, respectively.After 24 hours of incubation with glucose the values were3.7 X 10-17 and 4.4 X 1017 mole per cell for defibrinatedand EDTA cells, respectively. Thus, the concentrationsof EDTAemployed did not seem to remove cellular Ca++.No influence of Na2EDTA vs. defibrination was encoun-tered in 24-hour studies of autohemolysis of either nor-mal or HS cells, nor was there any influence of themethod of collection on critical hemolytic volume meas-urements made immediately or after 24 hours' incubation.Therefore, the results for 24-hour cell volume autohemoly-sis, critical hemolytic volume, and cation content includedata on both defibrinated and Na2EDTA blood. How-ever, the decrease in critical hemolytic volume that oc-curred in normal cells incubated without glucose for 36to 48 hours, which were similar to HS cells incubated for24 hours, seems to be prevented to some extent by thepresence of Na2EDTA; therefore, all of the incubationsthat were carried out for longer than 24 hours employeddefibrinated blood.

pH. In both the Na2EDTA and defibrinated normaland HS blood the initial pH was 7.7 to 7.8, falling at 24hours to values of 7.0 to 7.2 in incubations to which glu-cose had been added and remaining at 7.3 to 7.5 in incuba-tions to which no glucose was added. Thirty-six-hour in-cubations of normal cells resulted in a further pH drop to6.9 to 7.0 in glucose-supplemented incubations. Murphy(18) has called attention to the fact that red cell anaerobicglycolysis may be partially inhibited by lowered pH. How-ever, in the present studies pH was not controlled becausethe pH remained at or close to 7.4 in the incubations towhich no glucose was added. It was these incubations thatproduced the pathologic cellular alterations. In addition,one purpose of the study was to investigate the mechanismof hemolysis in the conventional autohemolysis test inwhich pH is not controlled (7).

In some studies, pH of glucose-supplemented cells wasmaintained at 7.4 by utilizing a low hematocrit (20%)and checking the pH of the blood at 8-hour intervals, re-adjusting plasma or serum pH with 0.1 N NaOHwhennecessary. These studies demonstrated that the protec-tive effect of glucose in preventing the changes in criticalhemolytic volume was evident at pH 7.4, as well as thelower pH.

Incubation mixtures. Experimental incubation mixtureswere composed of red blood cells that occupied 38 to 45%of the relative volume of the suspension, autologous serumor plasma containing 5 X 10' M Na2EDTA, and either30 mMglucose, 30 mMglucose + 1 X 10' Mouabain, 1 X10 ' Mouabain, or an equivalent (I) volume of 1%o NaCladded instead of glucose. Incubations were carried out inpolypropylene flasks under sterile incubation conditions at370 C.

Cell sizing and critical hemolytic volume determination.Mean cell volumes at varying external tonicities were

138

METABOLIC DEPENDENCEOF CRITICAL HEMOLYTICVOLUME

determined by analysis of volume distribution curvesobtained with a model B Coulter electronic particlecounter and either a model B plotter or a 400-channelanalyzer. The red cells were diluted into phosphate-buffered NaCl solution to give a final concentration of20 to 40,000 per 0.5 ml. In each case, the mean cellsize was calculated from the mean "window" or channelof the plot by using a calibration factor as described byBrecher and associates (19). The calibration factor wasevaluated independently each day by use of the micro-hematocrit in conjunction with the total red cell count.Because of the extreme rapidity of water movementthrough the membrane and the marked dilution of thesecells (1 to 50,000) a new osmotic equilibrium volume isachieved almost immediately. This volume remainsstable for at least 10 to 15 minutes in salt concentrationsabove those which produce hemolysis. However, in NaClconcentrations that are hemolytic, lysis secondary to theosmotic swelling beyond the critical volume results inloss of hemoglobin and cellular ion content resulting inan initial shrinkage. The maximal immediate mean cellvolume that is measured in the various NaCl concentra-tions is taken as the critical hemolytic volume. In Fig-ure 1, A, B, C, and D represent, respectively, the vol-ume distribution of fresh normal cells in 1% bufferedNaCl, their maximal volume in 0.425% NaCl, and thevolume distribution of lysed and shrunken cells in 0.4 and0.3%o NaCl. In 0.4%, with more than 50% hemolysis,the decrease in mean cell volume is composed of a popu-lation of small lysed cells and a population of swollen un-lysed cells, but the upper limit of the latter has not in-creased from 0.425%. Therefore, selection of the volumein 0.425% as the critical hemolytic volume by virtue ofits having a larger mean than that for 0.40%o NaCl doesnot obscure further swelling, even though the mean vol-ume in 0.40% is based on a bimodal distribution. How-ever, since repeated osmotic lysis is necessary to removeall the hemoglobin from red cells (20), the residual he-moglobin in ion permeable, once hemolyzed cells resultsin reswelling and "rehemolysis" after the initial shrinkage.This is a predictable phenomenon based on the colloidosmotic effect of the residual hemoglobin in the leakycell and is illustrated by E in Figure 1, which shows re-swelling 30 minutes after lysis in 0.30% NaCl. Therefore,to avoid variability associated with reswelling and to de-tect the salt concentration that represented the peak ofthe osmotic swelling curve, all of the measurements weremade immediately upon dilution of the blood into thesalt solution. Although there is considerable variabilityin the values of the cell volumes in salt concentrations thatare lytic in contrast to nonhemolytic salt solutions, theformer fall below the maximum if determined within 30seconds.

The instrument provides a true measure of the volumeof erythrocytes that has undergone osmotic lysis. Forexample, the volume of normal cells and ghosts, at atonicity of 0.3% NaCl, was accurately determined by theCoulter counter. This was established by comparing theratio of mean cell volume in 0.30% NaCl to mean cellvolume in 1.0% NaCl by use of the Coulter counter, by

1.0% A

0.425% B

0.40%/C

0.30%(30 min.) E

0 40 80 120 160

CHANNELNUMBER

FIG. 1. VOLUMEDISTRIBUTION CURVES OF FRESH NOR-

MALERYTHROCYTESAT DIFFERING TONICITIES OF NACL-P04BUFFER. The blood was diluted 1: 50,000 and the distri-bution analyzed with a Coulter counter and a 400-channelanalyzer. The curves represent relative particle fre-quency vs. channel number at various dilutions of oneblood sample. A = 1%, immediate plot; B = 0.425%,immediate plot; C = 0.40%, immediate plot; D= 0.30%,immediate plot; and E = 0.30%, plotted after standing for30 minutes at room temperature.

the microhematocrit method, and by measurement of thedilution space of radioactive 'I-iodinated serum albu-min. For these studies, to avoid the problem of chang-ing volume with reswelling of the cells hemolyzed in0.3% NaCl, all of the readings were obtained at approxi-mately 30 minutes when the cells had reswollen to theircritical volume. Values for this ratio obtained with asample of normal erythrocytes completely hemolyzed in0.3% NaCl were, respectively, 1.79, 1.77, and 1.71.

Cation composition and mean cell volume. Mean cellvolume as expressed in Table III was determined bymicrohematocrit and red cell counts using the Coultercounter. These determinations were carried out on freshblood as well as incubated samples at the time of re-moval for measurement of cation content. Cation deter-minations were carried out by measurements of sodiumand potassium with a Baird-Atomic flame photometer af-ter washing the cells with a cold (40 C) solution con-taining 0.1 M MgCl2+0.05 M Tris buffered at pH 7.4.Although Valberg, Holt, Paulson, and Szivek (21) sug-gested loss of Na' by washing of red cells with isosmolarMgC12, the values obtained in the present study for freshnormal cells are in substantial agreement with their val-

1139

ROBERTI. WEEDAND ANTHONYJ. BOWDLER

ues, perhaps because a hypertonic wash solution was em-ployed in the present studies.

ATP. Adenosine triphosphate was measured by thefirefly tail, luciferin-luciferase system described by Mc-Elroy and Seliger (22). We added 10 al of red bloodcells washed in 1% NaCl to 10 ml of distilled water,placed them in a boiling water bath for 1i minutes,chilled them in an ice bath, and either measured immedi-ately or froze them. The assay itself was carried out ina Nuclear-Chicago liquid scintillation counter accordingto the method of Tosteson, Cook, and Blount (23) andTal, Dickstein, and Sulman (24) modified to use standardvials and to record counts between 10 and 34 secondsafter mixing.

Photographs. Phase photomicrographs were taken offresh and incubated spherocytic erythrocytes. Samples tobe photographed were prepared before incubation by cen-trifugation twice at 250 X g for 10 minutes to removeplatelet-rich plasma. The latter was then freed of leuko-cytes, platelets, and particulate matter by centrifugationat 20,000 X g for 30 minutes. The red cells were washedtwice with 1%o NaCI to complete removal of leukocytesand platelets, then resuspended in the clear plasma.Rouleaux formation promptly occurred in both normaland HS samples treated in this fashion. Before photog-raphy one drop of blood was mixed with a drop of 1%NaCl to break up the rouleaux, and the preparation wasexamined wet between siliconized slides and coverslips.Electron micrographs were prepared by gluteraldehydethen osmium fixation of the blood cells and mounting inepon and sectioning.

Results

Autohemolysis and osmotic fragility. Table Isummarizes the autohemolysis tests carried out onspherocytic blood in this study after 24 hours ofgentle mixing and after 48 hours of incubation insterile test tubes without shaking.

In all of the described experimental situationsusing blood from five normal donors, hemolysiswas less than 1% in all samples incubated for 24hours with gentle agitation and less than 3%o inthe 48-hour tube autohemolysis tests. The HSincubations, however, were in accord with the

TABLE I

Per cent autohemolysis of hereditary spherocytes

24 hours* 48 hourst

Glucose 1.3 =1 0.5t 3.0 i 1.8Glucose and ouabain 2.3 i 1.1 5.4 i 2.9No additive 3.9 + 1.7 17.1 : 2.3Ouabain alone 16.4 + 4.6

* Four determinations.t Five determinations.$ Mean values + standard error are given.

OSMOTIC FRAGILITY CHANGESINHEREDITARY SPHEROCYTESAFTER INCUBATION

CJ)

K2

1.0 .80 .60 .40 .20

PERCENT NACLFIG. 2. EFFECT OF ADDITIVES ON OSMOTICFRAGILITY OF

A SAMPLEOF HEREDITARY SPHEROCYTOSISBLOODINCUBATED

24 HOURS. Added glucose concentration was 0.03 moleper L and ouabain, 1 X 10-' mole per L.

previous experience of others (5-7) in that theaddition of glucose protected the spherocytic cellsagainst the hemolysis seen in the incubations with-out added metabolic substrate. Although the ad-dition of ouabain appears to increase slightly the48-hour hemolysis of HS blood with glucose,this effect at both 24 and 48 hours is minimal com-pared with the significant protection against he-molysis conferred by glucose even in the presenceof ouabain. The latter finding is at variance withthe findings of Mohler (25), who demonstrated noprotection by glucose in the presence of ouabain.Two of Mohler's experimental conditions differedfrom ours, continuous mixing of the blood and alower concentration of ouabain (2.5 X 10-5 moleper L). These may contribute to the discrepancy.Defibrinated blood was used for the autohemolysisin both studies.

Figure 2 illustrates a typical pattern of osmoticfragility of spherocytes, which occurs after 24hours of incubation. Identical relative patternswere seen in studies of blood from five other HSpatients. The fragilities do not correspond to thefindings in the autohemolysis test. The cells towhich glucose was added appear to show a greaterincrease in osmotic fragility than the cells towhich no glucose had been added. This increaseis related presumably to a combination of increasein cellular cation content (Table III) and to in-creased cellular Cl-, which will be anticipated be-

1140

METABOLIC DEPENDENCEOF CRITICAL HEMOLYTICVOLUME

cause of the decreased ionization of hemoglobin as

pH falls. This observation of increased fragilityupon incubation with glucose was previouslypointed out by Selwyn and Dacie (6). Additionof ouabain does not seem to influence these find-ings significantly in either circumstance.

ATP levels. Mohler (25) presented evidencethat ATP utilization by HS cells is greater thannormal even in the presence of ouabain; therefore,these levels were compared after 24 hours of incu-bation. Table II summarizes these results alongwith data from parallel incubations of normalcells. The presence of glucose appears to havemaintained ATP levels in both the spherocytesand the normal cells between 50 and 68% ofpreincubation values. This was true whetherouabain was present or not, although the variabil-ity precludes any precise comparison. In thenonsupplemented cells, however, the ATP haddisappeared at 24 hours. The actual activity ofthe nonsupplemented incubations by the firefly as-

say ranged from 0 to 5 % at 24 hours. Becauseof the possibility of a small reading from ADP,the values have been reported < 5%. The simi-larity of the relative 24-hour ATP values fornormal and HS cells supplemented with glucoseis consistent with observations of Keitt (26).

Cation content. Table III compares the cationcomposition per cell of normal and HS cells beforeand after 24 hours of incubation in the presence

and absence of glucose and ouabain. The initial(preincubation) HS values were not significantlydifferent from values for K+ and Na+ of normalcells (p > 0.2). This observation agrees with thestudies of Selwyn and Dacie (6) on HS cells fromsplenectomized patients. In both normal and HScells, incubation without glucose, with ouabainonly added, and with glucose plus ouabain re-

sulted in highly significant (p < 0.001) losses ofK+ and gains of Na+ by comparison with the re-

spective fresh cells. The normal cells treatedwith ouabain alone or metabolically depleted ap-

peared to be unchanged in total cation contentwith the gain of Na+ equaling the loss of K+.The values for total cation content are not sig-nificantly different from the fresh values. By con-

trast, incubated but nonsupplemented HS cellsincubated without glucose lost more K+. Thisdifference between K+ content per cell of HS andnormal cells incubated without glucose is highly

TABLE II

Erythrocyte A TP levels

24 hours

Normal HS*

Glucose 68 1 13t 62 i 25(N=8) (N=4)

Glucose + ouabain 58 ± 13 54 d 35(N=5) (N=4)

No additive <5 <5(N=8) (N=4)

* HS = hereditary spherocytosis.t Values are expressed as the mean per cent of initial

ATP levels 4 standard error. The mean preincubationvalue for normal cells was 2.1 d 0.3 (SE) X 10-16 moleper cell and 1.8 i 0.4 for the HScells. N is the number ofdeterminations.

significant (p < 0.01), whereas the difference be-tween their total cation content is significant atthe p < 0.05 level. The addition of ouabain toHS cells incubated without added glucose resultedin a cation and volume pattern similar to thatseen in the nonsupplemented cells. The HS cellsto which both glucose and ouabain were addedgained sodium but appeared to lose less potassiumthan the nonsupplemented cells (p < 0.05), andthus their total cation content exceeded that ofthe nonsupplemented cells, with or without oua-bain (p < 0.001).

Mean cell volume. Table III also indicates themean cell volume of the spherocytic cells undereach of the incubation circumstances as measuredby hematocrit and red cell count done on thewhole blood initially and at the end of each incu-bation period. Analysis of the volume versustotal cation data of Table III reveals a highlysignificant (p < 0.001) correlation between themean cell volume of fresh and incubated normalcells as well as fresh and incubated HS cells andthe total cation content of the cells. The indi-vidual correlation coefficients (r = 0.60 and 0.32,respectively, for normal and HS), however, sug-gest that the cation content was not the only factordetermining cell volume. The differences betweenthe correlation coefficients for the HS versus nor-mal populations or the regression lines (b = 3.36and 1.4 for normal and HS, respectively) are ofborderline significance (p = 0.05) but suggest apoor correlation of volume and cation content inHS cells. In this regard, it is quite clear thatthe spherocytes to which glucose had been added

1141

ROBERTI. WEEDAND ANTHONYJ. BOWDLER

TABLE III

Cation content and mean cell volume of normal and HSerythrocytes*

Volume in- K+ Nat Na+ + K+ plasma N

NormalFresh 9.38 + 0.54 1.02 i 0.44 10.4 +t 0.54 97.9 i 3.0 1124-hour glucose 9.24 ± 0.44 1.20 A 0.93 10.8 i 0.43 99.8 ± 2.3 1024-hour glucose 7.70 i 0.36 3.44 i 1.49 11.1 ± 0.44 103.7 i: 2.7 8

+ ouabain24-hour no additive 7.64 i 0.57 3.04 i 0.22 10.7 i 0.44 102.0 ±- 2.9 1024-hour ouabain alone 6.81 ± 0.63 3.62 ± 0.32 10.6 ± 0.92 95.3 i 3.3 3

HSFresh 8.47 ± 1.07 1.23 + 0.18 10.06 ± 0.65 92.9 :1: 2.2 824-hour glucose 8.84 i 0.22 1.61 :1 0.11 10.67 4 0.36 98.2 i 1.6 924-hour glucose 6.72 :1: 0.47 3.86 i 0.10 10.56 :1 0.16 103.1 :1: 2.4 9

+ ouabain24-hour no additive 5.61 :1 0.45 3.12 :1: 0.22 8.76 i 0.45 96.3 i 3.2 924-hour ouabain alone 5.20 ± 0.54 3.55 i: 0.30 9.04 i 0.48 87.4 i 4.5 3

* Cation values are expressed as moles per cell X 10-11 ± SE. Mean corpuscular volume in plasma is expressed inmicrons i SE. The determinations were made on blood from ten normal subjects and five patients with hereditaryspherocytosis. Two separate determinations were made on some of the HSpatients. N is the number of determinations.

(p < 0.01) as well as the glucose plus ouabaincells (p < 0.01) were larger than the HS cells thathad not been supplemented with glucose. The lat-ter had apparently lost sufficient potassium to re-sult in a decrease in total cation content fromthe fresh cells.

100- .1 0 GLUCOSE - ieA .... & NO ALGIVE0--@ OUAUAIN

GLUCOSE.+ UA800 ,30---0 PRE-INCUBATIO*

80.. 0,.0.6.. 50.I.0 0.8 0.7 0.6 0.5 0.4 1.0 0.8 0.7 0.6 0.5 0.4

PERCENT Na Cl

FIG. 3. MEAN CELL VOLUMESCALCULATED FROM VOL-UME DISTRIBUTION CURVES AT DIFFERING TONICITIES OFNACL-PO4 BUFFER. The left portion represents normalblood mean values determined on six normal blood sam-ples examined fresh, after 24 hours of incubation withoutadditive, with glucose, and with glucose plus ouabain.Normal bloods incubated without additive or with onlyouabain added were not different and, therefore, thelatter is omitted from the graph. The right portion rep-resents mean values from similar studies of blood fromfive hereditary sperocytosis patients. MCV=meancellvolume; RBC= red blood cells.

HEREDITARYSPHEROCYTES

Critical hemolytic volume. The left section ofFigure 3 represents the mean cell volume at vari-ous tonicities of normal red blood cells, both freshand incubated, as derived from the volume distri-bution data provided by the Coulter counter.The right section of the Figure represents similar

1.0 %

0.80% B

0 5 I I I

0.55%

0.55 %(30 min.) / D

r- -I., a -I.

0 40 80 120 160

CHANNELNUMBER

FIG. 4. VOLUMEDISTRIBUTION CURVES IN NACL-P0&BUFFER OF A HEREDITARY SPHEROCYTOSISRED CELL SAM-PLE INCUBATED 24 HOURSWITHOUTADDEDGLUCOSE. Thecurves represent relative particle frequency vs. channelnumber. A = 1.0%, immediate plot; B = 0.80%, imme-diate plot; C= 0.55%, immediate plot; and D = 0.55%,plotted after standing for 30 minutes at room temperature.

1 142

METABOLIC DEPENDENCEOF CRITICAL HEMOLYTICVOLUME

TABLE IV

24-hour change in critical hemolytic volume (CHV)*

Normal HSCHV CHV

Glucose +5.5 i 1.0 +1 i 3Glucose + ouabain +6.9 :i 1.1 +2 1: 4Noadditive +5.5 ± 1.3 -15 ±4

* Mean (4i standard error) changes in critical hemolyticvolume of normal and HSblood after 24 hours' incubation,expressed as per cent of preincubation value.

data obtained by examination of the volume dis-tribution of HS cells, both fresh and incubated.Since it has been shown that the Coulter counterreflects a true estimate of cell volume, even afterosmotic lysis of cells in 0.30%o sodium chloride,the maximal or critical hemolytic volume is indi-cated by the point at which the curves reach amaximum. Beyond that point the curves decreaseas a consequence of loss of cellular contents, sec-ondary to hemolysis. The maximal value repre-sents the mean of the volumes of cells still swell-ing plus those which have hemolyzed. Thus, thepeak of the fresh normal cell curve falls within anarrow range of salt concentrations, in contrastto the flattened peak of incubated HS cells thathemolyzed over a wider range of salt concentra-tions. Figure 4 represents the volume distributioncurves of HS cells incubated for 24 hours withoutglucose. Comparison of B (0.80%), in whichhemolysis begins, with C (0.55%o), in which he-molysis is nearly complete, reveals little change involume or distribution. These patterns explainthe flat HS curve in Figure 3 and, in contrast tofresh normal cells (Figure 1, C), no inhomo-geneity is seen in partially hemolytic salt concen-trations. In Figure 4, D, the reswelling afterlysis is seen at 30 minutes. The volume, however,although slightly larger than Figure 4, B, is stillmarkedly decreased as anticipated from the lossof lipid (9).

Three features of the curves in Figure 3 deservespecial comment. 1) The critical volume of freshHS cells is decreased, as pointed out by Guest in1948 (27). 2) It is evident that incubating nor-mal cells for 24 hours results in some increase intheir volume in 1% buffered saline, but the swell-ing response to hypotonicity is essentially un-changed after this period of incubation, regardlessof the addition of glucose and ouabain. The

critical hemolytic volume of normal cells incubatedfor 24 hours with or without glucose does notappear to change or may actually increase slightly.Similarly, after 24 hours of incubation the glucoseand glucose plus ouabain-supplemented HS cellsappear to demonstrate a slight increase in criticalhemolytic volume. 3) The ouabain-treated andnonsupplemented HS cells, however, demonstrateda striking decrease in critical hemolytic volume,as indicated in the lower part of the right portionof Figure 3. Table IV summarizes the 24-hourcritical hemolytic volume data. Figure 5, whichrepresents the mean of three determinations, illus-trates that after 36 hours of incubation withoutsupplementary glucose normal cells also show adecrease in critical hemolytic volume, which isprevented by the addition of glucose.

Morphologic changes. Figure 6 is a phasephotomicrograph of fresh HS cells and illustratesthat many of the cells are biconcave discs with anevident light central area. In contrast to normalcells, however, several spherocytes appear crenated,even in freshly drawn blood. Incubation for 24hours with glucose produces an increase in thenumber of crenated forms without a decrease involume. Incubation without added metabolic sub-

h-),~4..

160-

140

120-

100'

1.0 0.8 0.7 0.6 0.5 0.4

%NaCIFIG. 5. MEAN CELL VOLUMESAT VARYING TONICITIES

OF NACL-P04 BUFFER OF NORMALERYTHROCYTESINCU-BATED FOR 36 HOURS. Values represent means of threedeterminations.

1143

ROBERTI. WEEDAND ANTHONYJ. BOWDLER

FIG. 6. PHASE PHOTOMICROGRAPHOF FRESH HEREDITARY SPHEROCYTOSISRED BLOODCELLS. Note some normal biconcave discs as well as crenation.

strate produces changes that appear to correspondto Ponder's (28) description of the disc-- crenateddisc -* crenated sphere -* sphere transformation.Crenated discs are transformed to crenated spheresand then to end stage spherocytes at 24 hours,smaller in diameter and volume and without cre-nation. Figures 7A and B represent HS bloodincubated without glucose for 5 hours. Note themarked crenation and also the extension of crena-tion to bud-like processes, which separate from thecells yielding. platelet-sized fragments. Underhigher magnification, smaller filamentous "myelinforms" can be seen coming off the tips of thecrenations. Figure 8 illustrates end stage sphero-cytes incubated for 24 hours without glucose.Figure 9 is an electron micrograph of normal cellsincubated for 36 hours without substrate; frag-mentation similar to that seen at an earlier stagein HS cells is to be seen. The end result of suchmembrane loss from normal cells is the productionof microspherocytes that appear like the HS cellsseen in Figure 8. Normal cells incubated in glu-cose or glucose plus ouabain for 48 hours showsome crenation, but many biconcave discs can stillbe seen.

Discussion

The main determinant of in vitro hemolysis isthe volume of the cell at any given time in relation

to its maximal possible membrane surface area.In vitro osmotic fragility is dependent on: a) thesuspending medium, whose pH and tonicity arecontrolled in the osmotic fragility test; b) the totalnumber of intracellular osmotically active con-stituents, which determine cell volume in anygiven external environment; and c) the criticalhemolytic volume, a complex parameter dependenton quantitative and qualitative factors associatedwith the membrane lipid and protein. Therefore,the important relationship determining osmoticfragility is the ratio of the critical hemolytic vol-ume to the internal osmotic contents of the eryth-rocyte. If we assume a continuous bilipid layerof membrane, the lipid loss after in vitro incuba-tion as demonstrated by Reed and Swisher (8, 9)and Prankerd (10) would result in a decrease inthe surface area. If this decrease of surface areais in excess of the loss of osmotically active cellcontents, increased osmotic fragility would in-evitably result. The corollary of this is that anyincrease of osmotic contents would also predisposeto increased sensitivity to osmotic lysis. Such anincrease in the total cation content of the cell withsecondary accumulation of anions but without achange in surface area has been demonstrated inthe HS cells incubated with supplemental glucoseand in normal cells incubated without glucose.Such cells will approach their critical volume at

1144

METABOLIC DEPENDENCEOF CRITICAL HEMOLYTICVOLUME 1145

FIG. 7. PHASEPHOTOMICROGRAPHOF HEREDITARY SPHEROCYTOSISBLOOD INCUBATED 5 HOURSIN ABSENCEOF GLUCOSE.Note fragmentation.

FIG. 8. PHASEPHOTOMICROGRAPHOF HEREDITARYSPHEROCYTOSISBLOODINCUBATED FOR24 HOURSWITHOUTSUBSTRATE. Note decreased size and loss of crenations.

ROBERTI. WEEDAND ANTHONYJ. BOWDLER

FIG. 9. ELECTRONMICROGRAPHOF NORMALERYTHROCYTESHOWINGFRAGMENTATIONAFTER 36 HOURSOF INCUBATIONWITHOUTSUBSTRATE. Cells were fixed with gluteraldehyde and osmium tetraoxide, mounted in epon, and magnified16,000 times.

a higher external tonicity than will cells with nor-mal intracellular osmotic content. Similarly, invitro autohemolysis in the absence of any externalphysical or chemical injury to the membrane maybe considered analogous to osmotic lysis and re-sult from a decrease in the ratio of critical hemo-lytic volume to cellular contents. Autohemolysiscan be produced by any alteration that producesa marked gain in intracellular osmotic activity

with swelling secondary to water gain. In theabsence of osmotically dictated water gain, how-ever, autohemolysis may result from a decreasein the critical hemolytic volume with the achieve-ment of the hemolytic ratio of surface area toosmotic contents, even at the tonicity of plasma.Obviously, osmotic swelling plus decreased criticalvolume can combine to produce hemolysis. Auto-hemolysis secondary to a decrease in critical hemo-

146

METABOLIC DEPENDENCEOF CRITICAL HEMOLYTICVOLUME

lytic volume is readily apparent in the HS cellsthat were incubated without the addition of glu-cose. Metabolically depleted HS cells were notsignificantly greater (p > 0.05) than fresh HScells in volume or total cation content, but theyunderwent only slight swelling upon introductioninto the buffered sodium chloride solutions ofdecreasing tonicity, indicating that many of thecells were close to their critical volume at an ex-

ternal tonicity equal to that of plasma. Conse-quently, in terms of the ratio of critical hemolyticvolume to osmotically active cell contents, the de-crease in surface area is primarily responsible forthe autohemolysis of nonsupplemented, metaboli-cally depleted HS cells. These observations pro-

vide direct support for the suggestion of Selwynand Dacie (6) that the primary abnormalityaccounting for in vsitro autohemolysis of non-

supplemented HS cells is a membrane alterationwith decrease in surface area.

The addition of ouabain, which inhibits thesodium-potassium pump, resulted in a net accu-

mulation of sodium but a lesser loss of potassiumin the HS cells incubated with glucose plus oua-

bain, with a resultant increase in the cation contentand volume of these cells in plasma. The glucoseplus ouabain HS cells did not demonstrate any

significant decrease in critical hemolytic volumeand were no different in this respect from thecells that had glucose alone added. On the otherhand, the HS cells incubated without added glu-cose also had both an increased sodium contentand decreased potassium content, but the totalcation content of these cells and in particular thepotassium content was decreased. Both the K+(p < 0.01) and the Na+ (p < 0.01) were sig-nificantly lower than like values for the glucoseplus ouabain HS cells. Thus, although swellingof the cells at 24 hours might be anticipated byvirtue of a decreased activity of the sodium-potassium pump with a secondary increase in totalcation content in line with the suggestion of Toste-son (29) for the role of the pump in the mainte-nance of normal cell volume, this does not appear

to be the case. By contrast, normal cells incu-bated under identical circumstances do appear toconform with the pump-leak hypothesis, showingsome gain in total cation and little change incritical hemolytic volume after 24 hours of in-cubation under identical circumstances. It is ap-

parent, however, that normal cells do undergo adecrease in critical volume after 36 to 48 hourssimilar to those found with HS cells after 24hours of incubation.

Thus, protection against autohemolysis of glu-cose or glucose plus ouabain-supplemented HScells appears attributable to maintenance of criticalvolume. Theoretically, even in the face of a de-crease in critical volume as seen with depletedHS cells, if the cell water (or effective osmoticactivity) were lowered sufficiently, autohemolysiscould also be prevented by maintaining the ratioof critical volume to osmotic contents below thepoint of autohemolysis. Observations of Jacoband Jandl (12) on the protection of depleted HScells against autohemolysis by added sucrose orby substitution of choline for sodium are consistentwith this alternative mode of protection againstautohemolysis.

Reed and co-workers (8, 9, 30) have shown thatthe exaggerated loss of lipid from HS cells in-volves all the phospholipids and cholesterol in theproportions in which they are found in the intactred cell membrane. This proportionate loss ofmembrane lipid and the apparently paradoxicalexaggerated loss of both potassium and total cat-ion are consistent with the morphological findingof fragmentation after crenation. Fragments maynot only contain lipid but possibly cation as welland thus provide an explanation for cell volumebeing unchanged at 24 hours in spite of the earlyincrease in volume secondary to Na+ accumulationdescribed by Jacob and Jandl (12). Althoughmetabolically depleted normal cells will undergoa similar disc-sphere transformation with loss oflipid and reduction of critical volume after incuba-tion for 36 to 48 hours, the hereditary spherocyteseems to manifest these changes more rapidly.The interchangeability of adenosine, inosine, andglucose as substrates to maintain the integrity ofhereditary spherocytes, as well as the demonstra-tion of Jaff6 and associates (31) that adeninenucleotides help protect the normal red cell againstosmotic lysis when incubated in vitro, suggests thatATP is essential for maintenance of the mem-brane lipid and critical hemolytic volume.

The addition of ouabain in no way interfereswith this protective action of glucose and there-fore, presumably ATP in terms of stabilizing thestructure of the membrane. Thus the sodium-

1 147

ROBERTI. WEEDAND ANTHONYJ. BOWDLER

potassium pump ATPase does not appear to beinvolved in this process. In considering the shapechanges, however, Nakao and co-workers (32, 33)have presented evidence to indicate that the disc-sphere transformation of normal cells may beproduced by lowering of cellular ATP levels.Since HS cells have an increased requirement ofATP for transport (12), they may also have asimilarly increased requirement for maintenanceof shape.

Fragmentation of normal erythrocytes afterprolonged in vitro incubation was first describedby Meltzer and Welch (34, 35) and characterizedin great detail by Ponder (28). A postulatedsequence for the in vitro events described for theHS cell would be initiated by ATP depletion.Initial swelling secondary to accumulation of Na+as described by Jacob and Jandl (12) would beaccompanied by crenation as ATP disappeared.Since a biomolecular layer of lipid is physicallythe most stable configuration (36), marked crena-tion may actually lead to small areas having, atthe tips of crenations, quadrimolecular lipid layersleading to submicroscopic losses of lipid. Thefact that normal cells incubated for 24 hours withglucose show some crenation and lipid loss but nodecrease in critical hemolytic volume is consistentwith such a notion. Maintenance of critical vol-ume, in spite of some lipid loss, must mean thatthe critical volume also depends on the structuralmembrane protein, the organization of which inturn may relate to ATP levels. Finally, grossfragmentation of the depleted HS cell becomes evi-dent with further losses of lipid and K+. How-ever, the relative importance of fragmentation evi-dent by light microscopy versus that not evidentwith the light microscope remains to be deter-mined. In any case, decrease of surface area anda steady decrease in critical hemolytic volume oc-cur and finally lead to the smooth microsphero-cyte. Thus, the well-known changes in osmoticfragility and autohemolysis of metabolically de-pleted HS cells would appear to be related tothe marked changes in the cell membrane as pos-tulated by Selwyn and Dacie (6). In additionto an increased Na+ influx (11) and increasedpump flux at normal intracellular Na+ levels (12),the fresh HS cell from splenectomized patientshas a decreased critical hemolytic volume and in-creased predisposition to undergo disc-sphere

shape changes in spite of a normal lipid and totalcation content (8, 9). Thus, the basic defect mayreside with the membrane protein and its ouabaininsensitive as well as ouabain sensitive ATPaseactivities. Tosteson (37) has called attention tothe genetic linkage and correlation between pumpATPase activity and cation leakiness in sheepcells. The increased permeability of HS cells mayrepresent a similar example of such a geneticcorrelation between increased cation leakiness andincreased pump ATPase activity involving theouabain insensitive ATPase as well.

Summary

Alterations in osmotic fragility and in vitroautohemolysis observed in both normal erythro-cytes and hereditary spherocytes can best beunderstood in terms of the relationship betweencritical hemolytic volume and intracellular osmoticactivity. Evidence has been presented for aouabain insensitive membrane requirement forglucose. Both normal cells and hereditary sphero-cytes when metabolically depleted undergo thedisc-sphere transformation described by Nakaoand his associates. These shape changes are as-sociated with a concomitant loss of membranematerial manifested as a physical fragmentation.

The hereditary spherocyte differs from the nor-mal cell in that this degenerative loss of mem-brane material is much accelerated compared withthe normal when both are incubated without addedglucose. Wesuggest that the primary defect maybe an abnormality in the membrane protein ofhereditary spherocytes.

Acknowledgments

We wish to thank Dr. Charles Yuile for his electronmicrographs of normal cells incubated for 36 hours andMiss Ann Gregory, Mrs. Sonja Valkenburg, and MissDonna Meisenzahl for their technical assistance in carry-ing out the experiments.

References

1. Haden, R. L. The mechanism of the increased fra-gility of the erythrocytes of congenital hemolyticjaundice. Amer. J. med. Sci. 1934, 188, 441.

2. Castle, W. B., and G. A. Daland. Susceptibility oferythrocytes to hypotonic hemolysis as a functionof discoidal form. Amer. J. Physiol. 1937, 120,371.

148

METABOLIC DEPENDENCEOF CRITICAL HEMOLYTICVOLUME

3. Dacie, J. V., and J. M. Vaughan. The fragility of thered blood cells: its measurement and significance.J. Path. Bact. 1938, 46, 341.

4. Sharpsteen, J. R., Jr. Physico-chemical mechanismsin the pathogenesis of certain hemolytic anemias.Amer. J. med. Sci. 1955, 229, 506.

5. Dacie, J. V. Observations on autohaemolysis in fa-milial acholuric jaundice. J. Path. Bact. 1941, 52,331.

6. Selwyn, J. G., and J. V. Dacie. Autohemolysis andother changes resulting from the incubation invitro of red cells from patients with congenitalhemolytic anemia. Blood 1954, 9, 414.

7. Young, L. E., M. J. Izzo, K. I. Altman, and S. N.Swisher. Studies on spontaneous in vitro auto-hemolysis in hemolytic disorders. Blood 1956, 11,977.

8. Reed, C. F., and S. N. Swisher. Abnormalities of invitro behavior of structural lipids of red blood cellsfrom patients with hereditary spherocytosis (ab-stract). J. clin. Invest. 1960, 39, 1019.

9. Reed, C. F., and S. N. Swisher. Erythrocyte lipidloss in hereditary spherocytosis. J. clin. Invest.1966, 45, 777.

10. Prankerd, T. A. J. Studies on the pathogenesis ofhaemolysis in hereditary spherocytosis. Quart. J.Med. 1960, 29, 199.

11. Bertles, J. F. Sodium transport across the surfacemembrane of red blood cells in hereditary sphero-cytosis. J. clin. Invest. 1957, 36, 816.

12. Jacob, H. S., and J. H. Jandl. Increased cell mem-brane permeability in the pathogenesis of heredi-tary spherocytosis. J. clin. Invest. 1964, 43, 1704.

13. Parpart, A. K., P. B. Lorenz, E. R. Parpart, J. R.Gregg, and A. M. Chase. The osmotic resistance(fragility) of human red cells. J. clin. Invest.1947, 26, 636.

14. Garby, L., and C. H. de Verdier. Glucose metabo-lism in normal erythrocytes. I. Kinetics of thehexokinase reaction in intact cells. Scand. J.Haemat. 1964, 1, 150.

15. Simon, E. R., and P. Ways. Incubation hemolysisand red cell metabolism in acanthocytosis. J. clin.Invest. 1964, 43, 1311.

16. Hoffman, J. F. Cation transport and structure of thered-cell plasma membrane. Circulation 1962, 26,1201.

17. Lepke, S., and H. Passow. Effects of temperature,alkaline earth metal ions, and complexing agentsof the hemolytic medium on potassium permeabilityof erythrocyte ghosts (abstract). Pfluigers Arch.ges. Physiol. In press.

18. Murphy, J. R. Erythrocyte metabolism. II. Glucosemetabolism and pathways. J. Lab. clin. Med. 1960,55, 286.

19. Brecher, G., E. F. Jakobek, M. A. Schneiderman, G.Z. Williams, and P. J. Schmidt. Size distributionof erythrocytes. Ann. N. Y. Acad. Sci. 1962, 99,242.

20. Weed, R. I., C. F. Reed, and G. Berg. Is hemoglo-bin an essential structural component of humanerythrocyte membranes? J. clin. Invest. 1963, 42,581.

21. Valberg, L. S., J. M. Holt, E. Paulson, and T.Szivek. Spectrochemical analysis of sodium, po-tassium, calcium, magnesium, copper and zinc innormal human erythrocytes. J. clin. Invest. 1965,44, 379.

22. McElroy, W. D., and H. H. Seliger. The chemistryof light emission. Advanc. Enzymol. 1963, 25, 119.

23. Tosteson, D. C., P. Cook, and R. Blount. Separationof adenosine triphosphatase of HK and LK sheepred cell membranes by density gradient centrifuga-tion. J. gen. Physiol. 1965, 48, 1125.

24. Tal, E., S. Dikstein, and F. G. Sulman. ATP deter-mination with the Tricarb scintillation counter.Experientia (Basel) 1964, 20, 652.

25. Mohler, D. N. Adenosine triphosphate metabolism inhereditary spherocytosis. J. clin. Invest. 1965, 44,1417.

26. Keitt, A. S. Changes in the content and 'P incorpo-ration of glycolytic intermediates during incubationof normal and hereditary spherocytosis erythro-cytes. Brit. J. Haemat. 1965, 11, 177.

27. Guest, G. M. Osmometric behavior of normal andabnormal human erythrocytes. Blood 1948, 3, 541.

28. Ponder, E. Hemolysis and Related Phenomena.New York, Grune & Stratton, 1948, p. 168.

29. Tosteson, D. C. Regulation of cell volume by so-dium and potassium transport in The CellularFunctions of Membrane Transport, J. F. Hoffman,Ed. New Jersey, Prentice-Hall, 1964, p. 3.

30. Weed, R. I., A. J. Bowdler, and C. F. Reed. Meta-bolic dependence of erythrocyte membrane struc-ture. Clin. Res. 1965, 13, 284.

31. Jaffe, E. R., B. A. Lowy, G. A. Vanderhoff, P.Aisen, and I. M. London. The effects of nucleo-sides on the resistance of normal human erythro-cytes to osmotic lysis. J. clin. Invest. 1957, 36,1498.

32. Nakao, M., T. Nakao, and S. Yamazoe. Adenosinetriphosphate and maintenance of shape of humanred cells. Nature (Lond.) 1960, 187, 945.

33. Nakao, M., T. Nakao, S. Yamazoe, and H. Yoshi-kawa. Adenosine triphosphate and shape of eryth-rocytes. J. Biochem. (Tokyo) 1961, 49, 487.

34. Meltzer, S. J. The effects of shaking upon the redblood cells. Johns Hopk. Hosp. Rep. 1900, 9, 135.

35. Meltzer, S. J., and W. H. Welch. The behaviour ofthe red blood-corpuscles when shaken with indif-ferent substances. J. Physiol. (Lond.) 1884, 5,255.

36. Thompson, T. E. The properties of bimolecularphospholipid membranes in Cellular Membranes inDevelopment, M. Locke, Ed. New York, Aca-demic Press, 1964, p. 83.

37. Tosteson, D. C. Active transport, genetics, and cel-lular evolution. Fed. Proc. 1963, 22, 19.

1149