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Journal of Clinical InvestigationVol. 41, No. 7, 1962

EFFECTS OF SULFHYDRYLINHIBITION ON RED BLOODCELLS.II. STUDIES IN VIVO *

BY HARRYS. JACOBAND JAMESH. JANDL

(From the Thorndike Memorial Laboratory and Second and Fourth (Harvard) MedicalServices, Boston City Hospital, and the Department of Medicine,

Harvard Medical School, Boston, Mass.)

(Submitted for publication January 17, 1962; accepted February 26, 1962)

A failure of metabolic processes in red cells isbelieved to initiate changes leading to their seques-tration and destruction in the reticuloendothelialsystem, principally in the liver and spleen (1, 2).Since red cells derive virtually all of their energyfrom glucose, it appears that the decline in activ-ity of certain glycolytic enzymes during normalred cell aging may determine the life span of thecell (3-5). The rate of "senescence" is somewhatincreased in red cells of subjects whose oxidativeshunt (pentose phosphate pathway) is impairedby a deficiency of the enzyme, glucose-6-phosphatedehydrogenase (G6PD) (6, 7), and is greatlyincreased in red cells defective in the main gly-colytic pathway, as by a deficiency of pyruvatekinase (8). Applying stress to such defective redcells, e.g., by administering a potentially oxidantcompound to G6PD-deficient subjects, markedlyaccelerates the rate of cell damage and destruc-tion (9, 10).

The way in which defective glycolysis causesthe red cell to be recognized, trapped, and de-stroyed by reticuloendothelial cells is not known,although it is reasonable to assume that ultimatelythe red cell membrane must be altered, since itis the surface of the cell that comes in contact withthe erythroclastic cells.

Subsequent to the work of others (11-14) in-dicating that sulfhydryl compounds are involvedin certain hemolytic mechanisms, evidence waspresented in a preceding paper (15) that theshape and viability of red cells in vitro dependupon the maintenance of membrane sulfhydrylgroups. When these groups are blocked by spe-cific inhibitors, the red cells undergo the followingsequence of events: 1) cation gradients across thecell membrane are disrupted, with a cellular loss ofK+ and a gain of Na+; 2) the resulting unopposed

* This work was supported by Grant RG3507 (C8)from the National Institutes of Health, Bethesda, Md.

oncotic pressure of hemoglobin and other intra-cellular macromolecules causes an influx of waterleading to cellular swelling and spherocytosis; and3) ultimately the cell membrane becomes dis-tended, permitting the leakage of hemoglobin (i.e.hemolysis). It was suggested that this sequenceof events, observed after the addition of specificsulfhydryl inhibitors, may be likened to the se-quence of cellular injury in various hemolyticprocesses. Accordingly, studies were made, andare presented here, of the behavior of sulfhydryl-inhibited red cells in man and in rats, the findingsin vivo being correlated with the physical and bio-chemical alterations produced. A preliminary re-port has been published (16).

METHODS

As in the previous studies (15) the sulfhydryl inhib-itors employed were p-mercuribenzoate (PMB),' whichdoes not penetrate the intact red cell membrane (15, 17),and N-ethylmaleimide (NEM),2 which rapidly permeatesthe normal red cell (15, 18). The inhibitors were dis-solved in physiologic saline and were rendered sterile bySeitz filtration shortly before use. Although quite dif-ferent chemically, both compounds react rather specif-ically with sulfhydryl groups, and both cause very similardamage to the red cell membrane (15). Accordingly,their effects will be attributed throughout this paper tospecific reactions with sulfhydryl groups and will bereferred to as involving sulfhydryl "inhibition" or "block-ade." It should be recognized that this inference is basedupon circumstantial evidence, as is the case with otherinhibitors.

Techniques applicable to studies in man. Unless other-wise stated, 25 ml of blood from hematologically normalsubjects was collected with sterile precautions in acid-citrate-dextrose (ACD) solution, and the cells labeledwith Cr`1 by incubation for 30 minutes at room tempera-

1 PMBwas obtained as the Na salt of p-hydroxymer-curibenzoate from Sigma Chemical Co., St. Louis, Mo.This chemical has been shown by the manufacturer to befree of Hge+ by the HgS precipitation method.

2Obtained from Schwarz Bioresearch, Inc., Mt. Ver-non, N. Y.

1514

EFFECTS OF SULFHYDRYLINHIBITION ON RED BLOODCELLS

NORMALSUBJECT

T50-22 MIN.

RED CELLS

SPLENECTOMIZED. SUBJECf

RED CELLS

T50-II HRS.

I-

41

hit

l-L

Co

a

0

4c

I-->4t

-J4

0_

hI

IL

5 0

HOURS

LIVER

"SPLEEN"1 2 3 4 5

FIG. 1. DESTRUCTIONOF RED CELLS TREATED WITH NEM. In the normal subject (left) red cells labeled withCr51, and treated with a concentration of NEMsufficient to inhibit cellular GSH, are rapidly removed in an exponen-tial fashion from the circulation (left, top). These cells are selectively trapped in the spleen (left, bottom). In thesplenectomized subject (right) survival of similarly treated red cells is prolonged 30-fold without evidence of specificorgan sequestration.

ture with 100 to 200 Ac of Na2Cr5O4.3 The labeled cellsuspension, washed once and brought back to a 50 percent cell suspension in sterile physiologic saline, was

further incubated with an equal volume of inhibitor.Although incubation of cells with inhibitor for 15 minutesproduced near maximal effects on red cell survival, a

1-hour incubation period was used in most experiments.Thereafter the cells were washed once and resuspendedat a 50 per cent concentration in sterile saline. Thelabeled red cells were injected rapidly into the antecubitalvein of the donor. At various intervals blood was drawninto 3 per cent sodium citrate (9 ml blood-1 ml citrate)for measurement of whole blood and plasma radioactivityin a well-type scintillation counter. The plasma was alsoquantitatively analyzed for bilirubin (19, 20) and forhemoglobin by the benzidine method (21). At less fre-quent intervals blood was also collected in oxalate fordeterminations of osmotic fragility. The fragility of thecirculating Cr51-labeled cells was followed at various in-tervals after injection by comparing the radioactivity andhemoglobin concentration of the spun supernatants fromblood added to graded hypotonic salt solutions [the pre-viously described differential osmotic fragility technique(22, 23)].

Sites of cell sequestration were determined by body-

3 Abbott Laboratories, North Chicago, Ill.

surface monitoring over spleen, liver, and precordium aspreviously described (24).

In preliminary work the possible effects of NEMandPMBon the uptake and elution of Cr51 by red cells werestudied in vitro. Neither agent appreciably affected thecellular uptake of Cr51 as Na2Cr'O4, whether the inhibitorwas added to the cell suspension before or after the Cr'.The elution of Cr51 from red cells exposed to these in-hibitors was observed over a 48-hour period of sterileincubation in buffered saline and in serunm Althoughelution from inhibited cells somewhat exceeded that ofthe control cells in this period, this was largely accountedfor by their increased autohemolysis. When the ratio ofintracellular:extracellular Cr55 .as corrected for auto-hemolysis, the rates of elution of Cr' from control cellsand from sulfhydryl-inhibited cells were comparable.

Red cell dimensions and concentrations were measuredby standard techniques (21). The consumption of glucoseby red cells was measured by the Somogyi-Nelson tech-nique (25); their production of lactate was measured bythe method of Barker and Summerson (26). Red cellglutathione (GSH) levels were determined by the nitro-prusside method of Grunert and Phillips (27) as modifiedby Beutler (28) and by the "alloxan 305" method (29).

As noted in the first part of this study (15) the nitro-prusside method is unsatisfactory as a measure of mer-

captide formation. In experiments involving the meas-

a

in.

I~-0

z

0

0

z

0.

1515

HARRYS. JACOB ANDJAMES H. JANDL

urement of thiols in the presence of PMB, the "alloxan305" method was used.

Techniques applicable to studies in rats. The experi-ments were made on large (400 to 500 g) males from aBartonella-free strain (Charles River C.D.) of Sprague-Dawley rat. Small volumes (5 to 10 ml) of rat blood inACD were labeled with Na2Cr5104 as described above.The labeled cells were incubated with the appropriatesulfhydryl inhibitor for 1 hour, washed, and injected bytail vein into each of several other recipient animals. Theusual dose of injected cells was 1.0 ml of a 25 per centcell suspension containing 1 to 3 Acc of Cr51.

At various intervals after injection the animals weresacrificed; cardiac blood, spleen, liver, kidneys, lungs, andfemurs were removed and counted as described previously(30). Intact red cells were obtained from the spleens ofthese animals by gentle homogenization of that organ ina loosely fitting Potter-Elvejhem homogenizer. Thishomogenate, containing released, radioactive, splenic redcells, was added to hypotonic salt solutions and theosmotic fragility of the labeled cells determined as de-scribed above.

Phagocytosis studies. The phagocytosis of sulfhydryl-inhibited red cells was studied by means of the test-tubetechnique outlined by Packer and colleagues (31) andalso by a modified Rebuck and Crowley window technique(32) as follows. Sulfhydryl-inhibited human red cells infresh autologous serum were added to the forearm of thedonor 3 hours after sterile inflammation had been pro-duced by scarification. At various intervals thereafterthe cover glasses were removed and examined for eryth-rophagocytosis. Studies of phagocytosis in rats weremade by examining splenic imprints at various intervalsafter intravenous injection of sulfhydryl-inhibited cells.

RESULTS

Studies of NEM-treated cells in man. Autog-enous, Cr51-labeled red cells that had been treatedwith small, nonhemolyzing doses of NEMwererapidly removed from the normal circulation, as

TABLE I

Effects of various doses of NEMon red cell GSHand redcell survival in man

NEMconc. Red cell GSH Cr'1

pmoles/ml RBC mg/100 ml T6ored cells

0 60 *1 24 22 days2.3 7 26 days5 0 3 hr

20 0 22 min80 0 8 min

* Control observations on the half-survival of red cellsincubated with saline alone were not made. The survivalof cells treated with small amounts of NEMpresented inthe table is within the accepted normal range for the Cr5'technique (2).

shown in the left portion of Figure 1. The doseportrayed (20 MAmoles NEMper ml red cells)caused complete blockade of intracellular GSH,cessation of cellular glycolysis, a moderate leakageof K+, and a slight reduction in cell volume andosmotic fragility. The cells appeared normal mi-croscopically. The injected red cells were clearedin an exponential fashion with a half-clearancetime (T50) of 22 minutes. The osmotic fragility,and hence the spheroidicity, of the circulatinglabeled cells did not increase prior to sequestra-tion, as measured by the Cr5' differential osmoticfragility technique. The lower left portion of Fig-ure 1 demonstrates that the cells so treated wereselectively trapped in the spleen. This sequestra-tion was not followed by hemoglobinemia. How-ever, plasma bilirubin levels rose from a baselinelevel of 0.4 to 1.4 mg per 100 ml at 2 hours, and1.6 mg per 100 ml at 4 hours after injection of200 ml of a 50 per cent suspension of NEM-treated cells. There was no effect on either cir-culating white cell levels or body temperature ofthe injected subjects.

The special role of the spleen in trapping redcells so treated with NEMwas further studied ontwo occasions in an otherwise normal individualwho had been splenectomized several monthsearlier because of a traumatic splenic rupture.As shown in the right portion of Figure 1, NEM-treated cells survived over 30 times longer in thesplenectomized patient (T50 = 11 hours) than innormal individuals. During this slow clearanceof treated cells no increase in their osmotic fragil-ity was observed in vivo.

The effects of various doses of NEMon redcell GSHand on red cell survival are comparedin Table I. Amounts of NEMsufficient to blockas much as 90 per cent of cellular GSHhad noeffect on cell survival. At doses just sufficientto block all GSH, red cell survival was impaired,with a slow selective sequestration of labeled cellsby the spleen (Figure 2, left portion). Cells sotreated were of normal size, shape, and fragility.In contrast, much larger doses of NEM, in calcu-lated excess of that necessary to block all thesulfhydryl groups of cellular hemoglobin as wellas of GSH (15), caused some spherocytosis, in-creased osmotic fragility, and slight autohemoly-sis. Red cells treated with these large doses wererapidly removed from the circulation with seques-

1516


5)JMOLES NEM/ML RBC

T5o = 3 HRS

0

80 PIMOLES NEM/MLRBC

T50 = 8 MIN

LIVER

0 SPLEEN

10HOURS

FIG. 2. EFFECT OF VARYING DOSES OF NEMON RED CELL SEQUESTRATION.

Red cells treated with low doses of NEM, just sufficient to inhibit red cellGSH, are removed slowly from the circulation specifically by the spleen(left). Cells treated with high doses of NEM, sufficient to cause sphero-cytosis and subsequently slight autohemolysis in vitro, disappear very rapidly,being trapped by the liver as well as by the spleen (right).

tration in the liver as well as in the spleen (Figure2, right portion). This rapid sequestration was

also attended by hyperbilirubinemia, beginning1 hour after injection, but not by hemoglobinemia.

These observations, summarized in Table I, andindicating that relative decreases in intracellularGSHlevels were unimportant, per se, to cell sur-

vival as long as some measurable sulfhydryl activ-ity remained, were further investigated. Labeledautologous red cells, treated with NEMso that90 per cent of their original GSHhad been in-hibited, were administered to two patients on

moderately high doses of the oxidant drug pri-maquine 4 (60 mg of the base per day). In bothpatients normal cell survival was observed, indi-cating that these cells responded normally toadded oxidant-drug stress in contrast to con-

genitally GSH-deficient cells, whose lowered GSHlevels are secondary to a deficiency of G6PD (9).It is possible that the normal survival of theseNEM-treated red cells reflected a reversal of the

4 Primaquine diphosphate, generously supplied by Dr.George D. Wessinger, Sterling-Winthrop Institute, Di-vision of Sterling Drug, Inc., Rensselaer, N. Y.

inhibition of GSHor a restoration of GSHlevelsby new synthesis in vivo. On the other hand, theblockade of intracellular GSHby NEMwas notreversible in vitro, even in cells with continuingglycolysis or in cells that were subsequently re-

suspended in either cysteine, which permeates thered cell, or GSH, which does not.

Studies of PMB-treated cells in man. Red cellsincubated with small doses of the nonpermeableinhibitor, PMB(4 to 5 ,umoles per ml cells), were

shown to contain normal amounts of intracellularGSH, to consume glucose and produce lactate innormal fashion, and yet to leak K+ (15). Thesecells, although metabolically active, when rein-jected were rapidly removed from the circulation,being trapped mainly in the spleen (Figure 3).The osmotic fragility of these labeled circulatingcells remained normal during their brief period ofsurvival in vivo. Sequestration was followed bya bilirubin peak in the plasma but not by hemo-globinemia. These cells, therefore, despite theirnormal glycolytic activity and normal GSHlevels,behaved in a fashion analogous to that of red cellstreated with sufficient NEMto block all GSH.

ahiI-

eIL,w

z

e

0Mi.0I.-z

a.

1517

II III

a 4 6 8


As with NEM, doses of PMBgreat enough tocause uniform spherocytosis and slight autohemol-ysis caused more rapid cell disappearance accom-

panied by marked hepatic as well as splenic se-

questration. Doses less than that cited in Figure3 caused lesser rates of cell sequestration. Therewas no clear threshold dose as with NEM. Thusa dose of 1 ,umole PMBper ml red cells causedthe rapid destruction of a minor population of cells(about 30 to 40 per cent of those injected), whilethe remainder survived normally.

That the survival of Cr5' in sulfhydryl-inhib-ited red cells was not spuriously shortened in vivoas the result of a possible increased elution of theisotope is indicated by the following: 1) radio-activity accumulated in specific organs rather thanappearing in the plasma, other circulating redcells, or urine, as would be the case with freedCr51 (24); 2) in the absence of the spleen thesurvival of Cr51 in inhibited red cells was mark-edly prolonged; 3) the disappearance of Cr51-labeled cells from the blood stream was followedby hyperbilirubinemia and, with the bigger dosesof inhibitor, by hemoglobinemia; 4) in animals(see below) the injection of sulfhydryl-inhibitedred cells was followed by splenic engorgement

a

7a

:z

0

I&0

I-

z

hi

U-i49

0J

wn

RED CELLS

0 2 4 6 6

HOURS

FIG. 3. DESTRUCTION OF RED CELLS TREATED WITH

PMB. Red cells treated with PMB, 5 ,umoles per ml

red cells, have normal glycolytic capacity and normalGSH level, yet are removed rapidly, principally by thespleen.

T-m=50 MIN

i 40-o \

Z 20-

° 6I SPLEENz

40 /

0

0 4 6 8

HOURS

FIG. 4. DESTRUCTION OF SULFHYDRYL-INHIBITED RED

CELLS IN RATS. Rat red cells treated with NEM, 25/Amoles per ml red cells, disappeared rapidly from thecirculation. Sequestration of these cells was predom-inantly in the spleen. Cells treated with PMB (notshown) behaved in a similar fashion.

with red cells and, after a delay, by evidence oferythrophagocytosis.

Studies of phagocytosis of sulfhydryl-inhibitedred cells in man. Although autogenous A-positivered cells exposed for short periods in vitro to com-plement-fixing anti-A underwent erythrophago-cytosis when placed subsequently in skin-windowpreparations, this phenomenon was not observedwhen sulfhydryl-inhibited red cells were em-ployed. Similarly, phagocytosis of inhibited cellsby autologous granulocytes in the presence offresh serum was not demonstrable in the test tube,whereas in parallel preparations anti-A did induceerythrophagocytosis of A-positive red cells.

Studies of sulfhydryl-inhibited red cells in rats.The behavior of sulfhydryl-inhibited cells in therat circulation was similar to that found in man.Both NEM-treated cells (Figure 4) and PMB-treated cells disappeared from the circulationwithin 4 to 6 hours after injection. With thedoses employed, total spleen uptake was 2 to 5times that of the liver and splenic uptake was 30to 80 times that of the liver on a gram-for-grambasis. In addition. as in man, a marked prolonga-

1518

I


tion of survival of sulfhydryl-inhibited cells wasfound in splenectomized rats, despite enhancedhepatic uptake in these animals.

As in man, the trapping of sulfhydryl-inhibitedrat red cells by splenic tissue was not necessarilypreceded by their sphering, as measured by theosmotic fragility of these circulating, labeled cells.The gentle homogenization of rat spleens at vary-ing intervals after cell injection caused the releaseof intact labeled cells from this organ and allowedtheir fragility to be measured at such times. Fig-ure 5 illustrates the radioactive osmotic fragilitycurves so obtained. It is noted that the initial(16-minute) fragility of sequestered cells wasnearly normal. After 1 hour, at a time when 45per cent of injected cells had already been re-moved from the circulation, an osmotically fragilepopulation appeared. Still greater increases infragility were found in those few intact cellsresiding for more prolonged periods in the spleen(Figure 5).

Evidence that splenic trapping of these cells was

not primarily a function of erythrophagocytosiswas derived from direct microscopic examinationof imprints of spleen removed at varying timesafter injection of the altered cells. Initially, noevidence of abnormal macrophage inclusions couldbe demonstrated. However, after 1 to 2 hoursthere was a marked and consistent increase inerythrophagocytosis, mainly evidenced by the ap-pearance of large, colorless, cytoplasmic vacuolesin granulocytes and histiocytes.

DISCUSSION

These studies have shown that sulfhydryl-inhib-ited red cells are removed rapidly and completelyfrom the human and rat circulations. Appropri-ately inhibited cells, although of normal size andshape, are trapped and destroyed almost entirelyin the spleen by mechanisms unknown at present.

The spleen as a trap for sulfhydryl-inhibited redcells. The specificity with which the spleen trapssulfhydryl-deficient cells is striking and may evenexceed that of red cells coated with incomplete

'N)-I

.90 .80 .70 .60 .5;0 .40 .30 io2 . 0

NoC0, GM PER GENT

FIG. 5. CHANGESIN OSMOTICFRAGILITY OF NEM-TREATEDRAT RED CELLSDURING SEQUESTRATION IN THE SPLEEN. Labeled, NEM-treated red cellssequestered in the rat spleen show a progressive increase in osmotic fragilityas compared with similarly treated red cells incubated in zitro (shaded area).Sixteen minutes after injection red cells trapped in the spleen are still ofnormal osmotic fragility ( 16-min curve) . At I hour an increase in thefragility of part of the labeled cell population is apparent in the spleen (1-hrcurve, lower portion) although labeled cells still in the circulation are ofnormal fragility at this time (not shown). Progressively greater increasesin fragility are noted in the curves for 2, 4, and 7 hours, respectively.

1519


(e.g., anti-D) antibody. Antibody-coated cells(33) and sulfhydryl-inhibited cells are comparablyhalf-cleared by the normal spleen at a rate of 3 to4 per cent per minute, a rate that is presumablylimited by the rate of splenic blood flow (34). Inthe absence of the spleen the rate of clearance ofsulfhydryl-inhibited cells was reduced to %0 ofthat found in the normal circulation, as comparedwith antibody-coated cells whose rate of removalin splenectomized subjects is about %/0o of normal(35). It should be noted that, with both sulf-hydryl-inhibited and antibody-coated cells (36),the pattern of sequestration and thus the speci-ficity of the spleen are influenced by quantitativefactors.

The means by which the spleen so specificallyrecognizes altered cells remains enigmatic. Theability of the spleen to act as a "filter" for ab-normally shaped cells has been well documentedin conditions that occur naturally, such as hered-itary spherocytosis (37) and, possibly, hereditaryelliptocytosis (38). Similarly, experimentallyproduced spherocytes (39) have been effectivelyfiltered from the circulation by the spleen. Thecells in the present studies, however, appeared ofnormal shape and size upon microscopic inspec-tion, and did not show increased osmotic fragilityeither prior to or during their circulation and se-questration. Indeed, it was found that sulfhydryl-inhibited red cells trapped in rat spleens were ofnearly normal osmotic fragility for at least 16minutes after trapping. Only after more pro-longed sequestration was there an increase inosmotic fragility of spleen-sequestered red cells,whereas no such change occurred during incuba-tion of these cells for the same time in plasmaat 370 C in vitro. Thus, we found that sphe-roidicity was imparted by exposure of these cellsto the spleen. With sulfhydryl-inhibited red cells,as is probably the case with antibody-coated cells(40-42), sequestration is not preceded by a sphe-roidal change in the cells but, rather, induces it.

It has been proposed that the spleen recognizesglobulin-coated cells by a change in their surfacecharacteristics, for instance, by their propensityto agglutinate in rouleaux-forming media (43).In a similar fashion, the trapping of sulfhydryl-inhibited cells may be secondary to physicochem-ical alteration of the cell membrane itself. In sup-port of this contention is the finding that, at the

low doses used in these experiments, PMBdoesnot penetrate the intact red cell membrane (15);presumably, therefore, it induces rapid splenicsequestration by direct injury to this membraneonly. NEM, on the other hand, was shown toenter the red cell freely, thereby inhibiting gly-colysis and binding intracellular GSH. However,evidence that this sulfhydryl inhibitor also owedits in vitro hemolytic activity to direct membraneinjury was presented (15).

In order to assess the possible part, if any, thatthe inhibition of glycolysis, per se, might play inthe destruction of NEM-treated cells in vivo, thebehavior of fluoride- and arsenate-treated red cellswas studied in man. It was found that cellstreated with arsenate disappeared slowly from thehuman circulation over 24 hours, and weretrapped primarily in the liver, results confirmingthe data of Harris, McAlister and Prankerd (39).Cells treated with fluoride for 6 hours in vitro,under conditions shown to reduce cellular ATPto 30 per cent of original levels (44), disappearedas two separate populations. Approximately halfthe treated cells were removed from the humancirculation with a half-clearance of 24 hours, se-questration being equal in liver and spleen, whilethe remainder survived normally. The cells ofthis latter population presumably were able toexpel fluoride ion in exchange for chloride priorto the onset of irreversible cellular damage. De-spite such prolonged metabolic inhibition in boththe arsenate- and fluoride-treated cells, little tend-ency for these cells to accumulate in the spleenwas noted, in contrast to observations with NEM-treated cells. Thus it is clear that cessation ofglycolysis per se leads to a delayed injury to thecell in which several hours are required forminimal impairment of viability, in contrast tosulfhydryl-inhibited cells, in which irreversibleinjury occurs within a few minutes. These ob-servations in vivo support the earlier conclusion(15) that NEMand PMBdo not act merely toblock regeneration of high-energy phosphate com-pounds such as ATP. The delayed and relativelymild effects of fluoride and arsenate upon redcell survival also emphasize the specificity of sulf-hydryl inhibition in the mechanism of splenicsequestration.

Unlike antibody-coated cells, sulfhydryl-defi-cient red cells do not agglutinate in Coombs sera

1520


or in polyvinylpyrrolidone. Furthermore, sulf-hydryl-inhibited cells are not abnormally prone toform rouleaux as measured microscopically or bysedimentation rates. However, a change in elec-trostatic potential of the sulfhydryl-deficient redcell membrane might underlie its propensity to betrapped by the spleen. In this regard, it was sug-gested by the early studies of Robinson (45) thatdifferences in the affinity of the spleen for colloidalparticles might relate to their electrical properties.Such a hypothesis remains for study. In addition,a possible relation of membrane sulfhydryl activityto cell "stickiness" was shown in the studies ofPhilipson and Choppin, who blocked with sulf-hydryl inhibitors the attachment of viruses to redcells (46) and to tissue culture cells (47). Inaddition, it has been shown that modification ofleukocytes by PMB inhibits their "adhesiveness"to siliconized glass beads (48). However, therelevance of these in vitro alterations of cellularadhesiveness to the mechanism of red cell trappingby the spleen in the present studies remains con-j ectural.

Enhanced phagocytosis of sulfhydryl-inhibitedcells could not be demonstrated in the test tube,in skin-window preparations, or by early exam-ination of rat splenic imprints after intravenousinjection of these cells. It seems unlikely thatphagocytosis alone, unaccompanied by other trap-ping devices, was responsible for cell removal;one would have to postulate that splenic macro-phages were 180 to 240 times as plentiful or asavid for red cells as were hepatic macrophages.since hepatic clearance was only %O that of thespleen while its blood flow is probably 6 to 8 timesas great.

Relation of biochemical alterations in vitro tobehavior in vivo of sulfhydryl-inhibited red cells.With both NEMand PMBthe minimal dose thataffected cell survival in vivo was the same as theminimal dose that caused K+ leakage in vitro. Inaddition, with NEM-treated cells there was alsoa correspondence of diminished cell survival invivo to impaired glycolysis and to complete in-hibition of GSH. No such association existedwith PMB-treated cells, whose survival was di-minished despite unaltered cellular glycolytic ca-pacity and GSH concentration. The possibilitythat Ki loss was primarily involved in this loss ofviability was discounted by the fluoride experi-

ments in which red cell survival was relativelylittle impaired despite marked loss of cellular K+.A second association was that the propensity ofaltered red cells to be sequestered in the liver, andto undergo autohemolysis upon prolonged incuba-tion, was coincident with the tendency of thesecells to take up Na+ and to manifest spherocytosisand an increase in osmotic fragility. Again, it isprobable that the cation changes were correlativerather than causative. Finally, the close associa-tion of membrane sulfhydryl inhibition and dis-turbed cation control with diminished cell viabilityshould be emphasized, as should the lack of asso-ciation of intracellular sulfhydryl activity and gly-colysis with in vivo survival.

The present studies, in conjunction with theearlier findings of Beutler and colleagues (12, 28),suggest that, in the course of poisoning by oxidantdrugs such as phenylhydrazine and the antima-larials, the eventual product of the intracellularoxidation that critically affects red cell survivalis the destruction of membrane sulfhydryl groups.Other hemolytic agents, such as mercury andpossibly fava beans, as well as certain antibodies,may act by directly affecting these groups. Wheninjury is relatively mild, splenic sequestrationwould predominate. When injury is relativelysevere, hepatic sequestration and intravascularhemolysis would ensue.

The relevance of these studies to normal red cellsenescence is more difficult to evaluate. Althoughdefinitive studies of sulfhydryl activity of senes-cent red cells have not been reported, the expec-tation that such activity would be diminished,equally with the decreased concentration of nu-merous enzymes and glycolytic intermediates inaged cells, seems reasonable. The decreased ac-tivity of GSH in adult red cells when comparedwith reticulocytes (49) fits this conjecture. It issuggested that diminished sulfhydryl activity insenescent red cell membranes might lead to thesequestration and destruction of these cells. Thefinding that reduced GSH also protects hemo-globin in vitro from auto-oxidative breakdown(50, 51) leads one to speculate as follows: 1) afailure of glycolytic activity in senescent cells leadsto diminished intracellular sulfhydryl levels and anincrease in hemoglobin oxidized; 2) this leads todiminished membrane sulfhydryl activity and thusto cell sequestration and destruction. The circu-

1521


lation is thereby freed of cells containing inefficientheme protein through the common intermediate ofdiminished cellular sulfhydryl concentrations.

SUMMARY

Red cells of human or rat exposed in vitro tosublytic amounts of the sulfhydryl inhibitors N-ethylmaleimide (NEM) and p-mercuribenzoate(PMB) are nonviable in vivo. Loss of viability

is related to the inhibition of membrane sulfhydrylgroups but is unrelated per se to intracellularsulfhydryl levels or to the over-all rate of cellularglycolysis. An association, however, does existbetween the ability of these cells to maintain cationgradients in vitro and their ability to survivein vivo.

Red cells treated with small doses of sulfhydrylinhibitor were of normal appearance and fragilityand slowly lost K+; such cells on reinjection weretrapped by the spleen with remarkable selectivity.Sequestration does not appear to depend uponchanges in red cell shape, increased agglutinabil-ity, or erythrophagocytosis. After their seques-tration in the spleen these red cells showed aprogressive increase in osmotic fragility. Largerdoses of inhibitor caused Na+ and water ingress,spherocytosis, and a more rapid hepatic, as wellas splenic, sequestration.

Since inhibition of high-energy phosphate me-tabolism in red cells by arsenate caused a delayed,slow destruction of red cells in vivo, and sincecessation of anaerobic glycolysis and K+ depletionin red cells by exposure for several hours tofluoride had comparatively little effect upon theirsubsequent survival, it is concluded that mem-brane sulfhydryl groups are uniquely importantto cell viability.

It is suggested that a loss of membrane sulf-hydryl activity may be the decisive hemolyticevent common to a number of processes, includingthe Heinz body anemias and normal cellular aging.

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rocyte. Bull. N. Y. Acad. Med. 1960, 36, 79.2. Prankerd, T. A. J. The Red Cell; An Account of

Its Chemical Physiology and Pathology. Spring-field, Ill., Thomas, 1961.

3. Allison, A. C., and Burn, G. P. Enzyme activity asa function of age in the human erythrocyte. Brit.J. Haemat. 1955, 1, 291.

4. Marks, P. A., Johnson, A. B., Hirschberg, E., andBanks, J. Studies on the mechanism of aging of

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