Potassium Loss and Cellular Dehydration of Stored Erythrocytes following Incubation in Autologous...

Original Paper

Vox Sang 1993;65:95-102

Oliviero Olivieria Lucia de Franceschia Mnrzia de Gironcoli Dornenico Girellia Roberto Corrochera

a Institute of Medical Pathology, Chair of Internal Medicine, Polyclinic Borgo Roma, University of Verona, and

Polyclinic Borgo Roma, Verona, Italy " Transfusional Service,

Potassium Loss and Cellular Dehydration of Stored Erythrocytes following Incubation in Autologous Plasma: Role of the KCI Cotransport System

................................................................................................. Abstract We studied the regulation of cell volume and cation content in erythrocytes stored at 4°C under blood bank conditions for various lengths of time and subsequently incubated in autologous plasma at 37°C for 4 or 24 h. Cell swelled during storage at 4°C whereas marked K+ loss and cell shrinkage were observed when erythrocytes were incubated at 37 "C in autologous plasma. The cell shrinkage was inhibited only by the K'C1- cotransport-specific inhibitor, [(dihydroindenyl) oxy] alkanoic acid, and not by other specific inhibitors of cation transport systems such as ouabain (Na+-K+ ATPase pump), bumetanide (Na+-K+-Cl- cotransport) or carbocyanine (Ca++-activated K+ channel). Acidification and swelling of the erythrocytes are well known to be able to activate the KT1- cotransport; such conditions, which were demonstrated to occur during the storage, could lead to activation of the K+CI- cotransport in reinfused cells. These data strongly support the evidence that K T l - cotransport plays a role in K+ loss and dehydration of stored erythrocytes, when incubated in autologous plasma. .....................

Introduction

When stored erythrocytes are reinfused into the circulation, a variable fraction is removed within the first 24 h. A loss up to 30% of the transfused cells is tolerated; cur- rent regulations in the United States require that 75% of the transfused erythrocytes remain in the circulation after 24 h [l]. To date, the exact mechanism responsible for this loss is unclear, nevertheless its prevention may reduce transfusion requirements and blood usage. It has been suggested that the survival capability of stored erythro-

This work was supported by grants from the National Research Council No. 89.02512,04, Ministry of University and of Scientific and Technologic Research 60%, and Health Assessorship of Venetian Region.

Keceived: Dec. 1, 1992 Revised manuscript received: Feb. 13,1993 Accepted: Feb. 15,1993

cytes is associated with cell hydration [2, 31 and that cell dehydration reduces survival. Incubation of cells in fresh autologous plasma (in order to mimic the effects of in- fusion) produces cell shrinkage in erythrocytes stored for 21 days; these dehydrated cells have impaired cell filterability through nuclepore membranes [2] ; moreover, the storage lesion of rabbit red cells was reversed when erythrocytes were rehydrated using nystatin and a medium containing sucrose, sodium chloride and potassium chloride [3]. Recently, Stuart and Ellory [4] have observed that incubation of erythrocytes in blood bank conditions at

Prof. Roberto Corrocher Cattedra di Medicina Intema 0042-9007/93/0652-0095 Istituto di Patologia Medica $2.75/0 Policlinico Borgo Roma 1-37134 Verona (Italy)

D 1993 S . Karger AG, Basel

4°C leads to loss of potassium (K'), chloride (Cl-) and water and impaired erythrocyte rheology. In human erythrocytes two transport pathways are effective in pro- ducing potassium loss and cell dehydration: the Ca+'-activated K' channel and the volume-sensitive K'Cl- cotransport. The ca'+-activated K+ channel, first described by Gardos [ 5 ] , is activated by an increase in free cell Ca++ in conditions of relative energy depletion and it is inhibited by carbocyanine [6, 71 and charybdotoxin [S]. The K'Cl- cotransport has been described more recently [9]. This system plays an important role in the K+ and water loss characteristic of HbC, HbS diseases or during physiological cell aging [lo-131. K'Cl- cotransport is activated by acidification or swelling of the cells. [(Dihydroindenyl) oxy] alkanoic acid (DIOA) has been recently demonstrated to be a specific inhibitor of K'C1- cotransport and the shrinkage induced by activation of this cotransport in normal and sickle cells [14-161. The aim of our work was to investigate the physiological bases of the cation and water loss occurring in stored erythrocytes after incubation in autologous plasma at 37°C. For this purpose, we first studied the changes in cations, pH and cell density during storage to verify if and how such conditions influenced events occurring after incubation in autologous plasma. In order to better understand the mechanisms underlying these events and the eventual involvement of the cation transport pathways, we also performed experiments in- cubating plasma with specific inhibitors of cation transport.

Methods

Drugs and Chemicals NaC1, KCI, NaN03, albumin (bovine fraction V), Tris(hydroxy-

methyl)aminomethane(Tris), 3 (N-morphilo) propanesulfonic acid (MOPS), ouabain, bumetanide and sucrose: were purchased from Sigma Chemical Co. MgCI2, Mg(N0J2, phthalate and dimethylsul- foxide (DMSO) WerepurchasedfromFisher ScientificCo. DIOAwas a gift from Dr. Carlo Brugnara, Harvard Medical School, Boston, Ma., USA. All solutions were prepared using double-distilled water.

Blood Collection and Experimental Design Experiments were performed on erythrocytes of adult, healthy,

volunteer blood donors; 450 ml were collected and stored in sorbitol- adenine-glucose-mannitol, using citrate phosphate dextrose (CPD) as anticoagulant, according to the standard operating procedures of the Transfusion Service of Verona. Erythrocytes were separated by centrifugation from platelets and plasma. Experiments were also performed on erythrocytes stored as whole blood with CPD as anticoagulant. At specific intervals (0, 3,7,10,14,21 and 35 days), cells were collected from the storage bags through an injection port and cellular cation contents, phthalate cell density distribution (as an

indexofcelldensityand hydrationstatus), ATP, 2,3-DPG, andextra- and intracellular pH measurements were performed.

For the incubation experiments in autologous plasma, donor's plasma was separated by centrifugation under sterile conditions. It was divided in separated plastic bags and stored at -80°C if not employed the same day. Finally, cell density populations and cellular cation contents were again determined after incubation in autologous plasma with specific inhibitors of the principal cation transport pathways.

In vitro Measurements Intracellular Cation Content. At specific storage intervals (0,3,7.

10, 14, 21 and 35 days) and after incubation in autologous plasma, erythrocytes were passed through sterile cotton to remove white cells and were centrifuged at 3,000 gfor 10min at 4°C in a Kontron refriger- ated centrifuge. The supernatant was removed and the cells washed four times in a solution containing 144mM choline chloride, 1 mMMgC1, 10 mM Tris-MOPS, p H 7.4, at 4°C (choline washing solution). An aliquot of cells was suspended in a choline washing solution to obtain a hematocrit (Hct) of 3545%. This suspension was used to measure Hct, hemoglobin (Hb), cell Na' (1:SO dilution in double-distilled water)and cell K+ (1:lOO dilution in double-distilled water). The erythrocyte Na+ and K' contents were determined in triplicate with an Eppendorf flame photometer in double-distilled water.

Phthalate Cell Density Distribution. Density profile curves of the erythrocyte populations were performed by the phthalate ester method, as described by Danon and Marikovsky [17]. A mixture of di- methyl and n-butylphthalic acid esters was made, having a range of specific gravity from 1,045 to 1,190glml. A 3-mm column of this phthalate ester mixture was placed on the top of each packed red cell column in the Hct tubes. The tubes were then spun at average centrif- ugal force of 12,000 g for 30 min. The length of the column of packed cells beneath the ester layer was expressed as a percentage of the total length of the red cell column, which represented the percentage of cells of known density. These values were plotted against density and a distribution curve of cell populations was obtained.

Extracellular and IntracellularyH. Extracellular pH was directly measured on whole blood with an ABL4 Haemogasanalyzer (Radi- ometer, Copenhagen). Cell pH was measured by lysing 0.2 ml of packed erythrocytes in 2 ml of unbuffered double-distilled water and determining the pH of the lysate with a radiometer pH meter at room temperature, as described by Canessa et al. [18]. Double-distilled water and known p H standards (Hanna Instruments) were checked routinely.

ATP and 2,3-DPG. For nucleotide measurement, nucleotides were extracted by addition of 20% perchloric acid (1 ml) to 2 ml ice-cold whole blood. ATP was measured according to Beutler's method [19]. ATP-phosphorylated glucose in the hexokinase reac- tion, formingglucosed-phosphate, was measured by the reduction of NADPtoNADPH. Thechangeinopticaldensity at 340 nm, resulting from the reduction of NADP to NADPH, was measured in the presence of excess glucose and represented the amount of ATP in the mixture. ATP was expressed as WmoVl packed RBC.

2,3-DPG was measured according toBeutler's method [19]. Phos- phoenolpyruvate was transformed in 2- and 3-phosphoglyceric acid in the presence of enolase, monophosphoglycerate mutase and 2,3- DPG. The disappearance of phosphoenolpyruvate was followed spectrophotometrically at 240 nm and was proportional to 2,3-DPG concentration. 2,3-DPG was expressed as pmolll packed RBC.

96 Olivierilde Franceschilde GironcolilGirellil Corrocher

K+C1- Cotransport in Stored RBCs

Fig. 1. Total cation, K' and Na+ contents of erythrocytes stored in sorbitol-adenine-glucose-mannitol, as afunction of storage time at 4°C (mean * SD, n = 4).

Fig. 2. Density percentage curves of the erythrocytes stored in sorbitol-adenine-glucose-mannitol during the first 14-21 days of storage at 4°C (a single set of density curves is shown, representative of five others).

Incubation in Autologous Plasma. Plasma was added to the packed erythrocytes to a Hct of approximately 40%. Plasma pH values were 7.38k0.2 at room temperature. When erythrocytes were added, pH of the unbuffered suspension decreased to 7.10f0.05. Experiments were also performed at buffered pH of 7.10,7.20,7.40 by adding adequate amount of 1 M Tris to obtain required pH values, prior to addition of the red cells. In each experiment, we determined cell cation content and cell density distribution before and after plas- maincubationfor4or24 hat37"C. Sterility testingwasperformedon all units at the end of the storage period by the MicrobiologyDeparte- ment of the University of Verona.

Experiment.y with Specific Inhibitors of Membrane Cation Trans- ports. The following tubes containing plasma and stored erythrocytes were prepared: (1)control t = 0, beforeincubation; (2)controlt = 4 h, after 4 h incubation, without inhibitors; (3) t = 4 h with ouabain 0.1 mM; (4) t = 4 hwithouabain0.1 mMandbumetanide0.01 mM; (5) t = 4 h in the presence of ouabain 0.1 mM, bumetanide 0.01 mM and carbocyanine0.01 mM; (6) astube(5)plusDIOAO.l rnM[10-16]. The inhibitors were dissolved in an adequate volume of DMSO to obtain the final expected concentration (maximal volume 10 p1 of DMSO on 10ml of the final cell suspension), and the pH of the media was carefully verified. In the control tubes, 10 ~1 of DMSO without inhibitors was added. In each experiment, cell cation content and cell density distribution were determined before and after plasma incubation for 4 h a t 37°C.

Resu I ts

of storage, so that the total cation content of the erythrocytes was little changed for 14-21 days (fig. 1). For longer storage times (35 days), K+ loss exceeded Na' gain and total cation content decreased (fig. 1). Similar results were obtained in erythrocytes stored as CPD whole blood (data not shown) for a maximum of 21 days, which is the time limit for storage prior to transfusion. Density distribution curves of the erythrocyte population, during blood bank storage in sorbitol-adenine-glucose-mannitol, showed a bi- phasic pattern. After 14-21 days, cell density decreased and the curves shifted toward the left in the density diagram (fig. 2). Curves obtained on days 3 ,7 and 10 were interme- diate (not shown in the figure), whereas in some experiments no significant cell density differences were found between curves of 14 or 21 days. For storage times longer than 14-21 days, to the limit of conservation for sorbitol- adenine-glucose-mannitol (35 days), cell density progressively increased in parallel to the decrease of cell total cation content (data not shown). For whole blood, using CPD as anticoagulant, density curves of the period 0-21 days were similar to those of the sorbitol-adenine-glucose-mannitol preservative solution. As shown in table 1, storage induced a progressive reduction of intra- and extracellular pH and depletion of red cell 2,3-DPG and ATP.

Effect of Storage on Internal Cation Content and Red Cell Volume During storage, a progressive increase of cell Na' and a

progressive reduction of cell K+ were observed (fig. 1). Gain of Na' and loss of K' were balanced in the first period

Effect of Incubation in Autologous Plasma on the Cation Content and Density of Stored Erythrocytes Figure 3 shows the Na+ and K+ contents of stored

erythrocytes after incubation for 24 h in autologous plasma at different pH. Na+ content changed only slightly,

97

Fig. 3. Na+ (0) and K+ (m) contents (as percentage) in stored erythrocytes after incubation for 24 hat 37°C in autologous plasma at different pH (mean f SD, n = 3).

Fig. 4. Na+ and K+ contents of erythrocytes during storage in sorbitol-adenine-glucose-mannitol, at 4°C and after incubation for 24 h at 37"C, as a function of the storage time (a single set of curves is shown, representative of four others).

Table 1. Changes in the erythrocyte t = O t=7days t=14days t=21 days t=35days content of ATP 2,3-DPG, and intra- and

extracellular pH during storage in sorbitol- adenine-glucose-manitol ATP 1,639+ 179 1,446k391 1,014k 154* 975+176* 784+232*

NmoUl RBC 2,3DPG 3.39k0.78 1.23*0.63* 0.35+0.14* 0.2i0.1* 0.08f0.05*

IntracellularpH 7.05k0.08 6.89+0.01* 6.82f0.02* 6.73,0.04* 6.48+0.01* Extracellular pH 7. 18+0.02 6.92+_0.01* 6.83 k 0.01 * 6.82 +0.04* 6.58 t 0.0 1 *

pmol/l RBC

Values are means +_ SD, n = 3. *p<0.05 vs. t = 0.

while a considerable loss in K+ content was observed, which was pH dependent and maximal at lower pH. For this reason, the experiments of incubation in autologous plasma were performed at pH 7.10k0.05 in order to maxi- mize these changes. Na' content was not different after 24 h, indicating that plasma incubation did not substantially affect the transport mechanisms controlling cellular Na' (fig. 4). A marked loss of cellular K+ was evident after plasma incubation, particularly in cells obtained during the first 7 days of storage (reduction of 25-35% in comparison to the values before incubation) (fig. 4). These data suggested an activation or inactivation of one

(or several) transport mechanism(s) , selectively able to induce or inhibit K+ loss following plasma incubation. In the erythrocytes stored as CPD whole blood, modifications in both Na' and K+ were similar (data not shown). Density curves of the erythrocyte population, after incubation in autologous plasma for 24 h, showed an increase in cell density throughout 21 days of storage (fig. 5) . This behavior was constant in both preservative systems during the 21 days of storage. Incubation experiments were also performed within 4 h. Both shift of the density curves and cation modifications were similar to those described for 24-hour plasma incubation.

98 Olivieri/de FranceschVde Gironcoli/Girelli/ Corrocher


Fig. 5. Density curves of the erythrocyte population: day of collection (t = 0); after 14 days of storage in sorbitol-adenine-glucose- mannitol, at 4°C (t = 14); after incubation of 14-day-old cell sat 37"Cin autologous plasma for 24 h (a single set of density curves is shown, representative of three others).

Fig. 6,7. Na+ (6) and K+ (7) of erythrocytes: after 7 days of storage at 4°C in sorbitol-adenine-glucose-mannitol (m); after incubation in autologousplasmaat37"Cfor4 h of 7-day-old stored cells (0); after incubation of 7-day-old cells in presence of ouabain 0.1mM (El); after incubation of 7-day-old erythrocytes in the presence of ouabain 0.1 mM and bumetanide 0.01 mM ( incubation of 7-day-old erythrocytes in the presence of ouabain 0.1 mM, bumetanide 0.01 mM and carbocyanine 0.01 mM (0); after incubation of 7-day-old erythrocytes in the presence ofouabain0.1 mM, bumetanide 0.01 mM, carbocyanine 0.01 mM and DIOA 0.1 mM (a) (mean +_ SD, n = 3) .

6

9 40 I 3 - 30

+ E 20 = 10

0 Inhibitors

7

In stored 21-35 days erythrocytes, the dehydration, high Na' and low K+ contents were not further modified by plasma incubation (data not shown).

Effect of Cation Transport Inhibitors on Membrane Transport Properties and Volume of Stored Red Cells Experiments were performed to determine the effect

of specific cation transport inhibitors on the cation content and the shrinkage of the erythrocytes observed after incubation at 37°C in autologous plasma. Cellular Na+ slightly increased after incubation for 4 h in autologous plasma. In the presence of ouabain cellular Na' was slightly increased compared to the control cells (fig. 6). This minimal Na' increase was still observed when cells were also treated with burnetanide, carbocyanine and DIOA.

Inhibitors

Incubation in plasma induced a marked reduction of erythrocyte K' content (about 30%), which was not modified by the presence of ouabain, bumetanide and carbocyanine (fig. 7 ) . The only inhibitor able to counteract this reduction in cellular Kt content after plasma incubation was DIOA, the specific inhibitor of K'C1- cotransport (fig. 7). This shift toward higher values of cell density occurring after plasma incubation was inhibited by DIOA (fig. S), whereas ouabain induced a shift towards less dense values (data not shown); bumetanide and carbocyanine were ineffective.

09

Fig. 8. Density curves of erythrocyte population: after 7 days of storage in sorbitol-adenine-glucose-mannitol at 4°C (control); after incubation of 7-day-old erythrocytes at 37°C for 4 h (control-4 h); after incubation of 7-day-old erythrocytes at 37°C for 4 h in the presence of ouabain 0.1 mM, bumetanide 0.01 mM, carbocyanine 0.01 mMand DIOA 0.1 mM (DIOA). A single set of density curves is shown, representatitive of three others.

Discussion

Studies are reported of (1) events occurring during the storage of erythrocytes at 4°C; (2) events triggered by incubation of the erythrocytes in plasma at 37°C. During storage, erythrocyte cation contents change markedly with potassium loss and sodium gain as described in previ- ous reports [20,21]. However, the changes in Na' and K+ contents are balanced, and the total cation content of the erythrocytes remains relatively constant during the first 14-21 days of storage (fig. 1); beyond this limit, K+ loss became predominant and induced cellular dehydration. During the first period of the storage (0-21 days), erythrocytes conserved in sorbitol-adenine-glucose-mannitol swelled progressively with consequent cell density decrease, whereas after this time cells underwent shrinkage and increased their density. The data observed during 14- 21 days of storage suggest that the cell swelling observed is probably the result of changes in erythrocyte pH or concentration of nonpermeant ions (i.e. 2,3-DPG) and not the result of differences in erythrocyte cation content [22]. When the level of 2,3-DPG decreases, chloride moves into the red blood cell to maintain electroneutrality; since 2,3-DPG has 3.67 negative charges, 3.67 moles of chloride must be substituted for each mole of 2,3-DPG to maintain

electroneutrality [23]. This movement of chloride is ac- companied by water and produces cell swelling [22]. Mea- surements of intra- and extracellular pH and 2,3-DPG supported this view. In fact, decreases of cellular pH and 2,3-DPG are both known to induce chloride and water recall into the cells and consequent swelling through a Donnan-type effect [21]. The acid pH of citrate (in CPD anticoagulant), useful to counteract the marked rise of pH that occurs when blood is cooled to 4"C, and accumulation of lactate [23] may contribute to the observed acidification in stored erythrocytes. As a consequence of these water and ionic modifications, stored erythrocytes are swollen and acidic [2]. When erythrocytes were incubated in plasma at 37"C, a further K+ loss (reduction of 25-35% of cell content) and cell dehydration were observed. This phenomenon could be responsible for the shorter survival of a 'tail' of the 30% of cells, which is lost the first 24 h after transfusion 123,247. Turner et al. [3], after K' enrichment and cell rehydration by a nystatin procedure, demonstrated improved nuclepore filterability of stored human erythrocytes, and improved in vivo survival of stored rabbit erythrocytes [25].

In the past, ionic modifications occurring during storage have been ascribed to the inefficiency of the Na+-K+ ATPase pump at 4"C, thus failing to counteract the effects of passive leaks due to electrochemical gradients [20]. In- efficiency of the Na+-K' ATPase pump may also contribute to postinfusion phenomena: it has been observed that ATP, magnesium and Na+ of transfused erythrocytes return to normal within 24 h, whereas cellular K' does not return to normal until approximately 7 days after transfusion [26]. These findings in part agree with the hypothe- sis of ATPase Na+ pump ineffeciency; however, the recovery of K' appears delayed in comparison to that of Na', and an additional K+-selective (without effect on Na ') mechanism might account for this discrepancy. Similar conclusions are suggested by our results. Stored erythrocytes incubated in plasma became dehydrated and increased their density through a selective K+ loss, without modification of Na+ content, unlike what would be expected if ATPase pump inefficiency was the only mechanism involved. The experimental plasma incubation at 37°C assured greater efficiency of the ATPase pump than during storage at 4°C; nevertheless, further K' loss was evident. In experiments with inhibitors, with complete blockade of the pump by ouabain, Na+ content increased and cell density decreased, suggesting effective counter- action of the pump. In the presence of ouabain, the loss of K+ ions during incubation was not substantially affected, indicating an additional K' loss process, independent of

100 Oliviervde Franceschgde Gironcoli/Girelli/ Corroch er


the Na+-K+ ATPase pump activity. Na'K'Cl- cotransport also affects erythrocyte Na+ and Kf contents [27]. Thus, activation of this system following the increase of cell Naf content may reduce cellular Kf content; however, simul- taneous decrease of Na' should take place, which was not the case in our results. Moreover, the specific inhibitor bumetanide did not affect Kf loss or cell dehydratation. The Ca++-activated Kf channel may also lead to significant K+ loss and dehydration, but the specific inhibitor carbocyanine did not modify K+ loss in the plasma incubation experiments. Moreover, activation of this channel requires a rise in free cytoplasmic calcium, recently excluded by McNamara and Wiley [28] in cold-stored erythrocytes.

Due to its thermal dependency, K'C1- cotransport is inactive at 4"C, and thus it is not involved in cation move- ments during storage [29]. In mature normal human erythrocytes containing primarily HbA, exposure to pH<7.4 at 37°C induces cell swelling and not shrinkage, different from red cells in patients with HbS disease [29]. However, stored erythrocytes, which have become dehydrated and shrunken in plasma, can be compared to SS cells, which are known to extrude large amount of Kf via the K'CI- cotransport mechanism. Unlike normal AA cells, stored erythrocytes are swollen and acidified, with reduced 2,3-DPG content. It has been proposed for SS cells that cytoplasmic acidification secondary to the increased 2,3-DPG content [30] is partially responsible for the increased activation of K'C1- cotransport in these cells [29]. Stored erythrocytes show similar cytoplasmic acidification following the accumulation of anions as lac-

transport. In experiments with inhibitors, only specific inhibition of the K'CI- cotransport by DIOA preserved erythrocytes from potassium loss and dehydration, whereas other inhibitors were ineffective.

These data suggest that the 'membrane storage lesion' might induce a reexpression of K T l - cotransport activity, which is usually decreased during the cell aging process.

Although acidification of the magnitude used in our experiments cannot frequently occur in vivo, activation of KCl cotransport does not appear as an all-or-none phenomenon; K+ loss progressively increases when the pH decreases. Erythrocytes in vivo are acidified at every pas- age through actively metabolizing tissues in the micro- vasculature; this is exaggerated in clinical conditions where blood transfusion is needed, for example in hypo- volemic-hypoxemic emergencies. Thus, reduction of the survival of the transfused erythrocytes may paradoxically occur in situations where this is a particularly undesired event. In any case it could be useful to correct acidosis in transfused patients, in order to improve erythrocyte survival.

The present data strongly support the conclusion that the KfC1- cotransport plays a role in K+ loss and dehydration of stored erythrocytes when incubated in autologous plasma. However, plasma incubation does not entirely reproduce in vivo circulation conditions, further clinical studies will be necessary to define the practical consequences of the mechanism described in the present work.

Acknowledgements tate in the place of 2,j-DPG. Moreover, KT1- transport

by blood bank conditions could activate K'Cl- cotransport. Cytoplasmic acidification and cell swelling of stored erythrocytes may contribute to activation of K T - co-

is volume-sensitive [12, 13, 311, and cell swelling induced Wethank Dr. Carl0 Brugnara from the Department ofPhysit310- gy, Harvard Medical School, Boston, for helpful discussion and revi- sion of the manuscript.

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OlivierUde FranceschUde Gironcoli/Girelli/ Corrocher

K'Cl- Cotransport in Stored RBCs

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