ON RED BLOOD CELLS, HEMOLYSIS AND RESEALED GHOSTS

Joseph F. Hoffman Department of Cellular & Molecular Physiology Yale University School of Medicine New Haven, CT, USA

INTRODUCTION AND DEFINITIONS This paper aims to consider, with a historical perspective, the characteristics of red blood cells and their ghosts that are associated with osmotic hemolysis and its reversal. Since hemolysis refers to the process by which a cell becomes permeable to hemoglobin (Hb) the term "ghost" is used to describe the resultant envelope or cell-like structure (also referred to as post- hemolytic residue or stroma) that survives the transition. This definition of the term, ghost, emphasizes the functional involvement of the plasma membrane and is independent of, or at least not biased by, the circumstances leading to its production. This definition, based on a loss of Hb by a change in the membrane's permeability, excludes the types of changes in a cell's Hb content that occurs, for instance, during erythroid maturation or by age related changes in cell density. While this definition of a ghost is general and independent of Hb content (ghosts can range from being nominally Hb-free to containing almost the cell's original amount), it should also be understood that the resultant types/properties of ghosts reflect the conditions that attended This means that ghost the hemolytic step(s) and any subsequent treatment(s). characteristics are method-dependent and should be specified in every case. It would be of interest to know just how (and by whom) the term, ghost, came to be used. The English word "ghost" was already in use in this context around the turn of this centuryl and presumably its German equivalent (Schatten?) even earlier. One imagines that the term derives from the appearance of red cells hemolysed by water ("laked" blood) whereupon an opaque suspension becomes transparent, as observed either in the bulk or microscopically. That ghosts, as pale remnants of their former cellular selves, persist after hypotonic hemolysis was subsequently established by measurements of electrical conductivity of the suspension with the finding that ghosts were non-conductors l.2 , by centrifuging the suspension and noting the change in pellet size after the addition of saW as well as by the appearance (translucence) and properties of the suspension following salt addition (see below).

The Use of Resealed Erythrocytes as Carriers and Bioreactors Edited by M. Magnani and J.R. Deloach. Plenum Press. New York, 1992

REVERSAL OF HEMOLYSIS Stemming from the work of Bayliss4, the term, reversal, as in reversal of hemolysis, is now used in two ways: 1) to refer to changes in the composition of the medium following hypotonic hemolysis, and 2) to refer to various responses of ghosts to such alterations in composition. Generally, the former involves restoration of the medium to its original osmolality while the latter may be used to describe recovery of reversed ghosts in terms of changes in ghost volume, content and membrane permeability properties. It is unfortunate that early work on ghost reversal gave rise to the erroneous claim that the addition of salt to the hemolyzed suspension mixture causes outside Hb (Hbo) to reenter the ghost. s While it is known that with proper conditions Hbo can, in fact, be made to reenter (see below), this false claim took time to sort out and delayed understanding of the osmotic properties of ghosts. This is so even though Stewart 1,2, soon afterwards, in performing experiments analogous to those of Spiros correctly concluded that, instead of Hbo passing back into the ghost or being absorbed to its surface, the ghost response to salt addition was shrinkage, concentrating the Hb j that was trapped inside after the conclusion of hemolysis. These results indicated that not only did Hbi diffuse out of the cell at the time of hemolysis but since the concentration of Hb j in the swollen ghosts essentially equaled that in the medium (accounting for the translucence of laked blood) and that the Hbj concentration increased upon shrinkage, the permeability of the ghost to Hb and salt must be relatively low. Nevertheless, claims for Hbo reentry (see 6 for references) or Hb absorption7 persisted even after Adair et a1. 8 and Bayliss4 clearly showed that volume changes accounted for the illusion of Hbo reentry upon reversal of hemolysis. It is not evident why these differences in interpretation took so long to settle especially since methods were already available for harvesting ghosts by centrifugation and measuring the Hb concentration colorometrically.

HEMOGLOBIN ENTRY AND DIFFUSION EQUILmRIUM It was fIrst reported in 19549 that Hbo could validly reenter the ghost during hemolysis when the gradient of Hbo to Hb j was favorably set. This was achieved in a twostage hemolysis procedure in which the Hb j content of ghosts was lowered during the first hemolysis and raised during a second rehemolysis, provided that in the second step the concentration of Hbo > Hb j .9,10 After rehemolysis the ghost content of Hbi was found to be significantly increased (up to 50 percent) above the amount, set by the first hemolysis and dependent upon the Hb o concentration to which the ghosts were exposed at the second hemolysis. It was subsequently shown 1o that during the time of hemolysis, the membrane was permeable to s9Fe-labelled Hbo as well as 131 1- labelled albumin, the latter representing perhaps the first incorporation of a foreign protein. In fact a variety of Hb types have been hemolytically entrapped within ghosts including Met Hb and Hb-FII, Hb S 12, 13 and Hb c. 13 The results lO on the distribution ratio of s9Fe- labelled Hb between ghosts and medium after hemolysis (and before reversal) was essentially unity and agreed with the distribution ratio of the non-radioactive Hb, where HbjHbo was one. These results were important for they showed not only that all of the Hb j was exchangeable at the time of hemolysis (Le. no bound Hb detectable within ± 1 percent accuracy; see ref. 14) but also that Hbj diffused, during the time available, to concentration equilibrium (see also refs. 6, 15). Shortly after this equilibrium was reached the ghost, still at (or near) its hemolytic volume, becomes impermeable to Hb since s9Fe-labelled Hb could subsequently be shown to neither enter

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nor leave 10, even though the membrane remains permeable to ions such as Na, K and Cl (see below). HEMOLYTIC VOLUME, SURFACE AREA AND TENSION The studies in which Hbi was found to diffuse to equilibrium at hemolysis indicated a way to estimate certain physical parameters of the red cell membrane in addition to providing insight into the mechanism of hemolysis. Thus it was possible to estimate 16 the cell's hemolytic volume Vh (the average volume at which a population of cells hemolyze I7), by measuring, for a known hematocrit, the volume occupied by the ghosts after hemolysis relative to the volume the cells occupied initially. Vh was calculated from the change in Hbi before and after hemolysis (since it was redistributed at Vh) and found to average 1.65 times the cell's original volume. 16 Thus Vh would be 143 u m3 for cells whose average initial volume was 87 p m3 • Given that a cell reaches its Vh without a perceptible increase in surface area l6• 17 hemolysis would appear to result from a change in membrane structure associated with any increase (stretch) in surface area (see below). If Vh is equivalent to the spherical volume that cells can attain before hemolyzing then the average cell's surface area is 133 p m2• These red cell dimensions determined in this manner are similar to estimates obtained by other means (e.g. 18- 22). Another consequence of Hbi diffusion to equal concentrations across the membrane during hemolysis is that the concentration of Hbi can be varied by varying the volume of hemolyzing solution relative to the volume of cells. This provided a way to test the colloid osmotic mechanism of hemolysis 23 by determining the extent to which the rate of hemolysis of ghosts depended on the concentration of Hbi , when all other factors were equal. It was found lO that when the cation permeability was increased with butanol, not only was the resultant rate of rehemolysis inversely proportional to Hbi concentration, in direct support of the theory, but that the curve extrapolated to the rate at which intact cells hemolyzed under the same conditions. Studies of this type also provided a way to estimate the tensile strength of the membrane. Ghosts rehemolyzed when they contained more, but not less, than 3.3 percent of their original Hbi, even though the ghosts with less than 3.3 percent of their original Hb could be shown to swell. Since the osmotic or hydrostatic pressure exerted by this concentration of Hbi is about 7 x 103 dynes/cm2 (65 mm H20), the surface tension of the membrane, which must be overcome for rehemolysis to occur, is approximately 1 dyne/cm (see ref. 10 for details). HEMOLYSIS AND MEMBRANE HOLES Certainly the osmotic forces that bring a cell to its Vh are better understood than either the structural changes in the membrane that allows for the escape of Hb or the nature of the resultant diffusional pathways (holes). It seems clear that the osmotic pressure differential that brings a cell to its Vh is dissipated during the time of hemolysis with the loss by diffusion of the cell's permeant constituents. Since in hypotonic hemolysis the pressure differential is mainly due to the difference in salt (Na and KCl) concentration across the membrane and since the diffusion rate for salts is about 10 times that for proteins, it is evident that the relaxation of the membrane's altered structure to its initial state occurs at a rate slow enough for Hb to diffuse to equilibrium. It is also evident that the membrane recovers its impermeability to Hb before it does to salts and these changes in

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permeability depend on the conditions at and after hemolysis and on such factors as temperature, pH and ionic strength (see below). Studies have been carried out (24-26) in which the sieving properties of the membrane during hemolysis have been demonstrated by measuring the rate of appearance outside of intracellular ions and of proteins differing in size such as adenylate kinase and Hb. One method25 • 26 utilized a continuous flow centrifuge in which the cells, contained in a plastic tube that was threaded through the rotor, were pelleted against the peripheral wall of the tube and held there centrifugally while hemolyzing solution flowed past at a constant rate, for subsequent collection and analysis. The promise that this approach offers might be optimized by employing Charles A. Lindbergh's27 design in which cells/ghosts can be kept suspended and constantly washed by balancing the outward centrifugal force on the cells/ghosts with an opposing inwardly (centripetally) flowing bathing solution. This method might provide a more quantitative dissection of the molecular sieving characteristics of the membrane. Basic aspects of the hemolytic process concern the types and size of the holes, their number and the length of time the holes are open. Tetrameric Hb, the form comprising more than 99 percent of cellular Hb (see 28), has a spheroidal shape, 65 N x 55 Ao x 50 Ao29, and a radius of gyration approximating 30 N. This places a lower limit on the size of the hole whereas the upper limit(s) is more difficult to establish and/or to find agreement on. Normally, as already discussed, Hb j reaches diffusion equilibrium during hemolysis but circumstances can be developed in which this is not the case. Hemolysis is usually considered "all-or-none"3o in the sense that 50 percent hemolysis means that half of the cells have hemolyzed rather than that all of the cells have lost half of their Hb. But partial hemolysis has been observed when cells are hemolyzed in the presence of macromolecules of varying size, much as dextrans 31 -33 and albumin. 34 These studies show that as the size and/or concentration of the macromolecular species is increased (at constant osmolality to keep the driving force for hemolysis constant), the fractional loss of Hb j from the cells is decreased. These studies provide approaches for probing the size of the hole through which Hb passes and raise a question about the mechanism by which these macromolecules inhibit Hb exit. The cutoff point for dextrose32 entry seems to occur at a Mr - 3 x 105 but the average hole size in this situation is difficult to estimate. With regard to the inhibition of hemolysis. Lowenstein34 suggested that if hypotonic hemolysis contains a colloid osmotic component, the extracellular macromolecules could act, after the salt gradient was dissipated, to balance the remaining Hb j (colloid) and thereby inhibit hemolysis. While this idea is not without merit, it appears unlikely principally because it cannot account for the fact that the inhibitory potency of macromolecules, such as ficol and the dextrans, is proportional to the total amount of polymer per unit volume solution (weight concentration) and independent of the molecular weight of the different polymers used. 33 This implies that the polymers interfere with the way Hb diffuses through the membrane. Seeman35 has proposed that the inhibition of Hb escape might involve excluded volume effects whereby the macromolecules compete for solvent water within the holes (see 36).

HEMOGLOBIN PERMEABILITY It would be misleading to conclude from the above that there was general accord concerning the time and manner in which Hbj leaves the cell during hemolysis and in the number of holes involved. One approach used to estimate Hb j exit times has been to take high-speed motion pictures ofthe event (at known frames/sec) and measure, on single cells, the length of film associated with cell fading. 37-41 Observations made when hemolysis was induced by hypotonicity and/or by lysins, such as saponin, were carried out by ordinary37.38.40.41 or by interferometric39 light microscopy. In the latter instance the change

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with time in cellular mass (Hb j ) was quantitatively assessed. The reported fading times vary considerably ranging from fractions of a second37 to seconds 19.37 ,39,40,41 to minutes. 21 ,40 Another approach attempts to dissociate the swelling event, caused by water and solute entry, from the event of Hb j liberation. 41 , 42 It is not clear to what extent the variation in fading times are dependent on such factors as cell suspension preparation, chamber design or the methods employed for inducing hemolysis. What is really needed is a method to evaluate quantitatively the membrane's permeability to Hb (say in terms of a permeability constant, PHb, that takes into account the concentration gradient, the surface to volume ratio and the membrane potential, in units of cm/sec or in terms of a Hb flux in units of moleslcm2 per sec). This would not only help to solve the problem of the number of holes involved (see below) but also to establish the properties and determinates of PHb as well as evaluate the mechanism by which external polymers inhibit Hb exit. Perhaps a solution to this problem would be to isolate a cohort of cells from a normal population, in which the cohort displayed the same (or very narrow range) of initial and hemolytic volumes. Then the osmotically driven hemolytic event could be quantitatively followed spectrophotometrically, in bulk suspension, provided there was sufficient synchrony in the rates of Hbi diffusion. Whether this technique would resolve time-dependent changes in PHb and their cause is another issue.

HOLE SIZE AND NUMBER Estimates of the number of holes through which Hbj escapes during hemolysis have ranged from one40,43.50 to manyl9.21, 37·41, 51·53 the latter of which are said to be spread over the cell's entire surface. Two general methodologies referred to as fast and slow40, 54 have been used to induce osmotic hemolysis, the difference between the two being dependent upon the rate at which the osmotic pressure of the suspension medium of the cells is lowered (i.e. acutely or gradually). But there does not as yet appear to be any correlation between the rate of Hb j exit (fading time) and the rate of fast versus slow hemolysis. On the other hand, abrupt physical shifts (displacements) of whole cells/ghosts have been observed to take place during fast 50 and slow40 types of osmotic hemolysis and under circumstances when Hbo-precipitating agents were also present in the medium.43, 44 These abrupt movements are associated with a single membrane lesion and are th~ught to represent cell/ghost recoil from pressure ejected Hb j • This kind of propelled displacement behavior is in marked contrast to studies in which Hbj is seen to exit over the entire surface and in which the cell/ghost remains essentially in a stationary position. 19·21, 37·41, 51 In studies in which only a single hole per ghost is seen, estimates of hole size (diameter) can range from below 100 N (e.g. 48, 49) to as much as 1 mJ.l or more40, 43·49 the latter being visible by light and/or electron microscopy and are described as rents, fissures or tears in the membrane. Lieber and Steck48,49 studied ghosts prepared by an osmotic technique, and found that the size of the ghost's single hole was changeable depending on conditions such as pH, temperature and ionic strength, and thus could be modulated to assume stable diameters that ranged from about 14 N to more than 1 J.l m. The extent to which these results 48, 49 apply to the situation that obtains during the hemolytic event per se is not at present known. On the other hand, any single hole mechanism is difficult to reconcile with the results, mentioned before, on fading times and their characteristics and on the discrimination by probes and the cutoff size of the holes involved (see below). Clearly, while conditions can be found that produce hemolysis via a single hole, convincing

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evidence still needs to be provided that this is the preferred or dominant mechanism that operates in hypotonic hemolysis. The studies that document that during hemolysis Hbi escapes over the whole cell are basic for establishing that multiple membrane holes are involved. 19-21. 37-41. 51-53 Concomitant with estimates of fading times, referred to before, were observations of the changes in the microscopic image of a celVghost as it undergoes hypotonic or colloidosmotic (saponin) hemolysis. Typical of these types of observations is Parpart's38 in which free-floating cells (see ref. 55 for a description of the optics and chamber used) were seen, upon making the medium hypotonic, to ftrst become spherical (with a reduction in diameter) and, because of the optics employed, increase in central chromaticity. The onset of hemolysis occurs with the beginning of cell fading and the gradual appearance of a bright diffraction halo around the periphery of the cell that expands at a rate (for the conditions used) compatible with the diffusion constant for Hb. The fading times measured were consistent with Ponder and Marsland's result. 37 Fricke56 provided a solution to Fick's equation that Ponder (57, p. 249) used to estimate the number of holes associated with Hbi exit. Taking the diffusion constant of Hb as 7 x 10-7 cm2/sec, an average fading time of 4 seconds, an average hole size of 70 N, the calculated number of holes per ghost is 100. Obviously this type of analysis needs to be rigorously quantified to get an accurate fix on the number of holes per ghost developed. As pointed out before, a similar quantitation needs to be carried out to establish reliable estimates on the size of the holes as well. Nevertheless, mention should be made of electron microscopic studies 52, 53, 58 showing that ferritin and colloidal gold can, to different extents, enter a celVghost during hemolysis, a result yielding hole sizes that range between 100 to 1000 N in width. Interestingly, the length of time the holes were open for permeation by ferritin (room temperature?) was between 15 and 25 seconds. 52 On the other hand, the full significance of these types of holes is not clear since none were seen in ghosts that ferritin or colloidal gold had not entered. 52, 53 Also, ferritin contained in intact cells is evidently not lost during hemolysis 59 and it is not known whether Hb and say, ferritin traverse the same hole.

ORIGIN OF HOLES

There are many questions concerned with the membrane structural changes that underlie, the hemolytic event. What is the origin of the holes that develop to permit the passage of Hb? Are they newly created or did they preexist in a form that is changed by the osmotic forces that induce hemolysis? Any stretch of the surface at a cell's hemolytic volume could be the force that opens the membrane holes 6o or induces lipid/protein instabilities (structural rearrangements) that also result in holes. It is known that although the lipid composition of the membrane is not altered by osmotic hemolysis 61 there is evidence indicating that the normal asymmetry in the distribution of phospholipids across the membrane (bilayer) is lost as a result of hemolysis 62, 63 but reestablished when the ghosts were resealed (resealing is discussed below). These results imply that a major fraction of the membrane's surface undergoes structural rearrangements during hemolysis. Another parameter that may underlie hole development in the membrane are changes in the membrane potential. The initial event that occurs when cells are placed in a low ionic strength environment is that the membrane potential changes from inside negative to become inside positive (cf. 64, 65). If this occurs it could activate cation channels (see 65, 66) that could, in tum, serve as candidate holes for Hb release. But not enough is known about the nature or number of these types of channels to warrant further speculation. The last parameter to mention is membrane flicker or rather the membrane undulations associated with the phenomenon. 67-69 Here the thermal fluctuations responsible for the

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undulations represent an energetic basis for molecular transitions that could be involved with the locus for hole development and/or their recovery.

RESEALING GHOSTS It was understood early on 2. 70- 72 that hemolysis was associated not only with the loss of Hbi but also with electrolytes, such as Na, K and Cl. Ghosts were also known 70-72 to remain leaky to these ions even after the addition of salt to the medium (reversal) although some reduction in permeability must have occurred in order for the ghosts to respond osmotically to changes in medium tonicity, as discussed above. Nevertheless it was the benchmark study by Teore1l 73 that first indicated that ghosts could recover their permeabilities to Na and K and behave as osmometers. This together with work from Straub's laboratory51. 74 set the stage for the use of ghosts in the analysis of function of the red cell membrane. Basically, Teorell's73 method for preparing ghosts was to hemolyze cells with 10 volumes of a hypotonic solution and store them at 4° C overnight. The resultant ghost preparation was heterogeneous containing tight (resealed) and leaky ghosts (cf. 10) but it should be noted that, given time, those ghosts that did reseal to cations did so at low ionic strength (one-tenth the tonicity of plasma) and at low temperature. Later studies lO. 75. 76 showed that, with regard to Na and K transport, the heterogeneity of ghost types in the population were separable into three groups: 1) those that spontaneously resealed; 2) those that could be induced to reseal, and 3) those that remained leaky. The relative proportion of these different types was found to depend on the preparative conditions that obtained before and after hemolysis. 10. 75-77 Once these various conditions had been specified it became possible to prepare ghosts (group 2) that displayed minimal heterogeneity with regard to Na and K permeability (cf. 78). The most important factors that act in concert to optimize membrane repair were found to be the ionic strength of the medium both before and after hemolysis 10. 75. 76, the pH of the medium (pH 6.0) at which hemolysis takes place77 , the presence of Mg++ if the temperature at hemolysis is above 0° C79 but not if the temperature is at 0° C76 and the resealing temperature after hemolysis. 80 The ionic strength of the hemolyzing medium is important for when optimized it appears to lessen the damage of hemolysis as the hemolytic ratio (volume of cells to volume of medium) is decreased.lO. 75. 81. 82 Returning the ionic strength of the ghost suspension after hemolysis to the original osmolality (Le. reversal) maximizes the rate and extent of membrane resealing to ions (80, see also 76). Reduction of ghost heterogeneity is also maximized when hemolysis takes place at pH 6.0. 77 The presence of Mg++ at hemolysis is evidently required for resealing the membrane to ions 79 but this effect has been shown76 to be critically dependent on the temperature at which hemolysis takes place (see below). But after hemolysis the effect of temperature on accelerating the rate of membrane resealing could be optimized by incubating (annealing) the reversed ghosts at 37° C for 30 to 60 min.80 This technique of annealing ghosts is now widely used but the molecular basis for the membrane changes that accompany resealing remain just as obscure as the changes that occur when the holes were formed. While the effect of temperature on resealing can be considered a continuum, there is a critical transition point that appears to occur around 0° C that remarkably affects the length of time the hemolysis holes are open and perhaps their size. 76 Thus when hemolysis is carried out at 0° C, and the ghosts are maintained at that temperature afterward, the membrane remains permeable to Hb as well as to other proteins (e.g. albumin) whereas at slightly higher temperatures (2-4° C) the membrane quickly recovers its impermeability to proteins, as already discussed. This means that at 0° C it is possible not only to prepare Hb-free ghosts (see 78) but also to trap various enzymes and substrates (e.g. 83) within them prior to resealing and preparation for subsequent study. Systematic study has yet to

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be made of the membrane hole size at 0° C as well as the extent to which Hbj exit is inhibited, in this circumstance, by polymers, such as dextrans and ficol, mentioned before. In contrast to ghosts made by hypotonic hemolysis it is also possible to hemolyze cells osmotically by swelling them in an isotonic salt solution. 84-88 This is done, not by a colloid osmotic mechanism involving a lytic agent, but by preloading cells with a permeant solute, such as glycerol86 ,87 or dimethylsulfoxide. 88 Such pretreated cells, when suspended in a medium containing little or no permeant solute, swell to their hemolytic volume because of the solute concentration difference and the fact that the preloaded solute leaves more slowly than water can enter. Ghosts prepared by this method at 37° C reseal in seconds 88 presumably reflecting the importance of ionic strength. However, detailed characterization of the hemolytic event per se and functional analysis of the resultant ghosts is still needed for an understanding of the molecular processes involved.

SLOW HEMOLYSIS Slow hemolysis offers not only another approach to studying the determinants of the molecular events associated with hemolysis but also provides a more efficient method for the entrapment of foreign molecules inside ghosts during the time of hemolysis. While it is not clear that the hemolytic process itself differs between slow versus fast hemolysis, the two types do appear to differ in the size range and amounts of molecular types that can be incorporated (see later). As pointed out before, slow hemolysis, in which the ionic strength or degree of hypotonicity is gradually changed, can be carried out either in steps89,90 or continuously.40, 54, 91-93 In the former, ghosts are successively hemolyzed by the stepwise lowering of the ionic strength of the medium, whereas in the latter instance the cells are dialyzed against a lower ionic strength buffer. (It may be of interest that the first use of a dialysis method to hemolyze cells was evidently carried out by Adair et al. 8) Katchalsky et al. 54 point out that there is a major difference in the osmotic fragility of human red cells hemolyzed by slow compared to fast means. Thus in slow hemolysis the cells hemolyzed at a lower salt concentration (higher osmotic resistance) than cells hemolyzed in fast hemolysis. The explanation for this appears to lie in the fact that there is a prehemolytic loss of K (and Cl) by the cells when they are in the swollen state. 94-97 This means that a greater osmotic pressure differential across the membrane would be necessary to bring the cells to their Vh' where Vh is the same for cells hemolyzing by either means. The mechanism(s) underlying this prehemolytic salt loss is not known but may involve increases in diffusion leaks and/or swelling activated KlCl cotransport processes (see ref. 98). Since this prehemolytic K loss is known to occur in human but evidently not in rabbit red cells 99 it would be interesting to know the characteristics of rabbit red cells subjected to fast versus slow hemolysis. Perhaps more to the point, studies defining the membrane transport parameters of swollen cells (near their Vh) are needed in order to understand the basis for the KClloss and the extent to which this occurs in fast hemolysis. The presumed reason that cells shrink in this circumstance is that the cells contain high K relative to Na and that the leakage rate of K exceeds that for Na (100), but this explanation would have to be modified if cotransport were involved. These changes in cation permeability that evidently underlie the osmotic differences in fast/slow hemolysis presumably occur in opposition to a colloid osmotic swelling force that is likewise activated by increased cation permeability (cf. 23, 101-103). The extent to which this type of mechanism could be involved in either slow or fast types of hemolysis

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would again depend on the extent to which the KClloss was due to a diffusion or mediated (cotransport) type mechanism. There have been many studies concerned with refining dialysis methods for preparing ghosts by slow hemolysis as well as defining optimum conditions for hemolysis and the resealing of ghosts (e.g. 104-109). While these studies provide insights into many of the issues raised above, a detailed survey of this literature is beyond the scope of the present article.

ENTRAPMENT OF IONS AND MOLECULES Entrapment into ghosts of molecules that were normally impermeant began with studies showing that substances such as ferricyanide SI , ATP at pH 2-3 110 or fructosediphosphate 74 could be incorporated inside provided they were present in the hemolyzing solution. These studies were in general carried out using a hemolytic ratio of one volume of cells to two Sl , 110 or 1.274 volumes of solution at 0° C. It is not clear that the substances that entered did so at hemolysis for it can be shown (cf. 111) that at these hemolytic ratios and with the tonicities of the hemolyzing media used that a major portion of the cells, though swollen, remained intact. This raises the question as to whether these solutes penetrated during hemolysis or through the swollen intact cells or both. In the case of ATP for the conditions used llO it could be shown 11 I that the undissociated acid penetrated intact cells at their isotonic volume without hemolysis. On the other hand, nucleotides such as ATP could, under appropriate conditions, be shown to be definitively trapped within ghosts during hemolysis. 79 , III Thus a major point of this discussion is to raise a cautionary flag about hemolysis procedures and the mechanism(s) of foreign molecule entry. With regard to the entrapment of proteins other than Hb9 and albumin lO, the first enzyme appears to be yeast hexokinase (79, cf. 111 for details) but a variety of other enzymes have been incorporated as well, such as an ATP regenerating system involving creatine kinase l12 and B -glucosidase. ll3 These proteins were all entrapped in human red cell ghosts by hypotonic hemolysis of the fast type. On the other hand a variety of other proteins, enzymes and drugs have been loaded into ghosts by means of procedures involving dialysis and slow hemolysis as extensively documented in other volumes in this series 1l4- 1I6 including the present volume. The basic thrust of most but not all of this work is to develop therapeutic agents encased in resealed ghosts that can, in vivo, be delivered to specific targets. Because of the possibility of using autologous red cells and because the ghost parameters (e.g. volume, high Hbi, deformability, circulatory life span) are similar to the intact cells from which they were derived, the exploitation of this methodology offers considerable potential. In addition, some of the work on encapsulation is aimed at analyzing cellular functions that are uniquely approached in these ways. Finally, it should be noted that the term "erythrocyte" is used (e.g. carrier erythrocyte) to refer to resealed ghosts containing various incorporated pharmacologic agents. While these ghosts may look like normal red cells and behave like normal red cells, they are in fact ghosts, as stated at the outset, and one should always be mindful of the difference.

ACKNOWLEDGEMENT This work was supported by NIH grant HL 09906 and by an Alexander von Humboldt Foundation Award.

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