electrolyte solutions showed two phases: an early rapid phase was followed

Transcription

1 J. Physiol. (1969), 201, pp With 9 text-figures Printed in Great Britain THE TIME COURSE OF RED CELL LYSIS IN HYPOTONIC ELECTROLYTE SOLUTIONS BY A. J. BOWDLER* AND T. K. CHANt From the M.R.C. Group for the Study of Haemolytic Diseases, University College Hospital Medical School, London W.C. 1 (Received 9 October 1968) SUMMARY 1. Osmotic haemolysis of a standard suspension of human red cells was followed using a recording spectrophotometer at wave-lengths between 600 and 650 my. 2. Optical density changes in the cell suspensions were related to turbulence of the suspension, cell swelling and loss of haemoglobin-containing cells. 3. The time course of the loss of cells from suspension in hypotonic electrolyte solutions showed two phases: an early rapid phase was followed by a smaller phase of longer half-time. 4. The second phase was most prominent in the middle ranges of partial haemolysis and less when total haemolysis was at the extremes of the detectable range. 5. The second phase was eliminated by the inclusion of 20 mm sucrose in the suspension, and was slowed by the presence of % tannic acid without alteration of magnitude. 6. The magnitude of the second phase was dependent on the dominant external cation, becoming progressively greater through the series: Mg2+ < Na+ < Li+ < K+ < Rb+. 7. The slow phase is interpreted as arising from passive cation permeability in cells swollen to a volume close to that critical for haemolysis, with water influx secondary to the unopposed colloid osmotic pressure of intracellular protein. INTRODUCTION In isotonic media the red cell membrane has an area which is considerably in excess of the minimum required to enclose the volume of the contents. * Present address: College of Human Medicine, Michigan State University, East Lansing, Michigan 48823, U.S.A. t Present address: University Department of Medicine, Queen Mary Hospital, Hong Kong. 28-2

2 438 A. J. BOWDLER AND T. K. CHAN Conventional tests of osmotic fragility provide a method for examining indirectly the distribution in the cell population of the relationship between cell content and membrane area. The usefulness of such tests in the clinical context has been mainly in the recognition of conditions such as hereditary spherocytosis and thalassaemia in which the relationship of membrane area to isotonic cell volume departs markedly from normal. Standard methods of measuring osmotic fragility, such as that of Parpart, Lorenz, Parpart, Gregg & Chase (1947), eliminate variability arising from the effects of the liberated cell contents and the time course of haemolysis, by using very small volumes of red cells in relation to the total volume of the system, and by measuring haemolysis after a standard period of exposure to the hypotonic medium. The importance of the time course of haemolysis to an understanding of the processes leading to the liberation of haemoglobin in an osmotically unfavourable environment is shown, however, by the early work of Jacobs (1932) who attempted to use the rate of haemolysis as a measure of red cell permeability to water. Using hypotonic sucrose solutions, Jacobs found haemolysis to be complete after 1-8 sec, while haemolysis occurred more slowly in osmotically equivalent NaCl solutions (Jacobs & Parpart, 1932). In these circumstances the haemolysis time is in excess of the period required for water transfer (Sidel & Solomon, 1957) and has been suggested to include components related to membrane rupture and haemoglobin release. That the membrane may offer substantial resistance to rupture is suggested by the studies made by Rand (1964). However, even when these possible factors are taken into account, there remain to be explained differences in the time taken for haemolysis to proceed to completion, depending on the total fraction of the cells in the system which are haemolysed. Thus, when 100 % of the cells haemolyse the process requires 3 min or less (Jacobs, 1932; Lowenstein, 1960), while Parpart et al. (1947) found that 20 % haemolysis in hypotonic saline required 45 min at 200 C. The present study was undertaken in order to define more closely the time course of osmotic haemolysis and to identify the causes of the differences found with various media. METHODS The use of spectrophotometric methods to follow the change in particle density of a red cell suspension offers a means of following cell lysis which is dependent on the presence of intact haemoglobin-containing cells rather than on the detection of liberated haemoglobin. In the present study the time course of haemolysis was followed by means of a Unicam SP 800 self-recording spectrophotometer which provided a continuous recording of changes in optical density (O.D.) of a suspension of red blood cells under a variety of experimentally chosen conditions. Red cells were obtained from blood freshly drawn by venepuncture from normal subjects

3 SLOW PHASE OSMOTIC HAEMOL YSIS 439 into tubes containing dry heparin (1 mg to 10 ml. blood). Oxygenation was effected by gentle agitation in air until no further colour change was observed. A convenient dilution for spectrophotometric studies was obtained by diluting whole blood with an equal volume of 0 16 M-NaCl solution. One part of diluted blood was then added to 100 parts of the test hypotonic solution to give a suspension containing approximately 2-5 x 104 red cells per mm3. HypotoniC solutions were made from stock solutions prepared as follows: (1) Buffered NaCl containing 154 mm-nacl, 0-96 mm-na2hpo4, 0-16 mm-nah2po4. 2H2O. (2) Buffered KCl containing 154 mm-kcl, 0-96 mm-k2hpo4, 0-16 mm-kh2po4. (3) Buffered LiCl containing 154 mm-licl.h210, 0-96 mm-na2hpo., 0-16 mm-nah2po4. 2H20. (4) Buffered RbCl containing 154 mm-rbcl, 0-96 mm-na2hpo4, 0-16 mm-nah2po4. 2H2O. (5) Buffered MgCl2 containing 103 mm-mgcl2. 6H2O, 0-4 mm Tris (hydroxymethyl) methylamine. The ph was adjusted to 7-4 by the addition of concentrated HR. The measured ph of the solutions was 7-4 in all cases. 1 M sucrose was prepared and 0 05 ml. mixed with 2-5 ml. of the appropriate hypotonic electrolyte solution when required, to give a final sucrose content of 20 mm. 0 5 % tannic acid was prepared (500 mg per 100 ml.) and ml. mixed with 2-5 ml. of the appropriate hypotonic electrolyte solution whan required, to give a final tannic acid content of %. Spectrophotometric method For the study of the time course of osmotic haemolysis 2-5 ml. of the hypotonic solution was pipettedintoacuvette of lcm light path, and allowedto attain the temperature of the cuvette carriage, which was maintained at 250 C. Diluted blood (0-025 ml.) was added and mixed by stirring with a plastic spatula. O.D. changes were followed and recorded graphically. A continuous tracing of O.D. was obtained for at least 20 min in each experiment, and, for those reactions in which the O.D. was falling at a rate faster than in 1 min after 20 min, readings were obtained at intervals of 5-20 min until there was no fall in O.D. in two successive intervals of 20 min. The longest time required for following one reaction was 233 min. Since red cells tend to sediment on standing, stirring with a plastic spatula was performed at 5 min intervals during the continuous tracing, and for interval readings 2 min before each reading. Preliminary observations The opacity of a suspension of red cells was used to measure particle density as early as 1895 by Oliver. The decrease in O.D. of a red cell suspension was used to follow the time course of osmotic haemolysis by Jacobs (1930) and Lowenstein (1960). Love (1954) and Rideal & Taylor (1956) used similar methods in the study of detergent-induced haemolysis. The advantages of the spectrophotometric method lie in its simplicity, accuracy and promptness in response, but problems arise from the use of an unmodified spectrophotometer as a densitometer and the method adopted is therefore outlined in detail below. (1) Choice of wave-length. The wave-length riost suitable for recording the events of haemolysis was selected from the absorption spectra shown in Fig. 1. Curves A, B and C were obtained with the standard suspension of red cells in 0-16 M, 0-10 M and 0-09 M-NaCl respectively, and curve D with the same number of red cells in water. Curve E was obtained in the same manner as D, with the cell ghosts removed from the preparation by centrifugation. Curve F was obtained from a preparation using a suspension of cell ghosts in isotonic saline, the particle concentration being four times that of the red cells used for the previous curves. It can be seen that all the curves show the absorption peaks of oxyhaemoglobin. Curves A, B, and C, with the haemoglobin in intact cells, show the absorption spectrum of haemoglobin superimposed on a high base line. This high O.D. throughout the range of wavelength investigated is due to the light scattering effect of the cells. It is clear that the light scattering property of the cell is principally dependent on the high intracellular concen-

4 440 A. J. BOWDLER AND T. K. CHAN tration of haemoglobin since with the ghost preparation, shown in curve F, it is virtually eliminated. Comparison of curves A, B, and C shows a decrease in the optical density of red cells in sublytic hypotonic suspension, indicating a reduction in the light scattering pro. perties of red cells subjected to hypotonic swelling. It can be seen that there is little absorption of light of wave-lengths above 600 mp by haemoglobin and ghosts, while intact cells have a high scattering effect. This region is thus suitable for the study of haemolysis, since reduction in light transmission is dependent in,0 A B 0-5 o 1, Wave-length (mit) Fig. 1. Spectrophotometric absorption spectra of (A) red cells in 0-9% NaCl; (B) red cells in 0 65 % NaCl; (C) red cells in 0-56 % NaCl; (D) red cells haemolysed in distilled water; (E) red cell haemolysate with ghosts removed by centrifuging; (F) ghost suspension in 0-9 % NaCl with particle concentration quadrupled. Cells in: A- 0.90%; B- 0.65%; C- 0.56%; D-.-haemolysate inwater; E-- free Hb only;fi. ghost. this range on the presence of intact haemoglobin-containing cells. One disadvantage, however, is that cell swelling also results in a decrease in optical density, but the magnitude of this effect is relatively small by comparison with that due to loss of intact cells and can be allowed for empirically by the use of a calibration curve. (2) The effect of cell swelling on optical density. The relationship between cell swelling and the decrease in O.D. with wave-lengths above 600 mui was investigated quantitatively. Identical volumes of a suspension of red cells were added to each of a series of sublytic hypotonic buffered NaCl solutions. Optical densities were determined after 5-10 min by which time steady readings had been attained. The relationship between red cell volume and tonicity was shown by Ponder (1948) to follow the equation V = RW ) +

5 SLOW PHASE OSMOTIC HAEMOLYSIS 441 where V is red cell volume, R is a constant, W is the volume of cell water and T is tonicity, isotonicity being given the value of 1-0. Cell volume may thus be expected to have a linear relationship with 11T values from 0-92 to 1*77, corresponding to a range of osmolality from 315 to 163 m-osmole. The maximum decrease in the sublytic range is to about 80 % of the O.D. at isotonicity (Fig. 2). (3) The effect of cell concentration on optical density. One part of whole blood was added to 200 parts of isotonic saline, giving a suspension containing 2-5 x 104 cells/mm3 and a range of dilutions with isotonic saline was prepared. The O.D. of each suspension was read after a, ~ T Fig. 2. The relationship of optical density of a red cell suspension to thr. reciprocal tonicity (T) of the suspending solution, shown by regression line and 95% confidence limits. steady level was attained and the relationship between O.D. and suspension concentration is shown in curve A in Fig. 3. This relationship shows close conformity with the pattern of a rectangular hyperbola. (4) Empirical determination of the relationship between O.D. and the cell fraction remaining unhaemoly8ed. For the purpose of this study it was necessary to determine the fraction of the red cells which remained intact after hypotonic haemolysis, and to assess the residual fraction at intervals short of final hasmolysis by means of the O.D. Allowance had therefore to be made for the reduction in O.D. arising from the swelling of the residual intact cells. The relationship found above for cells in isotonic saline cannot be assumed to hold for suspensions containing partial populations of swollen cells. Calibration was therefore effected by measuring the fractional haemolysis of a standard red cell suspension in a descending series of hypotonic saline solutions by estimation of the haemoglobin liberated into the supernatant. These values were then related to the O.D. Of the suspensions after haemolysis. An example of this method of calibration is shown as curve B in Fig. 3, where the initial cell concentration ('100 %') was identical with that of curve A. It can be seen that curve B differs from curve A, principally in lying somewhat below it, a finding consistent with the superimposed effect of cell swelling. (5) Stirring and mixing effect. Optical density changes were found to accompany the

6 442 A. J. BOWDLER AND T. K. CHAN initial addition of the red cell suspension to buffered saline. On the addition of red cells, there was a reduction in O.D. which lasted approximately 2 min, followed by a variable rise above the ultimate steady level, which settled to a constant reading between 5 and 8 min from the time of the addition of the cell suspension. Subsequent stirring led to an immediate and transient fall in O.D. followed invariably by a positive rise above the steady level, which was re-established in min. The initial disturbance was less marked in sublytic hypotonic saline, and approximated more closely to the stirring disturbance. The initial reduction in O.D. was also of shorter duration than in isotonic saline and the secondary rise was less prominent. However, reliable readings of O.D. could not be obtained for at least 1 min after the addition of cells to hypotonic saline and no analysis of changes in this interval was attempted. 0-8A e ~~~~~~B Cells (%) Fig. 3. The relationship of optical density to cell concentration in suspension, expressed as a fraction of the O.D. of standard concentration of 2-5 x 104 cells/ mm3 in isotonic saline. Curve A (upper): cells suspended in 0-9 % NaCl. Curve B (lower): cells suspended in hypotonic salines. Cells in: 0-9% NaCi 0-0 A; hypotonic salines 0-0 B. RESULTS The change in O.D. with time in one experiment in which red cells were added to hypotonic sodium chloride solutions is shown in Fig. 4. A steady value of O.D., indicating completion of haemolysis, was reached after 20 min for the middle range of values for percentage haemolysis. For extreme values, with 95 % and 100 % haemolysis, final levels were attained within 2-10 min. Curves were analysed by converting the O.D. to percentage of cells remaining unhaemolysed by means of the calibration curve. The ratio of the number of cells remaining to be haemolysed at intermediate times to the total number of cells finally haemolysed ('fractional haemolysis') was

7 SLOW PHASE OSMOTIC HAEMOLYSIS 443 plotted against time semilogarithmically. An example is shown in Fig. 5. This shows haemolysis occurring in at least two phases. The later part of the curve can be represented by a straight line which when extrapolated to zero time by regression analysis gives the magnitude of the later or 'slow' phase, which may be converted to a percentage of the whole population of cells in the system after estimation of the total fraction of cells haemolysed. The rate of haemolysis in this phase is represented by calculated half-time of I I I I I Haemolysis 7 0~~~~3 0~~~3 0~~~0.5~~~~ Minutes Fig. 4. The time course of O.D. changes of red cells suspended in. hypotonic salines to give indicated final percentages haemolysed. The ordinate scale shows the change in O.D., with 1 vertical interval representing an O.D. range of The base line values for the curves are not identical as the curves have been placed in the diagram at suitable levels to show better the differences in shape. All are drawn to identical scale. the curve (ti). By subtracting values of the slow phase from the early values of the curve, a series of points is obtained which can also be fitted to a straight line, much steeper than that of the slow phase. The time course of the rapid phase is short, and includes the period of the mixing disturbance, so that further subdivisions of this phase cannot be excluded. However, within the limitations of the method it appears as a single phase, and its dimensions of magnitude and half-time are calculated on this assumption. Table 1 shows the dimensions of the two phases using as the suspending

8 444 A. J. BOWDLER AND T. K. CHAN medium buffered NaCl of differing tonicities. It can be seen that the magnitude of the fast phase varies inversely as the tonicity ofthe hypotonic medium while the magnitude of the slow phase is greatest in the middle range of haemolysis. The tj of the fast phase is approximately 0-3 min, while that of the slow phase is approximately 4-2 min Minutes Fig. 5. Fractional haemolysis of red cells suspended in hypotonic saline with analysis into two phases. Ordinate: cells remaining to be haemolysed as a fraction of the total cells finally lysed (logarithmic scale). Abscissa: time in min. Further experiments were made using buffered KC1 in differing tonicities: the results are shown in Table 2. The magnitude of the fast phase again varied inversely with the tonicity of the hypotonic medium and was similar to that for cells in buffered NaC1 of the same tonicity, while the magnitude of the slow phase was greater in the middle ranges of haemolysis and was greater than that found in buffered NaCl for a comparable magnitude of the fast phase. The greater slow phase in buffered KCI thus contributes to the increased fragility of cells in that medium as compared

9 SLOW PHASE OSMOTIC HAEMOLYSIS 445 with the fragility in buffered NaCi. The tj of the fast phase in KCl was between 0*3 and 0.5 min, which is similar to that in buffered NaCi while the tj of the slow phase was between 8 and 21 min, which was significantly longer than that found in buffered NaCl. Further experiments were designed to investigate the cause of the slow phase. The change in O.D. with time was followed with light of wave-length 416 mnu in one experiment using buffered hypotonic NaCl as described TABLE 1. Red cell haemolysis in buffered NaCi Fast phase Slow phase Tonicity Total, _ - haemolysis Magnitude ti Magnitude ti (m-osmole) (%) (%) (min) (%) (min) 124* * * * '5 TABLE 2. Red cell haemolysis in buffered KCI Fast phase Slow phase Total._ Tonicity haemolysis Magnitude Magnitude ti (m-osmole) (%) (%) (min) (%) (min) _ * previously, with the exception that the number of red cells added was one quarter of the number usually employed. The slow phase was still present and represented by a rise in O.D. (Fig. 6). At this wave-length, this clearly indicates haemoglobin release and cannot arise from cell swelling, thus showing that the second phase reflects events accompanying cell lysis. Figure 7 illustrates a further experiment in which red cells were added to hypotonic buffered KCl (110 m-osmole), to give a final percentage of cells haemolysed of 90 %. There was a clearly defined second phase of haemolysis. The experiment was repeated, but 2 min after the addition of the red cell suspension, sufficient 10 % KCI was added to give a final osmolality of 150 m-osmole. It can be seen that the second phase was promptly terminated with the final level of total haemolysis at 75 %. Experiments were also performed using as the suspending medium buffered LiCl, buffered RbCl and buffered MgCl2 of differing tonicities. The magnitudes of the slow phase in the respective solutions are shown in Fig. 8, together with those found with buffered NaCl and KCl using red cells from the same individual. It can be seen that the slow phase differs in

10 446 A. J. BOWDLER AND T. K. CHAN magnitude according to the dominant cation of the medium, and that there is a progressive increase in the order Mg2+ < Na+ < Li+ < K+ < Rb+. Similar time course experiments were performed using as the suspending medium 20 mm sucrose in buffered NaCI of differing tonicities, to give the I I P-( 9Id To I II Minutes Fig. 6. Optical density change of red cell suspension at 416 mt in lytic concentration of hypotonic saline (115 m-osmole). The second phase at this wave-length is accompanied by a rise in O.D. indicating haemoglobin release \ + ~~~10% KC1 added 0-2F 1, I I I Minutes Fig. 7. The time course of optical density change of red cells in hypotonic buffered KC1. The upper curve shows the effect of adding hypertonic KCl at the time indicated in a duplicate experiment. same range of osmolalities as previously employed. Figure 9 shows the result for three different final percentages of haemolysis in this hypotonic medium. It can be seen that the final O.D. was attained in less than 2 min and analysis by semilogarithmic plot as described above confirmed that there was only one phase with a tj similar to that of the usual fast phase. Experiments with buffered KCl, LiCl and RbCl containing 20 mm sucrose in each hypotonic medium gave similar results.

11 SLOW PHASE OSMOTIC HAEMOLYSIS 447 Experiments were performed with hypotonic KCl with and without the addition of tannic acid. The results are presented in Table 3, which shows that tannic acid in a concentration of 0*005 % has no effect on the magnitude of the slow phase but increases the tj by a factor of 3 to 4 times D m a~~~~~~b 0,Bil B Haemolysed, fast phase (%) Fig. 8. The relationship of the magnitude of the slow phase, expressed as a percentage of the total cells in the system, and the magnitude of the fast phase. The suspending media comprised a range of hypotonic solutions of different salts: (A) LiCl; (B1) and (B2) repeated experiments with NaCl; (C1) and (C2) repeated experiments with KCI; (D) RbCl; (E) MgCl2. A A-A LiCl, B 0-* NaCl, C 0-0 KCl, D *-* RbCl, E V-V MgCl2. DISCUSSION The present study has been directed towards the clarification of the time course of the changes in optical density following the addition of red cells to hypotonic electrolyte media, and to investigating the causes of the changes demonstrated. The spectrophotometric method employed a wavelength above 600 mt. Three principal sources of O.D. variation of a red cell suspension were shown. First, there were transient variations following the initial mixing of red cells with the hypotonic medium; similar changes accompanied subsequent stirring. Secondly, swelling of red cells without lysis produced a reduction in O.D., presumably due to the less effective reflexion of trans-

12 448 A. J. BOWDLER AND T. K. CHAN mitted light by particles of spherical form compared with cells with a shape closer to that of a disk. Thirdly, the elimination of cells from the suspension by lysis led to the increased transmission of incident light. Haemolysis (%) 0~~~~2 0~~~~2 A-II 0 0~~~~~ 5 10 Minutes Fig. 9. Optical density changes in red cell suspensions in hypotonic salines containing 20 mm/l. sucrose. The curves have differing base lines and are placed so as to show better the shape of the individual curves. TABLE 3. Haemolysis in buffered KCI with and without tannic acid KCI + tannic acid KCI Fast Slow Fast Slow Total phase phase haemo- -,ra~ Experi- Tonicity lysis X ti ment (m-osmole) (%) (%) (min) (%) (min) * * Total phase phase haemolysis - > ti t, ( %) ( %) (min) ( %) (min) * * The disturbance oflight transmission through a red cell suspension during the initial period of mixing was observed by Ponder (1941), who described a reduction in opacity of suspensions following the initial addition of cells. However, he described only a rise in O.D. on subsequent stirring, and did

13 SLOW PHASE OSMOTIC HAEMOLYSIS 449 not explicitly comment on the preceding transient reduction, probably due to the short duration leading to its not being observed with the method employed. Ponder suggested that the mixing effect was related to the orientation of masses of cells by the production of currents in the suspension, which would also account for the initial reduction in O.D. on stirring. The secondary rise in O.D. after stirring would imply a temporary orientation of the red cells in such a manner as to give a higher degree of attenuation of transmission compared with that of the cell suspension in a steady state. This is consistent with the finding that this phase was much less prominent in hypotonic media than in isotonic media (and may be absent), since variations in the orientation of cells close to the spherical in shape would produce much smaller differences in the effect on light transmission than would disks. The principal finding related to the time course of the disappearance of cells from the system in the presence of lytic concentrations of electrolyte solutions was that the process occurred in two distinct phases when total haemolysis amounted to less than 100 %. To decide whether the second phase was due to cell lysis or to a late change in the cell volume of unlysed cells, the experiment was repeated with the spectrophotometer set at a wave-length of 416 m~u, at which wave-length haemoglobin release produces a rise in O.D., while cell swelling produces a fall. This showed that the second phase was accompanied by the release of haemoglobin and must, therefore, be regarded as reflecting part of the lytic process. The work of Sidel & Solomon (1957) showed that unaccompanied water transfer due to a difference in the chemical potential between the environment and the cell interior is normally very rapid, and Jacobs (1950) found that red cell permeability to water was greater than in any other cell investigated. Small increases in red cell volume may, however, reduce the permeability of the membrane to water, although the extent of this effect with cells close to their critical haemolytic volume is not clear (Solomon, 1960). Nevertheless, a progressive decrease in the water permeability of each cell with swelling would not produce a two-phase process, and the effect would not be expected to disappear with 100 % haemolysis, so that variations in water permeability with swelling would be unlikely as an explanation of the second phase. A further possibility is delayed release of haemoglobin from the membrane of the cells after rupture. However, in this case it would be expected that the slow phase would increase in magnitude pari pass with the magnitude of the fast phase, and this is clearly not so. Likewise, if slow phase haemolysis were due to an unrecognized factor unrelated to the hypotonicity of the medium it would not be expected to have the specific distribution related to the percentage of cells lysed. The finding that the

14 450 A. J. BOWDLER AND T. K. CHAN slow phase is greatest in the middle range of haemolysis does suggest, however, that there is a correlation with the slope of the fragility curve. In this region the residual unhaemolysed cells have the highest proportion swollen to a volume close to that critical for lysis. It therefore appears that the slow phase is due to delayed haemolysis of a population of cells rendered susceptible by their initial degree of swelling. This is consistent with the finding that the second phase is abruptly terminated by making the environment less hypotonic. If the slow phase is not due to a change in the permeability of the membrane to water, two further alternatives arise. First, it is possible that the cells haemolysing in the slow phase may have already reached their critical volume but are capable of resisting tension in the membrane for a short period before rupture. However, in this case it would be expected to have the same magnitude in equiosmolar solutions of different salts, whereas the second phase is found to vary in magnitude and tj with the dominant external cation. Secondly, it is possible that cells swollen to a certain size may develop cation permeability, so that water enters the cell due to the unopposed action of the intracellular colloidal osmotic pressure. This possibility is supported by the suppression of the second phase by the inclusion of sufficient sucrose in the medium to balance the intracellular colloid osmotic pressure. The dependence of the second phase of haemolysis on environmental cation is also supported by the finding that magnitude and tj vary with the dominant external cation. The magnitude of the slow phase was least in MgCl2 and increased progressively through the series of chloride salts of Na, Li, K, and Rb. With the exception of the reversed order of Na and Li, this represents the order of increasing ion mobility in bulk solution (Boyle & Conway, 1941). This can be interpreted as representing a decreasing order of hydrated ion size, and provides a measure of the increasing capacity of the ions to move within the interstices of a water lattice. A widely accepted model, recently reviewed by Weed & Reed (1966), holds that the red cell membrane consists of a double lipid layer bounded internally and externally by layers of protein, traversed by water-filled pores. Two possible pathways for the penetration of the membrane by electrolyte are by a carrier system through the lipid-protein complex, and by diffusion through the water-containing pores. The finding that tannic acid in low concentration delays the slow phase is consistent with poremediated penetration (Edelberg, 1952). Although the question has not been directly investigated as to whether the characteristics of the second phase depend principally on the passage of cation or on the subsequent water transfer, the fact that water transfer in the tannic acid treated cell is relatively rapid by comparison with the time required for the slow phase

15 SLOW PHASE OSMOTIC HAEMOLYSIS suggests that the rate limiting factor is cation transfer through the waterfilled pores. The pores of the normal membrane have been estimated to be approximately 7 A to 8 A in diameter (Goldstein & Solomon, 1960). Increased cation permeability might arise from an increase in the diameter of the pores or a reduction in the effectiveness of the pore charge in repelling the ions. The fact that sucrose molecules, estimated to be 8'8 A in diameter, are capable of balancing the colloid osmotic pressure of the cell shows that the pores will not admit this non-electrolyte, which is not consistent with a significant increase in the diameter of the channels. The mechanism by which the effect of the positive charges of the channels might be attenuated must be conjectural, but the reduction in effectiveness must occur in some orderly fashion, since the degree of swelling required to admit cation of particular species appears to vary in a precise and predictable manner. It is therefore suggested that the differences in magnitude of the second phase arise from a change in the water-filled channels, probably related to their charge, induced by a near-critical increase in membrane tension. The least degree of the hypothetical change is sufficient to admit those cations of greatest mobility in water, while greater degrees are required to admit ions of lesser mobility. Thus, penetration of the membrane by highmobility ions, such as Rb+, occurs with lesser degrees of membrane tension than with low-mobility ions such as Mg2+, and will thus involve a larger population of the residual unhaemolysed cells remaining after the rapid phase of haemolysis, in which water penetration alone is capable of inducing lysis. The slow phase of haemolysis thus appears to affect a fixed population of cells at any given level of haemolysis in an electrolyte medium of specific composition. The limit of the population affected is determined by the degree of swelling short of haemolysis induced by water transfer, occurring during the first phase of haemolysis. Differences in the apparent osmotic fragility of red cells tested in hypotonic solutions of different electrolytes were attributed by Ponder (1948) to variations in the haemolytic volumes of red cells due to environmental effects. However, part at least of such differences may be attributable to variations in the penetration of the membrane of the swollen cell by different cations in the suspending medium. It is not possible at present to exclude further effects, including perhaps a protective leak of K from the cell within the time limits of the fast phase in K-poor solutions, and also possibly qualitative changes in membrane properties induced by external cations, and these remain for further investigation. 29 Phy

16 452 A. J. BOWDLER AND T. K. CHAN REFERENCES BOYLE, P. J. & CONWAY, E. J. (1941). Potassium accumulation in muscle and associated changes. J. Phyriol. 100, EDELBERG, R. (1952). The action of tannic acid on the erythrocyte membrane. J. cell. comp. Phy8iol. 40, GOLDSTEIN, D. A. & SOLOMON, A. K. (1960). Determination of equivalent pore radius for human red cells by osmotic pressure measurement. J. gen. Phy8iol. 44, JACOBS, M. H. (1930). Osmotic properties of the erythrocyte. I. Introduction. A simple method for studying the rate of hemolysis. Biol. Bull. mar. biol. Lab. Woods Hole 58, JACOBS, M. H. (1932). Osmotic properties of the erythrocyte. III. Applicability of osmotic laws to the rate of hemolysis in hypotonic solutions of non-electrolytes. Biol. Bull. mar. biol. Lab. Wood8 Hole 62, JACOBS, M. H. (1950). Surface properties of the erythrocyte. Ann. N.Y. Acad. Sci. 50, JACOBS, M. H. & PARPART, A. K. (1932). Osmotic properties of the erythrocyte. V. The rate of hemolysis in hypotonic solutions of electrolytes. Biol. Bull. mar. biol. Lab. Wood8 Hole 63, LoVE, W. E. (1954). The hemolytic and antihemolytic action of dodecyl ammonium chloride (cationic detergent). J. cell. comp. Physiol. 44, LOWENSTEIN, L. M. (1960). The effect of albumin on osmotic hemolysis. Expl Cell Re8. 20, OLIVER, G. (1895). A new method of estimating the percentage of the blood corpuscles. J. Phyriol. 19, xvi. PARPART, A. K., LORENZ, P. B., PARPART, E. R., GREGG, J. R. & CHASE, A. M. (1947). The osmotic resistance (fragility) of human red cells. J. clin. Invest. 26, PONDER, E. (1941). Red cell counts, percentage volume and the opacity of suspensions. Am. J. Phyaiol. 134, PONDER, E. (1948). Hemolyai8 anrd Related Phenomena, pp London: J. and A. Churchill Ltd. RAND, R. P. (1964). Mechanical properties of the red cell membrane. II. Viscoelastic breakdown of the membrane. Biophy8. J. 4, RIDEAL, E. & TAYLOR, F. H. (1956). On haemolysis by anionic detergent. Proc. R. Soc. B 146, SIDEL, V. W. & SOLOMON, A. K. (1957). Entrance of water into human red cells under an osmotic gradient. J. gen. Physiol. 41, SOLOMON, A. K. (1960). Red cell membrane structure and ion transport. J. gen. Physiol. 43, suppl WEED, R. I. & REED, C. F. (1966). Membrane alterations leading to red cell destruction. Am. J. Med. 41,