Brit. J. Haemat., 1968, 14, 323.

The Effect of pH on the Volume, Density and Shape of Erythrocytes and Thymic Lymphocytes*

D. G. LEGGE AND KEN SHORTMAN

Wal te r a d Eliza H a l l Institute of Medical Research, Melbounie , Atrstralia

SUMMARY. Erythrocytes increase in volume, decrease in density and become more spherical as the pH of isotonic media decreases over the range 9.4-4.8. Swelling is rapid, reversible, occurs in a wide range of media and is independent ofgas exchange; there is strong indirect evidence that it is independent of metabolic activity. The swelling is largely the result of entry of anions in the cell as the pH falls, in accor- dance with the degree of ionization of haemoglobin and Gibbs-Donnan equilibria. Human erythrocytes show a complex response to the anion uptake, fail to act as perfect osmometers and ‘resist’ swelling in isotonic solutions. Rat erythrocytes differ, and swell to the extent predicted from their anion uptake.

By contrast, the volume of thymic lymphocytes does not normally vary with pH. Small changes in volume are observed in acid niedia containing acetate or phthalate, but no change occurs in albumin solutions used for density gradient analysis.

The results emphasize the need for control of pH as well as tonicity when using erythrocytes to calibrate Coulter Counter apertures, or when performing size range or density distribution analyses of erythrocyte populations.

In recent years the classical morphological description of a cell has been supplemented by measurements of certain physical parameters, such as volume and density. This permits a more precise description of the cell, and points the way to methods which may effect a separation of one type of cell from another. For example, the Coulter Cell Counter is widely used to determine the size range distribution of a cell population (Brecher, Jakobek, Schneider- man, Williams and Schmidt, 1962), and a density distribution may be obtained by centrifugation to equilibrium in an albumin density gradient (Leif and Vinograd, 1964; Shortman, 1968). Techniques have been developed to separate cells by size (Pradier, Holyoke and Sako, 1964; Fulwyler, 1965; Shortman, 1966), by density (Leif and Vinograd, 1964; Shortman, 1968), or by sedimentation rate, a function of both size and density (Mel, Mitchell and Thorell, 1965). In many of these techniques cells are subjected to a wide range of conditions and close control over any factor which could alter the cells’ physical state is an obvious requirement. The tonicity of the medium is a well-known factor which can alter both the volume and density of a cell (Ponder, 1948; Leif and Vinograd, 1964). This report concerns another factor not often recognized as affecting volume and density, namely the pH of the medium.

The red blood cell has been used to check the techniques mentioned above. This report

* This is Publication No. 1134 from the Walter and Eliza Hall Institute.

3 23

324 D. G. Legge atla Ken Shortman

derives from some apparently anomalous results obtained with rat erythrocytes. Coulter Counter size determinations by the procedure of Brecher et al. (1962) showed poor repro- ducibility, and the position of the band of cells in albumin density gradients varied from one albumin preparation to another. This variability was found to result from variations in the pH of the suspension media.

The effect of pH (in the form of a high CO, tension) on erythrocyte volume was noted as early as 1878. Ponder (1948) has reviewed the earlier literature, and later work is considered by Whittam (1964). Erythrocytes increase in volume with decreasing pH, and as a conse- quence of the work of Van Slyke, Wu and McLean (1923) and of Harris and Maizels (1952), an explanation for the swelling can be advanced (see Whittam, 1964, pp. 68 and 72): at pH values above the isoelectric point (6.8) the cells contain haemoglobin in the form of a polyvalent anion together with immobile K+ ions. As the pH is lowered, C1- (or HCO-3) enters the cells in accordance with Gibbs-Donnan equilibria, producing at the isoelectric point haemoglobin with no net charge, together with K+ and Cl-, and at lower pH values haemoglobin as a polyvalent cation, with K+ and more C1- . The net result is an increase in the number of ions in the cell, and the resultant osmotic pressure causes the cells to swell. The distribution of C1- in human red cells over the range 6-7-7-7 was in agreement with this hypothesis. However, volume changes have not been measured with sufficient accuracy to provide an adequate test. In the present report, erythrocytes are studied over the pH range 4.8-9.4. Two points were considered: (I) Whether the preceding hypothesis adequately accounts for the volume and density changes observed over this wide pH range. (2) Whether lymphocytes show a comparable behaviour. Apart from these theoretical questions, the results have direct bearing on all technical procedures listed above.

MATERIALS AND METHODS

Cell vohime determinations. Cell volumes were measured in duplicate at 18-20' C. using a Coulter Model B Electronic Cell Counter with a 50 or a I O O ~ diameter aperture and a Model J Automatic Particle Size Distribution Analyzer. The apertures (freshly cleaned, unsiliconed), were calibrated using a factor obtained from the haematocrit value of red cells, according to the procedures of Brecher et al. (1962), modified as follows: the calibration was performed with rat erythrocytes suspended in the buffered balanced salt solution given below. Cells were first equilibrated with 20 volumes of the solution for 30 minutes, then re-suspended in fresh medium to a haematocrit of about 30 per cent. After the haematocrit determination (in triplicate), samples were further diluted in the same medium for counts and size range distributions. Reproducibility of the calibration factors from day to day was - + 3 per cent S.D.

It was possible that mean cell volumes determined from the haematocrit values would be too high, due to the presence of extracellular fluid in the haematocrit pellet. The volume of extracellular space in the cell sediment was therefore estimated, using '1-labelled albumin to mark the extracellular suspension medium. This value was 2 per cent of the volume of the pellet. The calibration factors were corrected for this small error.

To check the possibility that shape changes could cause errors in the volume estimations,

pH Dependetlt Changes iiz Cell Voltrme 325

the calibration factors were checked using chick erythrocytes, which are larger and more elongated than rat erythrocytes. The calibration factors were unaltered, indicating that volumes were accurately determined, despite changes in size and shape of cells.

In some experiments the volumes of cells were determined in hypotonic media. This involved a change in the conductivity of the media and new calibration factors were therefore determined for each change in tonicity.

Size range distribution data may be expressed in terms of the mean cell volume (Brecher et al., 1962), or the mode or peak of the distribution curve may be used. For the erythrocyte studies, mean cell volumes were used, but results would be unaltered if the modal volumes had been employed. Thymocyte volumes are given as the mode of distribution curves, since slight aggregation of cells in some media complicated mean cell volume determina- tions. All values represented thus refer to the behaviour of the 'typical' cell of the population, the thymic small lymphocyte.

Buoyant density determinations. The buoyant density of human erythrocytes at various pH values was determined by centrifugation to equilibrium at 2300 g in isotonic bovine serum albumin density gradients, using techniques in general similar to those of Leif and Vinograd (1964). Full details of the method will be published elsewhere (Shortman, 1968).

Cell shape and diameter. The erythrocytes in suspension media were observed under oil immersion phase-contrast optics, magnification 1.250-fold. A true three-dimensional im- pression of cell shape was obtained by disturbing the sample sufficiently to produce movement of the cells. Cell diameters were measured using a calibrated eye-piece scale; high precision is not claimed for these measurements. Freshly cleaned and siliconed slides and coverslips were used, to avoid the shape changes induced by glass surfaces (Ponder, 1948).

Saline. 0.147 M-NaCl. Isotonic buffers. These were prepared in each case by mixing isotonic (0.294 osmolar)

solutions of the acid and the base forms of the buffers to give the appropriate pH. Bicfered saline. One volume of the appropriate isotonic buffer was mixed with 9 volumes

of saline. Bujered balanced salt solution. Isotonic solutions were mixed as follows : 0.147 M-NaCl,

121 volumes; 0.147 M-KC~, 3 volumes; 0.098 M-CaC1, 3 volumes; 0.147 M-MgSO,, I

volume; isotonic potassium phosphate buffer, pH 7.4, 2 volumes; isotonic Tris-chloride buffer, pH 7.4, 6 volumes. Erythrocytes and thymocytes suspended in this medium main- tained a constant volume over a period of at least 2 hours at 19O C. The volume of cells in this medium did not differ significantly from the volume in serum of the same pH.

Unbuffered balanced salt solution. This solution was used for preparing albumin solutions. It was prepared by mixing isotonic solutions as follows: 0.147 M-NaCl, 121 volumes; 0.147 M-KC~, 4 volumes; 0.098 M-CaCl,, 3 volumes; 0.147 M-KH,PO~, I volume; 0.147 M - M ~ S O ~ , I volume.

Bovine serum albumin solutions. Bovine plasma albumin (Fraction V, Armour, 20 per cent w/w) was dialysed against several changes of deionized distilled water. Samples were titrated with NaOH solution to provide a range of pH values, and the solutions of sodium salts so obtained were freeze-dried. The albumin samples were then dissolved to make 40 per cent w/ w stock solutions in a medium containing the following components :

(i) Su&cient deionized water to render the solution isotonic with respect to sodium and

3 26 D. G. Legge and Ken Shortman

albumin. This comprised 34.1 ml. HzO per 10 ml. I N-NaOH originally added, together with 5 ml. H,O per IOO g. albumin.

(ii) Sufficient isotonic salt solutions to balance the sodium ion added. This comprised per 10 ml. I N-NaOH; 0.147 M-KC~, 1.13 d.; 0.098 M-CaCI,, 0.85 ml.; 0.147 M-MgSO,, 0.28 ml.; 0.147 M-KH,PO~, 0.28 ml.

(iii) Sufficient unbuffered balanced salt solution to produce a 40 per cent wjw albumin solution.

The stock solution was diluted in unbuffered balanced salt solution to provide the media for density gradient centrifugation (20-38 per cent w/w), and the 10 per cent w/w isotonic albumin solutions for volume determinations. The final pH was checked with a glass elec- trode.

Foetal calfserum. This medium was used as a model of the normal physiological environ- ment. It was obtained from Commonwealth Serum Laboratories. The initial pH (glass electrode) was 7.2-7.3, and this rose to 7.9-8.0 after 8 hours standing at 19" C., presumably due to CO, loss. The pH was 7.4-7.5, when used in the present experiments.

Human erythrocytes. Blood was collected by cubital venepuncture from a single adult donor, spun in a heparinized tube and the buffy coat and plasma rejected. The cells were washed twice with buffered balanced salt solution before buoyant density studies.

Rat erythrocytes. Blood from 9-week-old male rats was spun in heparinized tubes and the plasma and buffy coat rejected. The cells were resuspended in a small volume of foetal calf serum, and maintained at 0-4" C. until dilution in the appropriate medium for size distri- bution studies.

Rat thymocytes (rat thymic lymphocytes). The thymus was removed from 5-week-old male rats and teased with needles into foetal calf serum. After filtration through a stainless steel sieve to remove debris, the cells were spun down, resuspended in a small volume of foetal calf serum and maintained at 0-4" C. until dilution in the appropriate medium.

RESULTS

Swelling of erythrocytes and thymocytes in acid media. The effect of the pH of a variety of isotonic suspension media on the mean cell volume of rat erythrocytes is presented in Fig. I. The following points should be noted:

(i) Rat erythrocytes swell in acid media and the extent of swelling increased from pH 7.6 to 4.8.

(ii) In the alkaline range (pH 7.65, 8.0, 9.4) the volume was relatively constant, and similar to the volume in serum. After a further 90 minutes incubation, the volume was exactly the same as in serum.

(iii) Swelling in acid media was rapid and almost complete within 4 minutes. After a further 90 minutes incubation the volume in all acid media increased by only about per cent.

(iv) Swelling was largely independent of the nature of the medium, acetate, phthalate and phosphate buffers producing roughly similar degrees of swelling at the same pH. In separate experiments saline acidified by small amounts of HC1 produced the same effect, and some swelling was noted in saline alone.

pH Dependent Changes in Cell Volume 327 80 -

L 4J a

2 0 - 10 -

0 - 0 v

-10- ; I I I I

6 7 8 9 P H

FIG. I . The effect of pH on the volume of rat erythrocytes in isotonic media. Cell volume was determined 4 minutes after suspension in the appropriate medium. The results are expressed as the percentage increase in volume above the volume in foetal calf serum. Ranges are given where two or more separate experiments were conducted. The media used were as follows: ., Buffered balanced salt solution; 0, bovine serum albumin buffered balanced salt solution; 0, acetate buffered saline; 6, phthalate buffered saline; A, phosphate buffered saline; v, tris buffered saline; 0 , glycine buffered saline.

(v) In separate experiments, buffered media containing 10-90 per cent serum gave results similar to those in serum-free media.

(vi) The actual mean cell volume of 9-week-old male rat erythrocytes in foetal calf serum, based on values from five animals, was 60+4 (S.D.) c u . ~ . The volume of a single sample in serum varied about 2 per cent over a period of 60 minutes.

z I

I I I 7 8 5 6 7 8

PH -10

5 6

FIG. 2. The effect of pH on the volume of rat thymocytes in isotonic media. See Fig. I for details. Note the change of scale from Fig. I . (a) Volume changes after 4 minutes; (b) volume changes after 90 minutes.

328

Fig. .(a) and 2(b) show the effect of pH on the volume of rat thymocytes. The following findings contrast with the rat erythrocyte results.

(i) Swelling of thymocytes at acid pH was a function of the suspension medium. No significant swelling (based on modal cell volumes) was observed when the buffering anion was albumin or phosphate, nor was there any change in the overall size distribution curve of the small lymphocyte population. Various degrees of swelling were noted with acetate and phthalate.

(ii) The extent of swelling, when it occurred, was much less than that observed in ery- throcytes.

(iii) Swelling, when it occurred, increased with time, the rate depending on the buffering anion.

(iv) The mean modal volume of 5-week-old male rat thymocytes in foetal calf serum based on values from five animals, was 125.5f 2.4 (S.D.) C U . ~ . The volume of a single sample

D. G. Legge and Ken Shortman

TABLE I

THE EFFECT OF PH ON THE SHAPE OF RAT ERYTHROCYTES

Medium

Foetal calf serum Balanced salt

solution Glycine buffered

saline

Phthalate buffered saline

PH

7.5

7.4

9.4

4.8

Mean cell volunre (c1i.p)

61

62

60

9s

Maxitnunr diameter ( p ) Typical appearance

. 6.7

6.7 1 5.7

6.6

I

Smooth biconcave discs Smooth biconcave discs

Crenated biconcave discs and crenated spheres

Smooth, slightly bicon- cave spheres

varied by about 2 per cent on standing in foetal calf serum. Volumes in bovine serum were 2-6 per cent higher.

Changes iti erythrocyte shape. Table I summarizes the changes in erythrocyte shape associated with volume changes. The normal appearance of rat erythrocytes in serum or balanced salt solution of pH 7.4 was the well-known smooth biconcave disc, with a maximum diameter of 6 . 7 ~ . In non-protein containing media at alkaline pH, the typical appearance was ‘crenated’ discs or spheres of reduced diameter but similar volume. In acid media, buffered by phthalate or by albumin, the cells maintained an approximately constant diameter, and the volume increase corresponded to a change towards a more spherical shape, the cells being smooth surfaced and only slightly biconcave.

Changes in erythrocyte density. As a corollary to the volume changes given in Fig. I, it would be predicted that the density of erythrocytes varies with pH. This was tested by equilibrium density gradient centrifugation of erythrocytes in albumin density gradients at various pH values.

Since the experiments were to be carried out over several weeks, a single human donor was used, Leif and Vinograd (1964) having shown that the peak density of an individual’s

pH Deperzdent Changes in Cell Vulurtie 3 29

erythrocytes shows a standard deviation of only +O.OOOS g./cu.cm. over a period of I

month. Table I1 shows that the prediction was confirmed, and the density of erythrocytes decreased with decreasing pH. The extent of the decrease was greater than predicted from the volume changes in buffered saline for the same human erythrocytes, as given in Fig. 3, but erythrocytes appear to show enhanced swelling in the albumin solutions (see Fig. I).

In media of constant pH, the peak density was quite reproducible over several weeks ( ~ 0 . 0 0 0 3 g./cu.cm. S.D.).

T h e reversibifity of erythrocyte swelling. A series of experiments was performed to determine the nature of the pH dependent volume changes. Table 111 illustrates the reversible nature of the swelling. Even after I hour at 19’ C. at acid pH, swelling in acetate or phthalate buffered solutions was almost completely reversed following recovery by centrifugation and resus- pension in serum.

The effect of inhibitors 011 pH induced vohrwie changes. To test the possibility that volume changes were in part related to the binding of respiratory gases by haemoglobin, pH-volume curves were determined for rat erythrocytes equilibrated with air, oxygen, carbon dioxide

TABLE I1

7.0 6.2 5.3 5.3

THE EFFECT OF PH ON THE BUOYANT DENSITY OF HUMAN

ERYTHROCYTES

I I

4 > l . I 4 1.0847+0.0003 (S.D.) 4 1.0690

25 1.0705

Tenryerature YH Density (8. /cu.cni .)

I I

Densities were determined by equilibrium buoyant density centrifugation (see ‘Methods’). The cells spun out of the gradient range at pH 7.0, and isotonic solutions of greater density could not bc prepared.

and carbon monoxide, the latter inhibitor being used to irreversibly block haemoglobin in the form of carboxy-haemoglobin. At all pH values erythrocyte volumes were 2-4 per cent higher in air and oxygen, but all conditions showed a similar response to pH. A variety of metabolic inhibitors were tested for their influence on pH induced swelling. Sodium fluoride ( I O - ~ M), sodium arsenate (IO-’ M), parachloromercuribenzoate ( I O - ~ M) and the carbonic anhydrase inhibitor acetazoleamide ( I O - ~ M), all had little effect on the mean cell volume of rat erythrocytes, at pH 5.1 or at pH 8.0 even after I hour’s exposure. In general, the volume changes seemed independent of gas exchange or metabolic activity.

The internal pH of erythrocytes. To understand the response of erythrocytes to media of different pH, it is important to know whether the internal pH of the cell follows that of the suspension medium. To check this point, I-ml. aliquots of packed rat erythrocytes were equilibrated with various buffered saline solutions, spun down to a firm pellet and lysed by adding I ml. of water, then freezing and thawing. The pH of the lysate was determined with a glass electrode. Previous equilibration of cells with buffered saline of pH 9.4 gave an ‘inter- nal’ pH of 7.9, 7.0 an ‘internal’ pH of 6.8, 6.0 an ‘internal’ pH of 6.0, and 5.1 an ‘internal’

3 3 0

Initial volume in foetal calf seruni

60 minutes in acetate-buffered saline, pH 5.1 60 minutes in acetate-buffcred saline, pH 5.1,

followed by 60 minutes in foetal calf serum

D. G. Legge and Ken Shortman TABLE I11

52.8

89.9

52.9

THE REVERSIBLE SWELLING OF RAT ERYTHROCYTES IN ISOTONIC ACID MEDIA

60 minutes in phthalate-buffered saline, pH 4.8

followed by 60 minutes in foetal calf serum 60 minutes in phthalate-buffered saline, pH 4.8,

Volume (cu.,~) I Experimerrfal corzditiowr

97.8

59.0

Results are means of two experiments.

pH of 5.0. Clearly lowering the external pH produced a corresponding drop in the internaI PH-

The effect o f p H oti the volunze occupied by cell haemoglobin. Volume changes in erythrocytcs could conceivably arise from variations in the actual volume occupied by haemoglobin itself. Two considerations prompted investigation of this possibility. (I) The high concen- tration and extremely close packing of haemoglobin in the cell (see Ponder, 1948), and (2) the pH dependent changes in crystalline haemoglobin, recently reported by Perutz

- 1 Y \ r \

I I I I I

5 6 7 8 9 PH

FIG. 3. The effect of tonicity on the response of human erythrocytes to pH. Each point was determined after 3 0 minutes incubation in the medium. A single donor was used, but the results are the means of two experiments. Volume changes are given as percentage increases above the volume in foetal calf serum (95.5 CU.,~). The osmo- larity of the medium was: 0, 0.29 osmolar, A, 0.24 osmolar; W, 0.21 osmolar; 0, 0.17 osmolar. The responsc of cells in serum to the changes in tonicity (assuming serum to be 0.29 osmolar) was: 0.24 osmolar, 8.2 per cent swelling; 0.21 osmolar, 17.4 per cent swelling; 0.17 osmolar, 24 per cent swelling.

pH Dependent Changes in Cell Volume 331

(1965). To test this possibility, rat erythrocytes (3 ml. of packed cells) were equilibrated at pH 9.4 or at pH 5.1, spun down, resuspended to a total volume of 5 ml. in saline and com- pletely lysed by freezing and thawing four times. The cell haemoglobin, together with the cell ghosts and other macromolecular constitituents which make up about 30 per cent of the cell were then completely sedimented to form a pellet by spinning for 49 hours at 197,000 g in the J rotor of a Spinco Model E ultracentrifuge. The pellets were then compared by volume estimation and by direct weighing. The size of the pellet at pH 9.4 was 6 per cent greater than at pH 5.1; the significance of a difference of this order is questionable. No evidence was obtained for an increase in the volume occupied by the macromolecular constituents of the cell at low pH.

The efect of tonicity on the pH response of human erythrocytes. The volume changes of ery- throcytes with pH resembled in many respects the passive osmotic response suggested by the hypothesis in the introduction. If the two were indeed equivalent, osmotic swelling and pH induced swelling should be additive phenomena. The relation between osmotic and pH induced volume changes of human erythrocytes was investigated by determining the pH- volume curves in media of different tonicity. The results are shown in Fig. 3. The following points are of particular interest.

(i) Human erythrocytes differed from rat erythrocytes in their response to pH in isotonic media (compare Figs. I and 3). At pH 4.8 the extent of swelling was about half that observed with rat cells.

(ii) The degree of swelling with pH varied with the tonicity of the medium, hypotonic conditions usually enhancing the effect. Osmotic swelling and pH induced swelling appeared to be synergic.

(iii) Lysis commenced after a volume increase of 60-65 per cent (compared to the serum volume). Beyond this point, the volume of the remaining cells dropped with decreasing pH. This may have resulted from a prelytic loss of potassium ion, as described by Ponder (1948).

The human erythrocyte as an osmometer. Experiments of the type given in Fig. 3 provided information on the conditions under which red cells will act as osmometers. The response of red cells to media of different tonicity has been extensively studied by various investigators and may be described by Ponder’s equation (1948)

V = R W --I +roo G ) where V = the final volume of the cell relative to a volume of IOO in isotonic media; W = the water content of the cell, assumed to be 65 per cent for the present calculations; T = tonicity, the ratio of the osmolarity of the medium to the osmolarity of serum. R is a factor introduced to measure the deviation of the cell from theoretical predictions; if R = I, the cell behaves as a perfect osmometer.

Some values for the factor R for human erythrocytes at different pH values are given in Table IV. The osmotic behaviour of the cell varied markedly with pH, and the cell acted as a good osmometer only under very restricted conditions. R values both above I (greater swelling than predicted) and below I (less swelling than predicted) were observed.

The anion uptake oferythrocytes. If swelling at low pH was a response to anion uptake, two explanatioix could be advanced for the difference between rat and human erythrocytes and

3 3 2 D. G. Legge and Ken Shortman

between human cells in media of different tonicities. The anion uptake could in all cases be similar, the anomalous volume changes resulting from a wide variation in the osmotic response (R value) of the cells. The results in Table IV made this a credible hypothesis. Alternatively, the cells might differ widely in their anion uptake at the same pH, due to

TABLE IV

THE OSMOTIC BEHAVIOUR OF HUMAN ERYTHROCYTES

Foetal calf serum Glycine buffered saline, pH 9.4 Phosphate buffered saline, pH 7.4 Phosphate buffered saline, pH 6.0

Mediuni

0.77 0.69 0.52 1.04 0.65 0.41 1.74 1.00 1.19 1.12 1.81 1.07

R values

0.251 0.168

variations in the accessibility of ionizable groups within the cell, or to differences in the internal pH. Another point requiring verification was whether the anion uptake was sufficient to account for the considerable volume increases observed under conditions of maximum swelling at low pH.

These questions were examined in the following experiments : Red cells (approximately

TABLE V

THE CHLORIDE UPTAKE OF RAT AND HUMAN BRYTHROCYTES

Erythrocyte species

Rat Human

Osnrolurity

0.294 0.294 0.252

0.168 0.210

I pH rutges

titrated

9.4-4.8 8.0-6.5 8.c-6.5 8.0-6.5 8.0-6.5

Moles H C l per 1014

cells

1.17 0.80 0.81 0.74 0.68

1 Calcvlated

possible sivelling

(%)

68

30 3 0 31

33

Actual swelling

(%)

72 14 19 29 36

Actual swelling is the per cent volume increase over the pH range titrated. The cal- culated possible swelling was derived from the volume of iso-osmolar chloride ion taken up by the cells, as described in the text.

3 ml. packed cell volume) were equilibrated with buffered saline at alkaline pH, and then were washed and suspended in NaCl of the appropriate osmolarity. The cells were then slowly titrated to the required pH (using a glass electrode to follow the pH of the external medium) with HC1 of double the osmolarity of the NaCl solution. This was the equivalent of adding C1- of the same osmolarity, changes in pH alone having no significant effect.

The experiments of Van Slyke et al. (1923) and Harris and Maizels (1952) may be cited

pH Dependent Changes in Cel l Volume 3 3 3 as evidence that the chloride actually entered the cell at low pH. Thus the maximum possible total volume increase of all erythrocytes duc to anion uptake was equivalent to the volume of HCl added. The initial total volume of all erythrocytes was determined from total cell counts and mean cell volume determiiiatioiis at the initial alkaline pH, using the Coulter Counter. The maximum possible swelling due to anion uptake, expressed as a percentage of the initial volume, was then compared to the actual percentage change in mean cell volume, as determined by the Coulter Counter.

The results given in Table V point to the following conclusions. (i) The chloride uptake was sufficient to account for almost all the volume increase ob-

served under conditions of maximum swelling. (ii) Rat erythrocytes in isotonic media increased in volume to the full extent predicted

from their anion uptake; human erythrocytes did not. (iii) The chloride uptake of human erythrocytes did not vary greatly with differences in

tonicity. The only effect noted was, in fact, a small drop in uptake as the tonicity decreased, not the predicted increase. Variations in the degree of swelling thus represented variations in the osmotic response of the cells.

DISCUSSION Theoretical Aspects

This paper presents a set of observations which strictly limits the possible explanations for pH dependent red cell volume changes. The variety of media in which the response was obtained indicates that the changes are a direct consequence of the low pH, and not related to the nature of the buffering anion. The reversibility shows that no irreversible cell damage is involved. The speed of the response and the changes at 4" C. (as shown by the buoyant density experiments) makes it unlikely that prolonged metabolic activity is required. In addition, several inhibitors of glycolysis were without effect. Blocking haemoglobin gas exchange, or inhibiting carbonic anhydrase activity were without effect. Direct volume changes of the macromolecular constituents of the cell seem unlikely to contribute signifi- cantly to the swelling. Swelling at low pH appears to be a passive phenomenon, resembling in many respects osmotic swelling. The shape changes induced by low pH are similar to those described by Ponder (1948) for osmotic swelling.

This strongly suggests that pH-induced swelling is indeed an osmotic response to anion uptake, as suggested in the Introduction. It was evident from other work that this would contribute to the size change, but the contribution of other factors was not excluded. It seems, however, that the C1- uptake of the erythrocyte at low pH is adequate to account for all, or very nearly all, of the approximately 70 per cent swelling possible over the pH range

A problem unresolved by this work is the anomalous behaviour of human erythrocytes, which appear to 'resist' both pH induced and osmotic swelling under certain conditions. While rat erythrocytes respond more nearly as predicted in a transfer of pH from 9.4 to 4.8, it is likely that there are regions in the pH-volume curves where they too appear to 'resist' the predicted volume changes. It seems that while the anion uptake hypothesis is adequate to explain the potential for volume changes, the response of the cell to the osmotic force (as measured by the R values) is a quite complex one. Several explanations have been offered

9.4-4.8.

D HAEM

334 D. G. Legge and Ken Shortman

for deviant R values. The presence of ‘bound’ water, not participating in osmotic phenomena, was an early explanation for low R values; such an explanation cannot apply with values in excess of I. A current explanation depends on the changes in the osmotic activity of haemoglobin with concentration. This must contribute to the complex volume changes, but it is not clear whether the effect is adequate to explain the wide range of R values encountered in this study. The simplest explanation would be a resistance of the cell membrane to shape changes at various stages of the swelling process. These possibilities could now be further explored, making use of the small volume changes detectable with the Coulter Counter.

The changes in thymocyte volume with pH in certain media clearly represent a different phenomenon. The approximately spherical shape of the thymocytes in suspension, repre- senting a shape of maximum volume, eliminates the possibility of a swelling process similar to that of erythrocytes, where a change of shape from a biconcave disc to a sphere gives a large volume increase without stretching the membrane. Swelling cannot be simply corre- lated with low pH, but requires the presence of certain anions. The swelling could be ex- plained by the penetration of the free acid form of acetate, and at a slower rate than that of the larger phthalate molecule, into the cell. Lowering the pH would thus raise the concentration of unchanged acetate or phthalate species, but would not facilitate the penetration of phos- phate or albumin.

Some Practical Implications Brecher et a!. (1962) have described the use of erythrocytes to calibrate the apertures of

Coulter Counters. They noted the swelling of erythrocytes in saline alone, and suggested the use of the salt-dextrose solution of Eagles basal medium (apparently without the 5 per cent CO, gas phase), to prevent this swelling. The present results indicate that the swelling they observed in saline was a consequence of the lower pH of this medium, and that maintenance of relatively constant volume in Eagles medium was the result, not of some particular balance of salts, but of the relative ‘resistance’ of human erythrocytes to volume changes at the alkaline pH. It is evident that close control over both tonicity and pH is required to obtain accurate calibration of apertures and accurate volume estimations. Human erythrocytes compared with rat erythrocytes offer the advantages of increased size together with a reduced susceptibility to volume changes should there be any variation in these factors.

Leif and Vinograd (1964) have established that the peak buoyant density is a highly reproducible parameter of an erythrocyte population, and this was confirmed in the present study. The buoyant density does, however, vary with pH and again control of both tonicity and pH is a prerequisite for reproducible density distribution analyses.

Finally, small lymphocytes from various sources have been found to separate into peaks when subjected to density distribution analyses in albumin gradients at pH 5.1 (Shortman, 1968). This raised the possibility that the density peaks were artifacts, produced by different degrees of swelling of some members of the population at low pH. However, the results presented show that the volume of thymic lymphocytes in albumin, buffered salt solutions at pH 5.1 differed very slightly, if at all, from the volume in serum, and was identical with the volume in a similar balanced salt solution, lacking albumin, and buffered at pH 7.4. Clearly, exposure of thymocytes to albumin solutions of low pH causes no change in the relevant physical parameters of the cell.

pH Dependent Changes in Cell Volume 335 REFERENCES

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