J. Physiol. (1953) I2I,

Transcription

1 470 J. Physiol. (1953) I2I, THE RATE OF SODIUM EXTRUSION FROM HUMAN' ERYTHROCYTES BY E. J. HARRIS* AND T. A. J. PRANKERD From the Department of Biophysics, University College, London (R?eceived 26 January 1953) The experiments described here were carried out to see whether the rate of extrusion of Na from human erythrocytes is affected by their rate of production in the body and by their shape. The latter variable has to be introduced because cells obtained from those pathological conditions (haemolytic anaemias) in which cell turn-over is rapid are often spherocytic, and this change in shape might contribute to altered behaviour. A control for this point has been obtained by examining normal cells in iso- and hypotonic media. Cells from cases of pernicious anaemia were taken as typical of a low rate of turn-over in the body, but it is possible that these cells were abnormal for reasons connected with their distorted shape rather than with their low rate of production. As far as conditions permitted experiments have been made on subjects before and after therapy in order to see whether a change towards a more normal rate of cell production caused the activity of the cells, as measured by the rate of Na excretion, to approach that of cells drawn from healthy individuals. As the validity of any comparison which may be made between the results of successive experiments depends upon the reproducibility of behaviour of normal cells this has also been studied. METHODS Tracer 8Odium effiux Cells were prepared containing a trace of the radio-active isotope "Na. 2-5 ml. (depending upon the age of the "Na preparation) of isotonic NaCl, prepared from irradiated NaHCO3, was added to 5-10 ml. of whole, heparinized blood. The mixture was incubated at for 2 hr except when otherwise stated. A shorter time of incubation could be used without affecting the results, but longer times, as described later, introduced uncertainty on account of deterioration of the cells. The cells were spun down in a graduated tube at 1500 rev/min (14 cm radius) for 10 min. In order to reduce the quantity of radio-active extracellular solution the cells were re-suspended in about 10 ml. of warm isotonic solution (the composition is specified later) and centrifuged again. * In receipt of a grant from the Medical Research Council for scientific assistance.

2 SODIUM EXTRUSION FROM ERYTHROCYTES 471 About 1-5 ml. of cells were added to 100 ml. of the saline medium. The suspensions of cells plus medium were contained in inverted polythene bottles, the normal openings of which were closed by means of rubber bungs; two holes were drilled in the bases so as to permit sampling and the introduction of a tube carrying gas for oxygenation and stirring. The gas used was 95% 02 and 5% CO2. The introduction of the gas kept the cell suspension stirred, but before withdrawing a sample by means of a calibrated syringe pipette, the pipette was filled and emptied several times in order to ensure mixing. 10 ml. samples of the suspension were withdrawn at intervals and spun (4500 rev/min at 14 cm radius for 15 min) in specially made haematocrit tubes having a 0-15 ml. calibrated capillary joined to a wider tube of volume 10 ml. The supernatant fluid was decanted, the upper part of the tube wiped dry and the part of the capillary not occupied by cells was thoroughly rinsed out by the introduction of isotonic glucose solution from a Pasteur pipette. To the measured volume of cells sufficient water to make a total volume of 3 ml. was added in portions by means of a Pasteur pipette in such a way that the haemolysate could all be transferred to a sample tube. Of these 3 ml., a portion of 2 ml. was used for assay of radio-activity, after which the total was washed into a silica flask and ashed by means of redistilled nitric acid. To the dry contents of the flask, 25 ml. water were added, and after allowing time for solution the fluid was used for analysis. This was effected by means of a flame photometer, which was standardized by use of Na + K mixtures of composition similar to those provided by the cells. The accuracy of figures mentioned is probably better than 5 %, but none of the conclusions depend to an important degree upon the exact values; the principal reason for obtaining them was to see whether the cells were maintaining steady K and Na contents during the runs. The cell Na has been corrected for the Na in the trapped extracellular solution. This quantity was estimated by comparing the analyses of samples which had been treated as described, with those of others which had been re-suspended in Na-free isotonic solution and re-centrifuged before ashing. The difference (mean of eight determinations) was 3-4 m.equiv/na in the trapped fluid per 1. cells. This quantity corresponds to the presence of 2-2% extracellular fluid, a figure in agreement with those found by Maizels (1945) and Jackson & Nutt (1951). Any changes taking place in cell Na and 24Na during centrifugation have been assumed to apply proportionately to all samples, so that the ratios used in evaluating the rate constant are not in error. The ph of the suspension was checked at intervals. After cooling to room temperature the value was ; on keeping the sample the ph drifted towards the alkaline side on account of C02 loss, but throughout the course of experiments lasting 4-6 hr the ph of freshly drawn samples remained constant. Exposure of cells to hypotonic media Two methods were used in assessing the effect of hypotonic solutions on the behaviour of red cells: (a) Whole heparinized blood, which had been treated for 2 hr with radio-active Na solution, was divided into two portions, and to one was added water to produce the requisite hypotonicity. The cells were spun off, and then washed with isotonic solution, after which they were again spun off and finally transferred to the soaking solution. The volume of cells recovered from the second spin was taken as a measure of the proportion that had survived haemolysis if any had occurred. This involves making the assumption that restoration to isotonic solution causes the cell volume to revert to its original value. (b) The cells after exposure to 24Na were spun off and washed as usual, but the tonicity of the non-radio-active solution in which they were suspended during the soaking-out period was varied during the run, first by addition of water, and later by addition of sufficient NaCl to restore isotonicity. Assay of radio-activity; errors The 2 ml. samples of haemolysate were assayed by putting them in a Perspex container which fitted beneath an end-window counter tube. The radio-activity of the haemolysate, even at the end of the experiment, was sufficient to reduce the statistical error of the count (in a 2 min period) to less than 3%. A more serious source of error was the measurement of cell volume; this was

3 472 E. J. HARRIS AND T. A. J. PRANKERD made difficult by the presence of variable amounts of fibrous material and buffy coat, and, in addition, in certain experiments debris from haemolysed cells was also mixed with the intact cells. Because of this effect, and possibly also of incomplete homogeneity of the suspension, two samples taken in quick succession would sometimes give cell volumes, and hence radio-activities, differing by up to 3 %, though usually better duplication could be achieved. Solution The composition of the solution used for the soaking-out of the labelled sodium was in m.mole/l., Na 146, K 5, Ca 0-9, Mg 0-9, C1 120, HC , SO4 0-9, HP RESULTS Constancy of behaviour of an individual's cells It was shown by Harris & Maizels (1951) that the exchange of cell sodium could be described in terms of two opposed first-order processes. In the present experiments the cell sodium was labelled by use of a proportion of radio-active sodium, and the sodium in the solution remained nearly free from activity, for 1-5 ml. cells contained about 16,uequiv of the 23Na + 24Na mixture, whereas the 100 ml. solution contains about 1000 times this quantity of inactive sodium. Therefore the loss of activity by the cells will be nearly a simple, first-order, process down to 1% or less of the original radio-activity remaining, provided that the cells all behave in the same way, and that they do not deteriorate during the experiment. A number of runs was made at different dates using the cells from a particular individual's blood. The time course of the content of labelled sodium during exposure to the soaking solution at C has been plotted in Fig. 1, using a logarithmic scale. Most experimental points fall on a single straight line corresponding to a rate constant for Na output of 0-3 hr-1. It therefore appears that the cells from a healthy individual's blood maintain a steady value of outward transfer constant over a period of months. It is important to stress that the results become variable if unequal times of incubation are used; for example, after incubation for 2 hr values of 0-37 and 0-46 hr-1 were obtained for two different bloods, whereas the figures for the same two bloods after incubation for 5 hr were 0-31 and 0-29 hr-1. A much greater diminution occurred after incubation for 15 hr; the rate constant fell to as little as O-lhr-1. The reason for this change is still being sought. This outward rate constant is higher than most of the values found by Harris & Maizels (1951) using a chloride-phosphate medium. Comparison of one sample of cells in the two media showed that the rate constant was greater in the bicarbonate solution. This might be explained by the greater ease with which the ph could be kept at the optimum value (7-4), but some specific influence of bicarbonate is not excluded.

4 SODIUM EXTRUSION FROM ER YTHROCYTES Hours Fig. 1. The time course of the 24Na output from the cells of an individual (I. W. Y.) blood. Temperature The experiments were made on the following dates: Q 17 Sept * 7 August 1952 (the last point is off the line, probably because this sample became contaminated) May ( 19 Feb The figures in brackets are the Na concentrations in cell water at various times. Sodium efflux from naturally abnormal cells If normally active cells were released into a blood containing a high proportion of abnormal, less active, cells it would be expected that the mixture would show a high rate of "Na output at the commencement of their exposure to the tracer-free solution followed by a slower loss when the active cells had exhausted their supply of tracer (by exchanging it for ordinary Na drawn from the solution). The slower loss should ultimately have a rate constant equal to that of the less active cells. Cells from the blood of patients with pernicious anaemia were examined before and after administration of vitamin B12 (Table 1). Before therapy the Na rate constant was about 023 hr-1, and the points (Fig. 2) did not fall exactly on a straight line. A week after giving the vitamin the rate had increased, as had the haemoglobin content of the blood. One patient (Table 1,

5 474 E. J. HARRIS AND T. A. J. PRANKERD line 2) had originally 1*6 million cells/mm3, and after 1 week this increased to 2*28 million, so of the latter at least 30% were new cells. After this time the points found for the time curve of 24Na loss could be fitted by a mixture of about 20% cells with rate constant 1 hr-' and the rest approximating to the original value, 0*23 hr-1. The anaemia of the other patient was more severe (line 1), the initial cell count was 0 9 million/mm3, and after 1 week this rose to 2-8 million. Thus, after a week at least 68 % of his cells were new ones, and 20 E z 0 c 0 Hour; Fig. 2. Output of 24Na from cells from blood of pernicious anaemia patients before and after administration of vitamin B12. * =Patient 0, 0 = patient D (lines 1 and 2, Table 1) in fact the points for the 24Na loss from cells at this time do not give any indication of there being any of the original cells present. It must be stressed that it would require more information about the keeping properties of the cells to be sure that one is justified in interpreting a slight curvature of the logarithmic time plot as showing the presence of certain proportions of cells with given rate constants. Any deterioration of the cells such as has been shown to take place during their exposure to the artificial medium would give

6 SODIUM EXTRUSION FROM ERYTHROCYTES 475 the curve a similar appearance. It will be clear, however, that the rate after therapy is higher than before, even if the exact value or values are not quite certain. In a third example (Table 1, line 3) there was not a marked reticulocyte response, although the haemoglobin increased. The rate constant did not rise immediately after commencement of therapy, but had increased TABLE 1. Diagnosis 1. Pemicious anaemia 2. Pernicious anaemia 3. Pernicious anaemia 4. Resistant haemolytic anaemia 5. Congenital haemolytic icterus 6. Acquired haemolytic anaemia with hypothyroidism 7. Haemolytic anaemia secondary to a reticulosis 8. Leucoerythroblastic anaemia secondary to a reticulosis 9. Ankylosing spondylitis (control experiment) Outward rate constants and permeabilities of erythrocytes for sodium at 37.50, measured before and after therapy applied to the blood donors Treatment after 1st sample Vitamin B.2 Haemolysis Vitamin B12 ± Vitamin B,2 i Splenectomy + + Splenectomy + + Thyroid and + transfusion Cortisone Cortisone Cortisone % reticu- Hb k Date locytes g/loo ml. MCD MCTh (hr-') 27. ii iii or {80 % % xii. 52 0* ii iii xii xi or f63% 0-25 G37% xi * xii ii iii xi ii xii * ix * * ix K (cm hrx * * * ix xi or 4-06 S55 % % 0*43 4. xi ix or 1-86 j73% % xi xi * xi or % % xii Results of experiments which furnished curved plots of log 24Na versus time have alternatively been given as the proportions of two fractions of cells of unequal rate constants, or the single rate constant best fitting the experimental points. Abbreviations: k, transfer constant; K, permeability constant; MCD, mean corpuscular diameter (,u); MCTh, mean corpuscular thickness (,O. by the time the third sample was taken. It is noteworthy that cells from the first two patients taken over a year after beginning vitamin administration had rate constants as high, or higher, than at the time of the second sample, although reticulocytosis had fallen to a normal value. This indicates that the increase in the rate of Na excretion to what seems to be a usual value (as given in Table 2) is not merely confined to the period of rapid cell production, and is more probably associated with presence of normal cells.

7 476 E. J. HARRIS AND T. A. J. PRANKERD Before describing results obtained from the cells of patients with other diseases it is necessary to consider how much of the change of rate which is observed may be a consequence of an altered surface/volume ratio. If a surface which transports a constant flux of Na per unit area encloses unequal volumes of fluid, differing values of the transfer constant per unit volume of fluid will be observed. Therefore, if the relative activities of the surfaces of two cells of unequal dimensions are to be compared, as occurs when an abnormality is alleviated, it is necessary to compare the permeability constants. These are found by multiplying the transfer constant by the ratio (cell fluid volume)/(cell surface area) (cf. the discussion of this point by Harris & Maizels, 1952). It is not easy to obtain a satisfactory figure for the ratio on account of the peculiar shape of the erythrocyte, which in pathological states may become changed from the usual biconcave disk. The figures we had. available were mean cell volume and mean cell thickness. From these an approximation to the surface was calculated from the formula of Emmons (1927). Ponder (1948) states that this gives a value 10-20% too high, but at least these figures should be comparable; a change from biconcave to biconvex disk would not alter the surface area. The fluid content of the cells separated from isotonic media was assumed to be 65 % (Ponder, 1948); actual determinations using both normal and abnormal cells furnished values between 64-5 and 65-5%. The figures for the permeability constant calculated on the assumptions mentioned are included in Table 1, and show that there is a change of outward permeability following response to therapy. The magnitude of the change is about seven times the standard deviation holding between different normal individuals. Examples of alterations in the reverse sense, towards a decreased Na efflux, were found in the therapy of some haemolytic anaemias. Splenectomy in one case and tberapy of associated myxoedema in another led to diminution in the proportion of reticulocytes as well as to reduction of Na rate constant and outward permeability (lines 4 and 6). The patient mentioned in line 4 suffered a relapse, and reticulocytes and rate constant again increased. In the condition described in line 5, a congenital haemolytic icterus treated by splenectomy, the spherocytosis persisted and the high rate constant for Na extrusion remained unaltered in spite of a fall in the reticulocytes. Haemolytic disease brings about a reduction in mean cell life; this was 24 days at the time of the first sample taken from the first patient. Less clear results were obtained following therapy of haemolytic states by administration of cortisone (lines 7 and 8). In a case of haemolytic anaemia secondary to a reticulosis (line 7) the hormone reduced but did not stop haemolysis, and the proportion of reticulocytes fell (Fig. 3 A). The rate constant applying to Na excretion underwent a temporary increase, which is contrary to the previous results, but which could be the result of cortisone exerting an effect of its own upon the cells.

8 SODIUM EXTRUSION FROM ERYTHROCYTES 477 A case of leucoerythroblastic anaemia responded to cortisone by a reticulocytosis (Fig. 3B) and the rate constant of the Na excretion increased just as in the therapy of pernicious anaemia (line 8, Table 1). It will be noticed that so 40 4' ~30 A st sample 3rd sample 2nd sample 10 B Days after commencement of cortisone Fig. 3. The time course of reticulocyte proportion in bloods of haemolytic states after commencement of cortisone therapy. A, haemolytic anaemia (Table 1, line 7); B, leucoerythroblastic anaemia (line 8) W 0-4._- Ix 0-3 E IV Z C 0._ - L.t Hours Fig. 4. Time curves of 24Na content of cells from haemolytic states, with an indication of the range of curves obtained using normal cells. () Congenital haemolytic icterus; 0 Leucoerythroblastic anaemia with cortisone; 0 haemolytic anaemia after relapse; C haemolytic anaemia during cortisone therapy. the rate constants for Na output from cells taken from these haemolytic states before therapy were invariably higher than the usual; this is illustrated in the activity-time curves of Fig. 4.

9 478 E. J. HARRIS AND T. A. J. PRANKERD A number of runs was made with normal cells to test the possible influence of cortisone. Three pairs of comparisons were made between cells with and without cortisone (adding 1 mg/100 ml. of medium); these yielded indefinite answers to the question. The plots obtained in presence of the hormone were more linear than the controls, as if deterioration of the cells was prevented. A similar effect could be produced by the addition of a proportion of plasma to the soaking solution. A test of the effect of administration of cortisone was made by drawing blood from a patient with ankylosing spondylitis before and following cortisone administration (Table 1, line 9). This showed little change, and it must be concluded that cortisone does not produce significant effects on normal erythrocytes. TABLE 2. Values of rate constant (k) and outward permeability constant (K) for bloods from healthy persons and pathological conditions not all known to alter blood behaviour. Temperature C. k K Condition (hr-1) (cm hr-1 x 10-5) 1. Healthy (Fig. 1) Healthy Healthy Healthy Healthy Atypical pneumonia, cells normal Acute haemorrhagic anaemia Disseminated sclerosis: Before vitamin B After vitamin B, Refractory hypochromic anaemia Polycythaemia (Fig. 6) Behave as a mixture of 75% % Polycythaemia Mean for cells from five healthy individuals 0 30±0 04 hr-1. Other results obtained using either normal or abnormal cells The rate constant of Na excretion from cells drawn from a number of individuals, either healthy or in pathological conditions, are summarized in Table 2. The rate constant for normal cells found under our experimental conditions is between 0O26 and 0-35 hr-', with a corresponding variation in the outward permeability constant. Cells from a number of pathological conditions also give values which fall within this range; one polycythaemic blood furnished the lower curve of Fig. 6, and probably corresponded to a mixture of cells of differing behaviour. This experiment is discussed again later. A blood with a relatively high reticulocyte content (10%) resulting from gastro-intestinal haemorrhage (Table 2, line 7) did not provide cells with a high extrusion constant, showing that reticulocytosis, when leading to production of cells of normal life span, is not associated with a high rate constant.

10 SODIUM EXTRUSION FROM ERYTHROCYTES 479 Attempts to alter the properties of originally normal cells Before describing the results of rate-constant measurements made on cells which survived haemolysis some results will be given which show that exposure to hypotonic media does not seriously influence the rate of Na output. The experiments were made in two ways (see Methods). In (a) the cells were exposed temporarily to hypotonic solution before making the experiment in isotonic solution and in (b) the tonicity of the solution was changed during the soaking-out period. Procedure (a) induced little, if any, difference in behaviour towards Na; thus cells after having been exposed to a mixture of isotonic solution (1 part) plus water (0x8 part) and then restored to isotonic solution, had a rate constant 0-28 hr-1 compared with 0 30 hr-1 for untreated cells; in another comparison between cells pre-treated in a mixture of isotonic solution (1 part) plus water (0.5 part) and untreated cells the respective figures were 0-26 and 0-27 hr-1. An experiment in which the procedure (b) was applied is illustrated in Fig. 5, together with the control. After 80 min soaking-out under isotonic conditions the fluid in one vessel was diluted to 1-4 x original volume with warm water. The volume of cells recovered from an aliquot of the suspension, with allowance for the dilution, permitted estimation of the degree of swelling. This came to about 1-3 x original volume (by calculation a perfect osmometer wyith 30% by volume solids would swell by [(70 x 1-4) + 30]/100 = 1-28 times). The swelling brought about a reduction of cell K concentration, but not of the Na concentration, which in fact increased during the remainder of the period. The rate constant for Na output fell from 0 30 to 0-22 hr-1. This diminution corresponds rather closely to the reduction in surface/volume ratio caused by swelling. The surface area in fact changes but little, if at all (Ponder, 1948), but the volume of cell water becomes 1-4 times greater (assuming a perfect osmometer). If Na output per unit area is constant, then swelling will reduce the rate constant per unit volume of cell water by a factor of 1-4; the factor found experimentally is 0 3/0-22 = 1F36. The next operation in the experiment, namely, restoration of isotonic conditions, appears, within the limits of error of observation, to restore the rate constant to its original value. A similar experiment was made on a different sample of cells in which the fluid was diluted by a factor of The cells swelled to 1x18 x original volume (calculated [(70 x 1-28) + 30]/100 = 1-20) and the rate constant fell from 0-27 to 0-24 hr-1. If output per unit area remained constant the swelling would reduce the rate constant per unit volume of cell water by 1x28 times; experimentally, the ratio 0.27/0.24 yields 1x13. Attention must be drawn to the fact that in these experiments a considerable volume of water enters the cells at the time hypotonicity is induced, and it

11 480 E. J. HARRIS AND T. A. J. PRANKERD leaves again when NaCl is added to restore isotonicity. Movement of this water does not have any obvious influence on the Na transfer. In the experiments just mentioned, hypotonicity was maintained for only about an hour, and it might be objected that insufficient observations had been taken to establish that the rate constant had become reduced to a new steady value. Accordingly a pair of runs each lasting 5 hr was made on a batch [126]K I IK 09 [18-1] Na 0*8- Water added 07-07[131]K 0-6 [136] Na [91] K \ 13-2] Na NaCI added E z j K[133]\ Na [10.9] I I I I I Hours Fig Na output from cells. The control line is obtained from one of the experiments of Fig. 1, and the experimental points refer to a sample of the same cells to which water was added (at 1 hr 20 min) sufficient to increase the volume of fluid by a factor of 1-4 and subsequently NaCl was added (at 3 hr 8 min) to restore isotonicity. The figures for K and Na are the concentrations in cell water at various times. of cells, one run being made as usual, and the other in hypotonic solution (volume 1-39 x original isotonic solution). The rate constants in the two runs were well maintained, the one in the isotonic solution being 0-25 hr-1 and in hypotonic solution hr-1 (ratio 1-35). The effect of previous haemolysis of part of the cells upon the rate of Na excretion from the remainder was studied. In certain runs during which haemolysis of some cells occurred (for no known reason) it was found that the

12 SODIUM EXTRUSION FROM ERYTHROCYTES 481 content of 24Na did not continue to diminish according to the logarithmic law, i.e. the rate constant applying to cells more resistant to haemolysis appears to be lower than that applying to the more fragile cells when a mixture is present. This conclusion has been confirmed by direct experiment. A sample of cells from a healthy individual was examined both directly before and after removal of about half the cells by hypotonic haemolysis. The time course of the Na extrusion was the same in each case, only the last pair of points (at 4 hr) [ 3] 1 [24] [ > \4t! 07~~~\[241] 0/ 0~~~~~~~~~~~6~~~~ 1 0*5 ~~~~~~~[45]I9 o oeh [41] 0-2 -[27.5] Hours Fig. 6. The time course of the 24Na output from untreated cells (from a case of polycythaemia) with curvature indicative of heterogeneity; and from cells surviving hypotonic haemolysis of 38% of the total cells. showing a slight divergence; presumably all the cells in the sample had nearly equal rates of Na extrusion. This, however, is not so if the blood does not behave in a homogeneous way, i.e. the "Na does not diminish according to the logarithmic law. For instance with blood from the case of polycythaemia, removal by haemolysis of 38 % of the cells has a marked effect, as is shown in the two curves of Fig. 6. The cells surviving haemolysis seem to be more uniform, and the rate constant is lower than that applying even at the latter PH. CXXI. 31

13 482 E. J. HARRIS AND T. A. J. PRANKERD end of the run made on untreated cells. The interpretation placed on this finding is that the more active cells are more sensitive to the hypotonicity than are the less active ones. It is known that partial hypotonic haemolysis preferentially removes young cells. Cruz, Hahn, Bale & Balfour (1941) and Stewart, Stewart, Izzo & Young (1950) reached this conclusion by studying dogs' cells labelled with radioactive Fe. We examined the relative fragility of reticulocytes by plotting the fragility curve of cells from a patient with a haemolytic disorder and determining the proportion of reticulocytes at each degree of haemolysis (Fig. 7). It appears that in the early stages the haemolysis is virtually restricted to the, o - % haemolysis 10 % reticulocytes * Tonicity, % NaCI Fig. 7. The degree of haemolysis and proportions of reticulocytes in cells exposed to solutions of diminishing tonicity. reticulocytes. Haden (1934), studying the relation between shape and fragility, showed that the more spherical cells, as are found in haemolytic anaemia, are the more fragile, apparently because less water can be accommodated before bursting point is reached. It therefore appears likely that the more active cells, which are also the more fragile, are the younger cells in the population. One might expect that in pathological states with reticulocytosis the extrusion constant should invariably be raised, but this was not always so, as shown in two cases, one of an haemorrhagic anaemia (Table 2, line 7) with a reticulocytosis of 10% and normal extrusion constant, and a second of a haemolytic anaemia (Table 1, line 7) in which the reticulocytes and the extrusion constant do not change in a parallel manner. It is possible that in these cases other properties of the cells besides their age have been affected. DISCUSSION The results show that the rate constants associated with sodium output from the cells of a healthy individual remain the same over a long period of time, provided experimental conditions are kept constant. Modifications of the

14 SODIUM EXTRUSION FROM ERYTHROCYTES 483 state of the cells and of their turn-over in the body such as are brought about by disease are reflected in changed rate constants. A simple logarithmic law usually describes the time-course of the 24Na content of cells which have previously been exposed for a short time (say 2 hr) to a solution containing the tracer, and which are then transferred to a tracer-free solution. When the time course of the 24Na output does not follow a simple exponential law, there are two possible explanations of behaviour: (a) the cells may have deteriorated during the experiment or (b) the cells may have been heterogeneous at the start, so that at first there is a rapid loss from the active cells and later a slower loss from the less active ones. Our technique biases the observed rate towards the faster if two or more constants apply to fractionof the cells. This is because the limited time of exposure to the tracer solution allows the more active cells to become more fully exchanged than the less active cells. As described, longer times of exposure (e.g hr) lead to a reduction of the observed rate constant of the subsequent loss of "Na. This cannot be explained by the presence of a proportion of less active cells in the original sample because such a mixture would give a complex time course having still a fast component due to the more active cells, whereas the time course found after long incubation still follows a simple exponential law but is slower than that of the sample after short incubation. It therefore appears necessary to suppose that a uniform deterioration of the cells takes place. However, when a curvature of the time course is found in short-term experiments (as in two curves of Fig. 4 and in the control curve of Fig. 6) it seems more reasonable to attribute it to cells of differing activity being present initially. Recently the exchange of Na and K between erythrocytes and plasma to which a proportion of tracer had been added has been studied by Solomon (1952). Cell samples taken at intervals from the radio-active plasma had to be subjected to extensive washing to remove extracellular radio-active material, and this necessitated application of corrections to the cell Na and 24Na on account of the exchange taking place during washing. The rate constant thus found at 370 was hr-1, which is above our normal range. Solomon suggested that the difference between his and earlier figures of Harris & Maizels arises because some Na is inexchangeable, but this explanation does not seem adequate; in our method only exchangeable Na would be replaced by "Na during incubation, so subsequent movement of 24Na from the cells to solution would only relate to 'mobile' Na unless the immobilization occurs after entry of 24Na. The quantities of 24Na in our cell samples after short incubation were close to the quantities expected on the basis of all the cell Na undergoing exchange with rate constant equal to that found in the latter part of the experiments. It seems possible that the rate constants applying to the cells in Solomon's experiments changed during the course 31-2

15 484 E. J. HARRIS AND T. A. J. PRANKERD of the run. The Na flux calculated either from his high rate constant (0x6 hr-1) and low exchangeable Na content (5x2 m.equiv/l. namely, 3-1 m.equiv from each litre of cells, is close to ours derived from a rate constant of about 0 3 hr-1 and higher exchangeable Na content. Comparison of cells from pathological conditions with those from normals shows that the extrusion constant for "Na remains within the same limits in health and in a number of pathological conditions recorded in this paper (Table 2). In the cases of pernicious anaemia an increase of activity occurred during treatment. This increase might be related to the sudden discharge from a rejuvenated marrow of new cells which, compared with their predecessors, are normal morphologically and physiologically. The cells of pernicious anaemia show anisocytosis and may well function abnormally. Under treatment two stages in a return to a normal peripheral blood might be expected. First, one in which the cells are a mixture of original abnormal ones and new normal ones, and secondly, one in wbich all the original cells will have disappeared. These changes are apparent in our cases, but we have no explanation for the high constant in one case during maintenance therapy when the blood should be homogeneously normal. That vitamin B12 is not an important factor directly affecting the activity of mature erythrocytes is shown by the two values of rate constant for Na extrusion (before: 0-31 hr-1; after: 0-38 hr-1) from the cells of a case of disseminated sclerosis without anaemia, treated with this vitamin (Table 2, line 8). In many haemolytic disorders, the extrusion constant is raised above the usual limit, presumably either as a result of humoral factors affecting the cells, or as a consequence of excessive activity of the bone marrow and the high proportion of reticulocytes and young cells discharged into the peripheral blood. The effect of splenectomy producing a remission from haemolysis and a coincident fall in extrusion constant is recorded in one case of acquired haemolytic anaemia (Table 1, line 4). The falling of the constant could have resulted from the removal of a splenic agent from the circulation or a reduction of marrow activity. Later, haemolysis recurred, reticulocytes rose, and the extrusion constant again increased. In a case of congenital haemolytic icterus (Table 1, line 5), splenectomy led to a cessation of haemolysis and an absence of reticulocytes, but no change in cell morphology. The extrusion constant remained raised, and in this case presumably reflected an inherent function of the cells themselves. Cortisone therapy led to a temporary rise in extrusion constant in the cells from a case of secondary haemolytic anaemia (Table 1, line 7) in spite of a diminution of haemolysis. This, it was thought, might be a direct effect of cortisone on the cells, but it is discounted by the fact that when given to a case of ankylosing spondylitis (Table 1, line 9) without anaemia which was regarded as a control, cortisone produced no reticulocytosis and no change in the extrusion constant. In one case of a leucoerythroblastic

16 SODIUM EXTRUSION FROM ERYTHROCYTES 485 anaemia (Table 1, line 8) secondary to a reticulosis, cortisone led to an increase in erythropoiesis, judged by a rise in reticulocytes, and to a simultaneous increase in extrusion constant. The conclusion from these experiments would appear to be that the rate of extrusion of 24Na is related both to erythropoietic activity and to intrinsic properties of the red cells tbemselves. SUMMARY 1. The rate of output of 24Na from human red cells has been measured using a bicarbonate-chloride medium. The rate constants are higher than those previously obtained with use of a chloride-phosphate medium. 2. Cells drawn from a normal individual show constant behaviour over a period of months. 3. Cells from pernicious anaemia cases are caused to become more active upon administration of vitamin B12, whereas cells from haemolytic anaemias (with rapid turn-over of cells in the body) are highly active and may become less so upon treatment. Cortisone administration does not reduce activity, even though it may reduce the rate of cell production. 4. Normal cells can be caused to swell in hypotonic media either before or during observation of sodium output without significant effect upon their rate constant per unit area of cell. The rate constant per unit volume diminishes roughly in inverse proportion to the increase of cell fluid volume. 5. Partial haemolysis in hypotonic solution has little effect upon the rate constant found for the residue of cells when normal cells are used, but does have an effect if abnormal cells are used. It appears that the more actively excreting cells are the more fragile. We have to thank Prof. M. Maizels for having suggested the investigation of the properties of abnormal cells. One of us (E. J. H). is in receipt of an expenses grant from the Government Grants Committee of the Royal Society, and has also to thank the Central Research Fund of London University for providing some of the apparatus. Miss P. A. Wills assisted in some of the experiments. Mr I. W. Young's co-operation in providing a number of blood samples is greatly appreciated. These experiments were carried out on blood drawn for routine pathological investigations. We wish to thank the Physicians of University College Hospital for permission to study their cases. REFERENCES CRuz, W. O., HAHN, P. F., BALE, W. F. & BALFOUR, W. M. (1941). The effect of age on the susceptibility of the erythrocyte to hypotonic saline solutions. Amer. J. med. Sci. 202, EMMONS, W. F. (1927). The interrelation of number, volume, diameter and area of mammalian erythrocytes. J. Physiol. 64, HADEN, R. L. (1934). Mechanism of the increased fragility of the erythrocyte in congenital haemolytic jaundice. Amer. J. med. Sci. 188, HARRIS, E. J. & MAIZELS, M. (1951). The permeability of human erythrocytes to sodium. J. Physiol. 113,

17 486 E. J. HARRIS AND T. A. J. PRANKERD HARRIS, E. J. & MAIIELS, M. (1952). The distribution of ions in suspensions of human erythrocytes. J. Phy8iol. 118, JACKSON, D. M. & NUTT, M. E. (1951). Intercellular plasma and its effect on absolute red cell volume determination. J. Physiol. 115, MAIZELS, M. (1945). Intercellular plasma in the centrifuged erythrocytes of normal human blood. Quart. J. exp. Physiol. 33, PONDER, E. (1948). Haemolysis and Related Phenomena. London: Churchill. SOLOMON, A. K. (1952). The permeability of the human erythrocyte to sodium and potassium. J. gen. Physiol. 36, STEWART, W. B., STEWART, J. M., Izzo, M. J. & YOUNG, L. E. (1950). Age as affecting the osmotic and mechanical fragility of dog erythrocytes tagged with radioactive iron. J. exp. Med. 91,