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612. III. I7 THE EFFECT OF WASHING UPON THE RESISTANCE OF ERYTHROCYTES TO HYPOTONIC SOLUTIONS. BY GEORGE SASLOW. (From Washington Square College, New York University.

1 612. III. I7 THE EFFECT OF WASHING UPON THE RESISTANCE OF ERYTHROCYTES TO HYPOTONIC SOLUTIONS. BY GEORGE SASLOW. (From Washington Square College, New York University.) THE view that hypotonic haemolysis occurs as a result of simple osmotic exchange of water between an erythrocyte with an ideally semi-permeable membrane and its bathing solution cannot be maintained in face of the fact that the solutions of different substances which are found to initiate hypotonic hsemolysis are of widely differing equivalent concentrations [Ponder and Saslow, 1931]. Of the various explanations which have been suggested for this fact [Ponder and Saslow, 1931], the most promising, from the experimental point of view, seems to be that of Brinkman and van Dam [1920 a]. These workers observed that rabbit erythrocytes washed in Brinkman's "physiologically equilibrated salt solution"1 (isotonic) are more resistant to hypotonic Brinkman's solutions (made by reducing only the concentration of NaCl of the isotonic Brinkman's solution) than unwashed erythrocytes. The hypothesis that the washing fluid removes certain cell constituents (" lecithin")2 which normally keep the resistance to hypotonic solutions low was proposed to account for this result. Such constituents, on being recovered from the washing fluid, should, when added to washed cells, lower their resistance to that of normal (unwashed) cells. "Lecithin" was detected by Brinkman and van Dam in the washing fluid, and lowered the resistance of washed cells. It was concluded, therefore, that lecithin is in fact removed from the cells by the washing fluid (isotonic Brinkman's solution). Cells 1 As described originally [Brinkman and van Dam, 1920a], the isotonic "physiologically equilibrated salt solution" had the following composition: NaCl 0 7 p.c., NaHCO p.c., KCl 0-02 p.c., CaCl2. 6 aq p.c., [H'] = 0 45 x 10-7 N, Ca = ± 30 mg. per litre. It is difficult to maintain the [H'] of this solution (CO2 is used) unless it is more strongly buffered. In a later review of the subject of resistance to hypotonic haemolysis [H am b urge r, 1924], it is stated that the composition of the solution had therefore been changed to the following: NaCl 0-55 p.c., NaHC030-2 p.c., KH2P p.c., Na2HPO. 2 aq p.c., CaCl.. 6 aq p.c., and that exactly the same results were obtained with the wellbuffered solution as with the poorly buffered one. The well-buffered solution was used in the work described in this paper. 2 The word "lecithin" is used in this connection by Brinkman and van Dam between inverted commas as above.

2 WASHING OF ERYTHROCYTES. washed in sucrose solutions, on the other hand, were observed to decrease in resistance to hypotonic Brinkman's solutions, and this decrease appeared, as a result of experiments similar to those above mentioned, to depend upon a washing out of cholesterol from the cells; it was concluded that cholesterol increases the resistance of red cells to hypotonic hwemolysis. Cholesterol and lecithin thus act antagonistically, and the cell resistance depends upon their ratio, increasing with increase in the quotient cholesterol/lecithin. The results of Snapper [1912], who observed that cells washed in isotonic NaCl solutions decrease in resistance, could thus be understood in the light of the effects of changes in the cholesterol/lecithin ratio due to the washing fluid. In another paper, Brinkman and van Dam [1920d] state that mere suspension in a pure isotonic glucose (or similar) solution results in changes in resistance to hypotonic hemolysis, for the reason discussed, i.e. change in the cholesterol/lecithin ratio. The generalization of Brinkman and van Dam was soon extended by them to other more or less similar phenomena. Thus a modification of the usual clinical "fragility test" was introduced, in which the fragility is determined for both washed and unwashed cells. The results are stated to be more in accord with expectation than those of the usual test [Brinkman and van Dam, 1920a, 1920c]. Experiments were carried out also to determine the effects on red cell resistance of lipoid-rich and lipoid-poor diets [1920c], and to determine the relation of the water content of the tissues to the cholesterol/lecithin ratio [1920 c]. The ratio, in short, was considered an important " cell constant" for cell resistance and related phenomena, as water intake. Attempts by other workers to use this generalization have, however, met with considerably less success. For example, Rywosch [1907] showed that the order of resistance of the red cells of various mammals to hypotonic saline is the reverse of the order of resistance to saponin heemolysis; this work has been confirmed by Ponder [1927] and by Yeager [1928]. The generalization of Brinkman and van Dam that the cell resistance to hypotonic haemolysis increases with increase in the cholesterol/lecithin ratio thus implies that the resistance to saponin heemolysis increases as this ratio decreases. Ponder, Saslow and Yeager [1930], however, were unable to find any significant relation between the cholesterol/lecithin ratios of the red cells of various mammals and their order of resistance either to hypotonic saline or to saponin haemolysis. Again, Ponder [1929] could not confirm the conclusion of B rinkma n and va n D am [1920 b] that the assumption of the spherical PH. LXXIV

264 G. SASLOW. form by mammalian erythrocytes in saline solutions depends upon electrostatic charge and cholesterol; neither of these factors seems to have anything to do with the phenomenon.

3 264 G. SASLOW. form by mammalian erythrocytes in saline solutions depends upon electrostatic charge and cholesterol; neither of these factors seems to have anything to do with the phenomenon. Finally, Bodansky and Dressler [1927] report that the relation between red cell volume (or water content) and corpuscular cholesterol content in experimental anaemia is the reverse of that expected on the basis of the generalization under consideration. Every attempt by other workers to extend the generalization of Brinkman and van Dam has resulted in failure. It is thus desirable to reconsider the evidence upon which it is based. This evidence consists almost wholly in the detection of changes of resistance to hypotonic haemolysis produced by suspending or by washing red cells in an isotonic medium other than plasma. METHOD. The method used in the original work of Brinkman and van Dam [1920 a] is incompletely described in their paper, but it has been followed so far as possible. The isotonic and hypotonic solutions necessary are made from the following stock solutions [Hamburger, 1924]: NaHCO3 4 p.c., Na2HPO4. 2 aq. 3*634 p.c., KH2PO p.c., CaCl2. 6 aq. 0 4 p.c., 5 c.c. of the NaHCO3 solution and 10 c.c. of each of the others being used, and a varying number of c.c. of 5 p.c. NaCl added (to produce iso- or hypotonic solutions); finally the whole is made up to 100 c.c. with distilled water. If 11.0 c.c. of 5 p.c. NaCl be added, the isotonic Brinkman's physiologically equilibrated salt solution is obtained; if less than 11*0 c.c., hypotonic Brinkman's solutions are obtained. The composition of the Brinkman's solutions used is then as follows: NaHCO3 0-2 p.c., Na2HPO4. 2 aq p.c., KHI2PO p.c., CaCl2. 6 aq. 0'04 p.c., and NaCl 0.10 to 0*55 p.c. Pure C02 gas is added from a cylinder until a ph of 7.35 ([H-] = 0 45 x 10-7) is reached, the solution being itself alkaline owing to the presence of the phosphates. The ph is determined colorimetrically by the use of standard buffer solutions containing phenol red. The desired ph is easily reached after practice and easily maintained. All the Brinkman's solutions keep well in the refrigerator. The central fact of Brinkman and van Dam's work is that washed red cells show a different resistance to hypotonic haemolysis from that of unwashed cells. To compare the resistance to hypotonic haemolysis of untreated red cells (in their own plasma) with that of cells washed in isotonic Brinkman's solution (or treated in some other way), the following procedure is used. Blood is obtained by cutting the carotids of a rabbit stunned by a blow

WASHING OF ERYTHROCYTES. on the occiput. Potassium oxalate is used in the usual quantity, as anticoagulant. Most of the blood is poured into several graduated 15 c.c. centrifuge tubes and spun for 10 mi.

4 WASHING OF ERYTHROCYTES. on the occiput. Potassium oxalate is used in the usual quantity, as anticoagulant. Most of the blood is poured into several graduated 15 c.c. centrifuge tubes and spun for 10 mi. at 2000 r.p.m. The relative volumes of plasma and of packed cells are read off, and these figures are taken to represent the relative volumes in the whole blood which has not been centrifuged, and is to be used as the source of untreated cells. The supernatant fluid is now removed from each centrifuge tube and replaced by the fluid in which the cells are to be washed (e.g. isotonic Brinkman's fluid or isotonic NaCl). The tube is shaken vigorously, centrifuged again, and the above processes repeated as often as desired. Finally, 2 c.c. of packed cells are removed from the tube by pipette and to them are added as much of the suspension medium to be used (e.g. plasma, or isotonic NaCl, etc.) as is necessary to produce a suspension in which the relative volumes of cells and of suspension medium are the same as in whole blood. A suspension so prepared I shall call for the present suspension B, and the whole blood itself suspension A. To 5 c.c. of each of a series of increasingly hypotonic Brinkman's solutions (NaCl 0-20 to 0 10 p.c.), 4 drops (from a 1 c.c. Ostwald pipette) of suspension A are added; to the tubes of another such series (series B), 4 drops of suspension B (from the same pipette). The tubes are shaken and allowed to stand for 1 hour. At the end of this time each tube is centrifuged for 4 mi. (a convenient and sufficiently long time), the supernatant haemoglobin-coloured fluid poured off, and the percentage of hoemolysis determined colorimetrically with a micro-duboscq colorimeter and a previously prepared standard. The standard consists of 5 c.c. distilled water to which 2 drops of suspension A (or suspension B) have been added; it may, for convenience, be considered a 50 p.c. standard, for, although it is not precisely so, its absolute value is of no consequence, as will appear below. The supernatant fluids of the tubes of series A are matched against the 50 p.c. standard prepared from suspension A, and similarly, in the case of the tubes of series B. Should the percentages of htmolysis in all tubes of series A and series B agree within the experimental errors of the method, the experimental treatment would have produced no change in resistance of the red cells to the hypotonic solutions; should the percentages of hemolysis for series B be systematically lower or higher than for series A, the experimental treatment would have increased or decreased the red cell resistancel. These conclusions are 1 It is assumed that the amount of hoemoglobin liberated is proportional to the number of cells destroyed, and thus that percentages of hxemolysis may be used to measure relative resistance. The validity of this assumption has been demonstrated (Saslow, 1929]

5 266 G. SASLOW. correct, however, only provided that suspensions A and B are really comparable with respect to the number of cells in each. If suspension B, for example, contains fewer cells than suspension A as a result of the various manipulations involved in its preparation, the percentages of haemolysis in the tubes of series B will differ systematically from those of series A, but the differences will not of necessity indicate differences of resistance. A method for deciding upon the comparability of the suspensions A and B consists in matching the two 50 p.c. standards prepared from them against one another. If these agree within the limits of error of the method used to prepare such standards, the two suspensions may be considered comparable. In the experiments described below, all the 50 p.c. standards agreed satisfactorily. The accuracy of the method used to prepare the 50 p.c. standards is investigated by preparing five or six such standards from a particular suspension-2 drops of suspension being added from a 1 c.c. Ostwald pipette to 5 c.c. distilled water in each case. Care must of course be taken that the dropping pipette be held vertically and that the dropping rate be fairly uniform from tube to tube. One of the mixtures so made is arbitrarily assigned the value 50 p.c. and the others matched against this. The contents of the tubes (the cells are completely hwemolysed, and the mixture centrifuged) are matched without converting the liberated haemoglobin to acid haematin. The precision of matching heemoglobin solutions as such is not so high as that of matching acid-haematin solutions [Sa slow, 1929], but, as will appear below, is sufficiently so for these experiments. In two experiments of the type just described, the results, expressed as percentages of heemolysis in terms of tube 1 as 50 p.c., were: Tube,~~~~~~~~ Hamolysis p.c The errors introduced by the matching of haemoglobin solutions as such, and by the use of a drop method, are then generally less than ± 3p.c. haemolysis. Since the differences of resistance due to washing cells were foundby Brinkman and van Dam [1920a] to be of the order of from 10 to 30 p.c. haemolysis, the method used here is sufficiently precise. The differences between the 50 p.c. standards prepared from different suspensions are typically of the magnitude shown below. Suspension A consists of untreated cells in their own plasma; suspension B of cells

6 WASHING OF ER YTHROC YTES. centrifuged in their own plasma, and then resuspended in the proper volume of fresh plasma; suspension C of cells treated like those of B, but resuspended in the proper volume of isotonic Brinkman's solution; suspension D of cells treated like those of B, but resuspended in the proper volume of 1'1 p.c. NaCl: 50 p.c. standards Suspension A B C D Hemolysis p.c (arbitrary) Comparable suspensions can thus be prepared without great difficulty, even after the cells have been subjected to several manipulations. The pipetting of a given volume of packed cells apparently introduces no error of consequence. RESULTS. The possible effect on red cell resistance of centrifuging whole blood for 10 min. at 2000 r.p.m. and resuspending the cells by shaking (without changing the supernatant fluid) was first studied. Suspension A denotes whole blood, suspension B has been prepared as above stated. The suspensions are added to the hypotonic Brinkman's solutions, and the mixtures stand 1 hour, etc. as already described. The hypotonic Brinkman's solutions have the composition already mentioned, and only the p.c. of NaCl varies, as indicated in the table: Hypotonic solutions Haemolysis p.c. suspension _ 267 NaCl p.c. A B It thus seems that centrifuging and shaking do not affect the resistance, as will appear below. This is also true of repeated centrifuging and shaking. The effect of a change in the nature of the suspension medium appears in the next table. Suspension A denotes whole blood; suspension B is prepared by centrifuging whole blood once, adding to 2 c.c. of packed cells the proper volume of isotonic Brinkman's solution, and shaking; suspension C is prepared as B, but the new suspension medium is "isotonic" (1.1 p.c.) NaCl [Ponder and Saslow, 1930]. Four drops of each suspension are added to 5 c.c. of each of a series of hypotonic B rinkman's solutions, and the p.c. hoemolysis is found as usual after 1 hour.

7 268 G. SASLOW. Hypotonic Hemolysis p.c. suspension solutions A NaCl p.c. A B a O * * A change in the suspension medium from plasma to isotonic B rinkman's solution (at ph 7*35 and well buffered) or to isotonic NaCl (at ph 5*8 and not at all buffered) apparently has no effect on the red cell resistance. The cells in this experiment remained in isotonic NaCl or isotonic Brinkman's solution for 40 and 50 min. respectively (at 210C.) before being added to the hypotonic Brinkman's solutions. Since changes in red cell volume are closely related to resistance to hypotonic solutions [Ponder and Saslow, 1931], it appears that the volume changes in such solutions are not dependent in any important way upon the ph of the isotonic suspension medium within the range ph This is observed not only when hypotonic Brinkman's solutions, but also when hypotonic NaCl solutions, are used, the experiments being done in the same way. Hxmolysis p.c. suspension NaCl p.o., A (g. per loog. water) A B C ' ' * * The nature of the isotonic suspension medium (with respect to ph, buffering capacity and balance of salts) does not seem to affect the resistance of rabbit red cells to hypotonic solutions of pure unbuffered NaCl or to hypotonic (balanced and buffered) Brinkman's solutions. In this experiment the cells remained in their suspension medium for from 60 to 90 min. before being added to the hypotonic solutions. Further, one washing in the new suspension medium has no effect on the red cell resistance, as appears in the table below'. Suspension A is as usual whole blood, B denotes whole blood centrifuged once, supernatant 1 Brooks [1925] has also failed to observe any change in the resistance of erythrocytes subjected to one washing in either Ringer-Brinkman's fluid (which is very much the same as Brinkman's fluid) or in 0*9 p.c. NaCl. Thus one washing in 0 9 p.c. or 1*1 p.o. NaCl does not affect the resistance.

8 WASHING OF ERYTHROC YTES. 269 plasma removed, more plasma added, cells resuspended by shaking, washed once for 10 min. in the added plasma, this then removed, and the cells finally resuspended in the proper volume of fresh plasma (the whole process is "washing once"); suspension C is prepared as B, but isotonic B rinkman's solution is used instead of plasma afterthe first centrifuging; suspension D is prepared as C, but isotonic NaCl solution is used instead of the Brinkman solution: Hypotonic solutions NaCl p.c *100 Hoemolysis p.c. suspension, ~ ~ ~~~A A B C D The same results as above are observed if the red cells are washed a second time in the new suspension medium. A, B, C and D have the same meanings as above, but B, C, D have been washed a second time in plasma, isotonic Brinkman's or isotonic NaCl solution respectively. Hypotonic solutions Hvemolysis p.c. suspension NaCl p.c. A B C D 0X * * ' The following typical results are observed when the red cells are washed a third time in the new suspension medium (suspension B was omitted; C and D have the usual meaning): Hypotonic solutions NaCl p.c. 0* Haemolysis p.c. suspension A a D The nature of the isotonic washing fluid in these experiments thus exerts no influence upon the red cell resistance; since each experiment cited has been repeated several times with the same results, it appears 1 The experiments shown in these tables were done on the blood of various animals at different times. The tonicity for zero haemolysis accordingly varies. For the same animal, however, the results are easily reproducible.

9 270 G. SASLOW. fruitless to pursue the investigation further, for cells washed as many as three times, the usual number for many types of work (although often less than three washings are used, but rarely more) in buffered Brinkman's solution (ph 7.35) or in unbuffered NaCl solution (ph ) apparently show negligible changes of resistance to hypotonic solutions, and this is true whether the hypotonic solutions are Brinkman's or pure NaCl, for which the same results are observed. I have thus failed to verify the fact upon which the generalization of Brinkman and van Dam is based. It is obvious, further, that failure to observe any change in the resistance of washed cells to hypotonic solutions implies either (1) that if lecithin or cholesterol is extracted by the washing fluid, neither of these substances affects the resistance, or (2) that if lecithin and cholesterol do affect the cell resistance, they are not extracted from the cells by the washing fluid. It is thus extremely doubtful whether the hypothesis of Brinkman and van Dam has any significant relation to the facts upon which it depends. SUMMARY. I have been unable to confirm the observation of Brinkman and van Dam that washed and untreated rabbit red cells differ in resistance to hypotonic solutions. The resistance of these cells to a given hypotonic solution seems, within limits, independent of the ph, buffering capacity or salt balance of the isotonic washing fluids used to prepare the suspensions for the experiments described. REFERENCES. Bodansky, M. and Dressler, 0. G. (1927). Quart. J. Exp. Phy8iol. 17, 157. Brinkman, R. and van Dam, E. (1920a). Biochem. Z. 108, 35. Brinkman, R. and van Dam, E. (1920b). Biochem. Z. 108, 52. Brinkman, R. and van Dam, E. (1920c). Biochem. Z. 108, 61. Brinkman, R. and van Dam, E. (1920 d). Biochem. Z. 108, 74. Brooks, S. C. (1925). J. Gen. Phy8iol. 7, 587. Hamburger, H. J. (1924). Handb. biol. ArbMet. Abt. iv, Teil 3, p Urban and Schwarzenberg, Berlin and Wien. Ponder, E. (1927). Bio-Chem. J. 21, 56. Ponder, E. (1929). Brit. J. Exp. Biol. 6, 387. Ponder, E., Saslow, G. and Yeager, J. F. (1930). Bio-Chem. J. 24, 805. Ponder, E. and Saslow, G. (1930). J. Phy8iol. 70, 169. Ponder, E. and Saslow, G. (1931). J. Phy8iol. 73, 267. Rywosch, D. (1907). Pftueger8 Arch. 116, 229. Saslow, G. (1929). Quart. J. Exp. Physiol. 19, 330. S napper, J. (1912). Biochem. Z. 43, 266. Yeager, J. F. (1928). J. Gen. Phy8iol. 11, 779.

The Journal of General Physiology

The Journal of General Physiology THE TONICITY-VOLUME RELATIONS FOR SYSTEMS CONTAINING HUMAN RED CELLS AND THE CHLORIDES OF MONOVALENT CATIONS BY ERIC PONDER (From The Nassau Hospital, Mineola, Long Island) (Received for publication, September