(Received November 9, 1934.)

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32 6I2. II.22 THE MEASUREMENT OF RED CELL VOLUME. VI. The different "fragility" of the red cells of various mammals. By ERIC PONDER. (From the Biological Laboratory, Cold Spring Harbor.

1 32 6I2. II.22 THE MEASUREMENT OF RED CELL VOLUME. VI. The different "fragility" of the red cells of various mammals. By ERIC PONDER. (From the Biological Laboratory, Cold Spring Harbor.) (Received November 9, 193.) THIS paper is concerned with the second of the two principal difficulties which arise in connection with the classical theory of osmotic hsemolysis, viz. that the red cells of different mammils begin to haemolyse in different concentrations of the same electrolyte, e.g. NaCl. This difference in fragility has never been satisfactorily explained. The only suggestions of consequence are (a) that of Brinkman and van Dam [1920], who regard the fragility as dependent on the lecithin/cholesterol ratio in the cell membrane, and (b) the observation that large cells, such as those of man, are more resistant than smaller ones, such as those of the sheep [see Krumbaahr, 1928]. The importance of the lecithin/cholesterol ratio, however, does not appear to be as great as was once thought [Bodansky and Dressler, 1927; Ponder, Saslow and Yeager, 1930; Saslow, 1932], and it will be seen below that the suggestion that fragility is dependent on cell size can be stated much more definitely. As has already been pointed out [Ponder and Robinson, 193], the concentration of NaCl in which the red cells of any animal begin to h.tmolyse (i.e. the fragility, as measured in the usual way) depends on at least four factors: (i) the initial concentration of osmotically active substances in the cell interior, (ii) the amount of "free water" contained in the cell, (iii) the critical volume to which the cell can swell without haemolysing, and (iv) the perfection of the cell as an osmometer, as measured by the value of the constant R. In the following experiments these four variables were measured simultaneously with a view to finding which one of them is the most important in determining the different fragilities of the red cells of different animals.

2 THE MEASUREMENT OF RED CELL VOLUME. 33 I. METHODS. The animals used were the sheep, the ox, the rabbit and man, these being selected because the fragility of their cells in NaCl solutions differ considerably. Defibrinated blood and serum were obtained from each animal, and the volume of the cells when suspended in the undiluted serum was measured diffractometrically after the addition of lecithin [Ponder, 1933 a; Ponder and Robinson, 193]. The serum was then diluted so as to give a series of tonicities differing by 0-02 (the tonicity of the undiluted plasma being considered as 1.0), and to 1 c.c. of the various diluted sera was added 1 drop of the whole blood (weight about 20 mg.). The tonicity in which beginning lysis was observed was selected, lecithin emulsified in the supernatant fluid obtained by gentle centrifuging, and the cell volume determined diffractometrically. This gives the critical volume, VA, as well as the critical tonicity, TA. Finally, the quantity of water contained in the cells was found by weighing a quantity of packed cells before and after drying to constant weight at 600, the proper corrections being made for the extent of packing and for the water content of the serum left between the cells. If Vh is expressed as a percentage of the initial volume of the cells in undiluted serum, and if V is the volume which would be attained at equilibrium in the tonicity TA by a "volumetrically perfect osmometer" containing that amount of water found in the cells in any particular experiment, R = (Vh- 100)/( V - 100), V being expressed, like VA, as a percentage of the initial volume in undiluted serum. Hypotonic serum is used instead of NaCl as the suspension medium because by using it we can dispense with measurements of the depression of freezing-point, the tonicity of the serum, put = 1 0, being presumably the same as that of the red cell interior. Defibrinated blood and serum are used in preference to oxalated blood and plasma so that no leakage factor is introduced at the start [see Ponder and Robinson, 193]. II. RESULTS. Table I gives results for five typical experiments on each kind of cell. In every case brilliant diffraction-patterns were obtained, although good patterns are not always obtainable, especially in the case of ox cells, which are very difficult to turn into spheres whether by the addition of 23-2

3 E. PONDER. TABLE I. TW R Vow &3 VA,s 3 VA p.. Sheep 1 0 62 66-0 08 27-37*0 13 2 0.60 6-1 0*86 28*0 38-137 3 0 6 63*8 07 30*2 1*0 137 0-6 6-0-7 27*0 37*0 137 0-68-0 063 28-0 38*2 138 Ox 1 0.

3 3 E. PONDER. TABLE I. TW R Vow &3 VA,s 3 VA p.. Sheep * *86 28* * *2 1* *0 37* *2 138 Ox *0 Og90 6*0 68* Rabbit 1 0-6* 0* *70 61*0 92* * * 92*0 12 Man *7 O* * 0* lecithin or by placing them between slide and coverglass. The columns of Table I show (i) the critical tonicity, TA, (ii) the percentage of water present in the cells, W, (iii) the value of R, (iv) the initial volume of the cells, V., when measured in undiluted serum, (v) the critical volume, VA, and (vi) VA expressed as a percentage of VO. Table I shows a number of points. (1) There is considerable variation in the critical tonicity at which lysis occurs in the case of the cells of any one animal, but the order of the average critical tonicities is: sheep, ox, rabbit, man, that for the cells of mani being the smallest. This is the order usually described. (2) There are no important differences in the water contents of the various types of cell. (3) There is considerable variation in the value of R for the cells of any one kind of animal, but the average values are about the same (0-71-0*76) for the four kinds of animal. The average values, as well as some of the individual values, are rather large, but this is in accordance with what Ponder and Robinson have found for cells from defibrinated blood suspended in hypotonic serum from defibrinated blood. Jacobs and Parpart [1933] have pointed out that under certain circumstances there is evidence that the red cells of the rabbit and man lose salts into a hypotonic environment to a greater extent than do those of the ox, but, so far as the above experiments go, all the four types of cell appear to be about equally imperfect osmometers. The loss of salts referred to by Jacobs and Parpart is probably a slow loss which occurs on standing, and not connected with that loss of os-

4 THE MEASUREMENT OF RED CELL VOLUME. 3 motically active substances which determines the value of the constant R. () The critical volume, expressed as a percentage of the initial volume, is smallest for the cells of the sheep, greater for the cells of the ox, still greater for the cells of the rabbit, and greatest for the cells of man (average values: 136, 1, 1 and 162 respectively). There are differences in the critical volume for the cells of different sheep, rabbits, etc., but the differences are not large, and are of the magnitude usually encountered. III. DISCUSSION. It is clear that the principal factor determining the fragility of the red cells of these different mammals is their ability to assume different critical volumes, and that variations in water content and "osmotic perfection," as measured by R, affect the fragility in a secondary manner only. Now, since swelling of the red cell must be accompanied by stretching of its envelope, we can calculate the increase in surface area which has occurred at the moment the critical volume is reached, and the first four columns of Table II show the initial area, the area corresponding to the critical volume, the increase in area, and the extension ratio (stretched area-initial area/initial area): TABLE II. Sheep 1 23 Ox 1 23 Rabbit Man 1 23 Initial area * O * *0 9-0 Extended area * * 80* * Increase A *0 10* *0 1* * * 3* Extension ratio *36 0* * Initial Vol. I-3 27* * * * daivo X2 0X 0*7 0* The value of the extension ratio is least for the sheep cells, greater for the cells of the ox, still greater for those of the rabbit, and greatest for the

5 36 E. PONDER. cells of man (average values: 0-22, 0*30, 0 3, 0.38). At first sight it is difficult to explain this, unless we fall back on the unsatisfactory hypothesis that the structure of the membrane of one kind of cell is different from that of the membrane of another, and that the difference, whatever it may be, results in greater or lesser extensibility. The matter becomes more understandable, however, if we examine the figures for the initial volumes of the cells and for the absolute increases in area which can occur fl2 0, ) 30 B Initial cell volume, us Fig. 1. Line A, data for cells in hypotonic serum; line B, average data for cells in hypotonic NaCl. before rupture (columns and 6 of Table II). Dividing the one by the other gives as the mean value for da/v0, i.e. this ratio is constant to within + 10 p.c., and the result of plotting the increase in area against the initial volume is a very good straight line through the origin, as is shown in Fig. 1, line A. Line B shows the result of plotting mean values obtained some time ago by somewhat different and less satisfactory methods for red cells in hypotonic NaCl instead of hypotonic plasma'; the absolute increases in area are less than in hypotonic plasma, for the 1 A short account of these methods, now superseded, will be found in Ponder [1933 b].

6 THE MEASUREMENT OF RED CELL VOLUME. 37 critical values are smaller [cf. Ponder and Robinson, 193], but the relation is linear, as before. It is interesting to speculate on the meaning of this linear relation. We can think of the cell envelope as a fluid or semi-fluid film, the permeability of which is governed by the properties of a few layers of molecules, altogether about ju thick [Fricke, 192-6]. This layer, of course, is thinner than the morphological envelope as a whole, and may be situated on the surface. Let us stretch the envelope as a whole, and imagine that molecules can be drawn from various regions,of the envelope in order to augment, or even repair, the layer upon which the permeability depends. We shall then have a "reserve" of material available to enter the latter layer when the envelope is stretched, and it is likely that the quantity of such material will be proportional to the mass of the envelope, which again is probably proportional to the cell volume. As the thin layer upon which the permeability depends is stretched, its thickness will accordingly suffer no diminution, for the "reserves" will make good the deficiency caused by the extension. This will go on until the " reserves " are exhausted, after which the thin layer will rupture and lysis will occur, and under such circumstances we will obtain the linear relation between cell volume and maximal increase in cell area which is found. Moreover, during the stretching of the thin layer and its continuous repair by a redistribution of the "reserves," it is not unlikely that the layer should become temporarily permeable to small ions which ordinarily could nrot pass through it, and that this may be connected with the loss of cations into hypotonic solutions with which the earlier papers of this series have been concerned. This hypothesis may appear highly 'speculative, but it is supported by an unpublished observation communicated to me by Drs Fricke and Curtis of this laboratory, viz. that when the red cell area increases in hypotonic solutions, the capacity per unit area is unchanged. If the reasoning upon which our present estimate of the thickness of the semipermeable layer is based is sound, this constitutes unequivocal evidence that the layer is not thinned when it is stretched. In conclusion, it ought to be pointed out that the linear relation between cell volume and maximal possible increase in area is almost certainly true for the red cells of the mammalia in general, for if all the animals for which there are available data are arranged in order of their red cell volumes, they are also arranged in order of the degree of hypotonicity which just causes hemojysis [see Krumbaahr, 1928]. This is very strong evidence of a linear relation between volume and increase in area,

38 E. PONDER. since there is a linear relation between these variables in the case of four animals, two at the ends and two in the middle part of the range. SUMMARY. 1.

7 38 E. PONDER. since there is a linear relation between these variables in the case of four animals, two at the ends and two in the middle part of the range. SUMMARY. 1. The fact that the red cells of different mammals show different fragilities in hypotonic serum is shown to be due to the different types of cell assuming different critical volumes, i.e. swelling to a different extent without losing haemoglobin. Differences in water content and in the extent to which the cells behave as "perfect osmometers" play only a minor part in determining the fragility. 2. In the four types of cell studied (sheep., ox, rabbit and man), the maximum increase in cell area compatible with the integrity of the cell is a linear function of cell volume. This is accounted for by supposing that the thin layer of the envelope upon which permeability depends does not undergo thinning as it is stretched, but that it is augmented during its extension by a transference of materials from neighbouring regions of the cell envelope. REFERENCES. Brinkman, R. and van Dam, E. (1920). Biochem. Z. 108, 2. Bodansky, M. and Dressler, 0. G. (1927). Quart. J. exp. Physiol. 17, 17. Fricke, H. (192-6). J. gen. Physiol. 9, 137. Jacobs, M. H. and Parpart, A. K. (1933). Biol. Bull. Wood's Hole, 6, 12. Krumbaahr, E. B. (1928). Cowdry's Special Cytology, 1, 27. Ponder, E. (1933 a). Quart. J. exp. Physiol. 23, 30. Ponder, E. (1933 b). Cold Spring Ilarbor Symposia on Quantitative Biology, 1, 178. Ponder, E. and Robinson, E. J. (193). J. Physiol. 83, 3. Ponder, E., Saslow, G. and Yeager, J. F. (1930). Biochem. J. 2, Saslow, G. (1932). J. Physiol. 7, 262.

(From Washington Square College, New York University.)

6I2.III.22 THE MEASUREMENT OF RED CELL VOLUME. II. Alterations in cell volume in solutions of various tonicities. BY ERIC PONDER AND GEORGE SASLOW. (From Washington Square College, New York University.)