Mammalian Red Cells Research Articles

Temperature Coefficients of Hemolysis of a Few Types of Nucleated Erythrocytes.

Jacobs et al. have presented a series of data giving temperature coefficients for the hemolysis of various types of mammalian erythrocytes in non-electrolytes. These data have recently been utilized in an attempt to relate species differences in permeability to possible chemical differences in the cell membrane (Dziemian and Ballentine) Because of their possible use in future studies, and for comparative purposes, the data in Table I are presented giving temperature coefficients of hemolysis of nucleated erythrocytes of several species. The technic employed in these experiments was essentially that used by Jacobs et al. In general, the temperature coefficients of hemolysis for these nucleated erythrocytes are of the same order of magnitude as those obtained using mammalian red cells. These data are too fragmentary to justify a detailed analysis. However, certain comparisons can well be made with data described by Jacobs and Glassman. These authors point out that in fishes (elasmobranchs and teleosts) the permeability to ethylene glycol is high, in birds the permeability to ethylene glycol and glycerol is very great and nearly equal, and much less to urea, except in the duck and chicken, and permeability to urea is great in reptiles. The present data are in complete agreement. Ethylene glycol permeability is high in the four fishes studied, urea and glycerol permeability is high in the chicken, and the turtle exhibits a very high permeability to urea.

Swelling and shrinking of mammalian red cells: departures from ideal osmotic behaviour

The first page of this article is displayed as the abstract.

THE PARACRYSTALLINE STATE OF THE RAT RED CELL

When washed rat red cells are kept in 3 per cent sodium citrate at low temperatures (4-9 degrees C.), their resistance to osmotic hemolysis increases so that after several days they swell very little in hypotonic solutions (R = 0.15 to zero) and do not hemolyze even in distilled water. In this and in other respects they behave as if they were gelated or paracrystalline. The paracrystalline state is reversible, disappearing when the cells are warmed and rapidly reappearing when they are cooled, and the resistance to hypotonic hemolysis is not due to the cells reaching equilibrium with their environment by losing so much K that the concentrations become equal inside and out. The concentration of K remains about 25 times as great inside the cell as outside it in a hypotonic medium of T = 0.1, and the failure to swell and to hemolyze seems to be due to the activity of K in the interior of the paracrystalline cell approaching zero. The paracrystalline red cells are more resistant to saponin and digitonin hemolysis, and do not undergo the usual shape transformations, probably because they are too rigid. Hemolysis by saponin and similar lysins occurs without sphere formation, and after lysis is complete a granular debris is left behind. The paracrystalline cells show a diffuse birefringence with polarized light; on their being warmed, the birefringence disappears except at foci which are usually situated along the rim of the cell. The occurrence of the paracrystalline state accounts for the different amounts of swelling of red cells which have been observed in systems of the same degree of hypotonicity, and its relation to other metastable states of the red cell is discussed in connection with a tabulation of the metastable states of the mammalian red cell and their relation to one another. Changes in a membrane alone seem inadequate to account for the varied phenomena observed in connection with red cell behavior, the explanation of which appears to require a more detailed knowledge of the molecular architecture of the cell interior.

The Sickling Phenomenon and its Bearing on the Problem of Red Cell Structure

ABSTRACT In considering the various forms of disk-sphere transformation which mammalian red cells undergo, reference has repeatedly been made to the ideas of Norris, Gough, Teitel-Bernard and others who have suggested that the discoidal form of the cell is the result of an equilibrium between two sets of forces, one expansive, and the other (which includes surface tension) conducing to contraction of the surface (Norris, 1882; Gough, 1924; Teitel-Bernard, 1932; Ponder, 1929, 1934, 1936, 1937, 1939, 1941, 1942 a, b, c;Furchgott, 1940a, 19406; Furchgott & Ponder, 1940, 1941). When the disk changes into the sphere, we are dealing with a diminution or disappearance of the ‘expansive force’ which is probably due to a modification or even a destruction of a surface ultrastructure, and in the absence of anything to oppose it, the tension at the cell-fluid interface determines that the cell takes up the spherical form in which the surface is a minimum for the volume. As will be seen below, other forces are probably operative in the same direction. A re-establishment of the ultrastructure, with the ‘expansive forces’ inherent in its molecular arrangement, leads to the transformation of the sphere into the original disk, and the various conditions under which this reversal can be brought about have already been described. We have now to consider a situation in which the ‘expansive forces’ are so great, or the forces resisting them so small, that the red cell assumes a series of shapes which are characterized by extension, just as the spherical form is characterized by contraction.

MAMMALIAN RED CELLS AS A SOURCE OF "SMALL PARTICLES"

1. Small particles essentially similar to those previously isolated from other tissues have been isolated from mammalian red blood cells (horse blood). 2. About one-third of the dry weight of the particles is lipids. 3. The particles produce hemolysins against the homologous erythrocytes when inoculated into a foreign species. 4. The fact that the particles can be isolated from mammalian red cells which do not contain visible granules is taken to indicate that some at least of the particles isolated from whole organs represent disintegrated "stroma."

Quantitative Aspects of the Disk-Sphere Transformation Produced by Lecithin

ABSTRACT This paper is concerned with quantitative aspects of the disk-sphere transformation of the mammalian red cell produced by lecithin (Ponder, 1935 a). This shape transformation, brought about when lecithin is added to mammalian red cells in saline or in plasma, and reversed by washing off the lecithin or by adding an excess of plasma, is very suitable for quantitative study, since, unlike the shape transformations produced by rose bengal and other lysins, it is not soon followed by haemolysis. Exact work on the factors involved, however, has been very difficult, for two reasons. (1) An unrelated disk-sphere transformation occurs when red cells in saline, whether lecithin-treated or not, are placed between a slide and a closely applied cover-glass (Ponder, 1929), and so it has not been possible to examine the lecithin shape transformation of the cells in saline except in uncovered preparations or in hanging drops. Even under these circumstances, the two factors responsible for the slide-slip transformation, removal of anti-sphering substance by the glass and diffusion of alkali from the glass (Furchgott, 1940; Furchgott & Ponder, 1940) interfere with the observations. This difficulty can now be avoided by using plastic slides and cover-slips, between which these interfering phenomena do not occur. (2) The original method of adding lecithin to plasma or saline by mechanical emulsification is not satisfactory, as the amount of lipoid emulsified is usually unknown and its physical state is variable. Recently I have used sols of lecithin in saline, in which the dispersion approaches uniformity and a known quantity of lipoid is present.

On Properties of the Red Cell Ghost

ABSTRACT This paper is concerned with certain properties of the mammalian red cell ghost. The principal conclusions are: When red cells are haemolysed by water they increase in volume, changing their shape and becoming spheres. If the degree of hypotonicity is sufficient they haemolyse in the spherical form, but quickly resume the form of disks, even in hypotonic solutions. If the haemolytic systems are rendered isotonic by the addition of salts, the cells shrink temporarily, but finally assume their original volume and shape. It seems that when there are no osmotic forces acting on it, the watery ghosts tend to take up the volume and shape from the red cell from which it was derived. When a red cell haemolyses in a hypotonic medium and becomes a ghost, a considerable amount of haemoglobin is retained, perhaps by adsorption. It is doubtful whether this adsorption could be on a purely surface structure ; to account for the results we might have to postulate an internal structure in addition. After haemolysis by water, red Cells will not form spheres between slide and coverslip, nor by the addition of lecithin. If the watery ghosts are made by precipitation with CO2, no sphering can be observed between slide.and slip, with lecithin, with saponin, or with rose bengal. In the case of the watery ghost, the “form component” seems to be preserved, for the ghosts are biconcave disks; the surface, however, must have been altered, for disk-sphere transformations no longer take place.

The Spherical form of the Mammalian Erythrocyte

ABSTRACT The most suggestive observation which we have regarding the remarkable disk-sphere transformation which occurs when mammalian red cells in saline are enclosed between slide and coverslip is that the velocity and extent of the shape change depends on the distance between the surfaces, and upon whether or not both surfaces are wetted by the suspension medium (Ponder, 1929). Thus the transformation occurs rapidly and completely between two glass surfaces 50μ. apart, but slowly and incompletely between two glass surfaces 1 mm. apart, and paraffining of both surfaces, or the substitution of surfaces unwetted by saline, prevents the shape change completely. That the transformation is due to electrostatic charges on the surfaces, pressure,2 or diffusion of alkali from the glass, has been disproved experimentally (Ponder, 1929, 1936), and the only suggestion which still remains is that the surfaces have an orienting effect, of an obscure nature, on a “liquidcrystal” structure in the red-cell envelope.

The Spherical form of the Mammalian Erythrocyte

ABSTRACTIn a number of papers, I and my collaborators have considered various methods for measuring the volume of mammalian red cells, either in the discoidal 1 or in the spherical form (Ponder & Saslow, 1930a, b, 1931; Macleod & Ponder, 1933; Ponder & Robinson, 1934; Ponder, 1935 a, b). Some of these methods, such as the diffraction method, are applicable to spheres only, and others measure only changes in volume such as occur in hypotonic media. So far as this paper is concerned, the two most important conclusions of the investigations are (a) that the transformation from disk to sphere, whether effected by the addition of lecithin or one of the photodynamic dyes (Ponder, 1936), or by enclosing the cells between two closely opposed surfaces (Ponder, 1928-9), is accompanied by no change in volume, and (b) that at the “critical tonicity” in which haemolysis begins in hypotonic media, the percentage increase in volume is substantially the same irrespective of whether the cells are disks or spheres.

On the Spherical Form of the Mammalian Erythrocyte

ABSTRACT Some time ago I published an account of the change in shape, from disc to sphere, which occurs when mammalian red cells, suspended in saline, are placed between a slide and a closely applied cover-glass (Ponder, 1929). Later I briefly described a similar transformation which occurs in plasma or serum to which small quantities of lecithin have been added, the normally discoidal cells again becoming perfect spheres without change in volume (Ponder, 1933), and recently I have observed a disc-sphere transformation in systems containing red cells and dyes of the fluorescein series. To avoid difficulties associated with the peculiar shape of the mammalian red cell, several observers have worked with the spherical form in lecithin-treated plasma (Ponder, 1933, for finding volumes, 1935, in conductivity studies; Race, 1934, in sedimentation experiments; Fricke and Curtis, 1935, in conductivity and capacity experiments) ; no detailed description of the phenomenon itself, however, has yet appeared, and the disc-sphere transformation which occurs with the dyes of the fluorescein series is altogether new.

THE ELECTRICAL CHARGE OF MAMMALIAN RED BLOOD CELLS

From data on the surface area and electrical mobilities of mammalian red blood cells in M/15 phosphate buffer at pH 7.4, it has been possible, with the help of the Gouy and von Smoluchowski theories, to calculate the net surface charge per cell as well as the charge per unit area. It was found that a single mammalian red cell has a net surface charge ranging from four to fifteen million electrons, depending on the species. No clear relationship between zoological classification and surface charge is apparent. It is suggested that a mechanism exists which is capable of keeping the surface density of net charge constant when comparatively large changes in surface area occur in the anemias.

The mammalian red cell and the properties of haemolytic systems

The mammalian red cell and the properties of haemolytic systems

The Mammalian Red Cell and the Properties of Haemolytic Systems

The author is well prepared to present this subject by virtue of his extensive researches. It is not a book of applied data, and such subjects as the chemistry of hemoglobin and its rôle in respiration, the clinical significance of changed permeability of the red cell, and other subjects have been purposely omitted. The treatment is entirely in an academic manner, and while this textbook contains valuable data its use will be almost confined to reference purposes. The book covers the number, dimensions, shape, structure, chemical composition and metabolism of the mammalian red cells. Permeability and the phenomenon of osmotic hemolysis and the properties of simple hemolytic systems are next discussed. Various forms of hemolysis are considered as well as the fundamental factors governing their behavior. There is an extensive and well selected bibliography for the reader who wishes to go beyond the confines of the book. The facts presented

Rate of Escape of Haemoglobin from the Erythrocyte

The technical problem of finding the rate at which pigment leaves the mammalian red cell was proposed to me by Dr. Hugo Fricke, with the idea that if the rate were known it might be possible to calculate whether the pigment leaves the cell through one or two holes in the membrane, or by diffusing across the membrane generally.The method which I have used consists in taking motion pictures of the haemolysing cells.∗ The best cell to work with is the red cell of man, which is relatively large. A suspension is made by suspending the thrice-washed cells from 4 cc. of blood in 20 cc. of 1% NaCl, for a suspension of this strength, when diluted with equal volumes of a lysin solution, shows from 5 to 15 cells per frame of the film used (35 mm.). Saponin made up in 1.0% NaCl in such a dilution as produces complete lysis in about 3 min. is a convenient lysin. The optical system used should be one of the highest resolution practicable, e. g., an aplanatic condenser of N.A. 1.4 working at 2/3 cone and critically focu...

ELECTROKINETIC PHENOMENA. III

A survey of the published electrophoretic mobilities of certain mammalian red cells reveals that the isoelectric points accorded to these cells are the result of equilibria incidental to red cell destruction. The electrophoretic mobilities of normal washed sheep and human cells have now been studied in 0.85 per cent NaCl solutions from about pH 3.6 to 7.4. All measurements were made within 2 minutes of the preparation of the suspension of red cells. In no case was reversal of sign of charge observed under these conditions. Reversal of sign of charge occurred only after sufficient time had elapsed to permit sufficient adsorption of the products of red cell destruction. There is little change in mobility as the pH of the medium is decreased. Reversal of sign of charge does occur in the presence of normal and immune (anti-sheep) rabbit sera. The isoelectric point determined under these conditions does not appear to be connected specifically with the immune body but is perhaps associated with phenomena incidental to red cell destruction and the presence of serum. The characteristic lowering of mobility by amboceptor occurs, however, from pH 4.0 to pH 7.4. The curves of mobility plotted against pH for normal and for immune sera support the viewpoint that the identity of the isoelectric points for normal and sensitized sheep cells is not primarily concerned with the immune reaction. It is most unlikely that an "albumin" or a "globulin" surface covers red cells with a complete protein film. Although serum protein reacts with red cells in acid solutions, this is not demonstrable for gelatin. The lowering of mobility usually ascribed to anti-sheep rabbit serum may also occur, but to a lesser degree, in normal rabbit serum. This diminution of mobility is not, in the first place, associated with sensitization to hemolysis induced by complement. This supports the view that only a very small part of the red cell surface need be changed in order to obtain complete hemolysis in the presence of complement.

On the Spherical Form of the Mammalian Erythrocyte

ABSTRACT In 1895 Hamburger described a remarkable phenomenon which can be observed in suspensions of mammalian red cells in isotonic, hypotonic, or hypertonic saline and in sugar solutions, but not in serum or plasma. The normally biconcave discoidal cells become perfect spheres, without undergoing any apparent alteration in volume; the addition of serum to the suspension of spherical cells, however, converts the spheres back again into the typical discoidal form. Hamburger has no explanation to offer for the occurrence of this phenomenon, but suggests that it may be due to changes in surface tension. In 1920 Brinkman and van Dam published a study of the phenomenon, in which they record the changes undergone by the red cells of man and of the rabbit when placed in a haemocytometer chamber. They correct Hamburger’s statement in one important respect, for they observe that red cells in saline are not spherical but discoidal, and that they become spherical only when placed in the haemocyto-meter chamber. In becoming spheres they pass through an intermediate form, “the crab-apple form,” which is spherical but finely crenated. Brinkman and van Dam believe that the cause of the phenomenon is that the cells receive an electrostatic charge from the glass when they come into contact with the floor of the chamber. They confirm Hamburger’s statement that the change from disc to sphere is prevented by the presence of serum, and claim that it is the cholesterol contained in the serum which inhibits the change of form. Unaware of these two investigations, Gough in 1924 described the same phenomenon once gain, but fell into Hamburger’s error of believing that the red cells are spherical when suspended in a volume of saline. This error is very easy to make, and both Millar and I have made it also; indeed, as will be seen below, it is impossible to avoid it if an oil immersion objective is used for examining the cells. Gough adds two interesting observations to the previous descriptions: that ammonium oxalate and ammonium chloride act like serum in preventing the cells from becoming spherical in saline, and that the spherical form becomes crenated after a few hours, passing spontaneously into the discoidal form in about 24 hours. He does not give any detailed explanation of the phenomenon. Both Millar and myself (Millar 1925, Ponder 1925) made the same mistake as Gough and Hamburger, in believing that the spherical form is due to the immersion of the cells in saline and that cells are spherical when freely suspended in this medium. On such an assumption, I carried out many measurements of sedimentation velocity without receiving any indication that the assumption was wrong1. It appears, however, on a more detailed examination, that the essential condition under which the spherical form occurs has escaped all the observers who have worked on the subject, and that the phenomenon is much more complex than has hitherto been believed.

ON THE SUPPOSED PARTIAL LIBERATION OF HA; MOGLOBIN FROM THE MAMMALIAN ERYTHROCYTE

In the hæmolysis of mammalian red cells, whether by complement and amboceptor, freezing and thawing, saponin, or hypotonic saline, the amount of hæmoglobin liberated is determined by the number of cells hæmolysed. The cells give up no hæmoglobin until they hæmolyse, and then liberate all or very nearly all of it. There is no evidence that partial liberation of hæmoglobin occurs.

THE CATAPHORETIC VELOCITY OF MAMMALIAN RED BLOOD CELLS

1. No significant change with time (to 24 hours) in the cataphoretic velocity of certain mammalian red cells occurs when the cells are suspended in M/15 phosphate buffer at pH = 7.35. Neither successive washings nor standing effect a change. 2. In M/15 phosphate buffer at pH 7.35 +/- 0.03 the following order of red cell velocity has been obtained. The numbers in parenthesis are micro per second per volt per centimeter. See PDF for Structure The order, though not the absolute values, was the same in buffered isotonic dextrose. Human and rabbit cells showed similar differences when both were studied simultaneously in the serum of either. Under these conditions, there is no apparent relationship between zoological Order and cataphoretic velocity. 3. Cholesterol and quartz adsorb gelatin from dilute solution in the phosphate buffer. Red cells, on the other hand, even after 24 hours contact with gelatin solution, retain their previous velocity. 4. Pregnant and non-pregnant white female humans have the same red cell cataphoretic velocity. (The cells were not agglutinated.) 5. In a series of severe anemias no significant change in cataphoretic velocity was in general apparent, although marked changes in the morphology of the red cells were present.

Hæmolysis by brilliant-green and serum

In earlier papers of this series (1, 2) I have described the rapid hæmolysis which results when small quantities of normal serum are added to systems containing sodium taurocholate or sodium glycocholate as lysins. This obscure phenomenon depends entirely on the order in which the cells, the lysin and the serum are mixed. If the lysin is added to the cells first and the serum added afterwards, the acceleration of hæmolysis occurs in suitable cases, whereas if the serum is mixed with the cells first and the lysin added afterwards, no acceleration,but an inhibition of hæmolysis, is the result. The effect of the addition of serum is similar for all types of mammalian red cells. The component of the serum which is responsible for the acceleration appears to be a protein, for either serum albumin, serum globulin, or hæmoglobin can bring about the effect, although egg albumin and gelatin are in effective. There is also some evidence that the acceleration on the addition of serum occurs with the lysins sodium oleate and potassium oleate, as well as with the bile salts. (3). In 1914 Browning and Mackie (4) described an action which occurs in hæmolytic systems containing red cells, serum, and brilliant-green (tetra-ethyl-diamino-triphenyl- methane sulphate). The addition of small quantities of serum to systems containing red cells sensitised with the dye results in a very rapid hæmolysis; if, however, the serum is mixed either with the dye itself before the cells are added, or with the cells before the dye is added, inhibition of the slow hæmolysis which is produced by the brilliant-green occurs. The acceleration is therefore dependent on the order in which the components of the system are mixed. A further investigation of the problem will be found in a paper by Mackie (5), in which it is shown that the serum component responsible for the acceleration is a protein, and that each of the protein fractions produces an effect. Egg albumin and gelatin do not bring about the acceleration. Dobner's violet, malachite green, ethyl violet, and methyl violet can be subsituted for the brilliant-green.

Changes in the Form of Mammalian Red Cells due to the Presence of a Coverglass

IN 1924, Gough described a remarkable change in the form of mammalian red cells which can be observed in suspensions of these cells in isotonic saline (Gough, A., Biochemical Journal, 18, 202; 1924). The normally discoidal cells become perfect spheres, the volume of which can be shown to be the same as that of the discoidal forms. It has hitherto been believed that it is the immersion of the cells in saline which produces this change of form. This, however, is not the case, as the following experiments show.