Volume Measurement' METHUDS Suspending Media

Transcription

1 Alan R. Liss, Inc. Cytometry 4: (1983) The Mutual Effect of Hydrogen Ion Concentration and Osmotic Pressure on the Shape of the Human Erythrocyte as Determined by Light Scattering and by Electronic Cell Volume Measurement' Asher Porath-Furedi Rogoff Department for Biomedical Engineering, The Hebrew University, Hadassah Medical School, Jerusalem, Israel Received for publication February 8, 1983; accepted June 10, The measurement of the fluctuations of the scattered light were the source of information on the flatness of erythrocytes. Data on cell volume, together with the measure of scattered light fluctuations, defined the cell shape. The measurements were repeated in a wide range of osmotic pressures and ph values in order to affect both the hemoglobin and the cytoskeleton. Major volume changes were detected at low osmotic pressures and ph. The erythrocyte volume was determined by electronic volume measurement. The cell flatness is maximal at physiological conditions. Key terms: Cytometry, cell shape, osmotic pressure, hydrogen ion concentration 'l'he erythrocyte has been tor many decades the subject of thorough investigation. Despite the fact that many investigators offered various hypotheses to explain its peculiar shape, and showed that shape deviations can be caused by a number of factors, up to now the erythrocyte shape has not been utilized as a quantitative parameter due to lack of relatively simple instrumentation for quantitative measurements. It can be assumed that the erythrocyte shape is the result of the structure and the behaviour of the cell membrane and of the changes in the concentration and organization of the hemoglobin. The analysis of data on cell shape and volume makes it possible, under given experimental conditions, to determine which cell constituent is affected by which factor. In this communication we present data on the average shape of red blood cells in a suspension as evaluated from the amplitude of the rapid fluctuations of light shining through the system. The fluctuation of light is caused by the movement of the cells in the suspension. These measurements were carried out under various osmotic pressures in a wide range of hydrogen ion concentrations. The corresponding mean cell volume was determined by electronic cell volume measurement. The use of forward scattering measurement as a means for following up disc-sphere transition has been already established (4, 8, 10). METHUDS Suspending Media Sodium chloride suspensions were prepared to cover the range of milliosmo1. The range of the hydrogen ion concentration was 4.7 to 10 ph. The hydrogen ion concentration was set to the desired value by titration with hydrochloric acid and sodium hydroxide, after which the osmotic pressure was measured, corrected if necessary, and the hydrogen ion concentration measured again. The measurements and corrections were repeated until the desired values were achieved. The hydrogen ion concentration was measured before each experiment, Cross-values of the above formed an experimental set consisting of 80 solutions to be used as suspending media. Blood was taken by venipuncture in edetic acid (EDTA), and a complete set of experiments were carried out from the same blood samples. 'Supported by the Rogoff Foundation. Address reprint requests to Dr. Asher Porath-Furedi, Rogoff Department for Biomedical Engineering, The Hebrew University, Hadassah Medical School, P.O.B. 1172, Jerusalem, Israel.

2 264 PORATH-FUREDI Measurement of Cell Flatness by Light Scattering Fifteen microliters whole blood was added to the 4 ml suspending media contents of a cylindrical test tube, at 25 C, placed in the measuring instrument. The blood was added through a light trap. The optical density reading was corrected to a constant value by the addition of up to 2.5 pl whole blood to the suspension. The slight optical density variations were the result of the different ph and osmotic conditions. The addition of a few microliters whole blood did not affect the forward scattering measurements, as it has a very wide optical density plateau. The mean value of the fluctuations of the scattered light was used to indicate cell shape (flatness). The duration of the measurement was 25 s (the time needed for signal averaging); thus, the erythrocytes are affected by the media for the short period of 30 s including about 5 s for the initial adjustment. The measurements were carried out at 563 nm, in an instrument developed especially for that purpose (10). The technique used is based on the fact that suspended erythrocytes, when stirred in a cylindrical tube, move up and down on helical paths. The axis of rotation of these vertical helices is a sinuslike curve instead of a straight line. When flat cells move along the helical path, they align parallel to the test tube s wall, and as a result of the sideward fluctuations of the helix, they appear in the light beam alternatively parallel or transversal. This change of alignment versus the light beam results in fluctuations of light intensity. The rotation of spherical cells is meaningless from the point of view of its effect on light dispersion, and, thus, stirred suspensions of sphered cells do not cause fluctuations in the intensity of light passing through the system. The intensity of light coming through was measured, and the alternating component, i.e., fluctuations, was separated; its mean value was expressed in mv units and used to indicate cell flatness. This was made possible by the finding that the changes in light fluctuations of the system are directly proportional to the change of the osmotic pressure in the hypotonic (210 mos) to the isotonic (340 mos) range at ph 7.4. Within this range, the erythrocyte exhibits the phenomenon of the reversible disc-sphere transition. This technique has been described with slight variations by a number of investigators (4,8, 10). Electronic Volume Measurement Measurements were carried out in an electronic cell volume measuring system built by Grover. The channel number of the mode of the distribution was recorded and used as a measure of average cell volume (3). Three-Dimensional (3D) Models The models were made by mounting along the Y-axis, one after another, thin plastic sheets with the upper edge cut to the shape of a given curve in the X-Z-plane. The empty space between the sheets was filled with plasticine. The model was checked against a 3D graph obtained by computer graphics. RESULTS The Effect of Hydrogen Ion Concentration and Osmotic Pressure on Erythrocyte Volume Experiments were carried out on blood from five healthy donors. Mean values of the cell population represented by channel number (ch #) versus ph and versus osmotic pressure were measured by electrical particle sizing. These values were expressed on the Z-axis (height). The X- and Y-axes represent the ph and osmotic pressure, respectively (Fig. 1). Each point of the topographical model represents the mean value of channel numbers (as calculated from the values measured on five blood samples with each measurement repeated three times) at a given ph versus a given osmotic pressure. The cell volume is seemingly very sensitive to deviations of the hydrogen ion concentration from the normal physiological value in a wide range of osmotic pressure. The normal ph 7.4 seems to counteract the effect of osmotic pressure variations in the mOs range (Fig. 2). At this ph the cells maintain minimal volume and they withstand the effect of changes in osmotic pressure in a wide range. This stabilizing effect of ph is impressive in the topographical presentation (Fig. 1) as it appears as a valleylike depression at all osmotic pressure values. The topographical model also shows that at low ph values the protein cross-linkage in the membrane does not interfere with the cell s swelling. The results presented in Figure 2 fit Ponder s equation for the given ph values and tonicities, excluding the hypertonic range at ph 10 (Table 1). The fits are similar but somewhat better than those cited by Ponder in detail from Hampson and Maizels (9), who used a similarly unbuffered system. The data presented by them for ph 6.5 does not fit Ponder s equation. Cell Flatness Derived From Light-Scattering Measurements Cell flatness is affected both by osmotic pressure and hydrogen ion concentration as it can be seen from the Table 1 Fil of Results Presented in Figure 2 to Ponder s Equation: G 1 Osmotic pressure range (mos) V= RW - - I PH Partial Partial 7.4 No Yes 7.9 Close Yes 10.0 Partial No V represents the cell volume; W, the percentage of cell water; and T, the tonicity. The value of R is in the range of

3 ph AND OSMOTIC PRESSURE ON ERYTHROCYTE SHAPE 265 ch # J topographical presentation (Fig. 3). In order to facilitate a better view of the interaction of the governing factors, graphs of the effect of ph variations (Fig. 4) and graphs of the effect of osmotic pressure variations (Fig. 5), both versus the amplitude of fluctuations, are presented. These two figures are actually sections of the topographic presentation at planes perpendicular to each other, while curves within one graph represent parallel slices. The osmotic pressure and ph values in which the cells are biconcave fit in a closed area (Fig. 6) within which the maximal flatness is found at isotonic pressure and at ph 6. Points where the light scattering was considerably higher than that of the surrounding vicinity form ridges, which are shown by lines within the closed area. DISCUSSION The combination of the data presented here on cell volume with the amplitude of the scattered light fluctuations can be added to information published in previous works on the behaviour of the cytoskeleton and the hemoglobin under given osmotic pressure and ph values (2, 5-7). This combination can be utilized to show the interaction responsible for cell shape under given conditions. The measurement of fluctuations of the scattered light complement data on erythrocyte volume and provide additional information on cell shape. FIG. 1. Topographical presentation of the volume of the majority of cells at varying osmotic pressures and at varying hydrogen ion concentrations, as measured by electrical particle sizing. Channel number (ch #) is proportional to mean cell volume. This stabilizing effect of the hydrogen ion concentration is represented by a valley. rnv FIG 2. The effect of osmotic pressure on the cell volume (cv) at four ph values. The volume-stabilizing effect of ph 7.4 is clearly seen in the mOs range. Each point is the average value for five individuals measured in triplicate. The following cv values correspond to osmotic pressure of 300 mos: ph 6.5, cv 3.529; ph 7.4, cv 2.00%; ph 7.9, cv 4.27%; ph 10.0, cv FIG 3. Topological presentation of fluctuations of the measured light expressed at varying osmotic pressures and varying hydrogen ion concentrations. The flatness of the cells is proportional to the measure of the fluctuations in mv.

4 266 PORATH-FUREDI mv 200 mv m I \ 340dS 160 I I FIG. 4. Sections of the topographical model (Fig. 3) showing the effect of ph variations on the flatness of the erythrocytes at three osmotic pressure values. Points indicate the average of 15 rnwsiirernmts wit.h the standard deviation (samples from five individuals measured in triplicate). Our experiments show that erythrocytes in suspension retain their relative flatness at wide ranges of osmotic pressures at different ph values. Both normal ph (7.4) and isotonic pressure have a stabilizing effect; the biconcave cell shape is not altered significantly if either one of the two normal conditions remains constant. Under normal osmotic pressure, part of the cell population retains its flatness even at ph 10 (13). At this ph value, hypotonic conditions produce flat erythrocytes from the discocyte-echinocyte mixture by forcing water into the echinocytes, but the system withstands inflation and does not sphere. On the other hand, elevated osmotic pressure at ph 10 causes extensive shrinkage. At low ph (4.4) and under hypotonic pressure, the cells are small but capable of retaining their double concave shape up to 380 mos. Above this osmotic pressure they are practically spheres. At low salt concentration, at ph 4.4, the cells volume increases as water penetrates, but the high surface elasticity of the membrane (1) enables it to retain its normal shape. According to Johnson (5) the volume of ghosts is reduced by about 25% when the ph is lowered from 7 to 5, but in the ghosts case no cell content is present to oppose the shrinkage of the membrane. The volume PH FIG. 5. Sections of the topographical model (Fig. 3) showing the effect of osmotic pressure variations on the flatness of the erythrocytes at three ph values. Points indicate the average of at least 15 measurements with the standard deviation (samples from five individuals measured in triplicate). reduction at low ph can be the result of altered mechanical properties of the membrane due to cross-linkage between the spectrin molecules and covalent bond formation between spectrin and transmembrane proteins (5-7). This was shown by a decrease of extractability of spectrin at low ph and by freeze etching, which showed aggregation of protein molecules in a spectrin-depleted membrane. The cross-linkage of the spectrin molecules at low ph value is also accompanied by the linkage of the neighbouring hemoglobin molecules to the inner surface of the cell membrane (12), which is considered to increase further the membrane elasticity. In our experiments, the osmotic pressure was set by selecting the suitable NaCl concentrations, and, thus, along with the effect of the osmotic pressure, the protein molecules were also affected by chosen ionic environment. High osmotic pressure affects the cells-not only by causing water to escape from them-but also by inducing an even more dense packing of the hemoglobin than under normal physiological conditions. At lower than normal osmotic pressures, water penetrates into

5 i PH ph AND OSMOTIC PRESSURE ON ERYTHROCYTE SHAPE mos FIG. 6. Cross-sectional area of the topographical presentation of the light scattering at the height of 160 mv. All the points within this area represent values at which cells are double-concave. The lines within this area represent downward projections of ridges above the level of 160 mv and represent strings of values at which light scattering is prominent and very sensitive to changes both of the ph and of the osmotic prcoourc. Dcvintions from these seemingly critical values brings about immediate cell deformation. the cells reducing the hemoglobin concentration and increasing the charge of the molecules. This elevated charge causes bigger spacing and higher cell volume (2). In the same range of increasing ionic strengths as in our experiments, Johnson detected about 35% volume reduction in cell ghosts, which, undoubtedly, is a result of the reaction of the cytoskeleton. We assume that in our experiments with intact cells, the same effect is present as well as the effect exerted on the hemoglobin by the changing ionic strengths. The elevation of the ph from neutral to ph 9 brings about swelling [as shown also by Ponder (9)] with the biconcave disc shape retained; but with further rise to ph 10, if accompanied by increase of the osmotic pressure, the cell survival decreases markedly, and the surviving cells are echinocytes. Undoubtedly, changes in ph and in ionic concentrations affect all cell constituents, creating, simultaneously, a much more complex situation. This can be best seen from the sharp ridges in the three-dimensional presentation of the light scattering versus ph versus osmotic pressure (Fig. 3). These ridges indicate local shape-stabilizing interactions caused by distinct ph andor osmotic pressure values. The hydrogen ion concentration in the range of normal physiological value is found to act as the chief factor governing shape. The cells can maintain a more or less constant volume and shape despite wide variations of osmotic pressure. This stabilizing effect of the normal ph appears as a valley on the topographical model of cell volume versus ph and osmotic pressures. As both the light scattering and the cell volume measurements summarize a given situation of all the cells of various ages present in an experiment, these methods are to be used as a source for comparative data supplying mean values only. We suggest use of the cell volume, together with light scattering, in order to establish the shape of the erythrocyte as measures within a cell analyser system. Such a system will supply data, not only as a population mean, but also in differentiating between the different subpopulations present. Such information might be used as a research tool supplying information on the effect of chemical and biological substances on the erythrocyte and also for the medical profession as a convenient method in the determination of various kinds of anemias or other disorders affecting the cell population or cell shape. LITERATURE CITED 1. Crandall ED, Critz AM, Osher AS, Keljo OZ, Forster RE: Influence of ph on elastic deformability of the human erythrocyte membrane. Am J Physiol 235:269, Gary-Boho CM, Salomon AK. Properties of hemoglobin solutions in red cells. J Gen Physiol 52:825, Grover NB, Naaman J. Ben-Sasson S. Dolianski F: Electrical siz ing of particles in suspensions Rigid spheroids and red blood cells. Biophys J 12:1099, Hoffman JF: Quantitative study of factors which control shape transformations of human red blood cells of constant volume. Nouv Rev Fr Hematol 12771, Johnson RM, Taylor G, Meyer DB: Shape and volume changes in erythrocyte ghosts and spectrin-actin networks. J Cell Biol85:371, Liu S, Fairbanks CG, Palek J: Spontaneous, reversible protein cross-linking in the human erythrocyte membrane. Temperature and ph dependence. Biochemistry 16:4066, Lux SE, John KM, Ukena TE: Diminished spectrin extraction from ATP-depleted human erythrocytes. Evidence relating spectrin to changes in erythrocyte shape and deformability. J Clin Invest , Oster G, Zlusky R: Shape transformation of erythrocytes determined by light scattering changes associated with relaxation of particle orientation. Biophys J 14:124, Ponder E: Hemolysis and related phenomena. Grune & Stratton, New York, 1971, pp Porath-Furedi A: A novel method for the follow-up of changes in erythrocyte and other particles. Biochim Biophys Acta , Smith BD, La Celle PL: Parallel decrease of erythrocyte membrane deformability and spectrin solubility at low ph. Blood 53:1:15, Wang K, Richards FM: Reaction of dimethyl-3,3'-dithiobispropionimidate with intact human erythrocytes. J Biol Chem 250:6622, Weed RI, Chailley B: Calcium-pH interactions in the production of shape changes in erythrocytes. Nouv Rev Fr Hematol 12775, 1972.