Proo. Biophys. molec. Biol., Vol. 55, pp. 85--105,1991. Printed in Great Britain. All rights reserved.

0079-6107/91 $0.00+ .50 ~ 1991 Pergamon Preu plc

AN EPR STUDY ON ERYTHROCYTE DEFORMABILITY SUMIHARENon,* SHIGEHIKOTANIGUCHI* and HrDEO KON~ *Okayama University Dental School, Department of Biochemistry, 2-5-1 Shikata-cho, Okayama City 700, Japan ~fNational Institute of Health, NIDDK, Laboratory of Chemical Physics, Building 2, Room Bl-14, Bethesda, MD 20892, U.S.A.

CONTENTS 85

[. INTRODUCTION

II. THEORY 1. EPR Spectrum of Spin-labeled Erythrocytes 2. Deformation and Orientation of Erythrocytes Under Shear Flow 3. Simulation of the EPR Spectra

III. EXPERIMENTS

89 89 91

1. EPR Instrumentation 2. Red Blood Cell Preparation and Spin-labelin0 IV. APPLICATIONS

1. 2. 3. 4. 5. 6. 7. 8. 9.

86 86 88 88

Effects of Spin-labelino, Diamide Cross-linkino and Glutaraldehyde Fixation Factors Determining the Cell Deformability Time Course of Deformation Recovery Effect of Ca 2 + Ion and Stomatocytogenic Reooents Effect of Cu 2+ Ion Effect of Heinz Body Formation Effect of Hematocrit Effect of Hardened Cells on Deformation of Normal Cells Deformation and Orientation of Erythrocyte Ghosts

V. CONCLUSIONS AND PERSPECTIVES

91 91 93 95 96 97 98 99 I00 I01 104

ACKNOWLEDGEMENTS REFERENCES

105 105

I. I N T R O D U C T I O N The human erythrocyte has the unique cell shape of a biconcave disk and contains hemoglobin at a high concentration of about 22 mmol/cell. The cells circulate to carry oxygen and carbon dioxide between the lungs and other tissues through blood vessels and capillaries. The cell can go through capillaries having a diameter less than the cell dimension because of its unique deformability. Thus, the cell deformability is a very important factor to carry out the function in vivo. When a force is applied in one direction, the cells are known to become elongated by about two times the original diameter at maximum in the direction of the force. The ability of a cell to change its shape in response to an applied force has been termed as whole cell deformability (Mohandas et al., 1980a). In in vivo flow, the stress depends on properties of vessels, hematocrit ( l i t ) , and plasma viscosity, while the whole cell deformability depends on the cell surface area to volume ratio (sly), the intracellular viscosity, and the visco-elasticity of the membrane including the cytoskeletal part (Hochmuth and Waugh, 1987). The cell generates ATP molecules through the glycolytic pathway to maintain the physiological conditions, which probably are optimal for exhibiting the unique deformability. The deformability, thus, must be controlled through biochemical reactions (Chasis and Shohet, 1987). Although the mechanism of the high deformability of red cells has not been fully elucidated yet, interaction of cytoskeletal structure with the lipidbilayer has been suspected as an important factor. 85

86

S. NoJn et

al.

In in vivo situations, the properties of an erythrocyte are known to be affected by nonhematological disorders as well as hematological factors (Chien, 1987). Thus, it is probable that the deformability is also controlled by those factors. Actually, the change in deformability of erythrocytes was observed, for example, during pregnancy (Mitsui et al., 1990), in patients with eczema (EI-Saaiee and Meky, 1986) or diabetes (McMillan et al., 1978), or for canine septic shock (Puranapanda et al., 1987). To clarify the deformability change with respect to some physiological conditions, the first step is to measure the deformability in conditions close to those in blood flow. There are three existing methods to measure the individual cell deformability; microcapillary (Evans and Hochmuth, 1976), rheoscopic (Schmidt-Schrnbein et al., 1973), and ektacytometric methods (Bessis et al., 1980). Since erythrocytes flow in vivo at a hematocrit of 40--45%, the intercellular interaction should occur and may influence the deformation of the cells. However, no convenient methods have yet been established to measure whole cell deformability in physiological hematocrit where the cell-cell interaction takes place. As a measure of the cell deformability, filtration time of cell suspension has been measured. However, it is now generally accepted that the "filterability" is not identical with the deformability (Teitel, 1978; Bull et al., 1986). In 1981, Noji et al. (1981a) developed a new method using the electron paramagnetic resonance (EPR) and spin-labeling method in order to estimate the average of whole cell deformability. Since then, the new EPR method has been applied to investigate various factors affecting cell deformability. In addition to our group, Bitbol and Leterrier (1982) independently developed the same method as we did and investigated properties of red cells in flow (Bitbol and Quemada, 1985; Bitbol et al., 1985). This review focuses on the new EPR method for measuring the red cell deformability and its applications. II. THEORY 1. E P R Spectrum of Spin-labeled Erythrocytes Since Ohnishi and McConnell (1965) first applied the EPR spin-labeling method to biological systems, many investigations have been reported providing information on molecular structures and dynamics of various biopolymers and membranes. In one of the investigations, Hubbell and McConnell (1969) first reported EPR measurements of spinlabeled erythrocytes under a flowing condition. They demonstrated that the cells tend to orientate in the flow direction. They did not show, however, that cells also deform during the flow. When the cells are spin-labeled with 5-doxyl stearic acid (Fig. ld), which penetrates into the membrane, the cell suspension exhibits an EPR spectrum as shown in Fig. 2a (Noji et al., 1981a). When cells flow in a fiat EPR sample cell, in which cells orientate and deform

a

b

flow

t

".

¢

f

I

I

I

O,a-. ~m,o

0 d

o

:.0

FIG. I. Schematic illustrations of (a) random orientations of RBCs at rest, (b) deformation and orientation of cells under shear flow, (c) incorporation of spin-labels (white head) in membrane bilayer, and (d) the fatty acid spin-label 5-doxyl stearic acid. H, external magnetic field.

EPR study

on

erythrocyte deformability

87

I

t

1

3300

3350 Ho I Gauss

I

3400

FIG.2. EPR spectraofspin-labeledRBC observedat the shear stress of0 (......... ) and 10.9( ) Nm- 2. Dcxtranconcentration5%, hematocrit35%.h. the peak-to-troughamplitudeofthe spectrum in the absence of flow;Ah, the maximumheight of the differencespectrum.

(Fig. Ib),the E P R spectrum changes to a spectrum shown in Fig. 2b. In Fig. 2, the maximal spectralchange (Ah) and the peak-to-trough height (h)of the stillspectrum are indicated.The normalized spectral change (Ah/h) is used as an experimental measure of whole cell deformation henceforth in this article.The explanation of the E P R spectral change under flowing condition isas follows.In fattyacid spin-labeling(Fig. Id) used in most of the present study, the unpaired electron of the spin-label is located in the 2pn orbital of the nitroxide moiety. The resonance parameters such as g-factor, hyperfinc coupling constant depend upon the directionof the applied magnetic fieldwith respectto the 2pn orbital.The fattyacid spin-label is known to undergo rapid anisotropic rotation about the chain axis when incorporated in the cell membrane (Fig. lc) (Griffith and Jost, 1976). The motion averaged principal values of hyperfine coupling constants, AII' A±, and g factors, gll' g±' are related to the single crystal parameters, Tzx, Tyy, Tzz, gxx, gyy and gz~ as follows:

All= Tz~,

AI = ( T . + T.)/2,

gl[- gz~,

g± = (gu + gyy)/2.

Furthermore, a rapid wobbling motion of the long axis of the rotating spin label must be taken into account. The wobbling averaged magnetic parameters are: A'°ll----A± + (All- A±)W A~'I = AI + (All--At) (I - W)/2

where W= (l + cos ? + cos 2 ~)/3 and y is the half maximum angle of wobbling. Corresponding expressions hold for the g-factor. The resonant magnetic field H(J, 8) is calculated by the following equations:

H(,], 8) = h[v +.]A (p)]/g(,~)~,, a ( ~ ) = [(A ~'llcos ~)2 + (A~,± sin//)211/2

g(fl) = g,Oll cos 2 fl + gO,± sin 2 fl where J is the nuclear quantum number of 14N in the N - O group; 8, the angle between the external magnetic field and the z axis of the nitroxide group; h, the Planck's constant; v, the microwave frequency; tic, the Bohr magneton. The wobbling angle, ~,,was estimated to be 35 ° from the best fit of an observed spectrum of spin-labeled erythrocytes at rest CNoji et al., 1984). The motion averaged parallel and perpendicular hyperfine coupling constants were ,IN l~tl-S

88

S. NoJi et al.

estimated to be All= 28.82 G and A 1 = 8.43 G. Since the resonant magnetic field depends on the relative orientation of the spin-label with respect to the external magnetic field, the EPR spectral change occurs when the spatial distribution of the spin-labels changes, deviating from the complete randomness, with the cell deformation and orientation during flow. 2. Deformation and Orientation of Erythrocytes Under Shear Flow

It is necessary to model in some way the deformation and orientation of cells in order to predict the EPR spectral change in flow. According to the photomicrographic studies by Goldsmith (1971) and Goldsmith and Marlow (1972), the cells in Poiseuille flow behave as follows: (a) at a shear stress below 0.1 Nm -2, the cells rotate like rigid disks; (b) when the shear stress exceeds 0.1 Nm- 2, they deform to an ellipsoidal shape and orientate themselves with the long axis lying at an angle to the flow direction, and the extent of deformation and orientation increases with the shear stress at least up to 0.3 Nm- 2; (c) the deformation and the orientation of the cells are generally analogous to those of liquid drops with the flat surface of the deformed cell facing the wall of the tube as do the deformed liquid droplets. A similar phenomenon was also observed photomicrographically by using the rheoscope by Fischer et al. (1978b) and similarly by Kon et al. (1983). There are several theoretical studies on red cell deformation. A red cell may be assumed to be a microcapsule that consists of a thin elastic membrane enclosing an incompressible Newtonian viscous fluid. The behavior of the cells in shear flow can be described with a hydrodynamic theory, but the resulting equation must be highly non-linear (Barthes-Biesel, 1980). Thus, no truly satisfactory theory has yet been proposed to describe the flow behavior. In view of this circumstance, we adopted as a first approximation the theory for drop deformation in flow developed by Cox (1969). Keller and Skalak (1982) showed that the tank treading surface motion of an ellipsoid is a key factor determining a steady orientation or flipping of an ellipsoid membrane encapsulating an incompressible Newtonian liquid immersed in a plane Couette flow of another incompressible Newtonian liquid. Since, however, the frequency of such motion is much slower compared to the time scale of EPR (of the order of MHz), we ignore the tank treading motion in our simple treatment and adopt the Cox theory as a first approximation for drop deformation in flow. The description of deformation and orientation derived from the Cox theory can reproduce the observed behaviour of the cells in flow, especially at high shear rates where the deformation of the cells in shear flow is calculated to approach a plateau, as was observed. The measure of deformation used in the Cox theory is the deformation index, D, defined by D = ( L - B ) / ( L + B), where L and B are the lengths of the long and short deformed axes, respectively. The length of the third axis is assumed to remain unchanged and is the same as the radius of the original sphere. D is related to other parameters appearing in the Cox theory by O = 5(193. + 163.)/4(2 + 1) [(193.)2 + (2Or~G) 2] 1/2 3. = ~lolrl,

x = trl~la

where r/o is the drop fluid viscosity; r/, the viscosity of the suspending fluid; tr, the interfacial tension; a, the radius of the drop; and G, the shear rate. The orientation angle, ~, between the longest axis of the cell and the normal to the flow direction is given by ~t = n/4 + (I/2)tan - 1(193. G/2Ox).

In applying the Cox theory (Cox, 1969), we treated 2 and x as adjustable parameters to find the best-fitted EPR simulation spectra, disregarding at first the original physical meanings. 3. Simulation of the E P R Spectra

In order to simulate an EPR spectrum of a given spin-labeled deformed-oriented cell, we assume the shape of the deformed cell to be an ellipsoid having three unequal axial lengths as shown in Fig. 3 (Noji et al., 1984). We further assume that the fatty acid spin-labels are

EPR study on erythrocyte deformability

89

X II Ho

FIG. 3. Schematicmodel for deformation and orientation of a red cellin a two-dimensionalPoiseuille flow. (Left)The shape 0fthe deformed cells is described by an ellipsoid having three different axial lengths a(z), b(y) and c(x). (Right) The cell orientates with the y axis of the ellipsoid parallel to the Y axis of the EPR cell. The orientation angle ct is defined as the one between the X and the z axis. The arrows parallel to the Z axis indicate the parabolic distribution of the flow velocity. incorporated in the red cell membrane with the chain aligned approximately normal to the membrane surface (Fig. lc), and the hydrophilic end of the label molecule located at a point on the surface. The intensity of the E P R signal due to the spin-labels around the point is also proportional to the population of the spin-labels in a small area at the point. From the calculation of the intensity, we can predict an EPR spectrum for a given shape of the deformed cell oriented at a given angle to the external magnetic field. In order to characterize the EPR spectrum for simulation purposes, we adopt the ratio I'll of the peak-to-trough amplitudes of the low field and the center absorption as defined in Fig. 4. F r o m the simulated spectra for various values of 2 and x, the relation of I'll values to the two parameters are estimated. F r o m the observed I'll of 0.58. and 0.43 at the shear stress of 8 and 0.4 N m - 2, respectively, ;t and K are estimated to be 2.27 and 280, respectively. We simulated five observed spectra measured at different shear stress values using these parameter values (Noji et al., 1984). The simulated spectra are compared to the observed ones at various shear rates as shown in Fig. 4. The excellent agreement between them verifies that the assumptions made concerning the deformation and orientation are adequate for this approximation. The maximally deformed shape of a red cell under the given conditions is calculated to be an ellipsoid with a = 6.0 gm, b = 2.4 ;tin, and c = 1.9 #m, from which the deformation index is calculated to be 0.44. The D(max) values estimated from photomicrographic observations are in the range 0.5-0.6, depending upon the viscosity of the medium in the range 2-3 x 10 .2 Nsm -2. Since the viscosity in the present experimental condition is 8.9x 10-3 N s m - 2, the deformation index of 0.44 derived here is considered reasonable, because the D(max) increases with the viscosity of the suspending medium. We obtained a semiempirical relation of the averaged deformation index ( D } or the average orientation angle (~t} with the spectral parameter I'll as shown in Fig. 5. The relation ofl'/l to Ah/h was found experimentally as follows:

I'/l = a(Ah/h) + b where an average value of a is 0.86 ___0.11 and b = 0.21 or larger, depending upon suspension viscosity. F r o m these relations, we can estimate the deformation and orientation of erythrocytes in flow by the EPR method. III. EXPERIMENTS

1. E P R Instrumentation E P R spectra under flowing condition can be easily obtained with an instrument as illustrated in Fig. 6 (Kon and Kon, 1985). An EPR flat sample cell is fixed so that the flat

90

S. NoJl et al.

d

20 G

t

Flo. 4. Comparison of the simulated EPR spectra with the corresponding observed ones. Simulated spectra (. . . . . . . . . ) were calculated with the following parameters: g,=2.0088, gy=2.O061, gz = 2.0027; T , - 17.6, Ty= 16.73, Tz=93.8 (in MHz); y=35 °, 2=2.72, • = 280. The observed spectra ( ) were obtained at 25°C from a red cell suspension at 35% hematocrit in 5 mM isotonic phosphate buffer (pH 7.4) containing 5% dextran. The flow rate was adjusted to give the shear stress at the surface of the flow cell (a) 0.0, (b) 0.3, (c) 0.4, (d) 1.0, (e) 3.2 and (f) 8.0 Nm -2.

0.4]

,

,

.

m

,

i

.

80

0.3

70

0.1L 0

0.2

-

0.3

0/, a/a

0.5

0.6

FIG. 5. The relation of the average deformation index ( D ) (left ordinate, 0 ) or the average orientation angle (~) (right ordinate, • ) with the spectral parameter, I'/l (see Fig. 4), calculated with 2=2.72 and g--280. The shear stresses correspond to those shown in Fig. 4.

EPR study on erythrocyte deformability

91

'~Computer~-~ bMI ~._

_ J~4F

SY

M:. FIG. 6. A schematic illustration of the flow-EPR instruments for measuring deformation and orientation of erythrocytes. C, EPR flat cell; M, magnet; SM, steppermotor; SY, syringe; MF, external magneticfield indicated by arrows; B, suspensionof red blood cells. surface is perpendicular to the external magnetic field. The total volume of the cell suspension needed for a measurement is about 3 ml. A spin-labeled cell suspension was driven through a flat quartz cell by a computer regulated syringe drive (or pump) so that it takes 2 rain to obtain data by a stepwise change of flow rate from 0 to 0.1 ml/sec. The flow-cell is constructed from a pair of parallel quartz plates (length 50 mm, width 8 mm, air-gap 0.26+0.01 mm) attached to an inlet and outlet tube of 1.5 mm ID. The middle section (15 x 8 ram) of the lengthwise flow is in the EPR cavity resonator. In this cell, the volume flow rate of 0.1 ml/sec corresponds to the average linear velocity of 4.63 cm/sec. The Reynolds number for the lowest viscosity suspension used is calculated to be 2.1. To obtain the spectral change vs flow rate curve, the volume flow rate was increased stepwise from 0 to 0.1 ml/sec. The computer program for regulating flow rate was made so that the dwell time in each step and the data acquisition time therein are adjustable, depending upon the viscosity of the suspension to ensure that the system reaches a steady state before starting EPR data acquisition. In the actual measurement, the magnetic field is scanned first to obtain the maximum peak-to-trough height (h) in the absence of flow (Fig. 2). The magnetic field is then held at a point near 3370 G, at which the spectral change (Ah) due to flow is expected to be maximal, and the computer-controlled syringe drive (or pump) is started to observe Ah vs flow rate characteristics, followed by normalization to Ah/h on computer.

2. Red Blood Cell Preparation and Spin-labeling Human red blood cells for measurement of deformability have been prepared as follows (Kon et al., 1983; also see ICSH report, 1986). Fresh heparinized (20 units/ml) human blood was washed three times with an isotonic HEPES buffer solution (50 mu HEPES, 125 mu NaCI, 3 mM KCI, pH 7.4) after the buffy coat was removed. In this preparation, the erythrocyte to leucocyte ratio is normally of the order of 104 or higher. The whole blood sample was not refrigerated normally but kept at ambient temperature and tested not later than 4 hr after venipuncture according to the ICSH guidelines on blood rheology (ICSH, 1986). An appropriate amount of dextran-40 (M.W. 40,000 Sigma Chemical Co.) was dissolved in hypotonic HEPES buffer (50 mM HEPES, 3 mM KC1, pH 7.4), and the osmolarity was adjusted to 295 mosmol/kg with NaCI. The cell suspension was incubated for 10 min at 37°C in a tube coated inside with a thin film of a fatty acid spin-label (Fig. ld). Since no free spin-label was detected in the cell suspension or the supernatant by its characteristic sharp triplet-line EPR signal, the suspension was used for EPR measurements without further washing and centrifugation. IV. A P P L I C A T I O N S

1. Effects of Spin-labeling, Diamide Cross-linking and Glutaraldehyde Fixation In order to characterize the spectral change due to flow, Noji et al. (1981a) adopted the relative spectral difference, Ah/h, which is defined in Fig. 2. The dependence of the relative

92

S. Non et al.

spectral change on flow rate is shown in Fig. 7. The open circles are the data from the normal cell suspension, when the total spin-label concentration is 13/ha. At this concentration, the ratio of spin-label to phospholipids in the cell membrane is about one to 104. The spectral difference increases steeply with the shear stress, reaching a plateau at about 5.0 Nm - 2, and orientation--deformation of the cells becomes saturated at a relatively low shear stress. At a higher concentration (e.g. 182 #M) of the spin-label, the initial rise of the curve becomes gradual and the spectral difference approaches the plateau much more slowly (Fig. 7). This I

I

2.0

4o

I

I

I

0.3 "~O,2 0.1

o

~o T ( N-m2 )

8.0

~qo

FIG. 7. Dependenceof the relative spectral differenceAh/h on shear stress ~ in suspension of RBCs spin-labeled at the concentrations of 0.013 (©), 0.065 (e), and 0.182 (~) raM. Hematocrit 35%, dextran concentration 5%. suggests that there is a perturbation of intrinsic properties of the membrane by the spin-label. Indeed at this concentration, in the absence of flow, we observed that the cell shape changed from discocyte to echinocyte in which several parts of the membrane are protruded. The spinlabel concentration has been kept at 65/zM throughout deformation measurements at which level the EPR spectrum has a reasonable signal-to-noise ratio for data analysis, yet the effect of the label is small enough so that the results are a good approximation to that for the intact cell. In order to demonstrate that the observed relative spectral difference does indeed reflect not only the results of cell orientation but also the extent of deformation (elongation) which occurs under the flowing condition, the dependence of Ah/h on flow rate was observed for the cells treated with diamide (Noji et al., 1981a). Diamide cross-links the red cell cytoskeletal protein spectrin and reduces the cell deformability (Fischer et al., 1978a). It is interesting to note that the diamide treatment does not change the original biconcave shape observed in the absence of flow. The EPR spectral difference against shear stress curves (Fig. 8) clearly depends on the diamide concentration. The dependence of the Ah/h value on shear stress for the diamide-treated cells closely resembles that of the cell elongation on shear rate obtained by the rheoscope method (Haest et al., 1980). 0.4

)

I

1

~ o

[ o

o

0.3

"~0,2 0.1

0

m

2D

I

4.0

I

)

I

.__J

6.0 8.0 10.0 12~ "12(N.~2) FI~. 8. Effectof diamide treatment on Ah/h vs shear stress t characteristicsat 35% hematocrit and 10% dextran concentration. Diamide concentration:0 (©), 0.2 (~), 0.4 (qD),and 0.8 (O) raM.

EPR studyon erythrocytedeformability

93

Glutaraldehyde is known to cause non-specific cross-linkings involving membrane proteins, phospholipids and hemoglobin in the cell. This reagent makes the cell less deformable without any change in the cell shape (Heusinkveld et al., 1977). In this case, a decrease in the saturation value of the spectral difference with glutaraldehyde treatment time was observed as shown in Fig. 9 (Noji et al., 1981a). These results suggest that the spectral 0.4

1

i

I

o

i

o

o

i

~

I

~4l'

0.3 tr-

< 0.2

0.1

0

i

2.0

i

4.0

i

6.0

i

8.0

i

10.0

"c ¢..,~2) FiG.9. Effectof glutaraldehydetreatmenton Ah/h vs ~ characteristicsat 35% hematocritand 10% dextran concentration.Incubationtimes:0 (O), 0.5 (~), and 1 (O) hr.

difference reflects the cell deformation as well as cell orientation. An interesting fact is that in a low shear stress region, the shape of these curves is different from that of the previous diamide treated case: the Ah/h value increases steeply in the case of the glutaraldehydetreated cells. Glutaraldehyde also cross-links hemoglobin molecules causing an increase in the intracellular viscosity of hemoglobin solution. Thus, the Ah/h characteristics also reflect the increase in internal viscosity of the cell. This explanation is consistent with other data obtained by the optical method (Mohandas et al., 1980b). Thus, the spectral difference vs shear stress relation is very sensitive to the cell deformation and orientation, and we conclude that our EPR method is applicable to assess the cell deformability in various other situations.

2. Factors Determining the Cell Deformability It is well known that the degree of the overall deformation of a single red blood cell in a shear flow is determined by intrinsic and extrinsic properties. There are mainly three factors: (1) the cell surface area to volume ratio (sly); (2) the ratio of intracellular to extracellular viscosity; and (3) the membrane viscoelasticity (Mohandas et ai., 1980a). The dependence of Ah/b on each of the three factors has been investigated with the EPR method. As we have shown above, cross-linking of the membrane protein by diamide causes reduction of the observed Ah/h proportionately to the extent of cross-linking (Noji et al., 1981a). Similar results were obtained by heat-treatment of the cells at a temperature of spectrin denaturation (Noji et al., 1981a). Red cells having a reduced surface area were prepared by incubating with lysophosphatidylcholine (LPC) followed by washing with a solution of bovine serum albumin. In cells thus prepared, Ah/h measured over the range of suspension osmolarities 150-300 mosmol/kg showed decreases consistent with the LPC concentrations used, demonstrating Ah/h dependence on the s/v ratio (Kon et al., 1983). Interesting results were obtained in red cells which are dehydrated for an increased intracellular hemoglobin concentration (and a higher intracellular viscosity) while maintaining the original intracellular ion concentration (Kon et al., 1983). Dehydration was made in sucrose solutions of various osmolarities. The Ah/h values were measured on these samples in suspensions of varied osmolarity 150-300 mosmol/kg (Fig. 10). The results given in Fig. 10 show that the plateau values of Ah/h in the dehydrated cells are lower in an isotonic medium but recover dramatically as the medium osmolarity is lowered. The results of similar experiments are replotted vs the measured intracellular hemoglobin concentration. Thus in

S. NoJ1 et al.

94

Fig. 11, circles and triangles represent the control and the dehydrated cells suspended in the media of varied osmolarities, respectively. The points from the control are distributed mostly in the low hemoglobin-concentration range (12-21 mmol/l), while the dehydrated ones are in the higher range (16-25 mmol/l) with a maximum of Ah/h between them around 18-20 mmol/l. This plot shows that there are two determinants of Ah/h operating with opposite effects; the decrease of Ah/h in the medium with osmolarities higher than isotonicity is attributed to the increase in the intraceilular viscosity (case 2), whereas the decrease of Ah/h at lower osmolarities is due to the decrease of the s/v ratio (case 1).

0.4

0.3

0.2

!

.

0.1

0

I 150

I 200

I 250

I 300

08MOLALITY (mosmol/kg) FIG. 10. Dependence of Ah/h values at the plateau on osmolarity in dehydrated cell suspensions. Osmolarity (mosmol/kg) used for dehydration: 300 (©, • ), 490 ( • ) , 600 ( A ). The cell count in all the suspensions was adjusted to 3.3 x 109/ml.

0.4

0.3

r

,~ 0.2

."

0

I 12

t

I 18

I

I I 24 HB. CONC. (rnmoleall.eell)

FIo. I 1. Dependence of A h/h on intracellular hemoglobin concentration in the dehydrated cells. Data and the symbols are the same as in Fig. 10.

EPR study on erythrocyte deformability

95

It is noted that the highest Ah/h values occur in the region of the hemoglobin concentration (18-20 mmol/l cell), somewhat lower than in the control cells on the isotonic medium. This is probably due to the fact that the Ah/h is also affected by the cell--cell interaction, and under the conditions of this particular experiment in which the cell count in all the suspensions was adjusted to 3.3 x 109/ml, the cell-cell interaction apparently acts to suppress the Ah/h values in the isotonic medium. The EPR spectrum of the spin-labels incorporated into the cell membrane is thus proved to contain full information on the whole cell deformation similar to that obtained by other methods, e.g. the ektacytometry, in spite of the complete difference in physical principle the spin-label method is based upon. Furthermore, instead of the deformation of an isolated individual cell, an ensemble average of the whole cell deformation is measured by the present method.

3. Time Course of Deformation Recovery Noji et al. (1981 b) also investigated a dynamic response of the cell to the change of shear stress. When the flow is abruptly interrupted, the spectral difference disappears with a characteristic time course. Figure 12 shows a typical decay curve for the intact cells (averaging over 16 curves). The decay curves can be simulated by superimposing three exponential components (Fig. 12). The estimated relaxation times of each components are

I

I

I

10 Time/see

15

I

O.8

°o.6 .c:

°

~ 0.4 r-

0.2

~

.....

~

0

|

5

20

FIG. 12. Decayof EPR anisotropyAh in intact cell suspensionat 35% hematocritcontaining5% dextran. Volumetricflowrate is 5.2 ml/min.The solid lineis the simulationas the sum of the three componentexponentialterms (dottedlines).

0.3, 1.6 and 9.8 sec at 35% hematocrit and 5% dextran concentration. The time required for cells to achieve random orientation by rotational Brownian motion has been measured and estimated to be 2-3 min (Oster and Zalusky, 1974), which is much longer than the time scale involved in the present experiment. The observed decay was assumed to be mainly determined by the cell shape recovery process; the cells recover their intact biconcave shape with concomitant randomization of orientation. When the cells are treated with diamide, they show much faster decay curves than the intact; the decay process is completed within ca. 2 sec in the less deformable cells. This is expected, because they have a greater modulus of elasticity. In general, the shape recovery time is characterized by the membrane viscosity (~m) and the modulus of elasticity (~). Namely, part of the energy stored by an elastic deformation is to be dissipated on release by the viscous element thus retarding the recovery process. In the special case of the uni-axial extension of a cell, shape recovery time is proportional to the ratio oft/to/~ (Hochmuth et al., 1979). For the diamide-treated cells, the increase in ~ may be SSzZ-¢

96

S. NoJI

et al.

a major factor for the fast decay time. From the measurements of decay time, the information on the bulk membrane viscosity can in principle be obtained.

4. Effect of Ca 2 + [01'! and Stomatocytooenic Reaoents It is known that with increase in the intracellular Ca 2 ÷ ion concentration, the red cells suffer from a partial loss of the whole cell deformability (Kirkpatrick et al., 1975). Since the concentration of the intracellular Ca 2 ÷ is strictly maintained to be as low as 1/~M, and may be regulated by some signals from outside the cell, the Ca 2 + ion may be a candidate for the control of cell deformability. When the intracellular Ca z ÷ ion concentration is increased by a treatment with the calcium ionophore A23187, a series of complex biochemical reactions occurs including the activation of (Ca 2 +-Mg 2+)-ATPase by Ca 2 +-binding calmodulin, modification of the metabolism and composition of membrane lipids, and the efflux of K + ions and water (Gardos effect (Gardos, 1966)). The mechanism of the Ca-induced loss of deformability is explained by the cell dehydration due to the Gardos effect resulting in the high internal viscosity (increase in hemoglobin concentration), because no significant loss of deformability was observed in a high potassium medium in which dehydration and K ÷ efflux are prevented (Clark et al., 1981). Noji et al. (1987) measured the change of Ah/h by increasing the intracellular Ca 2 ÷ ion with use of the Ca 2 + ionophore A23187, and obtained the results as shown Fig. 13. The observed decrease of Ah/h in a high Ca 2 + solution and no

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0.05 0.1 Flow Rate (mils) FI~3. 13. Dependence of Ah/h on flow rate for intact cells (O), Ca2*-loaded cells (73), and Ca2÷-loaded cells in the presenceof 50/ZM(A) CPZ at 50 rain after addition of 20/~MA23187. change of Ah/h in a high K ÷ medium are consistent with the results from the ektacytometer (Clark et al., 1981 ). With increase in the Ca 2 + concentration, potassium ions leak out of the cell with concomitant loss of intracellular water. The loss of water results in the increase of intracellular viscosity, which makes cells less deformable. In addition, an interesting phenomenon was found (Noji et al., 1987); the loss of the Ah/h was reversed to a various extent by the presence of a proper amount of stomatocytogenic reagents, chlorpromazine (Fig. 13), trifluoperazine, W-7, or calmidazolium (R24571). Since they are also calmodulin inhibitors as well as potent stomatocytogenic reagents, we postulated at first that the inhibitors might act to suppress the Gardos effect and/or that Ca 2 + ion may modify the interaction between membrane and cytoskeletal protein complex through calmodulin (Anderson and Morrow, 1987). However, the recovery cannot be explained solely by suppression of the Gardos effect due to the reagents except for the case ofcalmidazolium at a high dose (ca. 75 #M). Furthermore, it is unlikely that the inhibitors can have access to calmodulin from outside the cell. A plausible explanation for the recovery effect may be found in the fact that an enhanced intracellular Ca 2 + concentration induces breakdown of polyinositides which was shown to be implicated in the process of echinocytosis (Allan and Michell, 1978; Anderson and Marchesi, 1985; Ferrell and Heustis, 1984). The deformability of an erythrocyte may be related to the balance of the lipid bilayer through a close coupling between the lipid and the cytoskeletal proteins (Anderson and M archesi, 1985; Haest, 1982). In the presence of a stomatocytogenic reagent in an optimal dose, intercalation of the reagent

EPR study on erythrocyte deformability

97

into the membrane inner layer can compensate for the imbalance and restore approximately the normal physical state of the membrane. This explanation is supported by the fact that incorporation of an echinocytogenic reagent into the membrane is known to have a more marked effect on the rheological characteristics in the low shear rate (Meiselman, 1978) and that the observed recovery effect of stomatocytogenic reagents show mainly in the low shear region.

5. Effect of Cu 2+ Ion Hemolytic anemia is a well-recognized complication in Wilson's disease and in other forms of copper intoxications. The precise mechanism of copper-induced hemolysis, however, is not completely understood. In addition to Cu 2 ÷ ion-induced direct chemical damages ofthe membrane, a loss of cell deformability due to copper has also been postulated to explain the premature destruction of erythrocytes and consequent anemic conditions. As shown in Fig. 14, the data obtained by the present method unequivocally demonstrated that the

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FtG. 14. Recoveryfrom Cu(ll)-induced change in AA/A by EDTA and dithioerythritol (DTI'). Cells were pretreated with iodoacetate to eliminate donor-dependent response to the Cu 2+ effect. (A) Erythrocytespretreated with 3 mMiodoacetate were incubated without (O) or with 0.2 mMCuSO, in the presence (A) or absence (Tq)of 2 mM EDTA; cells pretreated with 3 mMiodoacetate, incubated with 0.2 mMCuSO4, and washed and treated with 2 mMEDTA for 40 min at room temperature (,). (B) Iodoacetate pretreatment (5 ml~),CuSO4 concentrations 0 mM (O) and 0.2 mM ([]). Cells were then treated with 3 mM DTT for 40 min at room temperature (A).

whole-cell deformability in shear flow is decreased as the result of treatment with copper even under the mild conditions of a relatively low dosage and a short incubation time at ambient temperature (Ito and Kon, 1987). The Cu 2+ ion has been shown to induce echinocyte formation, which contributes to the decrease of Ah/h. Comparison of the morphological patterns and the flow data shows that a correlation exists between the degree of echinocyte formation and the loss of whole-cell deformability. When the cells, treated with Cu 2 ÷ ions and washed, were incubated with 1% BSA, almost normal morphology was restored, and at the same time a completely normal Ah/h was restored in the low flow rate region, but with still some residual loss of Ah/h in the high flow rate region. A similar trend was observed by using chlorpromazine, a stomatocytogenic agent, in an attempt to reverse the Cu 2 ÷-induced echinocyte formation. Thus, the Cu 2 +-induced deformability loss cannot be explained solely by Cu 2 ÷-induced echinocyte formation. The copper-induced cell alterations are caused by complex events involving more than one type of reaction site. The detection of an enhanced level of intracellular methemoglobin E P R signals only 5 min after exposure to copper demonstrated that Cu(II) can easily pass through the membrane to cause the coppermediated methemoglobin formation, thus also suggesting the possibility of Cu(II) reacting with other intraceUular components. Ito and K o n (1987) have found that there is a type of Cu 2 ÷ reaction for which copper chelating agents are ineffective for reversal, but which can be reversed by a reducing agent such as dithioerythritol (DTT) (Fig. 14). This reagent reverses

98

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disulfide bond formation by reduction. Thus, it is reasonable to assume that the part of the deformability loss which is not reversed by chelating agents but is reversible by DTT is caused by the Cu(II)-induced disulfide bonds or Cu e +-bridged coordination involving -SH and other residues. These results obtained under prelytic conditions can be extrapolated to a higher Cu(II) dose and a prolonged incubation at an elevated temperature (e.g. 37°C). The resulting severely reduced cell deformability could lead to premature cell destructions. 6. Effect of Heinz Body Formation The Heinz bodies in mammalian RBC consist of aggregated particles of denatured hemoglobin, which are detected in in vivo peripheral RBC after splenectomy (Weatherall, 1977). The mechanism of hemoglobin denaturation in vivo leading to Heinz body formation is not thoroughly clarified, but is generally accepted to be similar to that in model reactions studied in detail using oxidizing agents, most notably hydrazine derivatives such as phenylhydrazine (PHZ) and monomethylhydrazine (MMH). Although RBC with Heinz bodies was known to possess a reduced cell deformability, oxidative damage to red cells can in principle affect any of the above described three factors influencing the cell deformability. Fukushima and Kon (1990) examined the process of the deformability loss when the Heinz bodies were induced by either PHZ or MMH. As shown in Fig. 15, deformability of RBCs

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treated with PHZ or MMH at 37°C for 1 hr decreases dose-dependently. Both hydrazines cause roughly the same degree of deformability loss up to l mM, but beyond the concentration, the Ah/h value sharply decreases with increase in PHZ concentration. At 2 mu PHZ, PHZ is five times more effective than MMH. Since no Heinz body formation and morphological change are clearly recognized at the hydrazine concentration less than 0.5 mM, the decrease in whole cell deformability can be attributed to a small membrane perturbation such as binding of hemoglobins denatured by hydrazines. Heinz body formation begins to appear with PHZ above 0.5 m~l, and appears to line the cell inner surface, exhibiting echinocyte-like distortions. On the other hand, such distortions are not

EPR study on erythrocyte deformability

99

seen with M M H even when Heinz bodies are present. A relatively minor decrease in Ah/h compared to the corresponding PHZ,-treated cells suggests the different ways the two hydrazines interact with cellular hemoglobin and the denatured hemoglobins with the cell membrane. The hydrazine effects on Ah/h are completely suppressed in the absence of molecular oxygen, proving that the redox reactions involving oxyhemoglobin are responsible for the ultimate loss of deformability. The resealed ghosts prepared from red cells treated with 0.25 mM PHZ or 0.5 m~a M M H maintain biconcave discoid shape, but decrease in Ah/h is evident, although PHZ is again much more effective. Measurements of membrane fluidity by 12-doxyl stearate spin-labeling indicate that there are two distinct modes of spin label interaction in PHZ-treated cell membrane, the fraction corresponding to more immobilization gaining with PHZ concentration, while M M H has only minor effects on membrane fluidity. The motional freedom of membrane proteins probed with maleimide label showed that binding of hemoglobin denatured by PHZ causes more immobilization than by MMH. These observations and a comparison of Fe(III) EPR spectra of the treated cells and ghosts demonstrated the difference in denatured hemoglobins by the two reagents; the hemoglobins denatured by PHZ seem to include components that preferentially bind to the endosurface of RBC membrane, possibly coclustering band 3, resulting in a perturbation of the membrane viscoelastic properties, while MMH-denatured hemoglobins remain mostly in the cytosol as Heinz bodies or in polymeric forms increasing the intracellular viscosity. The latter effect evidently causes less effect on the whole cell deformability.

7. Effect of Hematocrit As erythrocytes flow in vivo at a high hematocrit of 40-45%, the interaction between cells can take place. In order to find the effect of their interaction on the deformation and orientation, the dependence of deformation and orientation on hematocrit was investigated by Kon and Kon (1985). The vaue of Ah/h was measured at a fixed flow rate (0.053 ml/sec) corresponding to the calculated wall shear rate of 540 sec- 1. In this condition, all suspensions showed Newtonian viscosity characteristics. Figure 16 shows the dependence of the spectral

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difference Ah/h upon the apparent hematocrit on normal red cells suspended in isotonic dextran solutions of different viscosities (2.2-11.1 x 10 -a Nsm-2). Each curve consists of two phases; as the hematocrit is raised, the Ah/h ratio increases at first to reach a peak followed by a declining stage. Raising the dextran concentration shifts an entire curve toward a higher Ah/h region with concomitant shift of the peak position to a lower hematocrit. The declining phase appears to fall in one narrow zone regardless of the dextran concentrations. The two-phase characteristics suggest that opposing factors are operating in the effect of changing the hematocrit, one promoting and the other disturbing the free deformation and orientation of the cells in flow. It is generally expected that a greater extent of cell deformation and orientation should result from a higher shear stress as a consequence of raising the suspension viscosity by increasing the dextran concentration and/or hematocrit. The observed result is a manifestation ofthe cell-cell interference occurring as the hematocrit is increased. The interference may entail loss of physical space surrounding a cell necessary for free deformation and orientation, and/or the disturbance of laminar flow dynamics around the cell due to the motion of other cells in close proximity. Although the hematocrit dependence was altered by extracellular viscosity (dextran concentration), it was found that there exists an optimum hematocrit for deformation for a given viscosity of suspension. Hematocrit may be an important factor in determining in vivo cell deformation. It is known that the hematocrit in pregnancy is about 30% which is less than the normal by 10% or more. Furthermore, the viscosity of blood in that period is known to be higher than the normal. As shown in Fig. 17, the dependence of Ah/h value on hematocrit for pregnancy differs from that for normal (Mitsui et al., 1990). The value of Ah/h for pregnancy becomes maximum at lower hematocrit than the value for normal. Thus, we might speculate that hematocrit is adjusted for optimal deformation in pregnancy. 0.4

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