Constant magnetic field influence on passive electrical properties of red blood cells

Bioelectrochemistry and Bioenergetics, 14 (1985) 495-502 A section of J. Electroanal. Chem., and constituting Vol. 192 (1985) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

495

704-CONSTANT MAGNETIC FIELD INFLUENCE ON PASSIVE ELECTRICAL PROPERTIES OF RED BLOOD CELLS

M.S. MARKOV Department of Biophysics, Biological Faculty, Sofia University, Bul.Dragan Tzankov 8, Sofia 1000 (Bulgaria) F. PLIQUETT Institute of Biophysics, Karl Marx University, Leipzig (G. D. R.) (Revised manuscript received May 29th 1985)

SUMMARY The behaviour of red blood cell membranes in blood prepared for transfusion has been studied during the period of its’ conservation. The pulse impedance method was used to estimate the conductivity and the capacity of erythrocyte membranes. The changes of these parameters depending on the induction of magnetic field were investigated during the whole period of conservation. A clearly expressed extremum of the parameters was observed in the control sample at the 18th day. It has been observed that the application of constant magnetic fields leads to the reduction of this extremum and thus prevents changes of the passive electric properties of the erythrocyte membranes.

INTRODUCTION

Investigation of the passive electric properties of biological objects gives information on their structural and functional state, on the membrane properties as well as on the influence of different physical and chemical factors. The existence of a frequency dependence of the conductance, impedance and dielectric properties is established. The dispersion of the above-mentioned parameters is closely connected with polarization phenomena. Schwan [l] finds three regions for the dispersion of the dielectric constant t, and specific resistance p of the tissues (Fig. 1). The electric properties of tissues are characterized by three major dispersion regions. Each dispersion is defined fairly well by either a single relaxation time or a small spectrum of relaxation times. y-Dispersion is a result of the dielectric relaxation of free water, &dispersion is a Maxwell-Wagner relaxation resulting from the charging of cell membranes. a-Dispersion is a result of the variability of the apparent outer cell membrane capacitance with the frequency [2]. Cole and Cole [3] propose the idea that in order to understand the mechanism of the changes of the impedance, conductance, conductivity and dielectric constant it may be very useful to study the frequency dependence of these parameters. In a parallel manner they propose to construct equivalent electric circuits which may mould the electric properties of the subject under investigation. The simplest 0302-4598/85/$03.30

0 1985 Elsevier Sequoia S.A.

4%

Fig. 1. Dispersion

of the dielectric constant C, and specific resistance p (according to Schwan [I]).

equivalent circuit includes a resistance and capacitance in series (it is a model of an artificial lipid membrane). For this circuit only the imaginary part of the impedance is frequency dependent, but for the conductance both parts are frequency dependent: Z=R--iw-‘C-’ -lc-1 R y=z-‘=

R= + w-=C-=

+i

w

R= + w-~C-~

where R = resistance, C = capacitance, Z = impedance, Y = conductance, i = d- 1 and w = circular frequency. The frequency dependence of the conductivity is shown in Fig. 2 This method gives the possibility of determining the resistivity and conductivity of the simplest membrane: the artificial lipid bilayer. More complicated is the equivalent circuit presented in Fig. 3. In this circuit we have two types of relaxation times: in the millisecond and in the microsecond range.

Fig. 2. The frequency dependence

of the conductance

in the real/imaginary

plane.

497

Fig. 3. The equivalent circuit of a red blood cell. R,, C, = resistance and capacitance of the electric of the membrane, R,. C, = resistance and capacitance scheme, Rg. Cs = resistance and capacitance connected with the properties of the double diffuse layer. R,, = resistance of the cytoplasm.

In the microsecond range the relaxation time is in P-dispersion; the a-dispersion and polarization phenomena have a milisecond relaxation time. Using this equivalent circuit in the classical Cole-Cole scheme of the study of passive electric properties of the cell membrane one may encounter many complications. The method is very slow and requires a complete technique for calculation. For these reasons it is not appropriate for everyday application in routine measurements. One quick method for screening is the method developed by Pliquett et al. [4] the so-called pulse impedance method. It has several advantages. In the first place, it allows one to obtain data for the sample in a few seconds and to fix the picture on the screen of the oscilloscope. In the second place, this method gives the possibility to investigate the dynamics of the changes of the passive electric properties of the

Fig. 4. Rectangular

electric pulse, applied

to cell suspension(left)

and modified

signal(right).

498

J Fig. 5. Spheric model of a red blood cell. r,d = all radius and thickness of membrane, K, = the conductivity of the environment, K, = inside cell conductivity, K, = membrane conductivity and capacity, respectively.

cell membrane under the influence of different physical or chemical factors. In the third place, it makes possible the study of the development of some pathological processes. The fourth advantage is the information obtained by this method on the concentration of cells in the definite suspension. The principle of the method consists of applying a rectangular electric pulse to the investigated sample. The response of the object, seen as a modified signal, may be visualized on the screen of the oscilloscope. The values of h,, Ah and c may be measured directly on the screen of the oscilloscope or after taking a photograph (Fig. 4). It is very important to consider the cells as more or less spherical (Fig. 5). If K, 4 K,, K, -c ~~ and de r the pulse impedance method may be described by the following equations: 2KU AU=>- lJ ROS, X K A 1 +exp(-f/r) Ki+2K,+2V(K;-K,)

x

K;+zK,-

-~ 1 -v

V(K,-K~)

I

q, R,.,, 2Ku h=K

A

1+exp(-t/7)

1+r$

b+K$ [

I

(1

1 (1) 11 (2)

499

of the oscilloscope, K, = where U, = amplitude of the pulse, R,, = resistance ~~ = conductivity of the cell, K,~= membrane conductivity of the environment, conductivity, I/= relative cell volume (hematocrit), r = cell radius, d = thickness of the membrane, C, = membrane capacity, t = length of the pulse, r = relaxation time, K = time constant of the measuring chamber and A = amplification factor of the oscilloscope. EXPERIMENTAL

has been used in the experiment. Every Blood prepared for blood transfusion solution L-12 (2 g acid bank contains 300 cm3 blood and 75 cm3 anticoagulating sodium citrate, 2 g glucose in 100 cm3 bidistilled water). The banks were stored at 4OC, measurements were taken after stabilization at 26’C. After removing the buffy coat the blood cells were washed twice in 0.85% NaCl solution (pH = 7.4 * 0.1, osmotic pressure 290 moosmol) at 2000 x g for three min. This preparation results in an erythrocyte suspension with a volume concentration of cells of 0.70 k 0.02 (determined by hematocrit centrifuge Janet&i THII) and a conductance of 2.5 mS (determined by Zeibold conductometer). Constant magnetic fields (CMFs) with an induction B in the range of 20-90 mTesla and exposure times of lo-120 min were used. The choice of the range of CMFs was determined by literature data and by our own experience, showing that applying a magnetic field with an induction of 40-50 mTesla is the most effective [5-71. Some authors relate this fact to the existence of specific values of the magnetic influence which are acceptable by biological systems [5,8]. We also investigated the magnetic field’s influence on the electric properties of erythrocyte membrane when several chemical factors had been applied, with the aim to modify the membrane surface charge. Protamin and heparin were used as such modifying factors. It is known that they change the membrane charge in different directions. Protamin is low-weight globular protein. It has a sufficiently high net of positive charges and therefore decreases the membrane charge. On the contrary, heparin has a negative charge and causes an increase of the surface charge of membrane when heparin is bound to it. The presence of sulfate and carboxyl groups gives the heparin high acid properties. RESULTS

AND DISCUSSION

The changes of the passive electronic properties of erythrocyte membrane with and without influence of CMF with an induction of 45 mTesla are presented in Fig. 6. It can be seen that the parameter Ah has an absolute minimum at the 18th day

65

k (days) 0

2

4

6

8

10

12

14

16

i8

20

Fig. 6. Dynamics of the change of the parameter Ah depending Control, (- - -) magnetically treated sample.

on the time of conservation

of blood.

( -)

(for the control sample) and that the values increase dramatically on the following day. This fact indicates at least two points: in the first place, it is an indication that the number of non-hemolyzed erythrocyte cells reaches a minimum. The maximum is connected with erythrocyte membranes reconstituted after the hemolysis (erythrocyte ghosts). In the second place, it is of great importance for the blood transfusion. The transfusion of blood between the 17th and the 21st day of conservation is not appropriate. Moreover, it is dangerous for the patients - the ghosts may take with them not oxygen but substances from the hemolysis and solution. Nakache et al. [9] proposed to study the evolution of the mechanical properties of red blood cells during their conservation in the blood bank. We also consider that it is necessary to study the changes of red blood cell electric properties and their modification during conservation in the blood bank. It is well known that aged red blood cells exhibit a decreased deformability and a change in shape due to a decrease in their membrane flexibility and an increase in their internal viscosity. In addition to this deformability reduction, alterations in the physicochemical properties of the surface of the red blood cells lead to a decrease in surface charge [lo]. Finally, all these alterations lead to lysis. Our data confirm this point of view. Similar, but reversible modification, can also be obtained by a change of temperature [9]. One other interesting result may be observed in Fig. 6. During the second half of the period of conservation the values of Ah are higher in the sample treated with CMF than in the control one. This is evidence for the protective role of CMF against spontaneous hemolysis. These data confirm our previous results [6], showing that the

501 TABLE 1 Calculated values of the membrane capacity and conductivity in control and under the influence constant magnetic field with an induction of 45 mTesla and an exposure time of 30 min 18th day

Control Experiment

of a

19th day

c (PF cmm2)

;B-t

1.61 f 0.03 1.19*0.04

5.72 + 0.05 5.22k0.11

cm-‘)

c (kF cm-‘)

YB-r cm-‘)

1.03 f 0.04 0.81+ 0.07

4.21 f 0.05 4.64kO.10

level of spontaneous hemolysis after the 12th day is lower in the magnetically treated sample than in the control. It was also shown in this study that the difference experiment-control is expressed better between the 16th and 18th day. Using the equations (l)-(3) and experimental data obtained for Ah,h, and r one may observe a dramatic decrease of the values of the conductivity and especially of the capacity of the erythrocyte membrane between the 18th and 19th day (Table 1). It is necessary to note that the above-mentioned results are obtained by applying CMF with an induction of 45 mTesla and an exposure time of 30 min. The changes of the conductivity and capacity of the erythrocyte membrane when CMF with an exposure time of 30 min and different values of magnetic induction were applied to the sample are shown in Table 2. It can be seen that CMF with B = 45 mTesla causes a decrease of the capacity and conductivity, while CMF with higher induction (B = 90 mTesla) has the opposite effect. We regard these facts as an indication that CMF does not influence the base dose effect as it is expressed in radiobiological experiments. On the other hand, we suppose that it is a manifestation of the existence of specific resonance values of magnetic induction, leading to definite effects, while other values do not provoke observable effects or produce opposite effects [11,12]. For this reason it is necessary to investigate very carefully the changes of the blood parameters (mainly) before deciding which value of magnetic induction to apply. At the same time, this may be used in magnetotherapy to produce an increase

TABLE 2 Changes in the membrane capacity and conductivity magnetic field at the 11 th day of the blood storage Control

C(pF cm-*) ~(a-’ cm-‘)

1.48 * 0.03 5.51 f 0.02

depending on the induction of the applied constant

Constant magnetic field 20 mTesla

45 mTesla

90 mTesla

1.45 + 0.03 5.45 f 0.05

1.38 kO.02 5.38dzO.02

1.61 f 0.07 5.72 & 0.05

502

or decrease of the capacity or conductivity of red blood cell membranes depending on the necessity. REFERENCES

5 6 7 8 9 10 11 12

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