_______________________

Received: April 2009;

in final form September 2009.

ROMANIAN J. BIOPHYS., Vol. 19, No. 3, P. 171–185, BUCHAREST, 2009

EFFECT OF GAMMA RADIATION ON SOME BIOPHYSICAL

PROPERTIES OF RED BLOOD CELL MEMBRANE

NABILA S. SELIM*, O.S. DESOUKY*, SEHAM M. ALI*, I.H. IBRAHIM**, HODA A. ASHRY*

*Biophysics Laboratory, Radiation Physics Department, National Center for Radiation Research

and Technology (NCRRT), EAEA, POB 29 Madinat Nasr Cairo, Egypt

**Physics Department, Faculty of Science, Ain Shams University, Cairo, Egypt

Abstract. The present work aims are to study the radiation effects on the red blood cell

membrane from three different but correlated properties: electrical, mechanical and chemical, and to

derive useful parameters for the evaluation of radiation effects. AC conductivity of cell suspension

was measured in the frequency range 40 kHz to 5 MHz, the osmotic fragility of the membrane and

solubilization of the membrane by detergent were also measured. Adult male rats were exposed to 1,

2.5, 3.5, 5, 7 and 9 Gy gamma radiation from Cs137 source. The results showed decrease in the AC

conductivity, average osmotic fragility and average membrane solubilization. The effect of radiation

on the red blood cell membrane was discussed.

Key words: Red blood cell membrane, radiation, osmotic fragility, conductivity, membrane

solubilization.

INTRODUCTION

Biological membranes possess important roles in cells' life, which exceed

being only an envelope for the cellular components. They rather regulate in and out

transport of ions and metabolites, and govern intercellular communications. The

general feature of the biological membranes is a phospholipid bilayer in which

proteins and protein complexes are immersed. They are classified according to

their position in the body and their functions. From the different types of body

cells, the red blood cell possesses a unique structure. The mammalian red blood

cells are anucleated, they are shaped like a disk with a biconcave cross-section,

dumbbell resembling. The discoid shape provides maximum surface area for the

same cell volume, to permit maximum gaseous change between tissues and the

cells. During its existence, it must withstand great shearing forces as it travels

many hundred miles through the circulatory system. During this circulation, it is

172 Nabila S. Selim et al. 2

forced through capillaries whose diameter, which is considerably narrower than the

diameter of a resting cell. To pass these capillaries, it must be deformed. A too

readily deformed cell would be easily injured under stress, resulting in hemolysis.

A rigid cell would greatly increase the blood viscosity. Thus, the deformability of

the red blood cell membrane is one of the conditions for its viability. This

deformability is determined by the molecular and osmotic state of the cell. The red

blood cell membrane is composed of a double layer asymmetrically organized lipid

molecules acting as a boundary, in which integral proteins embedded. The

structural and functional integrity of the lipid double layer and of the integral

proteins depend on the association with a network of peripheral proteins (the

cytoskeleton) attached to the inner membrane surface. The role of the cytoskeleton

is to restore the shape of the red blood cell after mechanical deformation during its

passage through the capillaries [9].

Red blood cell is not a very radiosensitive cell, thus choosing it is not a

reflection of cellular radiation damage in vivo [10]. However, it is a suitable

candidate for monitoring the radiation effect for many reasons. First of all, it is a

representative sample for the whole body exposure, since it circulates all over the

body, second its accessibility and ease in its separation to obtain cells with intact

membrane. Also, being anucleated, it represents a useful model for measuring the

membrane properties without the interference of intracellular membranes.

Gamma irradiation of red blood cells induces alterations at three different

functional units of the membrane: lipid bilayer, protein components and

cytoskeleton at the membrane surface [3]. In addition, radiation induces shortening

in the lipid fatty acid chains by lipid peroxidation [18]. The production of

hydroperoxides and cross-linkages in the membrane lipids can disorder the upper

region of the bilayer favoring penetration of water and ending by hemolysis [16].

This work intends to study the radiation effects on the red blood cell

membrane from three different but correlated properties: electrical, mechanical and

chemical, using the following techniques: electrical conductivity of the cell

suspension, AC conductivity, osmotic fragility and membrane solubilization of the

membrane. The conductivity, in the frequency range 40 kHz to 10 MHz, takes into

account the structural arrangement of the membrane, different transport processes

occurring between the inner and outer media and permeability properties of the

lipid bilayer [4]. The osmotic fragility of the membrane can be measured by

placing the red blood cells in hypotonic salt solutions, the osmotic pressure exerted

by the diffusion of water into the cells, makes them first swell and then hemolyse.

The osmotic fragility measures the capacity of the cells to withstand hypotonicity

and resist hemolysis, which is determined by their volume to surface area ratio

[13]. The solubilization of the membrane by detergent is an induced transformation

of the phospholipids bilayer and the proteins into mixed micelles of composed

3 Gamma radiation and biophysical properties of RBCs

173

detergent, phospholipids and membrane-bound proteins. This phase transformation

depends on the molecular structure of the detergent and the composition of the

membranes [12].

MATERIALS AND METHODS

GAMMA IRRADIATION

Adult male Wister rats weighing 200g were used. They were divided into 7

groups of 6 animals each. Rats were kept under standard conditions along the

experimental period, 12/12 h light-dark regimen. Food and water were supplied

daily ad libitum. All animals were housed according to the ethic rules in

compliance with institutional guidelines. The irradiation process was carried out in

the National Center for Radiation Research and Technology using Cs137 source for

animals. The dose rate was (0.883 cGy/sec) at the beginning of the experiment. The

animals were exposed to 1, 2.5, 3.5, 5, 7 and 9 Gy single doses. They were

dissected 24 hours after exposure. The blood samples were withdrawn from the left

ventricle of the heart using heparinized needles. The measurement was carried

on blood extracted from each animal separated and afterwards average values

were computed.

NORMAL RED BLOOD CELLS HEMOLYSIS

Normal red blood cells hemolysis was determined by measurement of

hemoglobin released from the cells relative to the total cellular hemoglobin

content. Ten μL of whole fresh blood was incubating in 5 mL normal saline for 30

min. The samples were centrifuged at 3000 rpm for 10 min, and the supernatant

was measured spectrophotometrically at 540 nm. The percentage of hemolysis was

taken against complete blood hemolysis [7].

100

sample

%lysis

% 100

= × (1)

where Asample and A100%lysis are the absorbance of the hemoglobin released from red

blood cells (RBCs) in normal saline and after complete hemolysis in distilled water

respectively.

DETERMINATION OF HEMATOCRIT

The determination of the hematocrit was performed as follows: blood

samples, in the hematocrit microcapillary tube (75 mm / 75 μL), were centrifuged

for 5 minutes at 11,500 rpm. Then the hematocrit values were determined by

means of the microcapillary tube reader [11].

174 Nabila S. Selim et al. 4

MEAN CORPUSCULAR VOLUME (MCV), AND MEAN CORPUSCULAR

HEMOGLOBIN CONCENTRATION (MCHC)

The mean corpuscular volume (MCV) in femtoliter expresses the average size

of the red blood cells. It is related to the hematocrit (Hct) by the following

relations [11]:

s count(million per litre)

MCV Hct

RBC

= (2)

while the mean corpuscular hemoglobin concentration (MCHC) in mg/mL is a

measure of the concentration of hemoglobin in a given volume of packed red blood

cell [11]. It can be calculated as follows:

MCHC Hb conc (mg/mL)

Hct

= (3)

The hemoglobin concentration was evaluated using Drabkin’s reagent and

hemoglobin standard, obtained from EAGLE Diagnostics, USA.

CONDUCTIVITY MEASUREMENTS

The conductance was measured using LCR meter type HIOKI 3531, Japan, in

the frequency range 40 KHz to 5 MHz. The measuring cell is a parallel plate

conductivity cell with platinum black electrodes with area 4 cm2 and separating

distance 2 cm. The measured parameters were the capacitance C and resistance R,

from which AC conductivity σ can be calculated as follows [17–19]:

C = Aε 'εo / d (4)

where A is the area of the electrode, d is the distance between the two electrodes, ε'

is the sample permittivity and εo is the vacuum permittivity. The dissipation factor

(tan δ) is given by:

tan δ = 2πfRC (5)

where f is the frequency of applied voltage in cycles per second.

It is related to the dielectric loss ε" by:

ε" = ε ' tan δ (6)

σAC = ωεoε" (7)

where ω is 2πf.

5 Gamma radiation and biophysical properties of RBCs

175

The blood samples were centrifuged at 3 000 rpm for 5 minutes. The plasma

and buffy coat were removed by aspiration. They were washed twice in buffered

saline and separated by centrifugation at 3000 rpm for 10 minutes. The red blood

cells were re-suspended in isotonic buffered sucrose (0.3 M sucrose in phosphate

buffer pH 7.4, and conductivity 0.223 S/m), and the hematocrit was adjusted at 3%.

The samples were incubated in water bath at 37 ºC during measurement.

OSMOTIC FRAGILITY MEASUREMENTS

The degree of hemolysis can be quantitatively evaluated from the osmotic

fragility test [6]. Whole blood samples were added to the hypotonic buffer saline in

the proportion of 1:100 respectively. Hypotonic saline buffered to pH 7.4, with

different concentrations (1, 2.5, 3.5, 5, 6.5, 7 and 9 g/L) was used. The samples

were incubated for 30 minutes at 37 ºC, and centrifuged at 3000 rpm for 5 min., to

precipitate the nonhemolyzed red blood cells. The osmotic lysis of red blood cells

is detected by the release of hemoglobin into the extracellular fluid. The amount of

hemoglobin appearing in media was determined colorimetrically according to the

method reported by Dacie and Lewis 2006 [5]. The experimental curves were

normalized to 100% hemolysis to facilitate the comparison between different

samples without the interference of the hematocrit changes. The fragility curve can

be evaluated by the average osmotic fragility (H50) (the NaCl concentration

producing 50% hemolysis). Other parameters can be obtained from the

differentiation of the fragility curve, which represents a Gaussian curve (the rate of

hemolysis dH/dC versus NaCl concentration) (Fig. 4). These parameters are

position, width, height and area of the peak. The position on the x-axis is

equivalent to the average osmotic fragility (H50). The width at half maximum

reflects the dispersion of hemolysis process (lower dispersion than normal indicates

sudden rupture of the RBCs, while higher values of dispersion reflect the abnormal

increase in the membrane elasticity). The peak’s height represents the maximum

rate of hemolysis (dH/dC)max reached by the sample. The area under curve

represents the rate of hemolysis of red blood cells.

MEMBRANE SOLUBILIZATION TEST

Triton X-100 is a non-ionic surfactant (commonly denoted detergent) most

frequently used in wide applications to biomembranes. Its high solubilizing

capacity is related to its ability to form mixed micelles with membrane lipids and

proteins. The choice of Triton X-100 as detergent for the solubilization of red

blood cells membranes lies on the fact that it protects the cells against hypoosmotic

lysis.

176 Nabila S. Selim et al. 6

It is known that triton X-100 becomes hemolytic above a certain concentration

range (about 0.01%, v/v), which provides the study of their interaction with

membrane, in the low concentration ranges, without interference of the lysis effect

[12]. The samples were prepared as for dielectric measurements. The turbidity (T)

is given by:

I = I e−Tl (8)

where I0 and I are the incident and transmitted light, respectively, and l is the length

of the light path through the scattering solution [13]. The transmittance was

measured at (600 nm) using UV-visible spectrophotometer CECIL-3041. Turbidity

measurement of the membrane as a function of the added detergent is analyzed in

terms of percent solubilization normalized to the turbidity of cell suspension

without detergent. The turbidity curve can be characterized by:

– solubilizing detergent concentration (Ds): the point at which the turbidity

starts decreasing markedly (in this study it was considered at 95% turbidity);

– complete solubilization (Dc): the point at which all the membrane was

transformed into mixed micelles yielding a transparent solution (in this study it was

considered at 5% turbidity);

– average membrane solubilization (D50): the concentration of detergent at

which 50 % of the lipid bilayer is solubilized.

DATA FITTING

The fitting of the experimental data (osmotic fragility and membrane

solubilization) was carried out by the Origin software. The applied two functions

are:

– Gaussian function:

2 ( )

x C

y y A e

−

= +

(9)

where yo is the offset, A, C and S are the area under the peak, the center and the

width, respectively, and

– sigmoidal function:

1 e( o )

i f

x x f

x x

y x − λ

−

= +

(10)

where xi and xf are are the initial and final values of hemolysis and turbidity (at

95% and 5% respectively), and xo is the center (H50 and D50, respectively) and λ is

a constant.

7 Gamma radiation and biophysical properties of RBCs

177

RESULTS

NORMAL HEMOLYSIS

Normal red blood cells retain intracellular hemoglobin. The hemoglobin

release from incubated cells in saline solution gives rough indication about

membrane damage. The exposure to gamma radiation with different doses resulted

in significant increase in the hemoglobin release from the red blood cells as the

dose increases (Fig. 1). Meanwhile, the mean corpuscular hemoglobin concentration

(MCHC) decreased significantly with dose, these results showing an increasing

damage in the cell membrane as a function of dose increase.

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 1 2 3 4 5 6 7 8 9 10

Dose (Gy)

% H

2.8

2.9

3.0

3.1

3.2

3.3

3.4

MCHC (g/l)

Fig. 1. The percentage of hemolysis (▲) and MCHC (●) versus dose for control

and irradiated groups.

AC CONDUCTIVITY

The conductivity of red blood cells suspension showed a significant decrease

as a result of exposure to gamma radiation. It decreased as the irradiation dose

increased up to the lethal dose, 9 Gy (Fig. 2).

OSMOTIC FRAGILITY TEST

The present study shows a shift to the right of the hemolysis curve with the

increase of the dose, indicating increase in the average osmotic fragility (H50) as

shown in Table 1. At the same time, the dispersion of hemolysis (S) decreased

significantly with dose increase.

(g/L)

178 Nabila S. Selim et al. 8

The results show an increase in the maximum rate of hemolysis (dH/dC)max

with concomitant shift of the peaks center (H50) toward higher values of NaCl

concentration. Also, there is an increase in the rate of hemolysis (A) of red blood

cells as the dose increases (Table 1).

Table 1

The width (S), height (P), area (A), and center (H50) of the Gaussian peaks

for control and irradiated groups

Dose (Gy) S (g/L) P (L/g) A (g/L) H50 (g/L)

Control 1.93±0.017 72.84±2.69 91.80±2.92 3.53±0.127

1.0 1.85±0.036 75.73±3.98 93.58±2.92 3.54±0.133

2.5 1.852±0.029 79.26±1.62 99.86±2.45 3.59±0.048

3.5 1.82±0.018 81.25±4.96 106.48±7.72 3.66±0.078

5.0 1.79±0.020 85.79±4.38 116.63±2.75 3.82±0.108

7.0 1.69±0.017 90.07±2.15 125.49±5.04 3.91±0.149

9.0 1.67±0.048 101.24±5.52 125.49±2.57 3.99±0.124

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

5.0 5.5 6.0 6.5 7.0

Log Frequency (Hz)

Conductivity (S/m)

Control

1.0 Gy

2.5 Gy

3.5 Gy

5.0 Gy

7.0 Gy

9.0 Gy

Fig. 2. AC conductivity for control and irradiated groups.

9 Gamma radiation and biophysical properties of RBCs

179

100

2 3 4 5 6

% Hemolysis

control 1 Gy

100

2 3 4 5 6

control 2.5 Gy

100

2 3 4 5 6

% Hemolysis

control

3.5 Gy

100

2 3 4 5 6

control 5 Gy

100

2 3 4 5 6

NaCl concentration (g/l)

% Hemolysis

control 7 Gy

100

2 3 4 5 6

NaCl concentration (g/l)

control

9 Gy

Fig. 3. Fragility curves for control (▲ and solid line) and irradiated (x and dashed line) groups. The

dot with error bar is the experimental data and the solid and dotted line are the fitted data.

180 Nabila S. Selim et al. 10

MEMBRANE SOLUBILIZATION

The changes in cell membrane were tested chemically by interaction with

detergent. The solubilization process of membrane is an induced transformation of

nearly flat phospholipid bilayers containing embedded proteins into mixed micelles

of composed detergent, phospholipids and membrane-bound proteins. For the

phase transformation (micellization) to occur, the added detergent distributes

between the membrane bilayer and the aqueous medium. It induces spontaneous

phase transformation when the ratio of the detergent to lipid bilayer exceeds a critical

100

110

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

NaCL concentration l)

dH / dC

Control

1.0 Gy

2.5 Gy

3.5 Gy

5.0 Gy

7.0 Gy

9.0 Gy

Fig. 4. The rate of hemolysis versus NaCl concentration for control and irradiated doses.

value [12]. In this study, this critical value will depend on the membrane structure

(all other factors: temperature, type and concentrations of the detergent were kept

constant). In the membrane solubilization curve (turbidity versus detergent

concentration) (Fig. 5), the turbidity is initially affected only slightly by adding the

detergent. Further detergent addition results in a large decrease of turbidity until

complete solubilization is obtained (a point when additional detergent has no effect

on the turbidity of the suspension). Throughout the range of detergent addition,

which causes large decrease of turbidity, it may be assumed that lamellar and

micellar structures co-exist.

NaCl (g/L)

11 Gamma radiation and biophysical properties of RBCs

181

100

110

0 0.002 0.004 0.006 0.008 0.01

% Turbidity

1 Gy control

100

110

0 0.002 0.004 0.006 0.008 0.01

2.5 Gy control

100

110

0 0.002 0.004 0.006 0.008 0.01

% Turbidity

3.5 Gy control

100

110

0 0.002 0.004 0.006 0.008 0.01

control

5 Gy

100

110

0 0.002 0.004 0.006 0.008 0.01

Detergent concentration (% v/v)

% Turbidity

control

7 Gy

100

110

0 0.002 0.004 0.006 0.008 0.01

Detergent concentration (% v/v)

control

9 Gy

Fig. 5. The percentage of turbidity versus detergent concentration for control (▲ and solid line)

and irradiated (x and dashed line) groups. The dot with error bar is the experimental data and the solid

and dotted line are the fitted data.

Exposure to gamma radiation resulted in shift in a the membrane solubilization

curve toward lower detergent concentration (Fig. 5). The detergent concentration

(Ds), complete (Dc) and average membrane solubilization (D50) exhibited a

significant decrease as a function of dose increase (Table 3). Since the value of Ds

depends to a large extent on the cell volume, an important conclusion can be

derived from plotting the radiation induced change in both Ds with the mean

corpuscular volume (MCV) (Fig. 6). The figure shows that as the MCV increased,

Ds decreased.

182 Nabila S. Selim et al. 12

Table 2

The average membrane solubilization (D50), solubilizing detergent concentration (Ds)

and complete solubilization (Dc) for control and irradiated groups

Dose (Gy) D50 (% v/v) Ds (% v/v) Dc (% v/v)

Control 6.34×10–3±1.90×10–4 4.64×10–3±3.45×10–4 8.38×10–3±1.59×10–4

1 5.85×10–3±3.25×10–4 3.66×10–3±3.44×10–4 8.24×10–3±5.60×10–4

2.5 5.38×10–3±2.24×10–4 3.44×10–3±2.20×10–4 7.46×10–3±2.21×10–4

3.5 5.36×10–3±5.42×10–5 3.35×10–3±1.05×10–4 7.42×10–3±2.05×10–4

5 5.11×10–3±3.81×10–4 3.33×10–3±1.67×10–4 7.08×10–3±1.41×10–4

7 4.59×10–3±3.01×10–4 3.23×10–3±1.67×10–4 6.67×10–3±3.34×10–4

9 4.47×10–3±2.66×10–4 2.91×10–3±1.21×10–4 6.35×10–3±1.71×10–4

2.5

3.0

3.5

4.0

4.5

5.0

0 1 2 3 4 5 6 7 8 9 10

Dose (Gy)

Ds x10-3 (% v/v)

100

110

120

MCV (f l)

Fig. 6. The solubilizing detergent concentration (Ds) (♦) and mean corpuscular volume

(MCV) (▲) for control and irradiated groups.

DISCUSSION

Gamma radiation affects biological membrane in different ways. To study

and monitor the effects of radiation one needs a series of analyses to explore the

different damaging events that may occur. Normal red blood cells retain

intracellular hemoglobin. The hemoglobin release from incubated cells in saline

solution gives rough indication about membrane damage. Free radicals formed

during irradiation can cause a variety of membrane changes including lipid

13 Gamma radiation and biophysical properties of RBCs

183

peroxidation, hydrolysis of phospholipids head groups, lipid-lipid crosslinks,

disulfide bridge formation and amino acid residue damage in membrane proteins

and lipid-protein crosslinks [11]. Changes in membrane structures can also affect

the cytoskeleton. The combined effects of free radicals on the red blood cell

membrane and cytoskeleton may contribute to the leak of hemoglobin out of the

cells. The hemolysis of the red blood cells reflects the loss of integrity of the cells

which can lead to the liberation of intracellular hemoglobin [18]. In addition,

ionizing radiation was reported to cause oxidation of the sulphydryl groups to the

corresponding dithiols and induce conformational changes of membrane proteins [8].

The AC conductivity, in the β-dispersion region, gives information about the

amount of freely movable ions inside cells, permeability properties of the lipid

bilayer, protein-mediated transport processes, and existence of bound charges on

the external and internal membrane surfaces. It describes the physico-chemical

effects occurring not only within the membrane, but also near the membrane

surface, where the protein organization appears to be one of the important factors

in the functioning and in the chemical reactions with the ionic environment [4].

The conductivity of red blood cells was shown to be sensitive to gamma radiation,

and this effect is enhanced by the presence of hemoglobin molecule [3]. The

decrease in conductivity observed in our study reflects the permeability damage of

the cell membrane with the subsequent loss of ions, electrolytes and intracellular

components.

Radiation was, also, shown to affect the biochemical structure of the red

blood cells membrane. It increases membrane cholesterol level, causes oxidation of

membrane protein, thiol groups and lipid peroxidation, and impairment of

membrane permeability barrier [20]. Radiation-induced changes in permeability

and membrane elasticity were examined by the osmotic fragility test. Osmotic

fragility is considered to be a function of the osmotic pressure gradient between

intra and extracellular media, initial surface area to volume ratio, membrane

tension of hemolysis and ionic content of the cell [1]. The observed decrease in the

dispersion of hemolysis (S) can be attributed to the presence of unusually flattened

red cells in which the surface area to volume ratio is increased [10].

It has been reported that alteration in the lipid composition of the red blood

cell membrane has only minor effects on the mechanical behavior, whereas

alterations in membrane skeletal proteins play a major role [14, 15]. Oxygen free

radicals also alter cation permeability and reduce red blood cells deformability,

disturbance in microrheological properties of red blood cell membrane (increase

membrane rigidity) [2]. Hence, the change induced in the osmotic fragility reflects

the damaging effects on the cytoskeleton. Our results show that the fragility of red

blood cell membrane increased significantly as a function of dose increase. This

appears from the increase in the rate of hemolysis (A), the maximum rate of

hemolysis (P) reached by each group. Also, the average osmotic hemolysis (H50)

and the center of the Gaussian peak (C) which shifted toward higher NaCl

concentration.

184 Nabila S. Selim et al. 14

The radiation induced damage in the membrane permeability can facilitate

the diffusion of detergent molecule within the cell membrane as we can see from

the average membrane solubilization (D50). The mean corpuscular volume (MCV)

showed a significant increase as the dose increased (Fig. 6), and the decrease in the

dispersion of hemolysis reflects the presence of unusually flattened cells (i.e. the

well-defined discoid shape vanished) as we have reported previously. The change

in the shape of red blood after exposure to 6 Gy gamma radiation altered cell

permeability, and developed echinocytes and spherocytes with the progressive

appearance of the regularly spaced spicules on cell surface [20]. The progressive

appearance of the regularly spaced spicules on cell surface can increase the

interaction of the detergent with membrane components and facilitate the

transformation phase, thus decreasing the solubilizing detergent concentration with

dose (Ds) as shown in Table 3. The decrease in the complete solubilization can be

attributed to the radiation induced damage in the red blood cells membrane, which

facilitate the membrane interaction and decrease the detergent concentration

needed to solubilize the membrane completely as can be shown from the shift in

the membrane solubilization curve toward lower detergent concentration (Fig. 5).

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