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The hemolysis kinetics of psoriatic red blood cells
Blood Cells, Molecules, and Diseases 41 (2008) 154–157
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Blood Cells, Molecules, and Diseases j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y b c m d
The hemolysis kinetics of psoriatic red blood cells A. Górnicki ⁎ Department of Biophysics, Collegium Medicum Nicolaus Copernicus University, Jagiellońska 13, 85-067 Bydgoszcz, Poland
a r t i c l e
i n f o
Article history: Submitted 9 March 2007 Revised 18 January 2008 Available online 3 June 2008 (Communicated by B. Bull, M.D., 7 April 2008) Keywords: Psoriasis Red blood cells Cell membrane Osmotic hemolysis
a b s t r a c t Psoriasis has been reported to be associated with several red blood cell (RBC) membrane alterations including: a membrane fluidity decrease, a significant elevation of Na+-K+and quantitative changes of erythrocyte membrane proteins that may indicate red cell cytoskeleton impairment. The aim of the present study was to analyse the hemolytic behaviour of psoriatic RBCs. The osmotic behaviour of RBCs was examined by analysing the kinetics of hemolysis. The method is based on the measurement of the transmitted light (λ = 700 nm) scattered by a suspension of red blood cells subjected to osmotic stress in the stopped-flow regime. The transmittance as a function of time, which describes the lysis kinetics, can be satisfactorily fitted with a mathematical model which assumes three cell populations in each sample: cells that do not lyse in the experimental conditions and cells that undergo fast and slow lysis. A comparison of the erythrocyte hemolytic kinetics of blood samples from psoriatic patients and healthy subjects showed distinct differences. The fraction of hemolyzed erythrocytes for control samples was about 20%; for psoriatic ones 12.6% (P b 0.001). In control blood samples the fraction of fast-breaking cells was greater (about 61% of lysed cells) than in psoriatic ones (about 56%). The parameter Tfast, describing the time of fast kinetics and Tslow, which reflects the rupturing time of cells belonging to the fraction of slow hemolysing cells were significantly higher for psoriatic erythrocytes than for control cells (P b 0.001). It was shown that the psoriatic erythrocyte has a low propensity for hemolysis and that its plasma membrane is distinctly more resistant to osmotic stress, probably related to decreased bilayer fluidity and low cell deformability. The results of this study showed that the kinetics of hemolysis may be a promising method for detecting erythrocytes with defective plasma membrane components and/or defective cytoskeleton organization and indirectly can provide some information on cell function. © 2008 Elsevier Inc. All rights reserved.
Introduction Psoriasis is linked to plasma membrane alterations in different types of cells and that pathological changes are not limited only to the cell membrane of keratinocytes [1–4]. Psoriasis is associated with several red blood cell (RBC) membrane alterations such as: membrane fluidity decrease [2–4], significant elevation of Na+-K+pump activity [5] and quantitative changes of erythrocyte membrane proteins, especially spectrin deficiency and increase in band 4 protein [6]. These changes may indicate red cell cytoskeleton impairment. Our previous results revealed that the decrease of membrane fluidity corresponded with exacerbation of skin lesions [4]. The biochemical composition and molecular organization of erythrocyte membranes are strictly related to their biophysical properties. Modifications in composition or molecular organization can affect rheological properties, such as cell deformability [7]. In fact, we found that in psoriatic erythrocytes the main erythrocyte rheology parameter-erythrocyte deformability
⁎ Fax: +48 52 5853308. E-mail address: [email protected]. 1079-9796/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2008.04.003
was affected [8]. Among different factors involved in RBC deformability, membrane fluidity has been assigned a major role [7,9]. The aim of the present study is to analyse the hemolitic behaviour of psoriatic RBCs. Using a variety of methods based on hemolysis one may obtain some information concerning cell membrane stability and flexibility that reflects erythrocyte deformability. Several hemolysis tests, e.g., estimation of the osmotic fragility and the autohemolysis tests have been used as the diagnostic standard for many years [10]. The osmotic fragility test is a commonly used technique to detect changes in the erythrocyte shape and membrane flexibility as well as being a rough index of red cell surface area-to-volume ratio [11]. Membrane rigidity (flexibility) is therefore considered to be one of the determinants of red cell osmotic fragility. On the other hand, flexibility of the cell membrane is also one of the major factors determining erythrocyte deformability [12]. Therefore, analysis of the osmotic behaviour of RBCs can provide information concerning red cell deformability. In the present experiment RBC deformability has been measured by osmotic gradient ektacytometry [13,14]. This technique allows cell deformability to be measured in response to applied shear stress as a function of medium osmolality. As was shown by Clark et al. [13] the measurement of the whole cell deformability in isotonic and various hypotonic media by the use of laser diffractometry is a
A. Górnicki / Blood Cells, Molecules, and Diseases 41 (2008) 154–157
155
sensitive method capable of detecting alterations in surface area-tovolume ratio. The osmotic behaviour of RBCs was examined by analysing the red cell kinetics of hemolysis. The method is based on the measurement of the transmitted light (λ = 700 nm) scattered by a suspension of red blood cells, while the cells are exposed to osmotic stress in the stopped-flow regime [15]. Material and methods Preparation of blood samples Venous blood samples were obtained from a group of 23 patients with moderate psoriasis aged 46 ± 9 years and from a control group of 45 healthy donors from a Blood Centre aged 40 ± 11 years. The samples were drawn in sterile vacuum tubes containing anticoagulant (K3EDTA). RBCs were separated from the blood by centrifugation at 1500 ×g for 15 min. and then washed three times with PBS buffered saline (pH 7.4). Each sample of washed erythrocytes was prepared for further experiments.
Fig. 2. The average values ± SD of parameters A, B and C for control and psoriatic samples. ⁎Values statistically significant in comparison with control RBCs, P b 0.001.
Kinetics of hemolysis Washed RBCs were diluted in PBS buffer to obtain a cell suspension with hematocrit of 0.1%. The cell suspension was mixed with an equal amount of distilled water, and kinetics of erythrocyte lysis was measured in a home-made stopped-flow device by continuous monitoring light transmittance at 700 nm [15]. Each experiment was repeated five times. The transmittance as a function of time, which describes the lysis kinetics, can be satisfactorily fitted with a mathematical model which assumes three cell populations in each sample: cells that do not lyse in the experimental conditions and cells which undergo fast and slow lysis [15,16]:
In experimental conditions, the light absorbance of lysed erythrocytes is very low. In these conditions, most of the absorbance is due to light scattering which is linearly related to the volume of intact erythrocytes and decreases dramatically during cell lysis. It was shown [15] that there is a good correlation between the final transmittance and the number of surviving cells. So, the final value of transmittance (after 45 sec.) indicates the extent of sample hemolysis and can be used to evaluate the size of red cell population that remained intact. Osmotic deformability studies
where t is time. Parameter A can be correlated with the extent of final haemolysis, since not all erythrocytes are lysed in the experimental conditions. The value of this parameter can be correlated with the overall propensity of the blood sample to hemolysis [15]. The fast kinetic component is described by the exponential B⁎exp(-t/Tfast), where parameter B reflects the number of cells that undergo fast lysis, whereas parameter Tfast describes the time required for lysis. Similarly, the slow kinetic component is described by the exponential C⁎exp(-t/ Tslow), where parameter C reflects the number of cells that undergo slow lysis and Tslow is the time required for lysis.
RBCs were suspended in a solution of 25% Dextran 40 at a hematocrit of approximately 0.5%. The medium used to dissolve the dextran was PBS in which the concentration of NaCl was adjusted to give the desired final osmolality. The viscosity of the dextran solutions was about 24 mPa⁎s. All measurements were carried out at room temperature. Each sample of RBC, high viscosity solution was pushed by a syringe pump through the narrow (d = 0.1 mm) long (l = 15 cm) flowchannel, at a constant shear stress of 17 Pa, the laser beam passed through the flow-channel perpendicularly. The diffracted laser beam was projected onto a screen and photographed with a CCD camera, giving the opportunity to select circles or ellipsoids of equal light intensity. A computer program written in LabView allowed to us to
Fig. 1. A comparison between the measured dependences of transmittance on time for the control and psoriatic sample.
Fig. 3. The time of fast (Tfast) and slow (Tslow) kinetics of hemolysis (average values ± SD) for control and psoriatic samples. Values statistically significant in comparison with control RBCs, ⁎P b 0.05, ⁎⁎P b 0.001.
TðtÞ ¼ A B⁎expðt=Tfast Þ C⁎expðt=Tslow Þ;
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A. Górnicki / Blood Cells, Molecules, and Diseases 41 (2008) 154–157
distinguish isointensity lines of diffraction images and computed deformability index (DI) expressed as a ratio of the difference between the major and minor axes of the ellipse to their sum. This method allowed us to determine RBC elongation index with accuracy of less than 0.5% [17]. Statistical analysis The data are expressed as means ± S.D. The statistical analysis used the unpaired Student's t-test. The value P b 0.05 was considered as significant. Results Selected examples of the experimentally-derived dependence of transmittance on time for control and psoriatic samples are presented in Fig. 1. The value of parameter A for psoriatic erythrocytes was lower than for control cells (Fig. 2). The fraction of hemolyzed erythrocytes for control samples was about 20% and for psoriatic ones 12.6%. The difference was statistically significant (P b 0.001). The fast kinetic component described by the exponential B⁎exp(-t/Tfast) of psoriatic erythrocytes was significantly different from control samples. The average values of parameters B and Tfast are presented in Figs. 2 and 3, respectively. Control blood samples showed higher values of B than psoriatic ones and revealed that about 61% of lysed cells were contributing to the fraction of fast-breaking cells. In the population of psoriatic erythrocytes about 56% of cells belonged to this fraction. The parameter Tfast, describing the time of fast kinetics, was significantly higher for psoriatic erythrocytes than for control cells (P b 0.01) (Fig. 3). The value of Tfast for psoriatic samples was very high in relation to the small number of fast hemolysing cells under experimental conditions (as parameter B describes) reached only 7.1%. The number of fast-breaking cells was higher (12.2%) and Tfast was lower for control samples than for psoriatic ones. Parameters describing the slow kinetics (C and Tslow) are presented in Figs. 2 and 3. The fraction of slow rupturing cells for psoriatic samples (5.5% of cells in the sample) was significantly lower in comparison with control samples (about 8%). However, the parameter Tslow which reflects the rupturing time of cells belonging to the fraction of slow hemolysing cells was demonstrably higher for psoriatic erythrocytes as compared to controls (Fig. 3). For psoriatic erythrocytes the time Tslow similarly to the time Tfast is very high in relation to the small number of cells from the fraction of slowly lysing cells (as parameter C describes). The relationship obtained from normal and psoriatic red cells between deformability index (DI) and suspending medium osmolality
Fig. 4. Osmotic deformability profiles for control and psoriatic erythrocytes.
is shown in Fig. 4. As was also shown by other authors [13,14] the curve for normal cells riches a maximum near 290 mOsm/kg, suggesting that normal erythrocytes deform optimally at physiological plasma tonicity. The deformability remains at a relatively constant level between 290 and 250 mOsm/kg, and then DI begins to decline, reaching a minimum at approximately 135 mOsm/kg [14]. As the suspending medium osmolality was increased above 290 mOsm/kg, the DI again decreased. It can be seen from Fig. 4 that psoriatic RBCs showed distinct loss of deformability. It should be noted that there is a characteristic profile asymmetry, with a greater reduction in DI on the hypertonic part of the optimum deformation region of the osmoscan curve than on the hypotonic side. Moreover there was no difference in the minimum value of deformability index (DImin) between normal and psoriatic red cells. Discussion A comparison of the erythrocyte hemolytic kinetics of blood samples from psoriatic patients and healthy subjects showed distinct differences which can be quantitatively evaluated. The fitting of experimental data to a mathematical model [15,16] allowed us to estimate the fraction of cells that undergo lysis (parameters A, B and C) and/or the haemolytic properties of the cell plasma membrane (parameters Tfast and Tslow). The value of parameter A can be correlated with overall blood sample propensity to hemolysis and expresses the extent of final hemolysis [15]. Not all erythrocytes are lysed in the experimental conditions. Therefore, erythrocytes both from normal and psoriatic blood samples consist of two populations: a small one which lyses in 75 mM salt solution and a dominant one which sustains such conditions intact. As was shown in the present study, the fraction of hemolysed erythrocytes from psoriatic patients was significantly lower in comparison with the number of hemolysed control red cells. This suggests that psoriatic erythrocytes have lower propensity to hemolysis and that the psoriatic red cell plasma membrane is more resistant to osmotic stress. It is not clear whether the changes in the properties of a psoriatic red cell membrane influence erythrocyte osmotic fragility. RochaPereira et al. [18] demonstrated that psoriatic erythrocytes are less resistant to the osmotic stress than normal control cells. On the other hand, Roder [19] reported a significant lowering of the red cell osmotic fragility in psoriatic patients. The results of the present study seem to support these findings. Roder [19] suggests that increased psoriatic red cell membrane osmotic resistance is a result of the disordered metabolism and liver disfunction. On the other hand, the decreased osmotic fragility may result from the altered red cell membrane cation metabolism. An impaired membrane permeability will result in the osmotic imbalance between the cell and the medium. It was shown that the enhanced monovalent cation permeability resulted in the increased osmotic resistance in human erythrocytes [20]. It was shown that abnormalities in the monovalent cation membrane transport are one of the alterations observed in red cells of psoriatic patients [5]. This resulted in a significant increase in intracellular K+content and increased Na+influx. It was demonstrated that the K+uptake is significantly affected by the fluidity of the cell membrane. In addition, the increased K+uptake is closely associated with the increased osmotic resistance [20]. The population of hemolysing cells consists of two fractions: a fastlysing cell population and a fraction of slow rupturing cells. The size of these fractions is described by parameters B and C, respectively. Fitting of experimental data to the mathematical model revealed that psoriatic blood samples showed relatively low values of parameter B, indicating that about 54% of lysed cells were contributing to the fraction of fast-lysing cells. The same parameter for control samples
A. Górnicki / Blood Cells, Molecules, and Diseases 41 (2008) 154–157
revealed that the majority of hemolysed cells belonged to this fraction (61%). The difference is even larger considering that a large fraction of psoriatic cells was not lysed at all. This indicates differences in plasma membrane properties between control and psoriatic erythrocytes. In the mathematical model describing kinetics of hemolysis, red cell membrane properties are characterised by parameters Tfast and Tslow. The time of blood sample osmotic hemolysis depends on the number of lysed cells and the haemolytic properties of cell membrane. As was shown [15] Tfast can be associated with plasma membrane properties responsible for the progress of hemolysis in the fraction of fast rupturing cells. Similarly Tslow describes membrane properties of cells belonging to the population with slow kinetics of hemolysis. Measurements of deformability as a function of the osmolality of the suspending medium showed that the minimum value of deformability index (DImin) for psoriatic red cells was not shifted to higher osmolalities in relation to DImin for control erythrocytes. Clark et al. [13] showed that the hypotonic osmolality at which DImin occurs provides a measure of the average surface area-to-volume ratio of the cell population. This indicates that in psoriatic red cells the surface area-to-volume ratio is unaltered. When the geometry of erythrocyte is unchanged, one of the most important properties of erythrocyte membrane bilayer related to erythrocyte osmotic fragility is membrane fluidity [21]. Araki and Rifkind [21] showed a good correlation between the changes in the rate of hemolysis and the lipid matrix fluidity; the decrease of membrane fluidity corresponds to the lowering of the relative rate of hemolysis. A number of experiments concerning membrane fluidity of erythrocyte in psoriasis univocally indicate fluidity decrease [2–4,8]. This fact may explain extremely high values of Tfast and Tslow for psoriatic red cells in relation to the number of fast and slow rupturing cells, respectively. The fact that the values of Tslow are higher than those of Tfast, both for control and psoriatic samples, suggests that the red cells from the fraction subjected to slow kinetics of hemolysis have lower membrane fluidity values than the cells belonging to the fraction of fast breaking erythrocytes. The decrease in the rate of hemolysis may also be associated with a decrease of erythrocyte membrane deformability [21]. The results of the present study indicate that this phenomenon can also be observed in the case of psoriasis. Measurements of psoriatic RBC deformability as a function of medium osmolality showed a distinct decrease in deformability, with the characteristic profile asymmetry in the optimum deformation region. Clark et al. [13] showed that this kind of osmotic deformability profile, with the depression of the maximum attainable DI and the asymmetry of the curve around its maximum, is characteristic for red cells with reduced membrane flexibility. This finding is also supported by our recent results on the mechanical properties of the erythrocyte membrane in psoriatic patients [8].We observed a significant decrease in erythrocyte deformability in psoriatic red blood cells in comparison with controls, which can be attributed to alterations in intracellular viscosity and membrane viscoelastic behaviour. Conclusions The results of this study showed that the kinetics of hemolysis may be a promising method for detecting erythrocytes with defective
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plasma membrane components and/or cytoskeleton. The measurement of this phenomenon can not only be informative with regard to the extent of membrane disturbance but also with regard to cell functioning, since changes of the membrane structure and properties result in the alteration of cell metabolism [18,22]. Furthermore, the biochemical composition and molecular organization of erythrocyte membranes are strictly related to their biophysical properties, such as osmotic behaviour and cell deformability [23]. References [1] L.M. DiCicco, J.E. Fraki, J.N. Mansbridge, The plasma membrane in psoriasis, Int. J. Dermatol. 26 (1987) 631–638. [2] G. Ferretti, A.M. Offidani, O. Simonetti, et al., Changes in membrane properties of erythrocytes and polymorphonuclear cells in psoriasis, Biochem. Med. Metab. Biol. 41 (1989) 132–138. [3] A.M. Offidani, O. Simonetti, G. Ferretti, et al., Membrane fluidity changes in polymorphonuclear leukocytes and erythrocytes in psoriasis, Acta Derm.–Venereol., (Stockh) Suppl. 146 (1989) 39–41. [4] A. Górnicki, A. Gutsze, Erythrocyte membrane fluidity changes in psoriasis: an EPR study, J. Dermatol. Sci. 27 (2001) 27–30. [5] M.G. Mozzato, A. Semplicini, G. Grosso, et al., Red blood cell membrane cation transport in normotensive psoriatics, Acta Derm.–Venereol. (Stockh) Suppl. 146 (1989) 45–47. [6] M.S. Goncharenko, G.A. Andrukh, V.V. Ryazantsev, The protein spectrum of human erythrocyte membranes in normal conditions and in psoriasis, Vestn. Dermatol. Venerol. 3 (1989) 4–7. [7] A. Chabanel, K. Flamm, K.L.P. Sung, et al., Influence of cholesterol content in red cell membrane viscoelasticity and fluidity, Biophys. J. 44 (1983) 171–176. [8] A. Górnicki, Changes in erythrocyte microrheology in patients with psoriasis, Clin. Exp. Dermatol. 29 (2004) 67–70. [9] E. Ademoglu, S. Tamer, I. Albeniz, et al., Cyclosporin A-associated changes in red blood cell membrane composition, deformability, blood and plasma viscosity in rats, Acta Haematol. 112 (2004) 184–188. [10] H.C. Godal, A.T. Elde, N. Nyborg, et al., The normal range of osmotic fragility of red blood cells, Scand. J. Haematol. 25 (1980) 107–112. [11] J. Stuart, G.B. Nash, Red cell deformability and haematological disorders, Blood Rev. 4 (1990) 141–147. [12] M.A. Srour, Y.Y. Bilto, M. Juma, et al., Exposure of human erythrocytes to oxygen radicals causes loss of deformability, increased osmotic fragility, lipid peroxidation and protein degradation, Clin. Hemorheol. Microcirc. 23 (2000) 13–21. [13] M.R. Clark, N. Mohandas, S.B. Shohet, Osmotic gradient ektacytometry: Comprehensive characterization of red cell volume and surface maintenance, Blood 61 (1983) 899–910. [14] N. Mohandas, M.R. Clark, M.S. Jacobs, S.B. Shohet, Analysis of factors regulating erythrocyte deformability, J. Clin. Invest. 66 (1980) 563–573. [15] G. Pazdzior, M. Langner, A. Chmura, et al., The kinetics of haemolysis of spherocytic erythrocytes, Cell. Mol. Biol. Lett. 8 (2003) 639–648. [16] P.M. Didelon, S. Muller, J.F. Stoltz, Osmotic fragility of the erythrocyte membrane: characterization by modelling of the transmittance curve as a function of the NaCl concentration, Biorheology 37 (2000) 409–416. [17] A. Górnicki, A. Kempczyński, Measurement of erythrocyte deformability by the flow-channel diffraction method, Proc SPIE 5945 (2004) K 1–K 4. [18] P. Rocha-Pereira, A. Santos-Silva, I. Rebelo, et al., Erythrocyte damage in mild and severe psoriasis, Br. J. Dermatol. 150 (2004) 232–244. [19] H. Roder, Erythrozytenveranderung bei Psoriasis vulgaris, Dermatol. Mon.schr. 157 (1971) 645–647. [20] P. Bognar, K. Sipos, A. Ludany, et al., Steady-state volumes and metabolismindependent osmotic adaptation in mammalian erythrocytes, Eur. Biophys. J. 31 (2002) 145–152. [21] K. Araki, J.M. Rifkind, The rate of osmotic hemolysis. A relationship with membrane bilayer fluidity, Biochim. Biophys. Acta 645 (1981) 81–90. [22] R. Alleva, G. Ferretti, B. Borghit, et al., Physico-chemical properties of membranes of recovered erythrocytes in blood autologous transfusion: A study using fluorescence technique, Transfus. Sci. 16 (1995) 291–297. [23] A. Chabanel, K. Flamm, K.L.P. Sung, et al., Influence of cholesterol content in red cell membrane viscoelasticity and fluidity, Biophys. J. 44 (1983) 171–176.
Contents lists available at ScienceDirect
Blood Cells, Molecules, and Diseases j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y b c m d
The hemolysis kinetics of psoriatic red blood cells A. Górnicki ⁎ Department of Biophysics, Collegium Medicum Nicolaus Copernicus University, Jagiellońska 13, 85-067 Bydgoszcz, Poland
a r t i c l e
i n f o
Article history: Submitted 9 March 2007 Revised 18 January 2008 Available online 3 June 2008 (Communicated by B. Bull, M.D., 7 April 2008) Keywords: Psoriasis Red blood cells Cell membrane Osmotic hemolysis
a b s t r a c t Psoriasis has been reported to be associated with several red blood cell (RBC) membrane alterations including: a membrane fluidity decrease, a significant elevation of Na+-K+and quantitative changes of erythrocyte membrane proteins that may indicate red cell cytoskeleton impairment. The aim of the present study was to analyse the hemolytic behaviour of psoriatic RBCs. The osmotic behaviour of RBCs was examined by analysing the kinetics of hemolysis. The method is based on the measurement of the transmitted light (λ = 700 nm) scattered by a suspension of red blood cells subjected to osmotic stress in the stopped-flow regime. The transmittance as a function of time, which describes the lysis kinetics, can be satisfactorily fitted with a mathematical model which assumes three cell populations in each sample: cells that do not lyse in the experimental conditions and cells that undergo fast and slow lysis. A comparison of the erythrocyte hemolytic kinetics of blood samples from psoriatic patients and healthy subjects showed distinct differences. The fraction of hemolyzed erythrocytes for control samples was about 20%; for psoriatic ones 12.6% (P b 0.001). In control blood samples the fraction of fast-breaking cells was greater (about 61% of lysed cells) than in psoriatic ones (about 56%). The parameter Tfast, describing the time of fast kinetics and Tslow, which reflects the rupturing time of cells belonging to the fraction of slow hemolysing cells were significantly higher for psoriatic erythrocytes than for control cells (P b 0.001). It was shown that the psoriatic erythrocyte has a low propensity for hemolysis and that its plasma membrane is distinctly more resistant to osmotic stress, probably related to decreased bilayer fluidity and low cell deformability. The results of this study showed that the kinetics of hemolysis may be a promising method for detecting erythrocytes with defective plasma membrane components and/or defective cytoskeleton organization and indirectly can provide some information on cell function. © 2008 Elsevier Inc. All rights reserved.
Introduction Psoriasis is linked to plasma membrane alterations in different types of cells and that pathological changes are not limited only to the cell membrane of keratinocytes [1–4]. Psoriasis is associated with several red blood cell (RBC) membrane alterations such as: membrane fluidity decrease [2–4], significant elevation of Na+-K+pump activity [5] and quantitative changes of erythrocyte membrane proteins, especially spectrin deficiency and increase in band 4 protein [6]. These changes may indicate red cell cytoskeleton impairment. Our previous results revealed that the decrease of membrane fluidity corresponded with exacerbation of skin lesions [4]. The biochemical composition and molecular organization of erythrocyte membranes are strictly related to their biophysical properties. Modifications in composition or molecular organization can affect rheological properties, such as cell deformability [7]. In fact, we found that in psoriatic erythrocytes the main erythrocyte rheology parameter-erythrocyte deformability
⁎ Fax: +48 52 5853308. E-mail address: [email protected]. 1079-9796/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2008.04.003
was affected [8]. Among different factors involved in RBC deformability, membrane fluidity has been assigned a major role [7,9]. The aim of the present study is to analyse the hemolitic behaviour of psoriatic RBCs. Using a variety of methods based on hemolysis one may obtain some information concerning cell membrane stability and flexibility that reflects erythrocyte deformability. Several hemolysis tests, e.g., estimation of the osmotic fragility and the autohemolysis tests have been used as the diagnostic standard for many years [10]. The osmotic fragility test is a commonly used technique to detect changes in the erythrocyte shape and membrane flexibility as well as being a rough index of red cell surface area-to-volume ratio [11]. Membrane rigidity (flexibility) is therefore considered to be one of the determinants of red cell osmotic fragility. On the other hand, flexibility of the cell membrane is also one of the major factors determining erythrocyte deformability [12]. Therefore, analysis of the osmotic behaviour of RBCs can provide information concerning red cell deformability. In the present experiment RBC deformability has been measured by osmotic gradient ektacytometry [13,14]. This technique allows cell deformability to be measured in response to applied shear stress as a function of medium osmolality. As was shown by Clark et al. [13] the measurement of the whole cell deformability in isotonic and various hypotonic media by the use of laser diffractometry is a
A. Górnicki / Blood Cells, Molecules, and Diseases 41 (2008) 154–157
155
sensitive method capable of detecting alterations in surface area-tovolume ratio. The osmotic behaviour of RBCs was examined by analysing the red cell kinetics of hemolysis. The method is based on the measurement of the transmitted light (λ = 700 nm) scattered by a suspension of red blood cells, while the cells are exposed to osmotic stress in the stopped-flow regime [15]. Material and methods Preparation of blood samples Venous blood samples were obtained from a group of 23 patients with moderate psoriasis aged 46 ± 9 years and from a control group of 45 healthy donors from a Blood Centre aged 40 ± 11 years. The samples were drawn in sterile vacuum tubes containing anticoagulant (K3EDTA). RBCs were separated from the blood by centrifugation at 1500 ×g for 15 min. and then washed three times with PBS buffered saline (pH 7.4). Each sample of washed erythrocytes was prepared for further experiments.
Fig. 2. The average values ± SD of parameters A, B and C for control and psoriatic samples. ⁎Values statistically significant in comparison with control RBCs, P b 0.001.
Kinetics of hemolysis Washed RBCs were diluted in PBS buffer to obtain a cell suspension with hematocrit of 0.1%. The cell suspension was mixed with an equal amount of distilled water, and kinetics of erythrocyte lysis was measured in a home-made stopped-flow device by continuous monitoring light transmittance at 700 nm [15]. Each experiment was repeated five times. The transmittance as a function of time, which describes the lysis kinetics, can be satisfactorily fitted with a mathematical model which assumes three cell populations in each sample: cells that do not lyse in the experimental conditions and cells which undergo fast and slow lysis [15,16]:
In experimental conditions, the light absorbance of lysed erythrocytes is very low. In these conditions, most of the absorbance is due to light scattering which is linearly related to the volume of intact erythrocytes and decreases dramatically during cell lysis. It was shown [15] that there is a good correlation between the final transmittance and the number of surviving cells. So, the final value of transmittance (after 45 sec.) indicates the extent of sample hemolysis and can be used to evaluate the size of red cell population that remained intact. Osmotic deformability studies
where t is time. Parameter A can be correlated with the extent of final haemolysis, since not all erythrocytes are lysed in the experimental conditions. The value of this parameter can be correlated with the overall propensity of the blood sample to hemolysis [15]. The fast kinetic component is described by the exponential B⁎exp(-t/Tfast), where parameter B reflects the number of cells that undergo fast lysis, whereas parameter Tfast describes the time required for lysis. Similarly, the slow kinetic component is described by the exponential C⁎exp(-t/ Tslow), where parameter C reflects the number of cells that undergo slow lysis and Tslow is the time required for lysis.
RBCs were suspended in a solution of 25% Dextran 40 at a hematocrit of approximately 0.5%. The medium used to dissolve the dextran was PBS in which the concentration of NaCl was adjusted to give the desired final osmolality. The viscosity of the dextran solutions was about 24 mPa⁎s. All measurements were carried out at room temperature. Each sample of RBC, high viscosity solution was pushed by a syringe pump through the narrow (d = 0.1 mm) long (l = 15 cm) flowchannel, at a constant shear stress of 17 Pa, the laser beam passed through the flow-channel perpendicularly. The diffracted laser beam was projected onto a screen and photographed with a CCD camera, giving the opportunity to select circles or ellipsoids of equal light intensity. A computer program written in LabView allowed to us to
Fig. 1. A comparison between the measured dependences of transmittance on time for the control and psoriatic sample.
Fig. 3. The time of fast (Tfast) and slow (Tslow) kinetics of hemolysis (average values ± SD) for control and psoriatic samples. Values statistically significant in comparison with control RBCs, ⁎P b 0.05, ⁎⁎P b 0.001.
TðtÞ ¼ A B⁎expðt=Tfast Þ C⁎expðt=Tslow Þ;
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distinguish isointensity lines of diffraction images and computed deformability index (DI) expressed as a ratio of the difference between the major and minor axes of the ellipse to their sum. This method allowed us to determine RBC elongation index with accuracy of less than 0.5% [17]. Statistical analysis The data are expressed as means ± S.D. The statistical analysis used the unpaired Student's t-test. The value P b 0.05 was considered as significant. Results Selected examples of the experimentally-derived dependence of transmittance on time for control and psoriatic samples are presented in Fig. 1. The value of parameter A for psoriatic erythrocytes was lower than for control cells (Fig. 2). The fraction of hemolyzed erythrocytes for control samples was about 20% and for psoriatic ones 12.6%. The difference was statistically significant (P b 0.001). The fast kinetic component described by the exponential B⁎exp(-t/Tfast) of psoriatic erythrocytes was significantly different from control samples. The average values of parameters B and Tfast are presented in Figs. 2 and 3, respectively. Control blood samples showed higher values of B than psoriatic ones and revealed that about 61% of lysed cells were contributing to the fraction of fast-breaking cells. In the population of psoriatic erythrocytes about 56% of cells belonged to this fraction. The parameter Tfast, describing the time of fast kinetics, was significantly higher for psoriatic erythrocytes than for control cells (P b 0.01) (Fig. 3). The value of Tfast for psoriatic samples was very high in relation to the small number of fast hemolysing cells under experimental conditions (as parameter B describes) reached only 7.1%. The number of fast-breaking cells was higher (12.2%) and Tfast was lower for control samples than for psoriatic ones. Parameters describing the slow kinetics (C and Tslow) are presented in Figs. 2 and 3. The fraction of slow rupturing cells for psoriatic samples (5.5% of cells in the sample) was significantly lower in comparison with control samples (about 8%). However, the parameter Tslow which reflects the rupturing time of cells belonging to the fraction of slow hemolysing cells was demonstrably higher for psoriatic erythrocytes as compared to controls (Fig. 3). For psoriatic erythrocytes the time Tslow similarly to the time Tfast is very high in relation to the small number of cells from the fraction of slowly lysing cells (as parameter C describes). The relationship obtained from normal and psoriatic red cells between deformability index (DI) and suspending medium osmolality
Fig. 4. Osmotic deformability profiles for control and psoriatic erythrocytes.
is shown in Fig. 4. As was also shown by other authors [13,14] the curve for normal cells riches a maximum near 290 mOsm/kg, suggesting that normal erythrocytes deform optimally at physiological plasma tonicity. The deformability remains at a relatively constant level between 290 and 250 mOsm/kg, and then DI begins to decline, reaching a minimum at approximately 135 mOsm/kg [14]. As the suspending medium osmolality was increased above 290 mOsm/kg, the DI again decreased. It can be seen from Fig. 4 that psoriatic RBCs showed distinct loss of deformability. It should be noted that there is a characteristic profile asymmetry, with a greater reduction in DI on the hypertonic part of the optimum deformation region of the osmoscan curve than on the hypotonic side. Moreover there was no difference in the minimum value of deformability index (DImin) between normal and psoriatic red cells. Discussion A comparison of the erythrocyte hemolytic kinetics of blood samples from psoriatic patients and healthy subjects showed distinct differences which can be quantitatively evaluated. The fitting of experimental data to a mathematical model [15,16] allowed us to estimate the fraction of cells that undergo lysis (parameters A, B and C) and/or the haemolytic properties of the cell plasma membrane (parameters Tfast and Tslow). The value of parameter A can be correlated with overall blood sample propensity to hemolysis and expresses the extent of final hemolysis [15]. Not all erythrocytes are lysed in the experimental conditions. Therefore, erythrocytes both from normal and psoriatic blood samples consist of two populations: a small one which lyses in 75 mM salt solution and a dominant one which sustains such conditions intact. As was shown in the present study, the fraction of hemolysed erythrocytes from psoriatic patients was significantly lower in comparison with the number of hemolysed control red cells. This suggests that psoriatic erythrocytes have lower propensity to hemolysis and that the psoriatic red cell plasma membrane is more resistant to osmotic stress. It is not clear whether the changes in the properties of a psoriatic red cell membrane influence erythrocyte osmotic fragility. RochaPereira et al. [18] demonstrated that psoriatic erythrocytes are less resistant to the osmotic stress than normal control cells. On the other hand, Roder [19] reported a significant lowering of the red cell osmotic fragility in psoriatic patients. The results of the present study seem to support these findings. Roder [19] suggests that increased psoriatic red cell membrane osmotic resistance is a result of the disordered metabolism and liver disfunction. On the other hand, the decreased osmotic fragility may result from the altered red cell membrane cation metabolism. An impaired membrane permeability will result in the osmotic imbalance between the cell and the medium. It was shown that the enhanced monovalent cation permeability resulted in the increased osmotic resistance in human erythrocytes [20]. It was shown that abnormalities in the monovalent cation membrane transport are one of the alterations observed in red cells of psoriatic patients [5]. This resulted in a significant increase in intracellular K+content and increased Na+influx. It was demonstrated that the K+uptake is significantly affected by the fluidity of the cell membrane. In addition, the increased K+uptake is closely associated with the increased osmotic resistance [20]. The population of hemolysing cells consists of two fractions: a fastlysing cell population and a fraction of slow rupturing cells. The size of these fractions is described by parameters B and C, respectively. Fitting of experimental data to the mathematical model revealed that psoriatic blood samples showed relatively low values of parameter B, indicating that about 54% of lysed cells were contributing to the fraction of fast-lysing cells. The same parameter for control samples
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revealed that the majority of hemolysed cells belonged to this fraction (61%). The difference is even larger considering that a large fraction of psoriatic cells was not lysed at all. This indicates differences in plasma membrane properties between control and psoriatic erythrocytes. In the mathematical model describing kinetics of hemolysis, red cell membrane properties are characterised by parameters Tfast and Tslow. The time of blood sample osmotic hemolysis depends on the number of lysed cells and the haemolytic properties of cell membrane. As was shown [15] Tfast can be associated with plasma membrane properties responsible for the progress of hemolysis in the fraction of fast rupturing cells. Similarly Tslow describes membrane properties of cells belonging to the population with slow kinetics of hemolysis. Measurements of deformability as a function of the osmolality of the suspending medium showed that the minimum value of deformability index (DImin) for psoriatic red cells was not shifted to higher osmolalities in relation to DImin for control erythrocytes. Clark et al. [13] showed that the hypotonic osmolality at which DImin occurs provides a measure of the average surface area-to-volume ratio of the cell population. This indicates that in psoriatic red cells the surface area-to-volume ratio is unaltered. When the geometry of erythrocyte is unchanged, one of the most important properties of erythrocyte membrane bilayer related to erythrocyte osmotic fragility is membrane fluidity [21]. Araki and Rifkind [21] showed a good correlation between the changes in the rate of hemolysis and the lipid matrix fluidity; the decrease of membrane fluidity corresponds to the lowering of the relative rate of hemolysis. A number of experiments concerning membrane fluidity of erythrocyte in psoriasis univocally indicate fluidity decrease [2–4,8]. This fact may explain extremely high values of Tfast and Tslow for psoriatic red cells in relation to the number of fast and slow rupturing cells, respectively. The fact that the values of Tslow are higher than those of Tfast, both for control and psoriatic samples, suggests that the red cells from the fraction subjected to slow kinetics of hemolysis have lower membrane fluidity values than the cells belonging to the fraction of fast breaking erythrocytes. The decrease in the rate of hemolysis may also be associated with a decrease of erythrocyte membrane deformability [21]. The results of the present study indicate that this phenomenon can also be observed in the case of psoriasis. Measurements of psoriatic RBC deformability as a function of medium osmolality showed a distinct decrease in deformability, with the characteristic profile asymmetry in the optimum deformation region. Clark et al. [13] showed that this kind of osmotic deformability profile, with the depression of the maximum attainable DI and the asymmetry of the curve around its maximum, is characteristic for red cells with reduced membrane flexibility. This finding is also supported by our recent results on the mechanical properties of the erythrocyte membrane in psoriatic patients [8].We observed a significant decrease in erythrocyte deformability in psoriatic red blood cells in comparison with controls, which can be attributed to alterations in intracellular viscosity and membrane viscoelastic behaviour. Conclusions The results of this study showed that the kinetics of hemolysis may be a promising method for detecting erythrocytes with defective
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plasma membrane components and/or cytoskeleton. The measurement of this phenomenon can not only be informative with regard to the extent of membrane disturbance but also with regard to cell functioning, since changes of the membrane structure and properties result in the alteration of cell metabolism [18,22]. Furthermore, the biochemical composition and molecular organization of erythrocyte membranes are strictly related to their biophysical properties, such as osmotic behaviour and cell deformability [23]. References [1] L.M. DiCicco, J.E. Fraki, J.N. Mansbridge, The plasma membrane in psoriasis, Int. J. Dermatol. 26 (1987) 631–638. [2] G. Ferretti, A.M. Offidani, O. Simonetti, et al., Changes in membrane properties of erythrocytes and polymorphonuclear cells in psoriasis, Biochem. Med. Metab. Biol. 41 (1989) 132–138. [3] A.M. Offidani, O. Simonetti, G. Ferretti, et al., Membrane fluidity changes in polymorphonuclear leukocytes and erythrocytes in psoriasis, Acta Derm.–Venereol., (Stockh) Suppl. 146 (1989) 39–41. [4] A. Górnicki, A. Gutsze, Erythrocyte membrane fluidity changes in psoriasis: an EPR study, J. Dermatol. Sci. 27 (2001) 27–30. [5] M.G. Mozzato, A. Semplicini, G. Grosso, et al., Red blood cell membrane cation transport in normotensive psoriatics, Acta Derm.–Venereol. (Stockh) Suppl. 146 (1989) 45–47. [6] M.S. Goncharenko, G.A. Andrukh, V.V. Ryazantsev, The protein spectrum of human erythrocyte membranes in normal conditions and in psoriasis, Vestn. Dermatol. Venerol. 3 (1989) 4–7. [7] A. Chabanel, K. Flamm, K.L.P. Sung, et al., Influence of cholesterol content in red cell membrane viscoelasticity and fluidity, Biophys. J. 44 (1983) 171–176. [8] A. Górnicki, Changes in erythrocyte microrheology in patients with psoriasis, Clin. Exp. Dermatol. 29 (2004) 67–70. [9] E. Ademoglu, S. Tamer, I. Albeniz, et al., Cyclosporin A-associated changes in red blood cell membrane composition, deformability, blood and plasma viscosity in rats, Acta Haematol. 112 (2004) 184–188. [10] H.C. Godal, A.T. Elde, N. Nyborg, et al., The normal range of osmotic fragility of red blood cells, Scand. J. Haematol. 25 (1980) 107–112. [11] J. Stuart, G.B. Nash, Red cell deformability and haematological disorders, Blood Rev. 4 (1990) 141–147. [12] M.A. Srour, Y.Y. Bilto, M. Juma, et al., Exposure of human erythrocytes to oxygen radicals causes loss of deformability, increased osmotic fragility, lipid peroxidation and protein degradation, Clin. Hemorheol. Microcirc. 23 (2000) 13–21. [13] M.R. Clark, N. Mohandas, S.B. Shohet, Osmotic gradient ektacytometry: Comprehensive characterization of red cell volume and surface maintenance, Blood 61 (1983) 899–910. [14] N. Mohandas, M.R. Clark, M.S. Jacobs, S.B. Shohet, Analysis of factors regulating erythrocyte deformability, J. Clin. Invest. 66 (1980) 563–573. [15] G. Pazdzior, M. Langner, A. Chmura, et al., The kinetics of haemolysis of spherocytic erythrocytes, Cell. Mol. Biol. Lett. 8 (2003) 639–648. [16] P.M. Didelon, S. Muller, J.F. Stoltz, Osmotic fragility of the erythrocyte membrane: characterization by modelling of the transmittance curve as a function of the NaCl concentration, Biorheology 37 (2000) 409–416. [17] A. Górnicki, A. Kempczyński, Measurement of erythrocyte deformability by the flow-channel diffraction method, Proc SPIE 5945 (2004) K 1–K 4. [18] P. Rocha-Pereira, A. Santos-Silva, I. Rebelo, et al., Erythrocyte damage in mild and severe psoriasis, Br. J. Dermatol. 150 (2004) 232–244. [19] H. Roder, Erythrozytenveranderung bei Psoriasis vulgaris, Dermatol. Mon.schr. 157 (1971) 645–647. [20] P. Bognar, K. Sipos, A. Ludany, et al., Steady-state volumes and metabolismindependent osmotic adaptation in mammalian erythrocytes, Eur. Biophys. J. 31 (2002) 145–152. [21] K. Araki, J.M. Rifkind, The rate of osmotic hemolysis. A relationship with membrane bilayer fluidity, Biochim. Biophys. Acta 645 (1981) 81–90. [22] R. Alleva, G. Ferretti, B. Borghit, et al., Physico-chemical properties of membranes of recovered erythrocytes in blood autologous transfusion: A study using fluorescence technique, Transfus. Sci. 16 (1995) 291–297. [23] A. Chabanel, K. Flamm, K.L.P. Sung, et al., Influence of cholesterol content in red cell membrane viscoelasticity and fluidity, Biophys. J. 44 (1983) 171–176.