Osmotic fragility and surf ace ultrastructure of red blood cells exposed hypotonic stress in MgCl2 and NaCl solutions

Osmotic fragility and surf ace ultrastructure of red blood cells exposed hypotonic stress in MgCl2 and NaCl solutions
G. Zahavil), R. GilgaP), I. ZahavP) and M. DjaldettP)
Zusammenfassung Die osmotische Fragilitlit peripherer men­ schlicher Erythrozyten wurde mittels Fra­ gilograph und Spektrophotometer unter hypotonischer Belastung in MgClr und NaCl-Losungen studiert. Beide MeB· methoden zeigten, daB bei vergleichbaren molaren Konzentrationen die Hlimolyse in Gegenwart von MgCI2 langsamer er­ folgte als in NaCl-Losung. Dieses Phano­ men kann darauf beruhen, daB Mg-Ion en langsamer durch die Erythrozyten-Mem­ bran penetrieren. Untersuchungen der Oberflachen-Ultrastruktur mittels elektro­ nenmikroskopischen Scannings zeigten, daB in beiden Medien die Zellen ihre zentrale Eindellung beim Anschwellen verloren und die Gestalt von Krenozyten und schlieBiich von Ghost·Zellen annah­ men. Diese Alterationen wurden friiher bei Verwendung von hypotoner MgClr Losung beobachtet. Erklarungsmogli­ chkeiten fiir die Diskrepanz zwischen langsamer einsetzender Hlimolyse und friiherem Auftreten morphologischer Veranderungen in hypotoner MgC12-
Losung werden diskutiert.
Summary The osmotic fragility of human peripher· al red blood cells (RBC) exposed to hy· potonic stress in MgC12 and NaCI solu­ tions was examined with a fragiligraph and a spectrophotometer. By both meth­ ods the hemolysis of RBC incubated in hypotonic MgCh solution proceeded slower than that of RBC incubated in NaCI solution of compatible molar con­ centration. This phenomenon may be due to the slower penetration of Mg2 ~ ions through the RBC membrane. Examina· tion of the surface ultrastructure of RBC with a scanning electron microscope
1) Hematologic Laboratory 2) Department of Medicine "C" and
Cardiology Service 3) Department of Medicine "B" 1.2.3) Hasharon Hospital, Petah-Tiqva, Tel
Aviv University Medical Schooi/Is­ rael
156
showed that in both solutions the cells lost their central pit due to swelling and presented a progressive crenation till ap­ pearance of red cell ghosts. These altera­ tions were observed earlier in RBC incu­ bated in hypotonic MgC12 solution. The possible explanation of the discrepancy between the slower proceeding of hemo­ lysis and the early appearance of altera­ tions in RBC incubated in hypotonic MgC12 solution is discussed.
Resume Les auteurs ont etudie la fragilite osmoti­ que d'erythrocytes (GR) humains peri­ pheriques exposes a un stress hypotoni­ que dans des solutions de MgCI2 et de NaCI, a l'aide d'un fragilligraphe et d'un spectrophotometre. Ces deux methodes ont mcmtre une hemolyse plus lente des GR incubes dans une solution hypotoni­ que de MgCI2 que dans une solution equimolaire de NaCI. Ce phenomene est peut·etre du a la penetration plus lente des ions Mg + + a travers la membrane erythrocytaire. L'examen de !'ultrastruc­ ture de surface des GR au microscope electronique a balayage a montre que, dans les deux solutions, les cellules ont perdu leur zone centrale par gonflement et presentaient un aspect crenele, jus· qu'au stade d'achromatocyte. Ces altera­ tions se sont produites de fayon plus pre­ coce dans la solution hypotonique de MgC12• Les auteurs discutcnt d'une even­ tuelle explication a cette hemolyse plus lente et a !'apparition plus precoce des al­ terations intra-erythrocytaires dans la so­ lution hypotonique de MgC12•
Introduction
It has been shown that hypotonic hemolysis is a multistage process independent of the permeant ap­ plied [ 4, 5]. During osmotic hem­ olysis the red blood cells (RBC) pass through a few stages such as
swelling, popping, reduction in volume and leakage of ions and hemoglobin [5]. The morphologi­ cal alterations of RBC during os­ motic hemolysis have been docu­ mented with an inverted micro­ scope [ 4, 5]. Since the osmotic pressure ex­ erted by different ions plays an important role in the mainte­ nance of the RBC integrity and shape, it was the purpose of the present study to examine the res­ istance of human peripheral RBC exposed to hypotonic stress in MgC12 and NaCl solutions using a fragiligrap)l and a spec­ trophotometer. In addition, changes in the surface architec­ ture of the cells in hypotonic so~ lutions were followed with the aid of a scanning electron micro­ scope.
Materials and Methods
Osmotic fragility of RBC
Three ml of venous blood were withdrawn from healthy volun­ teers with a heparinized syringe. The plasma and buffy coat were discarded and RBC were washed. 3 times with isotonic saline. 20 ~-tl were resuspended in 1 ml of ei­ ther MgC12 (100 mMol) or NaCl (150 mMol) solutions. The osmotic fragility of RBC was monitored using a fragiligraph and a spectrophotometer. A Fra­ giligraph Osmotic Test Recorder,
Mag.- Bull. 9 (1987)
Model D2 (Elmedix Ltd., Haifa Israel) has been applied follow­ ing a previously described method [2, 3]. Briefly, the method consists in obtaining a curve expressing the osmotic fra­ gility of a minute amount of RBC suspended in isotonic solu­ tion in which the salt concentra­ tion gradually decreases during the test. The instrument records changes in light transmission through the cell suspension oc­ curring in the course of time. For spectrophotometry, 20 J.L1 of RBC were resuspended in 150, 135, 120, 105, 90, 75, 45 and 15 mMol of NaCl or lOO, 90, 80, 70, 60, 50, 30 and I 0 m Mol of MgCl2, as well as in distilled water. These concentrations are comparable for both salts. The RBC suspensions were incubated at 37 oc for 30 min. Following centrifugation at 2000 rpm for 5 min the supernatants were exam­ ined in a Gilford spectrophoto­ meter 300-N (Ohio/USA) at a wave length of 575 nm.
Scanning electron microscopy
et~! 0 1/ I I <:::) ..
z: 0 \NaCl-! ,MgC1 7J I I 0
,... .....
.., ..... 0
I I I I I I I I I ::c In
Vl :z 0 I I I 11 I I ex: .... 0::: 0 1- M
1- 0
I I I l I I I I I I I J I I
:::J:: N
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I / I I I _y _).-/ I I I I ,... N M <:::: I.() !.0 ,...._
TIME, MIN. Fig. 1: Light transmission curves of red blood cells incubated in hypotonic NaCI and MgCh solutions obtained with the fragiligraph. The curve for the cells incubated with MgCI2 solution shows a marked deviation to the right indicating an increased resist­ ance to hypotonic stress
sion during the hemolytic pro­ cess. When the tangent of the angle between the abscissa (time) and the linear portion of the curves was calculated, the fol­ lowing results were obtained: for NaCl solution tga = 4.585 ±
0.633 (SE) and for MgCh tga = 2.057 ± 0.009 (SE) (p < 0.005). The maximum time in minutes for the light transmission through both cell suspensions was 3.75 ± 0.32 (SE) for NaCl and 7.37 ± 0.25 for MgCh (p< 0.0005).
Red blood cells suspended in 150,90,75 and 45 mMol of NaCl and I 00, 60, 50 and 30 m Mol of MgCh, as well as in distilled ~ water were incubated for 30 min ~ at 37 o C. Thereafter the RBC ~ were spontaneously sedimented ~ and air-dried on glass coverslips, coated with gold and examined with a Philips 500 PSEM scan­ ning electron microscope at an acceleration voltage of 25 kV.
w... 0
I
Results
Figure 1 show the light transmis­ sion curves obtained from RBC suspended in hypotonic NaCI and MgCl2 solutions as detected with the fragiligraph. The light transmission curve from RBC suspended in MgC12 solution showed a marked deviation to
! the right, expressing a longer time needed for light transmis-
Mag.- Bull. 9 (1987)
lO
10
0
~ m q ~ ~ ~ ~ ~ m • 100 90 80 70 60 50 40 30 20 10
CONCENTRATION, mf.to 1
0 N.CI • Ml,iCI&
Fig. 2: Light absorbtion curves for red blood cells suspended in hypotonic NaCI and MgCI2 solution obtained with the spectrophotometer. Also in this case, the cells in hy­ potonic MgCh solution are more resistant than those in hypotonic saline
157
Osmotic fragility and surface ultrastructure of red blood cells
Figure 2 shows the absorption curve of the light through both cell suspensions as obtained with the spectrophotometer (average of 9 experiments). The difference between both curves expressing the percentage of hemolysis of the RBC in both solutions was statistically significant. The curves permitted to calculate the molar concentration of the salt solution in which 50% hem­ olysis was achieved, i.e. the me­ dian corpuscular fragility (MCF), which for NaCI was 69 mM and for MgCI2 was 29 mM (compati­ ble with 43.5 mM of NaCI). Therefore, the MCF for NaCI was - 1.6 times greater in com­ parison with that for MgCI2•
Scanning electron micros­ copy
1. Effect of hypotonic NaCI solu­ tion
Incubation of RBC in isotonic saline did not induce any mem­ brane alterations (Fig. 3). The cells retained their central pit and their round or oval shape. Incubation in hypotoni c NaCI solutions caused a gradual change in the cell appearance. At 90 mMol NaCI they lost their central pit and became flat (Fig. 4). A further decrease in the NaCI concentration to 75 mMol induced the appearance of crena­ tion (Fig. 5) which persisted till the salt concentration decreased to 45 mMol (Fig. 6). Red cells in­ cubated with distilled water showed a complete lysis and only ghosts were observed (Fig. 7).
2. Effect of hypotonic MgC12 solu­ tion
In isotonic MgCl2 RBC retained their discoid shape (Fig. 8). With a decrease in th e salt concentra­ tion to 60 mMol the first signs of Fig. 5: Red blood cell s incubated in 75 mMol of NaCI so lution. The appearance of crenation were observed (Fig. 9). crenated cell s is obvious. Marker - 10 ~un
158 Mag.- Bull. 9(1987)
Osmotic fragility and surface ultrastructure of red blood cells
Fig. 6: Progress of crenation in red blood cells with a decrease in NaCl concentration to 45 mMol. Marker - 10 ~Lm
Fig. 8: Red blood ce lls suspended in isotonic (100 mMol) MgCI2 solution. The cell sha pe is well preserved. Marker - I 0 ~m
Mag.- Bull. 9 (1987)
The crenation increased progres­ sively with the hypotonicity of the solution (Fig. 10) and became maximal at a concentration of 30 mMol (Fig. I 1).
Discussion
The results of the present study indicate that RBC exposed to hy­ potonic stress in MgCI2 solutio n are more resistant than those ex­ posed to hypotonic NaCl so lu­ tion of similar molar concentra­ tion. This finding was observed by two different methods applied in the present work. The curves obtained with the fragiligraph showed that RBC exposed to hy­ potonic MgCh solutions begin to hemolyze later in comparison with those exposed to hypotonic saline solution. Furthermore, the course of hemo lysis in hypotonic MgCI2 solution appears to pro­ gress more slowly than the hemo­ lysis in hypotonic NaCI. One of the factors affecting the course of hemolysis is the coefficient of the membrane permeability to mole­ cules or ions in the solution [9]. The better the membrane perme­ ability for the particles in the so­ lution, the greater is the number of particles which will penetrate through the membrane, and which will increase the hyperton­ icity of the intracellular fluid in­ ducing an increased endoosmo­ sis. The result will be a shorter time for the onset of hemo lysis. Since the cell membrane is more permeable for Na+ than for Mg2+ [6], the osmotic process in hypotonic NaCI will progress earlier and faster as compared to that in hypotonic MgC12 so lu­ tion. According to Aikawa [ 1], there is no exchange of magne­ sium between the plasma and the red cells in vivo. Therefore, var­ iations in Mg concentration ob­ served in certain diseases are not expected to induce serious alter­ ations in the intracellular Mg content. The earlier appearance of RBC fragility in hypotonic NaCl sol u­ tion observed with the fragil i­ graph and with the spectrophoto­ meter has not been reflected in
!59
Osmotic fragility and surface ultrastructure of red blood cells
the results obtained by electron microscopy. On the contrary, the changes in RBC ultrastructure appeared earlier in cells incubat­ ed in hypotonic MgC12 solution. One possible explanation could be that the smaller N a+ ions can move easier through "pores" of the cell membrane, with a conse­ quent disturbance in the osmotic equilibrium without marked al­ terations in the cell membrane and its shape. A transformation of RBC from discocytes into crenated cell s has been achieved also by incubation in hyperos­ motic NaCI both in our labora­ tory (unpublished observations), and in previous ly reported stu­ dies [8]. The mobility and integr­ ity of membrane proteins may play an additional role in the al­ terations of RBC membrane [7].
References [I) Aikawa. J. K.:The role of magnesium
in biological processes. C. Thomas, Springfield 1963.
[2] Danon, D.: A rapid micro-method for recording red cell osmotic fragility by continuous decrease of salt concen­ tration. J. clin. Pathol. 16 (1963) 377-382.
[3] Danon, D., Y. S. Frey and A. Rimon: Simple rapid osmotic fragility test proposed as a routine in blood banks. Transfusion 4 (I 964) 339-342.
[4] Jay. A. W. L.: Permeability of indivi­ dual human erythrocytes to thiourea. J. Physiol. 262 ( 1976) 447-458.
[5] Jay, A. W. L.. S. Rowlands: The stages of osmotic haemolysis. J. Phy­ siol. 252 ( 1975) 8 17-832.
[6] Keele, Neil. Joels: Samson Writ's Ap· plied Physiology, 13 ed. Oxford i982.
[7] Marikowsky. Y.. C. S. Brown. R. S. Weinstein and H. H. Wortis: Effects fo lysolecithin on the surface proper­ ties of human erythrocytes. Exp. Cell Res. 98 ( 1976) 313-324.
(8] Marikowsky, Y .. J. K. Khodadad and R. S. Weinstein: Influence of red cell shape on surface charge topography. Exp. Cel l Res. 116 (1978) 191-197.
(9] Saari. J. T. and J. S. Beck: Hypotonic haemolysis of human red blood cells: a two-phase process. J . Membrane Bioi. 23 ( 1975) 213-226.
(Correspondence to: Prof. M. Djaldetti. MD, Department of Medicine "B", Has­ haron Hospital, Petah-Tiqva 49372/ l s­ rael)
160
Fig. 9: Red blood cells incubated in hypotonic (60 m Mol) MgC12 solution. Distorsion of the cell shape and appearance of crenated cells is visible. Marker- 10 !lm
Fig. 11: With a further decrease in the molar concentration of the MgCI2 solution to 30 mMol, the crenation of the red b lood cells is maximal. Marker- 10 !lm
Mag.- Bull. 9 (1987)