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pH EFFECT ON HUMAN ERYTHROCYTE SHAPE
The effect of pH on shape of intact erythrocyte was studied by suspending erythrocyte in phosphate buffer saline of pH outside the range 8.0 to 4.0. These were found to induced instantly two typical types of shape changes of human erythrocyte when added into the erythrocyte suspension. Generally, as the pH of the suspending media decreased toward acidity (pH 3.0 – 1.0) cup shape calls (invagmation) were formed. Subsequently, this was accomplished by cell lyrics. On the other hand, increasing pH of the suspending media toward – alkalinity (pH 8.0 – 12.0) induced crenation (externalization) on the erythrocyte membrane. Accompanying this type of shape change were a progressive increased in cell lyrics which began as pH of the media increased to pH 11.0. Ghost cells also formed at this pH
TABLE OF CONTENTS
Title Page i
Table of Contents vi
GENERAL INTRODUCTION AND LITERATURE REVIEW
1.2 Red Cell Membrane
1.3 Analysis of Membrane Constitution
1.4 Red Cell Shape Deformability
1.5 Scope of the Project
2.0 Materials and Method
3.1 Analysis of Shape of erythrocyte induced by change in pH Value
GENERAL INTRODUCTION AND LITERATURE REVIEW
The normal mature erythrocyte are biconcave disks having a means diameter of approximately 8 microns and a thickness at the point of 2 microns and in the center of 1 micron or less (Guyton 1981).
The red cells was thought to be composed of two main parts: a retaining membrane and a highly concentrated solution of hemoglobin (Cooper et al 1950). But with further analytical studies, it was suggested that the red cells is compose of 7% water, 28% hemoglobin, 70% membrane lipid such as cholesterol, lecithin, phospholipid (Moskowitz and Calvin 1982, Perutz et al 1960) and 3% sugars, salt, enzyme protein and membrane protein (Budewig 1960).
1.2 RED CELL MEMBRANE
Shape controlling factors have been placed both within the interior of the cell (Shrivastastar and Burton 1969) and in the membrane but the membrane was generally considered to be responsible for the biconcave shape (Weed et al 1963). Actually, the red blood cell is a bag that can be deformed into almost any shape (Guyton 1981). Furthermore, because the normal cell has a great excess of cell membrane for the quantity of material inside, deformation does not stretch the membrane and consequently does not rupture the cell as would be the case with many other cells (Guyton 1981).
1.3 ANALYSIS OF MEMBRANE CONSTITUTION
1.3.1 RED CELL MEMBRANE LIPID
The total lipid that can be extended from red cells (approximately 1.0ml of packed cells) is 5mg. the extracted lipid are subdivided into three main classes, phospholipids 60% neutral 30% and glycolipids 10% (Watkins 1974).
The phospholipids comprise two main classes, glycerophosphate and sphingomyelin. Reed (1968) demonstrated that 60% of lectithin and 30% of sphingomyelin were exchange between the plasma and the human red cell membrane over five days. Lysolecithin (the monoacyl form of lecithin) which can be formed in the plasma through the esterification of free cholesterol by the action of the enzyme lecithin – cholesterol acyl-transferase, a fatty acid being transferred, from lecithin to cholesterol, has been shown to rapidly equilibrate with the membrane (Waku and lands. 1968, Shohet and Nathan, 1970), the lysolecithin undergoing acylation by the action of acyl-transferase to form lecithin. Shohet in 1971 demonstrated the transfer off labeled fatty acids between phospholipids notably from lecithin to phosphatidyl ethabolamine. There is uncertainty as to whether the interconversion of labeled fatty acids between the different classes of diacylphosphoglycerides takes place as a result of direct transacylation or through the combined action of phosphatases and an acyltransferase which catalyzes the phosphate formed through the action of the phospholipase (Shohet 1972).
A defect in transacylation has been postulated to account for the increased phosphatidyl choline and diminished phosphatidyl ethanolamine observed in the red cell membrane of the members of a family with nonspharocytic heamolytic anaemia (Shohet et al 1971). More recently Wiley and co-workers (1973) have shown that a similar disorder in the relative proportion of these two phospholipids may be found in patients with hereditary stomatocytosis.
Unlike the phospholipid, the free unesterified cholesterol in the membrane exchanges rapidly with the unesterified cholesterol bound to plasma lipoproteins (Cooper 1951). There is evidence that not all red cell membrane cholesterol is exchange as the specific activity of the plasma cholesterol is higher than that of membrane (Bell and Schartz 1971).
Whereas the cholesterol in the membrane undergoes no metabolic changes, the cholesterol in the enzyme lecithincholesterol acyl transferase (LCAT). The fatty acid at the 2 – position of lecithin is transferred to cholesterol and lysolecithin is formed. There is evidence that the acyl transferase reacts preferentially with lecithin attached to certain specific lypo protein (Glomset 1968). The inherited deficiency of the plasma enzyme results in a raised cell membrane cholesterol content and the presence of target cell in peripheral blood filam (Gjone, Toravik and Norum 1968: Gjone and Norum, 1969).
Murphy in 1962 demonstrated that the sterile incubation of crythrocytes in vitro resulted in a fall in membrane cholesterol through the reduction in plasma free cholesterol due to esterification and that the loss of cholesterol caused a reduction in surface area of the cell with an increase in osmotic fragility associated with a tendency to sphere formation and susceptible to osmotic lyses.
Inhibition of lecithin cholesterol acyl-transferase (LCAT) by bile salts was initially thought to be principal reason for patient with obstructive Jaundice developing raised red cell cholesterol levels and targets cells (Cooper and Jandl 1958). These workers further demonstrated in 1972 that in patients with target cells and spur cells due to liver disease, red cell membrane cholesterol correlated with the ratio of cholesterol to phospholipid in low density lipoprotein.
Varying the cholesterol content of the red cell in vitro was accompanied by changes in membrane surface area, red cell morphology and the altered deformability characteristic of the cholesterol loaded spur cells observed in patients with liver disease (Cooper, Kinbal and Durocher 1974). Thus the primary cause of the altered red cells morphology in patients with liver disease is related to the excess of unesterified cholesterol to phospholipid bound to low density lipoproteins.
1.3.2 RED CELL MEMBRANE PROTEIN
In the analysis of the cell membrane it had been showed to contain two larger molecular weight polypeptide and glycoproteins (Anselseltar and Horstman, 1975). These largest molecular weight polypeptides located on the inner aspect of the membrane, correspond to the filamentous proteins seen on electron microscopy and term spectrin by marchesi and co-workers (Marchesi and Stears 1968, Marchesi et al 1970, Nicolson, Marchesi and Singer 1971). Recently evidence surgests that spectrin is compose of several subunits (Fuller Boughter and Morazzani 1974), finding confirmed in two dimentional electrophoresis (Anselsetter and Horstmann, 1975).
The filamentous state of spectrin has been shown to be dependent on its interaction with actin (Tilney and Detmars, 1975). The role of spectrin in determining red cell shape and deformability and the cyclic nucleotide dependent protein kinase of the red cell is hypothetical.
Of great interest in relation to the functional state of spectrin on the inner surface of the membrane has been the demonstration that phosphorylation of spectrin is mediated by a cyclicuncleotide dependent protein kinase. The presence of a protein kinase within the red cell was first reported by Guthrow, Allen and Rassmusen (1972) and confirmed by Rose and Appal (1973).
The degree of phosphorylation of spectrin may be in part responsible for the degree of association of this molecule with actin (Tilney and Detmars 1975).
Greenquish and Shohet (1973, 1975) have demonstrated impaired protein kinase mediated phosphorylation of spectrin by the red cells of patient with hereditary spherocytosis, support for the possibility that an abnormality in the physical state of spectrin may underlie defect in hereditary spherocytosis has been suggested by Jacob and co-workers (1972, 1975). These workers observed that agenta such as vinblastine colchicine and strychnine known denaturants of microfilamentous protein, alter red cell morphology, sodium permeability osmotic frafility and deformability of normal cells in a manner to render them indistinguishable from the red cells of patients with hereditary spherocytosis. Furthermore, the morphological changes could be inhibited by the addition of cyclic 3’5’ guanosine monophosphate (Jacob et al 1975).
In view of the well documented influence of ca2+ on red cell shape and deformability (Weed, Lacelle and Marrill 1969, Lacelle et al 1972, white 1976), ca2+ must influence the physical state of the spectrin actin interaction. ca2+ has been shown to active the phosphorylation of membrane proteins (Rege and Gerraham 1975).
In 1969 Marokovsky and Danon reported that the surface of the membrane of the erythrocyte lies of not negative charge which diminishes as the cell ages in vivo. Recent studies indicate that this negative charge is attributable to the carboxyl groups of sialic acid. Treatment of the erythron with neuraminidase removes sialic acid and increases the isoeletrio point from pH 2 to pH4 – 5 (Eylar et al 1962). This negative surface charges is probably sufficiently strong to produce an intracellular repelling force that prevent the cells from touching one another, reduction of this negative charges by enzymatic treatment of the red cells reduces the repelling forces as evidenced by enhanced agglutination by chemical agents (Marikovsky and Danon 1969).
Furthermore, Winzler in 1969 suggested that the sialic acid that contributed to the negative charge is localized in the glycoprotein of the exterior surface of intact erythrocytes. This surface charge decreases with age in vivo (Yaari 1969).
1.4 RED CELL SHAPE DEFORMABILITY
The problem of how mammalian red blood cells maintain the shape of biconcave discs has been the object of many speculations over since it was discovered that this ability is a property of the membrane itself. The shape of the erythrocyte seems to be the result of a delicate equilibrium between divergent forces. Disturbance of this equilibrium principally gives rise to the transformation of biconcave erythro into either crenated or cup shaped cells both intermediate in the process of sphering (Deuticke 1968).
In human erythrocytes cell shape change is an indication of a major dysfunction and is normally associated with hemolysis. It has been suggested that the main mechanism of hemolysis destruction probably involve an alteration of red cell membrane (Weed and Reed 1986) that may be related to intracellular or an extracellular abnormality or conditions.
Various studies have demonstrated that exposure of erythrocytes to a number of agents (in vitro) may cause an alteration of the disc shape of the red cells. Past studies has revealed that a relationship exist between the fragility of erythrocytes in hypolonic solution and the geomelric configuration of the cell (Castle and Daland 1937, Hoffman et al 1958, Williams et al 1959).
It is already well established that mature human erythrocyte undergo one of two type of membrane transformation and a resulting shape change of the cells under in vitro action of amphipathic compound (Tatsuzo Fujii et al 1978). Of many amphiphillic and amphipathic compound those with an anionic polar group were reported to induced crenation and those with a cationic polar group invagination (Deuticke 1968).
Recent studies proposed these anionic drugs intercalate mainly into the lipid in the exterior half of the bilayer expand that layer relative to the cytoplasmic half and thereby induce the cell to crenate while permeable cationic drugs to the opposite and cause the cell to form cup shape (Sheetz et al 1974).
However it was further reported that when cells are incubated at 370C in the presence of the crenating agents with glucose, they gradually (“between” 4-8 hour) recover the normal biconcave disc form (Eytan Alhanaty et al 1981). It is suggested that the conversion of cell from crenated to disc shape in the presence of the crenators represents an alteration or rearrangement of membrane components rather than a redistribution of the crenators within the membrane.
In 1986 Deuticke reported in his finding that suspending erythrocytes in isotonic solution of non-penetrating anions leed to the occurrence of cup cells, the extent of transformation becoming more pronounced with lowering of extracellular pH. Restoration of normal shape under this condition is accomplished by addition of penetrating anions (Deuticke 1978).
In vivo test and in vivo survival studies clearly indicate an intracurpuscular abnormality. The vitro studies have localized the basic abnormality in the erythrocyte by demonstrating that the cells although they reacted normally to osmotic and mechanical stresses (Castle and Daland 1937) lysed in action when incubated in patients or normal compatible serum at a slightly lowered pH (Ham 1937, 1939 Ham and Dingle 1939).
1.5 SCOPE OF THE PROJECT
The scope of this project is to further investigate the effect of pH changes on the scope of normal Haman erythrocytes. In the course of this work normal erythrocytes will be subjected to different pH medium in vitro and the effect of this on normal erythrocytes shape will be examine.