Seminar On Ph Effect on Human Erythrocyte
CHAPTER ONE
PREAMBLE TO THE STUUDY
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 were 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 are 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).
The red blood cell is unique in its ability to undergo elastic deformation as it travels through the microcirculation. This ability to change shape reversibly is integral to red cell function, allowing the cell to traverse capillaries with diameters considerably less than the normal red cell diameter (8 pm) while subsequently returning to its normal biconcave shape. Deformability properties of red blood cells are dependent
Human erythrocytes equilibrated in altered pH buffers at room temperature assume altered shapes. Below external pH 6.0, membrane curvature becomes negative (stomatocytic); above pH 8.0, curvature becomes positive (echinocytic) (Gedde and Huestis, 1997). This phenomenon appears to be related to the pH-dependent shape changes of red cells in electrical fields (Rand et al., 1965) and red cells exposed to glass (Furchgott and Ponder, 1940; Bessis and Prenant, 2012; Weed and Chailley, 2013). These shape changes have distinctive common features: l) they proceed at ambient temperature; 2) they can occur within seconds; and 3) they reverse upon normalization of the extracellular environment (Rand et al., 1965; Bessis and Prenant, 2012; Weed and Chailley, 2013; Gedde and Huestis, 1997). Taken together, these features suggest that the shape changes result from changes in weak, noncovalent bonds, rather than higher energy bonds. Because the plasma membrane determines red cell shape, the events that produce altered shape presumably take place in the red cell membrane. Expansion and contraction of the spectrin membrane skeleton upon change in cell pH are expected to strongly affect membrane curvature (Elgsaeter et al., 2006), but predicted curvature changes are in the direction opposite those observed in intact cells.
CHAPTER TWO
MATERIALS AND METHODS
Sample collection
Buffering compounds and nystatin were purchased from the laboratory market. Other chemicals were reagent grade. Red cells and solutions were prepared as described (Gedde and Huestis, 1997).
Buffer specifications predicted to vary cell pH, cell water, and membrane potential independently
The equations of red cell physiology (Jacobs and Stewart, 2015; Freedman and Hoffman, 1979) were used to calculate specifications for buffers predicted to produce targeted cell states. Cell states were defined by cell pH (pHin), membrane potential (AV), and cell water (cw). Results specified buffer pH (pHout), chloride concentration ([Cl]out), and osmolality (Osout). Buffer pH was calculated from cell pH and membrane potential:
CHAPTER THREE
RESULTS AND DISCUSSION
Results
The aim of the present study was to identify PH effect on human erythrocyte. Using several strategies, red cells were placed in a wide range of physiological states. Morphology and four physiological properties (cell pH, membrane potential, cell water, and cell chloride concentration) were assayed in each state. Morphology and the four parameters were correlated by a multivariate analysis sequence that included stratification and mathematical modeling. The results showed altered cell pH to be necessary and sufficient for discocyte-stomatocyte and discocyte-echinocyte transitions in altered pH solutions.
Choice of predictor physiological properties
External solution properties seemed unlikely to be primary predictors of morphology. Theoretically, external pH variation could affect membrane curvature by titrating and thereby altering the packing of the cell surface glycocalyx; however, the cell surface isoelectric point is dominated by strongly acidic sialic acid residues (PK about 2), and only minor change in surface charge occurs as the external pH varies between 5 and 10 (Glaser, 1979). External ionic strength has been shown to affect red cell membrane structure (Herrmann and Muller, 2006), but variation in external ionic strength does not perturb intact cell shape (Gedde and Huestis, 1997).
In the present study, three cell properties and one transmembrane property were hypothesized to predict red cell shape. Cell pH and cell water were chosen because preliminary data indicated that both had roles in determining shape. Membrane potential was chosen because it has been reported to affect shape. Cell chloride concentration was chosen as a marker for cell ionic strength, variation of which might affect shape by causing expansion or contraction of the membrane skeleton (Elgsaeter et al., 2006). (However, cell chloride concentration may be a suboptimal marker for ionic strength in the concentrated polyelectrolyte environment of the red cell cytoplasm.)
CHAPTER FOUR
CONCLUSION
In this context, it is interesting to note that several peripheral and cytoplasmic erythrocyte proteins have been shown to insert hydrophobically and in a pH-dependent manner into anionic lipid-containing model membranes. These proteins include spectrin (Mombers et al., 2010), hemoglobin (Shviro et al., 1982; Szebeni et al., 2008), band 4.1 (Schiffer et al., 2008), and actin (St-Onge and Gicquaud, 2010). It is interesting to speculate that increased insertion of a cell protein into the red cell inner leaflet at low pH, either electrostatically between lipid headgroups or hydrophobically between lipid hydrocarbon chains, could cause an increase in the inner monolayer surface area and result in low cell pH stomatocytosis. Reduction of such interactions at high pH could be the cause of high cell pH echinocytosis. Superposition of these putative protein-membrane interactions on the opposing effects of pH-dependent spectrin contraction and expansion might explain the strikingly nonlinear pH dependence of erythrocyte shape.
Cell pH has been shown to have a regulatory role in the metabolism and development of certain organisms (review: Busa and Nuccitelli, 2004), and there is increasing evidence that cell pH is involved in the modulation of cytoskeletal interactions (Busa, 2006). However, difficulty in measuring intracellular pH in most cells has impeded the understanding of its role in cell biological processes. As shown here, the mature erythrocyte’s intracellular pH is readily manipulated and measured; thus the red cell is a useful model for studying responses of biological membranes to changes in intracellular pH. Elucidation of pH-dependent events in the erythrocyte membrane could provide basic information applicable to other biological membranes.
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