Factors Affecting the Permeability of the Red Blood Cells

Abstract: The purpose of the experiment was to study the properties of membranes with the use of red blood cells. At first, the effects of tonicity of solutions on red blood cells were studied. The concentration of NaCl that creates hemolysis was determined as 0.062M. Moreover, isosmotic solution of glycerol was not isotonic to 0.15M NaCl since it causes hemolysis. Also, when blood suspension was added to distilled water it caused hemolysis whereas 0.15M NaCl did not. Secondly the time to hemolysis against lipid-water partition coefficient and molecular weight. This resulted in high lipid-water partition coefficient needed less time to hemolysis and chemicals with small molecular weight needed less time to hemolysis. Finally, the red blood cells were centrifuged for microscopic observations on hemolysis. When cells were suspended in hypertonic solution the red blood cells appeared to be shrunken. At the same time, in hypotonic solution cells were broken and only few were unbroken. Also, in isotonic solution the circular and unbroken cells were seen.

Introduction: Cell membrane is a biological partition between a cells interior and the exterior. It is made out of phospholipid bilayer, carrier proteins, cholesterol and carbohydrates.3 The membrane itself is selectively permeable which means it allows only certain molecules in and out of the cell. Osmosis is a special way of transport in cells for water molecules. Osmosis occurs across a semi permeable membrane down the water potential gradient.3 As a result of osmosis, osmotic pressure is generated. In addition, osmolarity is another term that comes to play when considering osmosis across a cell membrane. Osmolarity means number of solute particles (penetrating and non-penetrating) in a solution.1

One of objectives of this experiment was to observe how osmosis and osmolarity affects the movement of water across cell membrane. The red blood cells from a rabbit were used as a model solution to study the properties of membranes. When the red blood cells are surrounded by aqueous solutions of lower solute concentration there is an inward flow of water and the cells will increase in volume.5 The lower concentrated aqueous solution is called the hypotonic solution. When this occurs cells will burst and releases hemoglobin a process called hemolysis.5 Cells suspension turned into clear red solution if hemolysis occurred and the suspension was opaque if it did not occur. If the cells are immersed in a hypertonic solution there will be an outward flow of water and the cells will shrink. This process is called the crenation.5 There will not be any net movement if the solute concentrations in and out of the cell are same. The solution at this stage will be called the isotonic solution.5 Another objective of this experiment was to describe and account for the effects of hypotonic, isotonic and hypertonic solutions on red blood cells. Moreover, permeability of solute across the cell membrane depends on couple of factors such as molecular size, molecular weight and lipid solubility. Lipid water partition coefficient is the measure of lipid solubility for molecules. The higher the lipid water partitions the higher the lipid solubility. To study the factors that affect diffusion of molecules across plasma membrane, blood suspension was added to twelve different chemicals and their time to cause hemolysis was measured. Finally, to analyse chemical hemolysis saponin solution was added to blood suspension and results were taken.

Materials and Methods: The rabbits blood was used to study the membrane properties. The blood suspension was made at the beginning of the experiment which was then diluted three times to identify the concentration of NaCl that caused the hemolysis. In the second part of the experiment twelve different chemicals were used to investigate the factors that affect hemolysis. The final part of the experiment used the blood suspension suspended in different concentrations of NaCl and distilled water to observe the red blood cells under the microscope. The materials and methods were followed according to the lab manual. However, in step 3 of the experiment the solutions were timed for 3 minutes and 25 seconds and this was the only alteration that was made in this experiment.

Results: Table 1. Comparison of transparency between distilled water and NaCl with blood cell suspension Solution Distilled Water 0.15M NaCl Volume (ml) 5 5 Transparency Yes No

Table 1 shows the difference between the transparency of distilled water and NaCl with blood suspension. The distilled water was clear red in colour whereas the NaCl solution appeared to be opaque. Table 2. Preparation of 1st series of diluted NaCl concentrations and the required volume of distilled water from the initial concentration of 0.4M NaCl Initial concentration of NaCl (M) 0.4 0.4 0.4 Final concentration of NaCl (M) 0.05 0.1 0.2 Volume of NaCl for the dilution (ml) 0.25 0.5 1 Total volume of solution (ml) 2 2 2 Required volume of water for the dilution (ml) 1.75 1.5 1 Appearance of the solution Clear red Opaque Opaque

0.4

0.3

1.5

0.5

opaque

Table 2 shows the 1st series of NaCl dilutions that received three drops of blood suspension and they were observed for 3 minutes and 25 seconds. At the end of the timing the 0.05M NaCl solution turned red in colour and was transparent. However, 0.1M, 0.2M and 0.3M NaCl solutions were opaque and blurry. Note the 1st series of dilutions were prepared to find the molarity of the NaCl solution to the one decimal place that will cause hemolysis. Table 3. Preparation of 2nd series of diluted NaCl concentrations and the required volume of distilled water from the initial concentration of 0.4M NaCl Initial concentration of NaCl (M) 0.4 0.4 0.4 0.4 Final concentration of NaCl (M) 0.06 0.07 0.08 0.09 Volume of NaCl for the dilution (ml) 0.30 0.35 0.40 0.45 Total volume of solution (ml) 2 2 2 2 Required volume of water for the dilution (ml) 1.70 1.65 1.60 1.55 Appearance of the solution Clear red Opaque Opaque opaque

Table 3 shows the 2nd series of NaCl dilutions that received three drops of blood suspension and they were observed for 3 minutes and 25 seconds. At the end of the timing the 0.06M NaCl solution turned red in colour and was transparent. However, 0.07M, 0.08M and 0.09M NaCl solutions were opaque and blurry. Note the 2nd series of dilutions were prepared to find the molarity of the NaCl solution to the two decimal places that will cause hemolysis.

Table 4. Preparation of 3rd series of diluted NaCl concentrations and the required volume of distilled water from the initial concentration of 0.4M NaCl Initial concentration of NaCl (M) 0.4 0.4 0.4 0.4 Final concentration of NaCl (M) 0.062 0.064 0.066 0.068 Volume of NaCl for the dilution (ml) 0.31 0.32 0.33 0.34 Total volume of solution (ml) 2 2 2 2 Required volume of water for the dilution (ml) 1.69 1.68 1.67 1.66 Appearance of the solution Clear red Opaque Opaque Opaque

Table 4 shows the 3rd series of NaCl dilutions that received three drops of blood suspension and they were observed for 3 minutes and 25 seconds. At the end of the timing the 0.062M NaCl

solution turned red in colour and was transparent. However, 0.064M, 0.066M and 0.068M NaCl solutions were opaque and blurry. Note the 3rd series of dilutions were prepared to find the molarity of the NaCl solution to the three decimal places that will cause hemolysis.
Observation for isosmotic solution of glycerol and 0.15M NaCl: The glycerol solution turned into a clear red solution.

Molecular weight Vs time to hemolysis

400 350 Molecular weight (mol/g) 300 250 200 150 100 50 0 0 20 40 60 80 100 120 140 160 180 200 R = 0.2982

Molecular weight

Time (sec)

Figure 1: Graph of molecular weight Vs time to hemolysis This is the graph of molecular weight of twelve different chemicals and the times for them to cause hemolysis. The R2 value shows no significant correlation between molecular weight and the time to cause hemolysis.

Lipid -water partition coefficient Vs time to hemolysis

1 0.9 Lipid- Water partition coefficient 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 -0.1 0 20 40 60 80 100 120 R = 0.0749 140 160 180

Lipid -water partition coefficient

200

Time (sec)

Figure 2: Graph of time to hemolysis and lipid-water partition coefficient The figure 2 provides the graph of lipid-water partition coefficient for twelve chemicals and the time for the molecules to cause hemolysis. The R2 value shows no significant correlation between lipid water partition coefficient and time to cause hemolysis.

Table 5. Observations on Hemolysis when red blood cells were suspended in water and NaCl solutions of different concentrations Tube Solution Observation before Observation centrifugation after centrifugation Opaque solution Clear solution with large amount of cell deposit The solution was Light pink pink in colour and solution with very clear very small amount of red cell deposit Slightly opaque, Clear solution dark reddish with small orange in colour amount of red cell deposit Opaque and Clear solution reddish orange in with large colour amount of deposit Microscopic observation Circular shaped black dots

1ml suspension + 1 ml of 0.15M NaCl 5 drops of suspension + 2ml of distilled water

Small amount of round cells and large amount of broken cells Mostly broken cells and few amount of round cells Mostly shrunken cells and very few are unbroken

1 ml of suspension + 1 ml distilled water 1 ml suspension + 1 ml of 0.4 M NaCl

Table 5 shows the effects of suspending red blood cells in water and in NaCl solutions in different concentrations. Tube 1 and 4 were opaque prior to centrifugation but under the microscope after centrifugation tube 1 contained round cells and tube 4 contained shrunken cells. Based on the table 5, tube 2 was the only tube that contained clear red solution but all other three tubes had opaque solution before centrifugation. In addition, tube 2 contained pink supernatant solution after the centrifugation whereas the other tubes contained more clear solution.

Observation for chemical hemolysis: The solution remained clear red. The cells taken from this solution had mostly broken cells under the microscope.

Discussion: This experiment was conducted to learn about permeability of plasma membrane by the use of rabbit blood suspension. According to table 1, 5ml of distilled water and 5ml of 0.15M NaCl received 5 drops of blood suspension in order to compare the transparency of the two solutions. The distilled water solution was transparent and clear whereas the NaCl solution was opaque and not transparent. This concludes that distilled water solution had hemolysis. Hemolysis is easily identified because solution changes to a clear red solution due to the burst of cells that released hemoglobulin. Hemolysis must have occurred as water flowed into the red blood cells as it was hypotonic to red blood cell plasma.6 In addition, the 0.15M NaCl is concluded to be isotonic to red cell plasma since it did not show any sign of hemolysis. Three serial dilutions for 0.4M NaCl were conducted to determine the nearest three decimal place of concentration that causes hemolysis in red blood cells. Table 2 shows the volume of water and volume of NaCl needed to prepare the four dilutions of NaCl. It was identified that 0.05M NaCl had hemolysis compared to the other three diluted solutions. The 0.05M NaCl was clear red solution compared to the rest. Also, the red blood cells hemolyse in 0.05M NaCl but not in 0.1M NaCl which resulted in table 3 with new dilutions. Table 3 also shows the volume of water and NaCl needed to prepare the desired dilutions. Based on table 3, 0.06M NaCl showed hemolysis since the solution was clear red and transparent whereas the other three solutions were opaque and not transparent. The final set of dilution was done in order to identify the molarity of the NaCl solution to the three decimal places that causes hemolysis in red blood cells. The 0.062M NaCl solution was clear red and transparent which shows that hemolysis occurred in this solution (table 4). Therefore, table 2, 3 and 4 was prepared to determine the accurate concentration of NaCl that will just cause hemolysis in red blood cells.

Isotonic and isosmotic are two common terminology when considering osmosis in a cell. Isotonic means two solutions having the same solute concentration and there will be zero net movement of water. However, isosmotic means two solutions having the same number of dissolved solute particles (penetrating and non-penetrating molecules) that created the same amount of osmotic pressure. During the lab an isosmotic solution of glycerol was used to determine whether this solution is isotonic to 0.15M NaCl. When 0.3M glycerol was compared with 0.15M NaCl, the glycerol solution changed in colour resulting in hemolysis. As mentioned above, isotonic solutions will not make any changes in a cell with no net movement of water. Isosmotic solution of glycerol can be hypotonic when it has different concentration. This also means the isosmotic solution of glycerol could contain more water molecules than the solutes which may have caused the water movement from the solution to the cell.6 In order to understand the movement across plasma membrane knowledge about certain factors such as lipid solubility and molecular weight and how they affect the permeability is essential. In Figure 1 and 2, group B molecules (glucose, arabinose, xylose, sucrose) did not show any changes in the solution during the 3min time limit. Therefore, it was assumed to have the maximum time to hemolysis which was 180 sec (3min). The chemicals mentioned above have large molecular weight, uncharged, and polar molecules. Moreover, they are impermeable in lipid membrane. Glucose moves across the membrane via carrier protein molecule but molecule like sucrose cannot cross the membrane at all.1 The group B chemicals cause hemolysis by the influx of water molecules since they are non-penetrating non-electrolytes. Moreover, these group B molecules are polar and have hydrogen bonds with water. The group B chemicals didnt cause hemolysis within the time limit (3min) as predicted by the low lipid-water partition coefficient. Glucose could have causes hemolysis if the measured time was more than 3 minutes.

According to results for group A, urea needed more time for hemolysis compared to thiourea. However, this data is not supported by other literatures such as Hober (1985). Although urea has low lipid water partition coefficient compared to thiourea, the molecular weight matters in this situation. According to Hober (1985), the times for urea and thiourea to cause hemolysis are 2.7sec and 48.6 sec respectively. Alcohols in group A such as ethanol and ethylene showed results that were supported by literatures. Ethanol caused in hemolysis much faster than Ethylene glycol. Ethanol has one hydroxyl group and ethylene glycol has two hydroxyl groups. The more hydroxyl groups in a molecule will increase its ability to form hydrogen bonds with water and reduce its permeability through lipid bilayer. In group C, the molecular weight for monoacetin, diacetin and triacetin increased along with their time to cause hemoysis. The lipid water partition coefficient for mono, di and triacetin increased respectively. In this situation, the molecular weight overpowered the lipid water partition in determining the diffusion rate across the plasma membrane. The number of carbonyl groups increased when you go from monoacetin to triacetin. This increasing carbonyl may have caused to form stronger hydrogen bonds with water and reduced the diffusion. The final chemical in group C is glycerol which is an uncharged, polar molecule had around 9 sec to cause hemolysis. However, its lipid water partition coefficient was low as 0.00007. The results for glycerol didnt match with the previous studies. According to Hober (1985), glycerol needed around 22 minutes to cause hemolysis due to the hydroxyl group in glycerol. In respect to the results regarding the diffusion of given chemicals, lipid solubility is the most considered factor when comparing the diffusion rate for similar molecular weight chemicals. Meanwhile, large molecules with higher lipid water partition coefficient will have less diffusion rate when compared to small molecules with lower lipid water partition coefficient.

In order to observe the appearance of the red blood cells microscopic observations were made with the use of different NaCl concentrations and distilled water. The tube 1 was opaque because it contained 0.15M NaCl which is isotonic to the solution to the blood cell. This explains the appearance of the red blood cells being unbroken and circular. There were no net movement of water in this tube. Tube 2 had clear reddish pink solution due to the complete hemolysis. Moreover, under the microscope it had plenty of lysed red blood cells explaining the inside movement of water molecules. As a result of influx of water the tube 2 contained hypotonic solution. Tube 4 clearly contained hypertonic solution since the red blood cells under the microscope were shrunken and had lost their biconcave shape. In this case, water must have flowed outside the red blood cells due to higher solute concentration in the outside solution and this is called as crenation. Tube 3 was similar to tube 2 but it was more opaque than tube 2. Moreover, tube 3 had less lysed cells under the microscope when compared to tube 2. Tube 3 also contained hypotonic solution but compared to tube 2 it was less hypotonic. The final procedure of the experiment was done to learn about the chemical hemolysis. When the plasma membrane is dissolved away it will result in hemolysis which is known as chemical hemolysis. A drop of saponin was added to blood suspension to study chemical hemolysis. The main function of saponin is to interact with cell membrane and its components. The saponin solution causes hemolysis of red blood cells by interacting with proteins, cholesterol and phospholipids. The solution remained clear red when saponin solution was added to blood suspension because the saponin dissolved the cell membrane of red blood cells in suspension. Also, the red blood cells from the saponin added solution had mostly broken cell under the microscope. Therefore, chemical hemolysis occurred in the blood suspension due to the addition of saponin.2

Reference: 1. Alberts et al. (2000) Molecular Biology of the Cell. 4th Ed. Garland Publishing Co. 2. Baumann, E., Stoya, G., Volkner, A., Richter, W., Lemke, C., Linss, W. (2000) Hemolysis of human erythrocytes with saponin affects the membrane structure. Acta Histochemica. 102, 21-35. 3. Bretscher, M.S (1985). The molecules of the cell membrane. Sci. Amer. 253, 100-108 4. Collander, R. (1954). The permeability of Nitella Cells to non-electrolytes. Physiologia Plantarum,7:420445 5. Hober et al. (1985). The permeability of the cells to organic nonelectrolytes. Physical chemistry of cells and tissues. 229-242. 6. Randall et al. (2002) Animal Physiology Mechanisms and Adaptations. 5th Ed. W.H. Freeman and Company

Appendix: Table 2: Initial Concentration of NaCl = 0.4M Final Concentration of NaCl = 0.05M Final Volume of the diluted solution = 2ml Therefore Initial Volume of NaCl = ? C1 V1 = C2 V2 0.4M * V1 = 0.05M * 2ml V1 = (0.05M * 2ml) / 0.4M V1 = 0.25ml The Volume of water to be added to make this dilution = 2ml-0.25ml = 1.75ml