The Affect of Molecular Weight and Lipid-Water Partition Coefficient on Understanding Hemolysis with Rabbit Red Blood Cells

Abstract

 The concentration of the solute and size dictates the direction osmotic effect will occur. The experimental objective is to observe rabbit red blood cells in various solution of different tonicity, isotonic, hypotonic and hypertonic, and if hemolysis can occur or not. Eight organic solvents were used to observe hemolysis, Ethanol, Ethylene glycol, Urea, Thiourea, Glucose, Arabinose, Xylose, Sucrose, Glycerol, Monoacetin, Triacetin, and Diacetin. The time it took for hemolysis to occur for each solvent were plotted on two graphs, one molecular weight vs. time and the other lipid-water partition coefficient vs. time. The result of these findings suggest that smaller molecules hemolysis faster as compared to larger molecules and the stronger the lipid-water partition coefficient the fast hemolysis can occur regardless of the size of the molecule.

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Introduction

 The plasma membrane is a biological membrane that allows separation of the exterior environment from the interior environment of a cell (Cooper GM, 2000). The plasma membrane comprises of a lipid bilayer which has embedded proteins that allow regulated movement of small molecules and atoms across the membrane for selective permeability (Cooper GM, 2000). This movement can either be classified as passive or active (requires energy) (Cooper GM, 2000). Passive transport can be done by osmosis and diffusion which exists in organic and inorganic systems (Cooper GM, 2000). Diffusion is instigated by the movement of molecules and atoms from high concentration to low concentration (Cooper GM, 2000). Diffusion continues until a uniform dispersal of the molecules is present in the solution (Odom, 1995). In liquid solutions there is constant random motion even though it is slow compared to gas diffusion which is fairly rapid (Cooper GM, 2000). The rate of diffusion is influenced by the temperature, increase in temperature increase the collision of the molecules, and the concentration gradient which also has proportional effect (Odom, 1995). For passive diffusion the molecules travel across the phospholipid bilayer without the need for any membrane proteins but instead it is controlled by the concentration of the outside and the inside of the cell (Cooper GM, 2000). Only small hydrophobic molecules can diffuse across the phospholipid bilayer, such as O₂ and CO₂ (Cooper GM, 2000). Larger molecules are unable to passively diffuse across the plasma membrane but instead can through the use of facilitated diffusion, active transport and channel proteins (Cooper GM, 2000). This allows amino acids, carbohydrates, polar/charged molecules, ions and nucleosides to travel across the cell membrane (Cooper GM, 2000). This form of diffusion is done by the concentration gradient to allow the molecules to travel across the member but the unlike passive diffusion, that allows the molecules to dissolve in the phospholipid bilayer, this is accomplish by proteins (Cooper GM, 2000). There are two kinds of proteins that allow facilitated diffusion to occur, channel and carrier proteins (Cooper GM, 2000). Channel proteins allow diffusion through pores in the membrane for specific charge and size of a molecule that open and close (Cooper GM, 2000). Carrier proteins require a confirmation change through the binding of a substrate that allows a molecule to travel across the membrane from one side to the other (Cooper GM, 2000). Sugars, nucleosides and amino acids primarily cross the membrane through carrier proteins (Cooper GM, 2000). Another form of movement by molecules and atoms is through osmosis (Borg, 2003). Osmosis is the phenomenon where a solute of low concentration can travel across the semi-permeable membrane to an area of higher concentration (Borg, 2003). This allows us to understand the attraction of water, the most permeable, to other solutes in terms of osmosis (Borg, 2003). This is simply due to the theory of hydration, where the molecules of the solute are surrounded by water because of the attraction present between them (Borg, 2003). This phenomenon allows the water to move from one side of the cell to the other through a semi-permeable when there is not enough water on one side to allow for the solute-water attraction (Borg, 2003). The factors that govern this are: solute concentration, dissociation of the solute, plasma membrane permeability as well as the temperature of the solution (Borg, 2003). However, this is mainly governed by the plasma membrane permeability and it only last until an equilibrium is achieved between the concentrations of the environment outside and inside of the cell (Borg, 2003).  The red blood cells are used to understand the functions of the plasma membrane in terms of diffusion and osmosis, as it relates to hemolysis (Animal Physiology I, 2018). Hemolysis is the rapture of red blood cells (erythrocyte) that causes hemoglobin to be released from the red blood cells into its surrounding aqueous solution (Animal Physiology I, 2018). This rapid hemolysis can be attributed to the membrane holes that appear after osmotic hemolysis has started (Seeman et al., 1973). Another reason hemolysis can occur is if the red blood cells are exposed to hypotonic or hypertonic solutions, which is the objective of this experiment (Sowemimo-Coker, 2002).  The objective is to understand three known phenomena of solutions – isotonic, hypertonic, and hypotonic – in eight different organic solutions (Animal Physiology I, 2018). Isotonic is when the solution maintains same volume inside and outside the cell having the same osmotic pression that is across the cell membrane (Animal Physiology I, 2018). When the volume inside the cell increases then a hypotonic (swelling of the cell) solution is expected while if the volume inside the cell decreases a hypertonic (shrinkage of the cell) solution is observed (Animal Physiology I, 2018). The eight different organic solutions are split into three different groups (Animal Physiology I, 2018). Group A consists of Ethanol, Ethylene glycol, Urea, and Thiourea (Animal Physiology I, 2018). Group B consists of Glucose, Arabinose, Xylose, and Sucrose (Animal Physiology I, 2018). Group C consists of Glycerol, Monoacetin, Diacetin, and Triacetin (Animal Physiology I, 2018). It is hypothesized the larger molecules will have a slower time to hemolysis compared to smaller molecules.

Methods and Materials

The experiment was conducted as per the lab protocol for Lab 1, Animal Physiology 1 SC/BIOL 3060. See lab protocol for Lab 1 (Animal Physiology I, 2018).

Results

Table 1: All the organic solutions used with their corresponding hemolysis time, lipid-water partition coefficient and molecular weight.

Organic Solution

Time to Hemolysis (min)

Lipid-Water Partition Coefficient

Molecular Weight

Ethanol

0.03

0.04

46

Ethylene glycol

0.25

0.0007

62

Urea

0.17

0.0002

60

Thiourea

0.5

0.002

76

Glucose

0

0.00003

180

Arabinose

0

0.00003

150

Xylose

0

0.00003

150

Sucrose

0

0.00003

342

Gylcerol

0.58

0.00007

92

Monoacetin

0.012

0.01

134

Diacetin

2.5

0.09

176

Triacetin

0.03

0.9

218

Figure 1: The time taken for each organic solution to undergo hemolysis compared to their lipid-water partition coefficient for their respectively. The lipid-water partition coefficient: Group A consists of Ethanol (0.04), Ethylene Glycol (0.0007), Urea (0.0002), Thiourea (0.002), Group B consists of Glucose (0.00003), Arabinose (0.00003), Xylose (0.00003), Sucrose (0.00003), and Group C consists of Glycerol (0.00007), Monoacetin (0.01), Diacetin (0.09), Triacetin (0.9)

Figure 2: The time taken for each organic solution to undergo hemolysis compared to their molecular weight respectively. The molecular weight: Group A consists of Ethanol (46), Ethylene Glycol (62), Urea (60), Thiourea (76), Group B consists of Glucose (180), Arabinose (150), Xylose (150), Sucrose (342), and Group C consists of Glycerol (92), Monoacetin (134), Diacetin (176), Triacetin (218)

The factors investigated are: lipid-water partition coefficient and molecular weight, both plotted against the time it takes for the organic solutions to undergo hemolysis (Figure 1 and 2 respectively). The lipid-water partition coefficient and time for hemolysis (in minutes) correspondingly are: Group A consists of Ethanol (0.04 and 0.03), Ethylene Glycol (0.0007 and 0.25), Urea (0.0002 and 0.17), Thiourea (0.002 and 0.5), Group B consists of Glucose (0.00003 and 0), Arabinose (0.00003 and 0), Xylose (0.00003 and 0), Sucrose (0.00003 and 0), and Group C consists of Glycerol (0.00007 and 0.58), Monoacetin (0.01 and 0.012), Diacetin (0.09 and 2.5), Triacetin (0.9 and 0.03). The molecular weight and time for hemolysis (in minutes) correspondingly are: Group A consists of Ethanol (46 and 0.03), Ethylene 3Glycol (62 and 0.25), Urea (60 and 0.17), Thiourea (76 and 0.5), Group B consists of Glucose (180 and 0), Arabinose (150 and 0), Xylose (150 and 0), Sucrose (342 and 0), and Group C consists of Glycerol (92 and 0.58), Monoacetin (134 and 0.012), Diacetin (176 and 2.5), Triacetin (218 and 0.03). For the first figure the equation of the line and R² is: Group A the equation of the line y = -6.7979x + 0.3104, R² = 0.4533 and Group C the equation of the line is y=-0.93x + 1.013, R² = 0.1184. For the second figure the equation of the line and R² are: Group A the equation of the line y = 0.0158x – 0.7247, R² = 0.964 and Group C the equation of the line y = 0.002x + 0.4712, R² = 0.0085.

Discussion and Conclusion

 The R² is used to correlate the relationship between two variables plotted on a graph. For Figure 1 the R² for Group A is 0.4533 and Group C it is 0.1184. This indicates that the lipid-water partition coefficient for Group A has a relatively strong correlation with the time it takes to hemolyze, Group C does not have a strong correlation since the R² value is small. For Group A the relative strong correlation is due to the polar compounds that are in that group which allows them to diffusion into the red blood cells to cause hemolysis (Burgen, 1962). For Group B there is no relationship between the two as the time to hemolyze is zero for all of the organic solutions in that group. The reason for this is all of the organic solutions are sugars. Sugars require facilitated diffusion and do not readily diffuse into a cell unless the need occurs (Cooper GM, 2000). They are also known to decrease the osmotic pressure inside the cell since sugar molecules to do not readily diffuse through the membrane and thus the hemolysis rate is non-existent or is significantly lower as compared to the other groups (Grosicki and Husas, 1954). Group C molecules have a higher lipid-water partition coefficient than the other groups which indicated they are more willingly to diffusion through the membrane of the red blood cells. The reason behind this is because organic molecules are known to have an attraction towards solvents where predominately a non-polar hydrocarbon structure exists (Jacobs, 1950). Therefore, greater the partition coefficient is between the organic solvent and water, the more easily the organic solvent is able to enter the cell (Jacobs, 1950). Although for polar solvents it is the opposite effect in terms of permeability into the cell (Jacobs, 1950). So, there is an increase in the rate of diffusion into the red blood cell as the lipid solubility increases even though the molecular weight is higher (Jacobs, 1950). For Figure 2 R² for Group A is 0.964 and Group C it is 0.0085. There is no R² value for Group B since the organic solvents in that group did not undergo hemolysis. This shows that the molecular weight of the organic solvent has an impact on the time it takes to undergo hemolysis after the blood suspension was added, the smaller the molecule the faster the hemolysis occurs (as shown in Figure 2). This is due to the simple fact the plasma membrane is generally more permeable to smaller molecules as compared to larger one as they enter the cell effortlessly (Jacobs, 1950). So, the hemolysis rate is dependent on the size of the molecules (Shalel et al., 2002). Although the permeability of red blood cells varies from species to species for small molecules (Jacobs, 1950). For Group B in both figures no hemolysis had occurred (see Table 1). This can be attributed to the chance that the concentration of the cell and the solution is the same which would create an isosmotic solution, preventing hemolysis from occurring since there is no diffusion taking place. This can be prevented by changing the concentrations of the sugars to either higher or lower than the stated concentration to see if there is a possibility of hemolysis to occur or not.

 In conclusion, the larger the lipid-water partition coefficient the organic molecule has the more readily it can diffusion into the red blood cell and cause hemolysis, regardless of the size of the molecule. For the molecular weight of an organic molecule it is found the larger the molecule the harder it would be to diffuse through the cell membrane of the red blood cell as compared to a molecule with a smaller molecule weight the time to hemolysis is smaller.

References

–          Animal Physiology I. Laboratory 1: Properties of Membranes. SC/BIOL 3060, 2018. York University.

–          Borg, F.G., 2003. What is osmosis? Explanation and understanding of a physical phenomenon. arXiv preprint physics/0305011.

–          Burgen, A.S.V., 1962. The structure and function of cell membranes. Canadian journal of biochemistry and physiology, 40(9), pp.1253-1260.

–          Cooper GM, 2000. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; Transport of Small Molecules. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9847/

–          Grosicki, T.S. and Husas, W.J., 1954. Isotonic solutions. III. Amino acids and sugars. Journal of the American Pharmaceutical Association, 43(10), pp.632-635.

–          Jacobs, M.H., 1950. Surface properties of the erythrocyte. Annals of the New York Academy of Sciences, 50(8), pp.824-834.

–          Odom, A.L., 1995. Secondary & college biology students’ misconceptions about diffusion & osmosis. The American Biology Teacher, pp.409-415.

–          Seeman, P., Cheng, D. and Iles, G.H., 1973. Structure of membrane holes in osmotic and saponin hemolysis. The Journal of cell biology, 56(2), pp.519-527.

–          Shalel, S., Streichman, S. and Marmur, A., 2002. The mechanism of hemolysis by surfactants: effect of solution composition. Journal of colloid and interface science, 252(1), pp.66-76.

–          Sowemimo-Coker, S.O., 2002. Red blood cell hemolysis during processing. Transfusion medicine reviews, 16(1), pp.46-60.