How Oryctolagus Cuniculus Red Blood Cell Membrane Properties Influence Hemolysis In Organic Molecules

Abstract

Hemolysis occurs when hypotonic solutions lead to cell swelling and eventual lysis (Goodhead et al, 2017). From hemolytic disease of the new born (Dean, 2005) to sickle cell disease (SCD) (Milligan, 2013), hemolysis is the reason behind many chronic hereditary or acquired hemolytic anemias (Kato, 2017). In this experiment, Oryctolagus cuniculus red blood cells were utilized to observe how and why organic molecules effect hemolysis. It was hypothesized that when 5 drops cell suspension was added to 5mL of 0.15M NaCl the solution would be isotonic (opaque) and when added to 5mL of distilled H2O, the solution would be hypotonic (clear red) resulting in hemolysis. Both solutions appeared as predicted. 5 drops of cell suspension was added to 5 mL of 0.2M of several organic molecules to observe molecular weight vs average time to hemolysis and lipid-water partition coefficient vs average time to hemolysis. Conclusions about correlations between the factors were established. Thus, studying effects of various organic molecules on mammalian red blood cells gives insight on research that can provide us with beneficial information to assist us in finding cures for hemolytic diseases.

Introduction

Hemolysis is one of the most important performance parameters of blood pumps (Kozo et al, 1994). It occurs when hemoglobin bursts from red blood cells due to increased osmotic pressure. (Schaer J et al, 2013). Hemolysis occurs in many hematological and non-hematological diseases. (Schaer J et al, 2013). Hemolytic anemia occurs when the production of new RBCs (red blood cells) from bone marrow fails to compensate for the loss of blood cells during hemolysis (Orf, 2015). Studying hemolysis and its effects on red blood cells plays a crucial role in finding cures for its related diseases. The plasma membrane plays an important role in hemolysis and red blood cells prove useful when it comes to studying the barrier. (Backx P, 2019). The structure of the plasma membrane consists of a phospholipid bilayer that has proteins embedded within to carry out specific functions, including selective transport of molecules. (Cooper GM, 2000). The bilayer acts as a barrier between the interior and exterior environment of a cell, in turn forming a concentration gradient. This gradient is responsible for diffusion and osmosis of contents in and out of the cell. Diffusion is the tendency of molecules to move from an area of high concentration to an area of low concentration. (Backx P, 2019). Osmosis is a process in which the concentration difference between two solutions creates pressure difference across a separating semipermeable membrane. Solvent transport takes place from the more diluted solution to that of higher concentration, until equilibrium is reached (Minkov et al, 2013).

Plasma membranes are more permeable to water than they are to solutes. (Backx P, 2019). A solution in which cells maintain the same volume which they have in the plasma/ tissue fluid is isotonic. (Backx P, 2019). However, plasma membranes tend to hemolyse and release hemoglobin with an increase in pressure. A cell that is hemolysed changes from opaque to a clear red colour in a solution. If the cell volume increases, the external solution is hypotonic, if it decreases it is hypertonic (Backx P, 2019).

The experiment conducted allowed us to observe how the red blood cells of Oryctolagus cuniculus hemolysed in various concentrations of NaCl, NH4Cl, urea and sucrose along with other organic molecules. We inspected relationships between molecular weight vs time to hemolysis as well as lipid-water partition coefficient vs time to hemolysis. We hypothesized that when 5 drops of the cell suspension were added to the 5mL of 0.15M NaCl the solution would be isotonic due to their identical concentrations and when 5mL of distilled H2O was added, the solution would be hypotonic and the suspension would hemolyse as the membrane is permeable to water and would flow into the cell, causing it to burst.

Materials and Methods

Detailed experimental procedure can be found in Animal Physiology I BIOL 3060 Laboratory manual (Backx P, 2019).

Results

The results for the first part of the experiment indicated that the solution that had the cell suspension added to 5 mL of 0.15M NaCl appeared opaque, while the addition of the cell suspension to 5 mL with distilled H2O showed the predicted clear red. We determined the minimal concentration of NaCl, NH4Cl, sucrose and urea necessary for the cell suspension to hemolyse. Obtained results are displayed in Table 2 (located in the Appendix). The lowest average concentration necessary for hemolysis was NH4Cl with 0.06M. The highest average concentration necessary was sucrose with 0.1465M. In the second portion of the experiment, we determined the amount of time it took to notice hemolysis in chemicals in Groups A and C. The cell suspension was added to 5mL of 0.2M of each solution. Table 3 (located in the Appendix) displays timings recorded by groups as to how long it took for each chemical to hemolyse the suspension. The data collected was utilized in Table 1 which shows the average amount of time it took for each chemical to hemolyse. Urea took the shortest average amount of time to hemolyse at 23.34 seconds while Triacetin took the longest average amount of time to hemolyse at 224 seconds. The graph in Figure 1 displays the relationship between molecular weight and time to hemolysis. There was a steady linear increase and a positive correlation for both groups. Chemicals in Group A hemolysed faster than chemicals in Group C. The graph in Figure 2 displays the relationship between lipid-water partition coefficient and average time to hemolysis. There is a linear increase for chemicals in Group C and a linear decrease for chemicals in Group A. Chemicals in Group A hemolysed faster than chemicals in Group C.

Table 1. The chemicals that were used to test for hemolysis in Oryctolagus cuniculus red blood cells along with their lipid-water partition coefficients and their molecular weights. 5 drops of cell suspension was added to 5mL of 0.2M of each chemical listed below. The chemical that took the longest average amount of time to hemolyse was Triacetin with 224 seconds while the chemical that took the shortest average amount of time to hemolyse was Urea with 23.34 seconds.

Chemical

Lipid-Water Partition Coefficient (P) / Molecular Weight (g/mol) / Average Time to Hemolysis (s)

• Group A: Ethanol
• 0.04 / 46 / 25.78
• Ethylene Glycol
• 0.0007 / 62 / 47.54
• Urea
• 0.0002 / 76 / 23.34
• Thiourea
• 0.002 / 96 / 50.54
• Group C: Glycerol
• 0.00007 / 92 / 80
• Monoacetin
• 0.01 / 134 / 44.5
• Diacetin
• 0.09 / 176 / 167.5

1. Triacetin
2. 0.9 / 218 / 224

Figure 1. Molecular Weight vs Average Time to Hemolysis. The graph above displays the relationship between the molecular weights of the chemicals in Groups A and C (listed in Table 1) and the average amount of time it took for them to hemolyse. 5 drops of Oryctolagus cuniculus red blood cell suspension were added to 5 mL of 0.2M of each chemical in the two groups. The blue points and linear trend line display results from Group A. The orange points and linear trend line display results from Group C. Both groups display a positive correlation between the molecular weight and the time it took for the chemicals to hemolyse. Both groups show a positive slope and R2 value – Group A has an R2 value of 0.57065 and a slope of 0.652 while Group C has an R2 value of 0.7682 and a slope of 0.5813. The chemicals in Group A hemolysed faster than the chemicals in Group C showing that molecular weight does have an effect on time to hemolysis – the lower the molecular weight, the faster a cell will hemolyse. SEM bars are displayed for each chemical in both groups.

Figure 2. Lipid-Water Partition Coefficient vs Average Time to Hemolysis. The graph above displays the relationship between the lipid-water partition coefficients of the chemicals in Groups A and C (listed in Table 1) and the average amount of time it took for them to hemolyse. 5 drops of Oryctolagus cuniculus red blood cell suspension were added to 5 mL of 0.2M of each chemical in the two groups. The blue points and linear trend line display results from Group A. The orange points and linear trend line display results from Group C. Group A displays a negative correlation between the lipid-water partition coefficient and the time it took for the chemicals to hemolyse while Group C shows a positive correlation. Group A has an R2 value of 0.24031 and a slope of -0.0007 while Group C has an R2 value of 0.68165 and a slope of 0.0044. The chemicals in Group A hemolysed faster than the chemicals in Group C showing that lipid- water partition coefficients do have an effect on time to hemolysis – the smaller the lipid-water partition coefficient, the faster a cell will hemolyse. SEM bars are displayed for each chemical in both groups.

Discussion

As mentioned earlier, one property of the plasma membrane is the selectively permeable nature of the cell membrane that allows for the movement of some solutes and prevents the movement of others (Goodhead et al, 2017). Studying the effects of the addition of red blood cells in different solutions can help with research regarding diseases that are affected by hemolysis of cells and finding potential cures. In this experiment, we hypothesized that the 5 drops of cell suspension would hemolyse in distilled H2O but would be isotonic when added to 0.15M NaCl. We observed that the cell suspension turned the solution a clear red when added to distilled H2O indicating hemolysis. By making the solution with distilled water, complete hemolysis occurred due to the hypotonic effects (Goodhead et al, 2017). The plasma membrane burst due to the flow of water into the cell, generating the release of hemoglobin. The cell suspension that was added to 0.15M NaCl was opaque indicating that the cell suspension remained unhemolysed and that it did not take place because the concentration of NaCl used to make the cell suspension and the concentration of the NaCl used to make the solution were of the same strength (0.15M). Therefore, we can accept our hypothesis – that the solution with distilled water would undergo hemolysis and the solution with 0.15M would be unhemolysed.

The second portion of the experiment we added drops of cell suspension to various organic molecules and seeing how long it took them to hemolyse and how molecular weight and lipid-water partition coefficient affected hemolytic activity. Figure 1 displays the relationship between molecular weight and the average time to hemolysis when drops of cell suspension were added to 5 mL of 0.2M chemicals in Groups A and C. The graph shows chemicals in Group A hemolysed faster than chemicals in Group C. The chemicals in Group C had higher molecular weights than the chemicals in Group A and since Group A hemolysed faster we can conclude that a lower molecular weight will lead to a faster hemolysis reaction.

Figure 2 displays the relationship between the lipid-water coefficient and the average time to hemolysis when 5 drops of cell suspension was added to 5 mL of 0.2M chemicals in Group A and C. The graph shows chemicals in Group A hemolysed faster than the chemicals in Group C. Chemicals in Group A had lower lipid-water coefficients than chemicals in Group C. Therefore, we can conclude that a smaller lipid-water partition coefficient will lead to a faster hemolysis reaction.

Chemicals in Group C took longer to hemolyse than chemicals in Group A which could be explained by their molecular weight and lipid-water coefficient – these chemicals had higher molecular weights and higher lipid-water partition coefficients. The results of earlier classical studies have already shown that permeability is governed by at least three factors: molecular size, lipid solubility, and the chemical nature of the solute (Sha’afi et al, 1971) which can explain why Group C was slower than Group A.

This experiment provided us with the opportunity to observe the effects of various organic molecules on Oryctolagus Cuniculus red blood cells through the processes of diffusion and osmosis. Researching and observing hemolytic activity can help develop cures for human (and animal) hemolytic related diseases to help us improve medicine for future generations.

References

1. Backx, P. (2019). Biol 3060 Animal Physiology I Lab 1: Properties of Membranes. York University, Toronto.
2. Cooper, Geoffrey M. (Jan 1970). “Structure of the Plasma Membrane.” The Cell: A Molecular Approach. 2nd Edition., https://www.ncbi.nlm.nih.gov/books/NBK9898/.
3. Dean, Laura. (2005). “Hemolytic Disease of the Newborn.” Blood Groups and Red Cell Antigens https://www.ncbi.nlm.nih.gov/books/NBK2266/.
4. Goodhead, Lauren K., et al. (May 2017). “Measuring Osmosis and Hemolysis of Red Blood Cells.” https://www.physiology.org/doi/full/10.1152/advan.00083.2016.
5. Kato, Gregory J, et al. (Mar 2017). “Intravascular Hemolysis and the Pathophysiology of Sickle Cell Disease.” The Journal of Clinical Investigation https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330745/.
6. Milligan, C., et al. (Jan 2013). “A non-electrolyte haemolysis assay for diagnosis and prognosis of sickle cell disease.” The Journal of Physiology https://www-ncbi-nlm-nih-gov.ezproxy.library.yorku.ca/pmc/articles/PMC3607166/
7. Minkov, Ivan L, et al. (Dec 2013). “Equilibrium and Dynamic Osmotic Behaviour of Aqueous Solutions with Varied Concentration at Constant and Variable Volume.” The Scientific World Journal https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3888744/
8. Kozo, Naito et al. (Nov 2008). “The Need for Standardizing the Index of Hemolysis.” Wiley Online Library https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1525-1594.1994.tb03292.x.
9. Orf, Katharine, and Aubrey J Cunnington. (June 2015). “Infection-Related Hemolysis and Susceptibility to Gram-Negative Bacterial Co-Infection.” Frontiers in Microbiology, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485309/.
10. Schaer, Dominik J., et al. (Feb 2013). “Hemolysis and Free Hemoglobin Revisited: Exploring Hemoglobin and Hemin Scavengers as a Novel Class of Therapeutic Proteins.” Blood Journal, http://www.bloodjournal.org/content/121/8/1276.
11. Sha’afi., et al. (Sept 1971). ‘Permeability of Red Cell Membranes to Small Hydrophobic and Lipophilic Solutes.” The Journal of General Physiology. http://jgp.rupress.org/content/jgp/58/3/238.full.pdf