Understanding Osmosis and its Effects on Red Blood Cells

LAB 2: OSMOSIS, OSMOTIC PRESSUE, AND HEMOLYSIS

Dierdra Renfroe

Biology 340-002

Lab Partners: Ale Sanchez, Luke Brown, and Abby Fox

September 15, 2016

INTRODUCTION

Erythrocytes, or what are commonly known as red blood cells (RBC) within our bodies are constantly being faced with a changing environment. Tonicity is referred to as the concentration of solutes, permeable and nonpermeable, as well as the concentration of water both influencing the water that will come and goe through the RBC, and the surrounding fluid of the RBC (Sherwood, 2013). Osmosis on the other hand is known as the movement of water from an area of low concentration to an area of high concentration and this will happen across a cell’s membrane until it reaches a state where it is isotonic.

This more blatantly means that the concentrations on both sides of the membrane are the same. Another state we can find cells in is called hypertonic, or the concentration of water is higher outside of the cell than inside of the cell, thus the cell will shrink due to the water movement outward. At other times a cell can be found in a state where the opposite is occuring and the concentration of water is much higher inside the cell than outside of the cell. In this case the water is moving inside the cell, causing the cell to swell. The process of both hypertonic and hypotonic can result in cell death is the case is severe enough, but there is a regularly natural occurrence of this fluctuation within cells normally (Williams, 2016).
In order for a cell to reach the isotonic state, and stay there, it will need to cancel the force being applied on it by osmosis and it does so by the osmotic pressure (π = CRTG). This equation takes temperature, gas constant, the concentration of solute, and the degrees of ionization of a solute. Osmotic pressure is often expressed in mOsm/kg. Also, as previously mentioned when a movement of water in or out of the cell is greater than or less than the osmotic pressure significantly then the cell will either expand or shrink until it hits its final capacity. If this cell is further pushed it will result in cell death, or hemolysis (Buffenstein et al, 2001). The processes thereafter for hemolysis is the release of the cell’s contents to the external environment and due to certain things released into the environment, we can visually see when hemolysis occurs because it shifts from being cloudy and thick to being able to see through the solution (Williams, 2016). We tend to use a spectrophotometer to measure the degrees of transmittance through calculating the degree of hemolysis of erythrocytes occurring within the various solutions. When in the lab, we routinely use a .9% saline solution to observe the erythrocytes because though of mammals have the same osmotic pressure as the solution. So in lab we are creating an ideal isotonic environment for the cells and from there we can test concentrations of solutes through variability (Williams, 2016).
We can understand the effects of and determine tonicity, osmotic pressure and osmolarity of hemolysis through the use of the red blood cells of the sheep.

In the first experiment we measured the amount of maximum absorbance of the RBC’s that were lysed and due to blood being red we expect to see a absorbance peak for the hemoglobin at around 540nm, but it could range between 500nm to the 550’s. We hypothesized that the solutions that contained minute NaCl (0.09,0.18,0.27,0.36,0.45,0.54,0.63, and 0.72% NaCl solutions) should have hemolysis readily occuring, and that it should also only occur slightly in solutions with higher concentrations of NaCl (like .81 and .9% NaCl solutions) because of the presence of the hypotonic solutions. We also hypothesized that hemolysis of cells would occur more quickly in the 0.9% NaCl plus one drop of saturated soap solution and that in the 0.9 NaCl (standard) solution there would be no hemolysis because it aids in the production of a isotonic environment. We also hypothesized that hemolysis of the 0.3 M ethylene glycol solution would be quick and 0.3 M glycerol and 0.3M glucose solutions would occur much more slowly based on their structure and hydroxyl

groups.
MATERIALS AND METHODS
For the first experiment we placed 100uL of the sheep blood into a tube with 5ml of distilled water and waited for hemolysis to occur. We then centrifuged the sample to remove the lysed cells (ghost cells). We then began measuring the percent of transmittance of the solution at differing wavelengths through the use of a Genesys spectrophotometer. We measured from 400nm to 650 nm in increments of 10 and from there the Optical Density (O.D.) was calculated. We used the O.D. – log (1/ % transmittance), where % transmittance was converted into a decimal form. From this a graph was constructed (O.D. vs. wavelength) so the maximum wavelength of light that is absorbed by hemoglobin could be found
For the second experiment a serial dilution was created. We made solutions with the concentrations of 0.9, 0.81, 0.72, 0.63, 0.54, 0.45, 0.36, 0.27, 0.18, and 0.09% NaCl. To these tubes we added 100uL of sheep’s blood to each tube, caped it and gently mixed. Then we again centrifuged so we could remove all the ghost cells. The supernatant contained the hemoglobin and for each solution was transferred into a new tube and inserted into the Genesys spectrophotometer. We turned the percent transmittance to 540nm. We again calculated the percent transmittance and converted it to O.D. by the same equation in part one. The O.D. was then used to find the percent hemolysis of each solution by using the following equation, (O.D. of Sample / Highest O.D.) * 100 = % Hemolysis). A hemolysis curve was constructed and it showed the percent hemolysis vs the saline concentration, was created to assess the amount of hemolysis that occurred in each solution.
For experiment three and four we used the same procedure, just different solutes. For part 3 we used a 0.9% NaCl (standard), a 1.8% urea solution, and 0.9% NaCl plus one drop of saturated soap solution, 2ml of each were added to an individal test tube. To each of these solutions we added 100uL of the sheep blood and waited for complete hemolysis to occur, or the solution to become a clear red liquid you can read through from the thick cloudy red color it initially was. If the solution hadn’t lysed completely within 30 minutes, then the test was stopped and it was assumed that it wouldn’t happen at all. For part 4 of the experiment, 0.3M glucose, 0.3M glycerol, and 0.3M ethylene glycol, were used to measure the amount of time it took for sheep erythrocytes to hemolyze and the same process for part three was used.
RESULTS

The O.D. of the hemoglobin from the RBC’s of the sheep that were hemolysed at various wavelengths were graphed in Figure 1. This depicts the peak absorbance of hemoglobin to be found at approximately 520nm with a second peak found around the 540nm. For Figure 2 we analyzed the percent hemolysis vs the osmotic concentration of NaCl solution. This figure depicts the complete hemolysis occurred around 90 mOsm/kg. We also show that little to no hemolysis occurred between 220-280mOsm/kg. Also taken from experiment 2 was Table 3. Which showed the approximate concentration of NaCl in solution when 10%, 50% nd 90% of sheep red blood cells underwent hemolysis. At approximately 220 mOsm/kg very little hemolysis occurred within the solution and at very high concentrations of hemolysis the concentration of the solution decreased to about 170 mOsm/kg.
Table 1 depicts three different solutions that were observed to view the time it took for a solution to completely hemolyse in seconds. The standard 0.9% NaCl shows that within a 30 minute window it did not fully hemolyse, but in 1.8% urea and 0.9% NaCl plus one drop of saturated soap solution showed hemolysis occurring very rapidly only a second apart. Table 2. shows a different set of solutions containing 0.3M glucose, 0.3M glycerol, and 0.3M ethylene glycol and their timed completion of hemolysis in minutes. Hemolysis did not occur in the 0.3M glucose solution over the 30 minute time frame. The 0.3M glycerol took 27 minutes to complete hemolysis and the 0.3M ethylene glycol reached completion sooner at only 2 minutes.

DISCUSSION
At a peak of optical density, the absorbance of hemoglobin was found to occur at 540 nm and holds true to our hypothesis. This could be to the red color of the hemoglobin, and the color across from red on the color wheel is green. This is more so the color green and how it can completely absorb the color of the hemoglobin (red). Also seen on the Figure 1 graph is another peak that is smaller peak around 520nm. This peak is also within the accepted range of 500-550nm and could be due to the blood cell’s absorbance to light at that particular wavelength also. The absorbance peak seen at 540nm was optimal and showed the wavelength of light used in experiment 2 to determine the amount of hemolysis that occurred in multiple varying concentrations of NaCl solutions (in mOsm/kg). In Buffenstein et al., a study was also done that analyzed the hemolysis of the blood cells of sheep using the same wavelength value of 540nm.
The red blood cells of the sheep show a decrease in fragility as the concentration of NaCl goes up and is depicted in Figure 2. It also shows that complete hemolysis (or 100%) occurred in a solution that was approximately 90mOsm/kg. The results attained by Buffenstein et al. also depicted by Figure 1, show a higher value of 100% hemolysis of the blood cells of sheep to occur around 150 mOsm/kg solution of the NaCl . Also in solutions that were hypotonic blood cells, solutions with low NaCl concentrations hemolysed the blood. Also the osmotic pressure of two solutions, 0.9% NaCl and the 0.81% NaCl, were found to be 686mmHg and 617mmHg and these had pressures that were greater than contrasting osmotic pressures of the RBC’s of sheep and this allowed the hemolysis to occur. These two solutions also had higher concentrations of NaCl. Figure 2 also depicts that at high concentrations of NaCl very little hemolysis is occurring, and as explained by Dr. Williams mammal RBC’s have relatively similar solute levels with solutions that are high in 0.9% NaCl (Williams, 2016). Our results show that hemolysis occurred at concentrations as high as 220mOsm/kg NaCl. This in comparison with Buffenstein et al, showed exactly the same results in Table 1 with their hemolysis occurring as high as 220 mOsm/kg. Our entire Table 3 resembles very closely to Buffenstein et al’s Table 1. With this our hypothesis again was correct and the hemolysis of blood cells of the sheep will strongly happen in solutions that are low in NaCl concentrations, which are depicted in Table 3 and Figure 2.
Our experiment 3 (Table 1), shows that hemolysis will happen more in solutions with membrane penetrating solutes. We expected no hemolysis to occur in the standard 0.9M NaCl because of the solution being in an isotonic relationship with the blood cell. In the 1.8% urea we did see hemolysis happening very quickly in about 5seconds. 16.632 grams of urea per liter (0.2769 mol/L) was found to be isotonic in the 0.9% NaCl solutions. The speed of the 0.9% NaCl plus one drop of saturated soap solution’s hemolysis was also extremely fast, coming in at 6 seconds. Due to the soap being able to dissolve membranes of the erythrocytes of the sheep to cause hemolysis, we believe our findings are correct.
Table 2 depicts a different set of solutions with membrane penetrating solutes, but these have different hydroxyl groups within their structures. We also found that this hypothesis was also supported. Both of the 0.3M glucose and 0.3M glycerol solutions did happen at very slow rates, glucose took longer than 30 minutes, and glycerol took 27 minutes whereas the ethyl glycol solution took only 2 minutes a much faster rate of hemolysis. For the glucose, maybe with an increased time it may have had a applicable time. Our data shows a trend similarly related to the amount of hydroxyl groups within a structure. The more hydroxyl groups, the slower the rate of movement is across the plasma membrane (Williams, 2016). The ethyl glycol solution presented with only 2 of these groups whereas the others had 3 or 4 hydroxyl groups. The benefits of the analysis of RBC’s of sheep were very rewarding regarding various solutions to help aid the understanding of concepts like osmotic pressure, osmolarity and tonicity and their relationship with hemolysis of the RBC’s.

SOURCES
Buffenstein R, McCarron HCK, and Dawson TJ (2001) Erythrocye osmotic fragility of red (Macropus rufus) and grey (Macropus fuliginosus and Macropus giganteus) kangaroos and free-ranging sheep of the arid regions of Australia. J Comp Physiol B 171: 41-47
Sherwood L, Klandorf H, and Yancey P. Animal Physiology: From Genes to Organisms. 2nd ed.
Belmont, CA: Brooks/Cole, 2013. Print.
Williams, Jason (2016) Physiology Laboratory Handout and Physiology Lecture Notes. Sept.2016.