A red blood cell is placed in a 0.6% NaCI solution, describe the effects on the cell--Homework Help Plz?

Answer 1

NaCl - Sodium Chloride (salt)

Introduction

The blood of animals is well designed as it changes its properties in order to facilitate gas exchange between the blood and the tissues. Blood serves as a transport mechanism for many different molecules such as hormones, those involved in metabolism, and many other chemical agents found in the bodies of animals. In certain conditions, the volume of the red blood cells can change (increase or decrease) as a physiological response to changing conditions, such as the concentration of extracellular solutes, or changes in pH. Blood will also change its volume properties when different levels of oxygenation occur, and with the effects of molecules such as epinephrine. The main objectives of this investigation are to design some experiments that will test the affect of varying NaCl concentration on red blood cell volume, and to discover some other factors that can alter the red blood cell volume.

Material & Methods

In this experimentation, the cell volume of the red blood cells in the sample of bovine blood were determined by measuring hematocrit (hematocrit (Hct) = % packed red cell volume). Measuring hematocrit indicates the percentage of a sample of blood that is composed of red blood cells.
Measuring Hematocrit

The hematocrit of the blood sample (any blood sample in this lab, regardless of treatment) was determined using the following method. First, the blood sample was well mixed. A hematocrit tube was dipped into the blood at a 45°C angle, allowing capillarity to fill the tube with blood. When the tube filled to around 80 - 90%, the filled end was capped using Critoseal. The tube was then placed in the hemafuge (with the Critoseal towards the outside). The tube is then centrifuged for 5 minutes @ 10,000 g. After the five minutes of centrifugation, the %Hct of the blood sample is measured using a reference card.

Effects of NaCl Concentration on Red Blood Cell Volume
1ml of the blood sample was placed in each of 3 plastic centrifuge tubes and centrifuged for 3 minutes. The centrifuge forces the red blood cells to the bottom of the tube, leaving plasma at the top. 200µl of plasma was then removed from each centrifuge tube, and replaced with 200µL of a salt solution of known concentration. The tubes were then shaken in order to mix the red blood cells, the plasma, and the added NaCl solution. Three Hct tubes were then made from each of the three blood samples, and the % Hct was then determined. This procedure was completed for salt concentrations (molarity of added NaCl) of 0.075, 0.15, 0.30, 0.60, 1.20, and 2.40 M solutions of NaCl.

This procedure was also done for the conditions of distilled water, and a condition where no NaCl was added to the centrifuged blood sample (control).

The following calculation was used in order to calculate the new dilutions :

New concentration = (200 µL • X M) + (300 µL • 0.15 M)
500 µL

Where X M = the concentration of the added NaCl solution

Effect of Oxygenation Status

After measuring the Hct of the stock solution of blood, the blood was treated with 02, N2, Air, and CO2. This was done by bubbling the various gases through the blood and taking Hct (in triplicate) values immediately after.

Effect of Adrenaline (Epinephrine) on Red Blood Cell volume

The stock solution of blood was measured for Hct value and then 0.1 m 10-5 adrenaline solution was added. Hct values were taken after 5, 15, and 30 minutes.

Results


Table 1 Results of adding varying concentrations of NaCl. Note that (*) indicates that lysis occurred (plasma was red at the end of centrifugation)


Table 2 Mean Hct results for distilled water, epinephrine treatments, and exposure to various gasses. Note that (*) indicates that lysis occurred (plasma was red at the end of centrifugation)


Figure 1 Illustration of the decrease in % Hct as concentration of NaCl (M) increases.

Discussion

Hematocrit values are a numerical representation of the cell volume of the red blood cells. An increase in the hematocrit number indicates that the red blood cells are increasing in size, and a decrease in % hematocrit indicates a decrease in red blood cell volume.

Effects of NaCl Concentration on Red Blood Cell Volume

A red blood cell (rbc) will attempt to regulate its volume when placed in a solution of impermeable substances dissolved in water (Randall, 1997). A solution is considered to be isosmotic when it has an osmolality equal to that of normal plasma (Freedman, 1998). The osmolarity is the osmotic pressure that a cell effectively has exerted upon it (Randall, 1997). The osmotic pressure is the pressure that is created by osmosis between different solutions that are separated by a semipermeable membrane (Randall, 1997). If a rbc is placed in an isotonic solution, the cell will maintain its normal volume because no osmotic pressure is developed. A hypotonic solution has a lower concentration of solutes than the interior of the cell. Therefore, a hypotonic solution will cause water to flow into the cell, as the concentration of solutes inside of the cell is higher. If this water influx continues for a long period of time (with ineffective (or absence of) volume regulation by the cell), the cell my lyse (burst) as the pressure builds up inside to a magnitude greater than can be handled by the cellular membrane. In a hypertonic solution, the solutes outside of the cell have a higher concentration than those inside of the cell. In a hypertonic solution, the water will leave the cell, which will cause it to shrink (crenate).

A red blood cell’s volume will increase until it reaches a point called the critical hemolytic volume. When the red blood cell swells, the surface are will remain constant while the cell gains water and changes from a biconcave shape to a spherical one (Freedman, 1998). When the critical volume is reached, the cell can stretch very little, so further pressure from influx of water will cause cell lysis. The ability of red blood cells to adapt to changing conditions is often related to their age. Old cells and very young cells often cannot adapt as quickly or as well to such changes and may undergo fatal lysis or crenation faster than mature cells (Randall, 1997). Human red blood cells rapidly adjust their volumes when placed in a hypertonic or hypotonic solution, In contrast, red blood cells from other species (including duck, salamander, dogs, trout, and skates) change the permeability of their membranes in response to fluctuating extracellular solute concentrations. This type of adaptation will actually change the intracellular solute concentration, which will change the cell volumes back to a normal level (Freedman, 1998).

There are a number of ways in which red blood cells (and animal cells in general) cope with changes in solute concentration on the outside of their membrane surfaces. There may be a volume regulatory decrease (RVD) or increase (RVI) depending upon the concentration of the solutes on the outside of the cellular membrane (Freedman, 1998). The response that is activated by the swelling of the cells usually involves an increase in the K+/Cl- transporters (see figure 2) in ducks, sheep, rabbits, and some other mammals (Freedman, 1998; Motais et al, 1997). The swelling response may also trigger the formation of a channel (in which band 3 is involved) that allows osmolytes such as taurine (and other small, organic, osmotically active solutes) to be transported out of the cell (Freedman, 1998; Motais et al, 1997). The shrink activated response in red blood cells involves the activation of Na+/K+/2 Cl- cotransporters (in general, and in the case of the duck (Freedman, 1998)).


Figure 2 The main transport systems of red blood cells. Starting with the Na+/K+ pump and the passive leakage pathways for Na+ and K+ on the right. Proceeding clockwise : K+/Cl- cotransport, Na+/K+/2 Cl- cotransport. Cl-/HCO-3 exchange, the Ca2+ pump with Ca2+ leakage pathway, the Ca2+ activated K+ channel, and Cl- conductance (Adapted from Freedman, 1998).

If a swelling or shrinking results in the influx of efflux of ions such as K+/Cl-/ and Na+, this will serve to increase or decrease the concentration gradient between the exterior of the cell and its interior. This will cause the cell to stop the influx or efflux of water molecules, which will subsequently curb the stresses caused by cell swelling or shrinking. Molecules such as taurine have also been found to be lost from the cells in the cells response to swelling. Motais et al (1997) suggest that the loss of these organic osmotically active solutes (through a specific channel called the volume-sensitive organic osmolyte anion channel (VSOAC)) likely accounts for close to half of the response during regulatory volume decrease of a blood cell. VSOAC channels have been implicated in the efflux of taurine, betain, inositol (Goldstein & Davis, 1994), as well as sorbitol and glycerophosphocholines (Musch et al, 1994) from a cell exhibiting a swell response mechanism These processes occur to a variable amount in different species, with mammals normally utilizing K+/Cl- transport, and blood cells in species such as fish utlilizing VSOAC channels. It should be noted that these are the main transporters used, but that the other transporters are also utilized, though to a lesser extent.


Figure 1 illustrates the effects on blood cell volume with varying concentration of NaCl ions. The molarity in plasma of NaCl is approximately 0.15M, therefore the concentration of 0.15M NaCl treatment would be considered isosmotic to red blood cell plasma. The change in concentration to 0.12 M NaCl would be a hypotonic condition, and the conditions of 0.21, 0.33, 0.157, and 1.05M NaCl would be hypertonic conditions. If the concentration in this blood sample was actually 0.15M, then the 0.15M treatment would show little difference compared to the control (no salt solution added) shown in table 2. The control showed a % Hct of 46.67 ± 2.60, while the condition of 0.15 M showed a % Hct of 49.78 ± 1.20, values which are statistically different using a students t-test (p < 0.001). Since the concentration of 0.12 is lower than 0.15M, one would expect the cell to be in a hypotonic situation, causing the cell to increase in size (and therefore increase % Hct). This is what occurred in this experimentation. The % Hct for the 0.12M concentration was 57.33 ± 0.71 (table 1) while for the 0.15M concentration it was 49.78 ± 1.20M. This difference is statistically significant (p < 0.001). The remainder of the trials were for hypertonic conditions, and as expected, the cells volume shrunk as water left the cells. Notice in figure 1 that the last point in the data (for a concentration of 1.05M NaCl) had a slightly higher hematocrit than the previous data point. The last data point was different in that the plasma in the hematocrit tube was turned red due to the red blood cells breaking. This was not a case of lysis (water was leaving the cell) but the cells seemed to have been ruptured anyway. One possible reason for this was that a large percentage of the cells’ water had left the cell, leaving it crenated. Possibly, the crenated condition also weakens the cells membrane, and when the rbc’s were centrifuged, the cell membranes ruptured due to the force of the centrifuge.

A mentioned earlier, cells can withstand a small change in extracellular solute concentration, and maintain normal cell volumes after regulatory processes are initiated. Motais et al (1997) mention that a volume regulatory increase or decrease requires the usage of ATP. It seems possible that there was no regulation noted in these particular samples of red blood cells because there were not much in the way of ATP available. In order for the cells to regulate their volume through the initiation of ion channels, ATP must be utilized. The efflux of organic molecules such as taurine through VSOAC’s must also be accompanied by the utlilization of ATP stores. Freedman (1998) suggests that there is a time period of swelling or shrinkage before regulation processes begin to function, without noting the time period actually involved. It would seem likely that this period of time would be minimal, in order to ensure that there was no permanent damage due to the stresses involved in shrinking or swelling. As there was no concentration of NaCl (other than possible 0.15 or 0.21M NaCl) in this experiment that allowed the volume of the red blood cells to remain relatively constant, there may be evidence for a long time delay in volume regulatory decrease or increase. However, it should be noted that the time given for the effects of the salt solution to become apparent varied greatly between the different concentration conditions. This was due to the need to share equipment and related problems. While it is possible that there is a large amount of time needed before the regularity decrease or increase occurs, it seems unlikely.

Effect of Oxygenation Status

Mammalian red blood cells utilize the hemoglobin molecule for gas transport and exchange. Each hemoglobin molecule can carry a maximum of four oxygen molecules (oxygenated hemoglobin is referred to as oxyhemoglobin. If there is no O2 bound, it is referred to as deoxyhemoglobin.

Addition of O2

The addition of oxygen gas to a sample of blood will cause the hemoglobin to bind with oxygen molecules. Blood from trout show increased hematocrit values when oxygenated, and this has been shown in mammals as well (Sorensen & Weber, 1995). In this experimentation, the blood exposed to oxygen gas showed a statistically significant increase (p< 0.01) in % Hct from that of the control. The O2 carrying capacity varies proportionally with flow resistance (and therefore Hct). This makes sense as a physiological advantage since oxygen exchange with the tissues occurs at the capillaries (and lungs), where there is a high resistance to flow. Since the red blood cells “want” to dump their oxygen at these locations (and pick up CO2), increasing the Hct at times of high oxygen saturation will aid in this exchange (Randall, 1997).
Addition of CO2

The transport of CO2 from the tissues of the body to the lungs is aided by bicarbonate ions produced in the red blood cells (figure 3). This is a cyclic reaction known as the Jacobs - Stewart cycle. Carbon dioxide reacts with water in order to form carbonic acid, and carbonic anhydrase catalyzes its hydration to carbonic acid. Carbonic acid will dissociate into carbonate and bicarbonate ions. Carbon dioxide also reacts with H+ ions to form bicarbonate (Randall, 1997). The subsequent decrease in pH will cause a swelling of a red blood cell, likely due to the influx of ions, mainly Na+ and Cl- (due to the activity of the transporters involved in the Jacobs-Stewart cycle), with water following behind (Lessard et al, 1995). The data for this experimentation showed red blood cell swelling due to the reduced pH (from addition of C02 gas), at statistically significant levels (p <0.001). As with the increased Hct from the addition of oxygen gas, the increased Hct with addition of CO2 also makes sense. It is advantageous to gas exchange to slow the blood flow at the point of the capillaries.


Figure 3 The Jacobs-Steward cycle for red blood cells (taken from Freedman, 1998).

Addition of N2

The addition of N2 gas to the blood sample caused an increase in Hct to a statistically significant degree (p <0.05). However, one would expect that N2 would have either no effect, or would decrease the Hct. If the partial pressure of N2 is high enough, the gas will force the CO2 and O2 from the hemoglobin molecule (Akers, 1998). Since it has already been established that CO2 and O2 gasses increase the size of the red blood cell, it seems reasonable to assume that removing these from the red blood cell will decrease the Hct.

Addition of Air

With the addition of air, one would expect no changes in Hct from the control. Since the solution is in contact with normal air regularly anyway, adding it would likely not cause any effect. In this experimentation, there was a significant difference between the control and the sample treated with air (p < 0.01). There does not seem to be any explanation for this, though there may have been some experimental error when measuring the control Hct.


Effect of Adrenaline (Epinephrine) on Red Blood Cell volume

Epinephrine, and other catacholamines cause a swelling of red blood cells due to the activation of the Na+/H+ pump (in mammals). The activation of this transporter will cause a decrease in the plasma pH and increase the intracellular pH (Sorensen & Weber, 1995). This will cause a similar effect to that of CO2 (Kaloyianni & Rasidaki, 1996). Since there will be Cl- and Na+ ions influxing into the cell, water will be brought along with them, causing the cell to swell. The data for this experiment shows that epinephrine increases (statistically significant after 30 minutes of exposure) the Hct of the blood solution (p <0.001. However, this is an eventual development. Although epinephrine is supposed to increase red blood cell volume, it appears that this is a time dependent effect. The first trial with epinephrine measured Hct after 5 minutes of epinephrine exposure. The data at this point seems to indicate that initially, the epinephrine decreased Hct. This seems unlikely, as the effect of epinephrine is likely a constant one. The second trial with epinephrine (after 15 minutes) showed an increase from the control, though not at a statistically significant level. The final trial (after 30 minutes) did show a significant increase in Hct. The first trial may have experimental error involved in the eventual decrease from the control.

A decrease in pH has been shown the decrease the affinity of hemoglobin for oxygen (Randall, 1997). The normal release of epinephrine is when one is excited or stressed. It would seem that at this point, one would want to increase the propensity for gas exchange, as well as not having effects upon hemoglobin’s affinity for oxygen. However, an overall positive gas exchange affect will likely occur even though the affinity for oxygen decrease. First, with the increased blood pressure and flow due to epinephrine, more blood per minute will be circulating over a specific tissue. Second, the decrease in affinity for oxygen is not likely to a very large extend, so the overall effect of epinephrine on gas exchange will be that of an increase.

It should be noted that the effects (after 30 minutes) of epinephrine were not statistically different from those of O2 or CO2.

References


Akers, T.K. (1998) Physiological Effects of Pressure on Cell Function. In : Cell Physiology Source Book (N. Sperelakis, ed) 2nd edition. Academic Press Inc.

Freedman, J.C. (1998) Membrane Transport in Red Blood Cells. In : Cell Physiology Source Book (N. Sperelakis, ed) 2nd edition. Academic Press Inc.

Kaloyianni, M., & Rasidaki, A. (1996). Adrenergic responses of R. ridibunda Red Cells. The Journal of Experimental Zoology. 279:175-185.

Goldstein, L., & Davis, E.M. (1994) Taurine, betaine, and inositol share a volume-sensitive transporter in skate erythrocyte cell membrane. American Journal of Physiology. 267:R426-R179.

Kaloyianni, M., & Rasidaki, A. (1996). Adrenergic responses of R. ridibunda Red Cells. The Journal of Experimental Zoology. 279:175-185.

Lessard, J., Val, A.L., Aota, S., & Randall, D.J. (1995). Why is there no carbonic anhydrase activity available to fish plasma? Journal of Experimental Biology. 198:31-38.

Motais, R., Fievet, B., Borgese, F., & Garcia-Romeu, F. (1997) Association of the Band 3 Protein With A Volume-Activated Anion and Amino Acid Channel : A Molecular Approach. The Journal of Experimental Biology. 216:361-367.

Musch, M.W., Leffingwell, T.R., & Goldstein, L. (1994). Band 3 modulation and hypotonic- stimulated taurine efflux in skate erythrocytes. American Journal of Physiology. 35:R65- R74.

Randall, D., Burggren, W., & French, K. (1997). Ekert Animal Physiology. 4th edition. W.H. Freeman and Company.

Sorensen, B. & Weber, R.E. (1995). Effects of Oxygenation and the Stress Hormones Adrenaline and Cortisol on the Viscosity of Blood from the Trout Oncorhynchus mykiss. 198:453-459.

Answer 2

1

Answer 3

The RBC will shrink as there is more water inside RBC compare to NaCl solution. So water will leave the RBC through osmosis. This will continue untill the water outside the cells and water inside the cells is same or equal.

Answer 4

The cell would shrink or look shriveled because it was placed in a hypertonic solution. The red blood cell is being "overcome" with extra sodium causing the cell to push out excess sodium chloride in the cell.

Answer 5

Actually red blood cells are approximately isotonic to 0.9% NaCl.
Thus, 0.6% NaCl is hypotonic, water would enter the cells which would swell probably until they burst.

Answer 6

The human erythrocyte has the biconcave disc shape to optimise the flow properties of the blood in the large vessels. It is linked to the pressure its pushing against it, and its necessity to easily flow through thin vessels, such as venules and arterioles, and also serves as an efficient shape to flowe through the large elastic vessels, the vena cana, aorta, pulmonary trunk, etc.

Answer 7

all of the water inside of the cell will exit the cell membrane which is semipermiable. the water is seeking to go from higher concentratrion to lower concentration osmosis is movement of water across a semipermiable membrane. the cell will eventually shrind a process called plasmoloptosis.