Lab 3: Red Blood Cell and Tonicity
Isotonic
If you have 2 solutions on either side of a semi-permeable membrane, and they contain equal concentrations of non-penetrating solutes, they are considered to be isotonic (same tonicity) to each other.
Q: 2. Consider the dialysis bag experiment (exercise 2, part III) we did previously: a. Describe the tonicity and osmotic concentration of the experimental bag to the solution of the experimental dish.
The bag contents are hypertonic & hyperosmotic to the dish solution.
Q: Imagine that a living human red blood cell was put into a hypertonic solution
Water moves out of the cell until both solutions are isotonic.
When RBCs are put into an isotonic solution
will experience no change in volume from "normal".
A hypertonic solution will
- Draw water towards itself, creating a pressure/force (osmotic force) across the membrane - This will occur until the water dilutes the hypertonic solution enough to bring the non-penetrating solute concentration on either side of the membrane to equality (equilibrium)
As cells metabolize they produce waste products which must be transported or diffused out of the cell
- However, the cell must keep most proteins and carbohydrates inside of it. - Since these molecules are polar they must be moved across the cell membrane by membrane transport proteins. - This is also the case for small ions.
Molar Concentration
- If a molecule dissociates into 2 ions when put in water, its osmotic concentration is twice that of its molar concentration. - If another molecule dissociates into 3 ions in water, its osmotic concentration is 3 times that of its molar concentration. - This is due to the fact that each individual ion contributes to the osmotic force of a solution. - A molecule which does not dissociate in water will have an osmotic concentration equal to its molar concentration.
Living cell permeability:
- Therefore, the living cell is selectively permeable to most molecules since it can control the number of transport proteins embedded in its membrane. - However, very small molecules like water (H2O), oxygen (O2), carbon dioxide (CO2)and non-polar organic molecules (e.g. urea & many lipids) can simply diffuse across the cell membrane without assistance because they freely pass through the phospholipid bilayer.
Lipid bilayers
- compose the boundaries of cells - These barriers prevent molecules in the cell from leaving the cell and keep unwanted molecules out of the cell - Lipid composition of the membrane determines the permeability of the membrane itself. - Non- polar, lipophilic molecules can pass through the membrane easily - In contrast, membranes have very low permeability to ions and large polar molecules.
RBC's are good osmometers
- instruments that change their volume if placed into solutions of differing tonicities. - Living RBCs make good "osmometers" because their shape and sometimes their diameter changes in response to change in the relative tonicity of the solution in which they are placed.
In order for osmosis to occur, the membrane must be:
1). permeable to water but 2). impermeable to some or all of the solutes; therefore, the membrane is considered semi-permeable (in living cells the term selectively permeable is used). **When you have a difference in the concentration of non-penetrating solutes on either side of the membrane, the solution with the higher concentration of non-penetrating solutes will draw water towards it**
So, in order to determine osmolarity, you must know two things:
1. The molarity of a solute when it is in solution, and 2. The number of particles that the solute will break into when placed in the solution.
Molarity (molar concentration)
Denotes the concentration of a single chemical compound that was put into the solution, disregarding any dissociations that may have occurred after the solute was added to the water. It is the number of moles of the chemical per liter of solution. - For example, a 1 L solution containing 150 milliMolar (0.15 Molar) of nonpenetrating NaCl (MW = 58.5g/mole) has an osmolarity of 300 milliOsmolar (0.3 OsM) because the Na & Cl dissociate in water.
Dialysis and Osmosis
In lab 2, you could tell that osmosis occurred because your dialysis bag gained weight
Q: 2. Consider the dialysis bag experiment (exercise 2, part III) we did previously f. Why did the glucose-control bag gain weight after 30 minutes when glucose is a PS (shouldn't glucose equilibrate by just leaving the bag?)? If we let the bag sit for 4 hours, do you think the dialysis bag would have still shown a weight gain? Why or why not?
It gained weight because water diffused faster than glucose. At 4 hours it would not have shown any weight gain.
Q: 6. Make 500 ml of a 150 millimolar (mM) CaCl2 solution. What is the mOsm of this solution?
Molarity = moles/liter We know the volume is 500 ml or 0.5 l (in molarity problems ALWAYS need to make the volume in L). Need to calculate # of moles needed to make this solution. - 0.150M = X moles/0.5L - X = 0.075 moles How much CaCl2 (in g) is in 0.075 moles? Use the M.Wt. of CaCl2 to calculate this: - 110.98g/1 mole = X g/0.075 moles - X = 8.32 g. - To make 500 ml of a 150mM CaCl2 solution mix 8.32 g of CaCl2 in 500 ml. To find the osmolarity of this solution multiple the molarity by the # of particles the CaCl2 dissociates into (3): - Osmolarity = 150 mM X 3 - Osmolarity = 450 mOsm
Molarity
Molarity is a term used to designate the concentration of a solute in solution, regardless of its ability to dissociate. - For example, take Sulfur (S); it has an atomic weight of 32.1. Therefore, one mole of sulfur (S) weighs 32.1g. If you dissolve 32.1 g of sulfur in water (with a final volume = 1 liter), you have a 1M solution of sulfur (you have 6.02 x 1023 actual atoms of S in solution).
Q: 1. What causes water to move in a certain direction, into or out of the RBC?
NPS and concentration gradient produce an osmotic force that moves water from a high concentration to a low concentration (down a gradient).
Q: 2. Consider the dialysis bag experiment (exercise 2, part III) we did previously: c. Define the term "chemical equilibrium". Would any of the dialysis bag contents ever come to "chemical equilibrium"? List the solutes, if any, which would come to chemical equilibrium in the dialysis bag experiment?
No net movement of solutes. Yes With time glucose would come to equilibrium.
Q: 2. Consider the dialysis bag experiment (exercise 2, part III) we did previously: b. Define the term "osmotic equilibrium". Would the dialysis bag ever come to osmotic equilibrium with the outside solution? Explain your answer.
No net movement of water. Probably not. The bag would exert back pressure, called osmotic pressure, which would oppose osmotic equilibrium.
Osmosis
Osmosis is the net diffusion of water across a semi-permeable membrane. -
Q: 2. Consider the dialysis bag experiment (exercise 2, part III) we did previously d. Which solutes were penetrating (PS) and which were non-penetrating (NPS) in the dialysis bag experiment?
PS were glucose and KI NPS were starch and protein. Cannot include water because while water did penetrate it is NOT a solute, it is a solvent.
Ionic bonds
Solutes that dissociate readily in water are usually held together ionically instead of covalently. - Ionic bonds are weak compared to covalent bonds and ionic bonds break in the presence of polar water molecules.
Q: 4. If you had a hypothetical cell in a solution of 0.3M NaCl/0.2 M Urea and there was no observable change in volume of the cell, what would you conclude about the cellular solute concentration? Explain your answer.
The cellular solute concentration must be 0.3M (or 300mM) because the volume did not change. (the cell and the solution were isotonic to each other, therefore the cell did not change volume).
Cells in your body are constantly subjected to changes in tonicity in their environment.
The chemical contents of the cell (in the ICF) differ from those of the external environment (the ISF)
Q: 2. Consider the dialysis bag experiment (exercise 2, part III) we did previously e. Explain why the experimental bag (filled with dialysis solution) and the glucose- control bag both gained significant weight, but this was not observed in the negative control bag?
The experimental bag gained weight because protein and starch were NPS and drew the water into the bag ... bag gained weight. The glucose control bag gained weight because while the glucose is a PS, in the 30 minute frame used, water diffused in faster than glucose could diffuse out ... the bag gained weight. At equilibrium that bag would not have gained weight.
Hypotonic
The more dilute solution, on the other side of the membrane, which contains a lower concentration of non-penetrating solutes - (lower tonicity; lower concentration of non-penetrating solutes)
osmotic pressure
The pressure that opposes the movement of - In LAB 2, water moved into the dialysis bag, until the bag swelled and could not stretch anymore. - The strength of the dialysis bag prevented it from exploding due to the volume of water moving into it. - When fully stretched, the bag resisted the movement of water; and therefore, osmosis stopped before water could reach an equilibrium (simply put, back pressure of the stretched bag resisted the osmotic force of the salts in the bag which are drawing water into the bag).
Q: 5. There have been stories about shipwrecked sailors who die quicker when their thirst became so overwhelming that they drink the salty ocean water rather than not drinking any water. With your understanding of osmosis, why does a sailor die quicker drinking salt water than not drinking any water?
The salt water bathes the cell in a hypertonic solution. The cell crenates and dies due to the harsh environment of the salt water. This ultimately leads to death.
Living mammalian cells do not have strong membranes
Therefore, the solute concentration in the ECF and ICF must be maintained in a relatively constant state of equilibrium at all times - If too much water is consumed and the amount of solutes in the ECF is insufficient, a hypotonic condition will be created in the ECF (relative to the ICF). - This could be very dangerous as water moves by osmosis into the cells of the body and causes them to lyse (rupture). - To counteract this, the urinary system acts to maintain a water and solute balance in the body.
When RBCs are put into a hypertonic solution
They will shrivel and may appear to exhibit spikes, in a process called crenation (wrinkled, shrunken appearance).
Hypertonic
To the solution on the opposite side of the membrane. - (higher tonicity; contains a higher concentration of non-penetrating solutes)
isoosmotic, hyperosmotic, and hypo-osmotic
denotes simply the osmolarity of a solution relative to that of any other solution without regard to whether the solute is penetrating or non-penetrating.
If RBCs are placed in a solution of lower NPS (called a hypotonic solution) (e.g., distilled water), water will move
into the cell causing them to swell. - The swelling will eventually become so great that the cell will open, releasing Hb into the cellular environment (hemolysis). - The ruptured cells are called "ghosts", and the solution becomes relatively transparent.
Osmolarity (osmotic concentration)
it is the number of osmoles per liter of solution. Denotes the additive concentration of all chemicals in a single solution (you need to know if any of the molecules placed into solution will dissociate and into how many pieces each molecule dissociates)
Tonicity
may only be used when comparing the concentrations of non-penetrating solutes of two (2) solutions.
If the RBC and the extracellular environment have the same concentration of NPS there will be
no net movement of water into or out of the cell; such a solution is said to be isotonic to the cell.
A living cell placed into a solution that is isotonic and hyperosmotic will
not exhibit a net change in volume/size when observed over a period of time.
Osmolarity
osmoles/liter = OsM - Osmotic concentrations of solutes in the ECF of people are small, so clinicians usually use milli-OsMolar (mOsM) units when analyzing blood chemistry on patients
Any compound that dissolves in water and then dissociates (breaks apart) will
produce an osmotic concentration which is greater than its MOLARITY (molarity = # mol/l).
RBCs are ordinarily found in
solute-rich plasma of approximately the same solute concentration as the interior of the RBCs.
When RBCs are put into hypotonic solutions they will
swell and may hemolyze (burst).
If living cells are placed into a solution which is 1. hyperosmotic because it contains more penetrating solutes (PS) than the cytosol of the cell, and 2. isotonic because it contains a concentration of non-penetrating solutes which are equal to the concentration of non-penetrating solutes (NPS) in the cell (an isotonic condition) It is acceptable to hypothesize that...
there will be no net change in the volume of the cell since there will be no net diffusion of water into or out of the cell. This is due to the fact that the ICF and ECF are isotonic. The solution into which the cells are placed is initially hyperosmotic but isotonic to the cytosol. Eventually, the PS's will come to equilibrium and the solution and the cytosol will become iso- osmotic.
Red blood cells function
to carry oxygen to the many tissues of the body and to carry carbon dioxide to the lungs. - A mature RBC has no nucleus and few internal organelles. - The RBC is essentially a bag of hemoglobin (Hb), which is the molecule that holds the gases being carried. - Because of their simplicity, ease of obtainability, uniform size, and relative simplicity of determining hemolysis (cell rupture); RBCs are ideal for studying the membrane dynamics of tonicity.
When RBCs are placed in solution of higher NPS . . .
water leaves the RBC, the cell collapses, and takes on a "spikey" appearance (crenated). A suspension of intact RBCs is cloudy. Use this terminology as appropriate.
