PRINCIPLES
A meaningful discussion of cellular and extracellular fluid compositions requires knowledge of the concentrations of several solutes. It generally is desirable to express these concentrations on a molecular and ionic basis. The student of physiology should become intimately familiar with the following units of measurement.
A. Molar - A mole of a substance is simply the molecular weight of that substance in grams (the gram molecular weight). For example, the hexose sugar glucose has the chemical formula C6H12O6 and thus a gram molecular weight of (6 x 12) + (12 x 1) + (6 x 16) = 180 grams. Thus, 180 grams of glucose represents one mole or 6.02 x 1023 molecules (Avagadro's number) of glucose. A one molar solution of a solute contains one mole of solute per liter of solution. Millimolar (mM), micromolar (µM), and nanomolar (nM) designations are most often used by physiologists. The following illustrates their relationships.
1 M = 1 mole of solute/liter of solution
1 mM = 0.001 moles/liter or 1 x 10-3 M
1 µM = 0.001 millimoles/liter or 1 x 10-6 M
1 nM = 0.001 micromoles/liter or 1 x 10-9 M
It is essential that you become familiar with these terms, as you will encounter them repeatedly during the semester in your laboratory exercises.
B. Osmolar - From a thermodynamic standpoint, an osmole is a measure of effective solute concentration and is dependent on the number of active particles in solution and not on the kind of particles. One millimole/liter (mM) of a non-electrolyte such as glucose, which does not dissociate in solution, is equal to one milliosmole/liter (mOsm). By contrast, NaCl, a strong electrolyte that dissociates almost completely in solution into Na+ and Cl- ions, contributes nearly 2 mOsm per mM. Similarly, Na2SO4 dissociates into Na+ +, Na+ +, SO4-2 and thus contributes 3 mOsm per mM. However, no salt dissociates completely.
The exact degree of dissociation of NaCl and other salts varies with the compound and with the conditions under which the compound is solubilized. Each compound has a specific dissociation constant which, when multiplied by the molar concentration, indicates the ionic activity.
C. Permeability of Cell Membranes - An exchange of materials is continually taking place between living organisms and their environments. At the cellular level, this depends on several physiological properties, collectively referred to as the permeability of the cell or plasma membrane. Two groups of factors are involved. The first consists of the intrinsic properties of the cell membrane itself, which include the presence or absence of particular carrier and transport proteins and the composition of the lipid bilayer in which they are embedded. The second group of factors affecting transport of materials across the cell membrane is of a purely physical nature and consists of the chemical and electrical gradients across the membranes. In this laboratory exercise, the effects of different solutes on cell membrane permeability will be determined using the mammalian erythrocyte, a simple anucleate eukaryotic cell.
Mammalian blood plasma has an osmotic concentration of about 300 mOsm. Intracellular erythrocyte osmolarities likewise approximate 300 mOsm, although the solute constituents of the extracellular fluid and cytoplasmic fluid are quite different. If erythrocytes are placed in distilled water, water molecules will enter rapidly across the cell membrane by osmosis and the cells will swell and rupture, releasing their cytoplasmic contents. This process is called hemolysis. By contrast, if erythrocytes are placed in a 300 mM solution of sucrose (a non-electrolyte), hemolysis will not readily occur because the membrane is nonpermeable to this sugar and because the osmotic concentration of the extracellular sucrose solution balances that of the intracellular fluid. 300 mM sucrose is thus isosmotic to erythrocytes and is also isotonic, as the cells do not change shape (at least over the short term). 300 mM urea is likewise isosmotic to the intracellular fluid of erythrocytes. However, if erythrocytes are placed in an isosmotic solution of urea, hemolysis rapidly occurs. The plasma membranes of mammalian cells including the erythrocyte are very permeable to urea, which can rapidly enter the cytoplasm from the extracellular fluid. Since the intracellular solutes penetrate the cell membrane at a much slower rate, the total osmotic concentration within an erythrocyte placed in a 300 mM urea solution will quickly approach 600 mOsm, effectively doubling the previous cytoplasmic osmolarity. The resulting osmotic gradient across the cell membrane is comparable to that of a cell in distilled water and hemolysis rapidly occurs. Therefore, although 300 mM urea is isosmotic to erythrocytes, it is not isotonic.
The rate of membrane penetration by a solute is dependent on a variety of factors. These include molecular size of the solute (permeability generally decreases with increasing size), lipid solubility (permeability usually increases with increasing fat or oil solubility), and degree of ionization (permeability generally decreases with increased ionization). The diffusion gradient for the solute across the cell membrane also is of great importance. Other factors that can influence transport of a solute include temperature and pH of the extracellular fluid. In this laboratory exercise, you will determine the concentrations of several electrolytes and non-electrolytes that cause hemolysis of sheep erythrocytes and the effects of temperature and molecular size on rates of hemolysis.
PREPARATIONS
A. Living Material
The Instructor will provide you with defibinated sheep blood.
This blood will be diluted into heparinized 150 mM NaCl and distributed
to each laboratory group in a large test tube.
B. Reagents
For Part A of the PROCEDURES section, 1 M stock solutions of
sodium chloride (NaCl), calcium chloride (CaCl2), and sucrose will be supplied.
You will make dilutions from these stock solutions as required. To
obtain 1 M stock solutions, bring over to the Preparation Bench a labelled
large test tube and half fill it from the appropriate stock bottle.
Never contaminate the stock solution by pipetting directly from it and
never return excess solution to this stock.
For Part B and C, isosmotic (300 mOsm) stock solutions of urea,
thiourea, glycerol, and ethylene glycol will be supplied.
C. Equipment
1. Pipetman automatic pipettors with disposable tips
2. Pasteur pipettes (disposable)
3. 18 X 150 mm glass test tubes
4. Test tube racks
5. Distilled water in squirt bottle
6. Labelling tape and pen
8. Water Bath (4 C)
9. Water Bath (37 C)
10. Parafilm and scissors
PROCEDURES
A. Determination of the Hemolyzing Concentrations of Solutes
In these exercises, the time required for the hemolysis of erythrocytes
will be used as a measure of their permeability. While various instruments
have been used to precisely measure rates of hemolysis in studies of erythrocyte
fragility and to compare hemolytic phenomena among species, the difference
in appearance of solutions of intact and hemolyzed erythrocytes is significant
enough that it can be detected readily with the naked eye.
1. To define the intact and hemolyzed states, add four (4) drops of the suspension of sheep erythrocytes to each of two small test tubes containing, respectively, 5 ml of distilled water and 5 ml of 0.2 M NaCl. Immediately invert each tube using a small piece of Parafilm to cover the tube opening. Observe the print on a page of your laboratory manual through the suspensions of erythrocytes and compare the degree of opacity.
2. Having established that you can recognize hemolysis when it occurs, determine the concentration of NaCl that just produces lysis of the erythrocytes in suspension by mixing appropriate amounts of 1 M stock NaCl and distilled water. In each instance, keep the final volume at 5 ml and test with 4 drops of erythrocytes. Use a standard time interval of two minutes from addition of erythrocytes before making your determinations. Since 0.2 M NaCl does not cause hemolysis, you will require a solution that is somewhat more dilute. Locate the critical concentration to two decimal places (i.e., 0.13 M). Remember, it is essential that you mix the solutions well by inversion after adding the water and stock NaCl solution to the test tube and that you gently mix the tubes again after adding erythrocytes.
3. After you have determined the critical hemolyzing concentration for NaCl, do the same for sucrose, and CaCl2, using the 1 M stock solutions provided. The values that you obtain should reflect the relative osmotic activities generated by particular molar concentrations of the various solutes as well as the selective permeability of the erythrocyte cell membrane to each.
B. Effect of Solute Size on Time to Hemolysis
As noted above, the rate at which solutes penetrate the plasma
membrane bilayer of intact cells is to some extent governed by their molecular
size. Theoretically, therefore, when two compounds of similar chemical
nature are tested for their ability to cause hemolysis of erythrocytes,
the smaller compound should cause a more rapid rate of lysis because of
its increased ability to penetrate the lipid bilayer. In the following
simple experiment, you will test this hypothesis by determining the time
for hemolysis of sheep erythrocytes placed in isosmotic solutions of urea,
thiourea, glycerol, ethylene glycol, ethanol and propanol. The structures
of these compounds and their molecular weights are shown in Figure 1.
H2N - C - NH2
H2N - C - NH2
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O
S
urea
thiourea
(60)
(76)
H
H
H H H
H H
H H H
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H - C - C - H
H - C - C - C - H H - C
- C - OH H - C - C - C - OH
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OH OH
OH OH OH
H H
H H H
ethylene glycol
glycerol
ethanol
propanol
(62)
(92)
(46)
(60)
Figure 1. Molecular structures of urea, thiourea, glycerol, ethylene glycol, ethanol and propanol.
1. Pipette 5 ml of the appropriate isosmotic solution into
a small test tube. Then add four drops of sheep erythrocyte solution
as above and quickly mix by inversion, starting the stopwatch at the time
of inversion. Determine the time (in seconds) to hemolysis.
Repeat this process for each of the four compounds.
C. Effect of Temperature on Time to Hemolysis
The lipid bilayer of the plasma membranes of intact cells is
in effect a two-dimensional fluid within which the movement of the molecular
components is governed by the composition of the bilayer itself and by
the temperature at which the bilayer exists. At the body temperatures of
homeothermic vertebrates (35-40 C), individual phospholipid molecules within
the bilayer undergo substantial lateral diffusion. However, when the lipid
bilayer is cooled, the structure of the membrane changes from a liquid
to a gel-like or semi-crystalline state. This change of state is
called a phase transition. For most mammalian cells, it occurs between
10 C and 20 C.
1. To determine whether the fluidity of the erythrocyte membrane has any effect on the rate of permeation of solutes, pipette 5 ml of thiourea solution into each of three small test tubes. Label each tube and place one in the ice bucket provided (4 C), leave one at room temperature (23 C), and place one in the water bath at 37 C. Incubate each tube for at least 5 minutes to allow the contents of the tube to equilibrate to the appropriate temperature. After the contents of each tube have come to the desired temperature, add five drops of sheep erthrocytes, mix quickly, and determine the time in seconds to hemolysis. Repeat this experiment until you have two times for each temperature that match fairly closely.
2. Repeat this experiment with ethylene glycol.
Data Tables
Critical Concentration of Solute for Hemolysis (mM)
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Effect of Solute Size on Time to Hemolyze (s)
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Effect of Temperature on Time to Hemolyze Erythrocytes (s)
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