Red Blood Cell Permeability
Eric Duitsman
Genevieve Roland
Plasma Membranes
• As discussed in MCB 252, cells contain a phospholipid bilayer.
It is amphipathic meaning it contains a hydrophilic and
hydrophilic layer. Cells also contain internal plasma
membranes. These membranes provide:
– Transport of nutrients inward and waste outward via selective
channels and pumps
– They act as sensors that respond to external stimuli
– They can increase in size without any loss of continuity simply by
adding new membrane
– They are flexible – they can expand and contract according to need
• Within the membrane surfaces are regions called membrane
domains
– These are where proteins are localized which creates a specialized
function
Osmosis
• Osmosis is the term used for diffusion of water. This occurs
only when two solutions are separated by a selectively
permeable membrane. The water will move across the
membrane in place of the solutes to create an equilibrium.
• Osmolarity is osmoles/liter of the total solute concentration
of a solution.
– This also determines the concentration of water in a solution.
• Water will move from an area of low to high osmolarity in order to reach an
equilibrium
• Osmolarity directly correlates with osmotic pressure
– Osmotic pressure is the pressure required to act on a solution in order
to prevent pure water from entering.
– The greater the osmolarity, the greater the osmotic pressure.
• The pressure depends on the number of particles in a solution
In an isosomotic cell, the total pressures of the two
solutions are equal ao there is no movement across the
membrane. An isoosmotic solution contains 300 mOsm of solute
regardless of penetrating or not. Hyperosomotic condition is
when the solution within a cell has a greater osmotic pressure
than its surrounding environment. This solution contains more
than 300 mOsm of solute. In a hypoosmotic state, the cell has
less osmotic pressure than the solution in the extracellular zone.
This solution contains less than 300 mOsm of solute in the cell.
Tonicity
• The environment surrounding the cell and the effect that
environment has on the cell is referred to as tonicity
– This depends on the concentration of non-penetrating solutes in a
solution. It does this while disregarding the penetrating solutes.
• In a RBC, the volume is inversely related to the solute
concentration in the extracellular medium.
– In a hypotonic situation there is a lower concentration of nonpenetrating solute in the extracellular fluid relative to the cell. This
causes water to move into the cell and increase volume
– In an isotonic situation there is an equal concentration
– In a hypertonic situation there is a higher concentration causing water
to move out and decrease volume
Urea
• Urea is special in osmosis because it is able to rapidly
diffuse across the cell membrane. This is opposite to
glucose which diffuses much more slowly. A solution of 300
mOsm of NaCl and 100 mOsm urea has a 400mOsm/L
osmolarity. This makes it hyperosmotic while still isotonic
since the urea moves across the membrane to create an
equilibrium between the cell and the extracellular fluid.
This does not change the volume of the cell.
• All hyposomotic solutions are also hypotonic however, a
hyperosmotic solution can be hypertonic, isotonic, or
hypotonic.
Blood
• A blend of fluid (55%) and cells (45%)
– Fluid consists of water, electrolytes, proteins, and nutrients
– Three types of cells – red blood cells (~45%), white blood
cells, and platelets (<1%)
• The different cell types perform various tasks in the
blood.
– RBCs transport O2 and CO2
• They also deliver nutrients to all areas of the body.
– Platelets form blood clots at wounds
– White blood cells fight infections
• Blood cells cannot reproduce as they do not contain a
nucleus
– Replaced by destroying old cells and creating new cells from
the cells located in the bone marrow
Blood Types
• Determined by blood types of parents
• 4 different types
– A, B, AB, and O
• Antigens are found on the surfaces of the
RBCs
– These determine what the blood type is
– All RBCs of a single individual have the same
antigens on their surface
Antigens and antibodies by blood type
Blood Type
Antigen on RBC
surface
Antibodies in
serum
A
A
Anti-B
B
B
Anti-A
AB
A and B
No antibodies
O
Lack of antigens on
RBC surface
Anti-A and Anti-B
• Antibodies (proteins) in the blood recognize antigens
on the cell surface and bind to them.
– Hemagglutination – agglutination of antibodies with the
blood type antigens on RBCs
• Anti-A will stick to A antigens resulting in
hemagglutination. The same for Anti-B and B antigens.
Blood Transfusions
Blood Type
Can Accept Blood From
Can Donate To
A
A or O
A or AB
B
B or O
B or AB
AB
A, B, AB, or O
AB
O
Only O
A, B, AB, O
Objectives
• Be able to describe the effects and distinguish between
hypotonicity and hypertonicity.
• Distinguish between electrolytes and non-electrolytes
– In regards to permeability
• Demonstrate ability and mastery in slide preparation
and use of a light microscope
• Explain how the molecular weight of a substance can
affect its permeability
• Be able to explain the effects of polar groups on
permeability
• Summarize and illustrate permeability.
Part I – Effect of Electrolyte and NonElectrolyte Concentrations on
membrane Permeability
• Materials
–
–
–
–
–
–
–
–
–
–
Gloves
12 tubes
Sharpie
Tube Rack
NaCl
dH2O
Sucrose
Red blood cell suspension
Printed letter sheet
Slides equal to number of tubes whose contents remained intact and
respective coverslips
– P-200
– P-1000
– Light microscope
Procedure – Part I
• Label your tubes 1-12 and place into the tube rack with #1-6
in the first rack and #7-12 in the second. NaCl used in 1-6 and
sucrose, 6-12
• Using the chart given in the manual, determine amount of
water and stock solution to combine to get desired NaCl and
sucrose concentrations and add calculated amounts to
respective tubes.
Final [NaCl]
0.16 M NaCl
dH2O
Final [sucrose]
0.32 M sucrose
dH2O
0.16 M
2.00 mls
0 mls
0.32 M
2.00 mls
0 mls
0.12 M
1.50 mls
0.50 mls
0.25 M
1.56 mls
0.44 mls
0.08 M
1.00 mls
1.00 mls
0.15 M
0.94 mls
1.06 mls
0.04 M
0.50 mls
1.50 mls
0.08 M
0.50 mls
1.50 mls
0.02 M
0.25 mls
1.75 mls
0.04 M
0.25 mls
1.75 mls
0M
0 mls
2.00 mls
0M
0 mls
2.00 mls
Part I - Continued
• In quick succession, add 0.5 ml of the red blood cell
suspension to tubes 1-12 using the P-1000. Make sure not to
let the pipet tip touch the solutions in the tubes.
• Cap the tubes and mix gently by swirling.
• Set a timer for twenty minuets. At each five minute interval,
hold the tubes against the printed letter sheet to see if
hemolysis has occurred. If you compare the tube to the page
and can see the letters through the solution, hemolysis has
occurred. If the letters are not visible, the red blood cells are
still intact.
Intact – non-hemolyzed
Lysed - hemolyzed
Part I – Continued
• After identifying the tubes in which hemolysis did not occur,
label a slide with the corresponding number of those tubes.
Place a 25 ul drop of the blood from these tubes on their
respective slides using the P-200. Place a coverslip on the
drop at an angle to reduce the amount of bubbles in the
sample. Observe the slides under the microscope and
determine the shape and size of the cells present from each
solution. A slide with the original blood solution should also
be made in order to compare any changes to.
Crenated
hypertonic
Normal
isotomic
enlarged
lysis
hypotonic
Osmolarity
Part II – The Effect of MW on
Membrane Permeability
• Materials
– 0.3 M urea
– 0.3 M ethylene glycol
– 0.3 M glycerol
– 0.3 M glucose
– 4 tubes
– P-1000
– Printed letter sheet
Procedure – Part II
• Label tubes #1-4 and place into tube rack
• Add 2 mls each to the respective tubes:
–
–
–
–
1. 0.3 M urea – MW = 60
2. 0.3 M ethylene glycol - MW = 62
3. 0.3 M glycerol – MW = 92
4. 0.3 M glucose – MW = 180
• Add 0.5 ml of chilled blood into the tubes one at a time and
time how long it takes for hemolysis to occur. Do this by
holding against the printed page and record in seconds time
elapsed
• Record the times obtained for each substance and dispose of
tubes in bleach tub
Part III – Blood Typing
• Materials
– Control ABO simulated blood samples
• Tubes A, B, AB, O
– Unknown blood samples of four patients
• P1, D1, D2, D3
–
–
–
–
–
Microtiter plate
Transfer pipets
Gloves
Toothpicks
Sharpie
Procedure – Part III
• Place microtiter plate on a white piece of paper.
• On cover of the plate, label the wells with the sample name
using a sharpie
A
B
AB
O
Patient
Donor 1
Donor 2
Donor 3
Anti-A
Anti-B
Anti-A
Anti-B
Part III - Continued
• Plate 10 ul of each control blood type and patient sample into
each of the corresponding labeled wells. Be sure to use a new
transfer pipet for each sample.
• Using a new pipet, add 10ul of anti-A serum into each of the 4
wells of the anti-A row. Repeat this for the bottom row
• Do the same procedure with anti-B transferring into the 4
wells of the anti-B row in the top and bottom sections
• Using a new toothpick for each sample, gently mix/stir the
sample together
• Allow plate to sit for 5-10 minuets
• Observe the wells for the presence of agglutination. This
occurs if the mixture has a granular texture rather than a
smooth one. Record the results