Download Effects of Solute Permeability on Hemolysis in the

Effects of Solute Permeability on Hemolysis in the Red Blood Cells of the
Domestic Sheep, Ovis aries
Lab section: 1B- Biology 3151
Date: 11/30/2012
o
Results:
y = -0.05x + 93.244
R2 = 0.7657
!
I·
I
Q,I
(.)
C
o
~
---5\70-+-----------------
~
·-·-~-401__+------------------·--······-
---.-.----
I
...
-··-·-·-·
---30~~-----------------------------~I--+--------
I
······--·10·00000j-----··..---···-··----·--·-·
o I
o
-100
100
...····...·...·-·----
200
...-------.------
400
300
600
500
[NaCl] (mOsm)
Figure 1: The mean cell response (measured in % transmittance, T) in relation to the
extracellular concentrations of Sodium Chloride solution (in milli-osmolar) in the erythrocytes of
Domestic sheep (Ovis ariesi. The error bars represent ±1 standard deviation. (R2 = 0.7657, pvalue <0.05)
,.
l,../
~~O~;----------------------.-.-ge~G-41!.~-----.-801__+1-----·
--7.~o-+-I------------------._--_._-----_._------_.
·_--60 -+i-,
1
__
...._-- ....•.
----5&O~---------------------------4~·~----------
----3~~------------------------20-1--
-
..
.-- ±o~.-- ------.---
----- ..-
.
-.---.--.-.--------." " -.
o !
-100
o
I
100
200
300
400
500
600
[Urea] (mOsm)
Figure 2: Mean Cell response (measured in % transmittance, T) in relation to extracellular
concentrations of Urea (measured in milli-osmolar) in erythrocytes of domestic sheep (Ovis
aries).
The error bars represent ± standard deviation. (p-value > 0.05)
1
V
/
//
1
3-5----
i
~
•...-_
•...••.•
30 +----1
['-l
'-"
['-l
-Vi 25
••••
"0
5
..::
o
.•...
Q,)
--.•...e
20
15
=10
~
Q,)
:;s 5
o
Cold
Room
Temperature Treatments
i
~-.-.~..-------
----
Warm
_____
-.1I
Figure 3: The average time to hemolysis (in seconds, s) of Domestic Sheep (Ovis aries) in
relation to various temperatures of Ethylene Glycol (cold, room, and warm). The error bars
represent ± 1 standard deviation. (p-value <0.05)
Discussion
I
There were great variances in solute permeability to the red blood cell membranes of
Domestic Sheep (Ovis aries). The Different concentrations of sodium chloride and urea and the
different temperatures of ethylene glycol caused the red blood cell to respond in a volume
change due to the movement of water into or out of the cell. In order to measure the effects of the
solute permeability to the membrane, the turbidity of cell suspensions were measured using a
spectrophotometef/rlvlanalis,
II"
1996) •..As the cells shrink, they reflect greater amounts of light.
fi
That light which :~ ~.eete'Lf;;;:dsorbed
by the cells does not pass through to the light sensor in
the spectrophotometer; therefore, the percent transmittance (%T) decreases as the turbidity of the
solution increases. (Manalis, 1996) Consequently the percent of transmittance increases as the
2
cell volume increases due to the movement of water into the cell. If the water from the
extracellular solution continues to move into the cell it will eventually cause the cell to burst in
an event called hemolysis. Hemolysis of the cell will greatly increase the percent of
transmittance.
When the concentrated sodium chloride solution is hypertonic to the red blood cell, water
moves by osmosis to the area of higher solute concentration (Sherwood et. al, 2005) from the
cell, through the aquaporins, to the outside of the cell and causes the cell to crenate. Crenation of
the erythrocytes is the cause of turbidity in the solution and as a result the percent transmittance
decreases. When the red blood cells were exposed to higher concentrations of sodium chloride
solution (measured in milli-osmolar) hemolysis occurred faster. Hemolysis occurs slower as the
concentrations of sodium chloride solutions are decreased.This effect is due to the
impermeability of sodium chloride and the high permeability of water to the cell membrane. The
membrane permeability of sodium is .00000001 11mper second, and the membrane permeability
for water is 30 11mper second. (Inouye, 2012) .
When the cells were exposed to a hyposmotic solution of sodium chloride, the water
moved down its concentration gradient into the cell. When the cell is in a hypotonic solution the
cell will swell and eventually hemolyze. (Inouye, 2012) This causes the transmittance percentage
to be higher because there is no longer a cell membrane present to block the light sensor in the
spectrophotometer.
In the second experiment we exposed the red blood cells to various concentrations of
urea. Urea is highly permeable across most plasma membranes due to the presence of numerous
urea transporters (integral proteins in the plasma membrane). (Inouye 2012) Based on our data,
3
which is expressed by Figure 2, we see that the transmittance percentage remains at around 99%
regardless of the concentration of Urea. As said by Goodman (2002), red blood cells lyse very
rapidly (too fast to measure the time) when placed in an isosmotic solution of urea. Both
compounds enter the cell rapidly causing the cell to swell and hemolyze instantly.
In the third experiment we were able to conclude that there is a relationship between
temperature and the time for the cells to reach hemolysis, see figure 3. We must first
acknowledge that there are no ethylene glycol membrane transporters and that ethylene glycol is
moderately lipid soluble. (Inouye 2012) We must also keep in mind that membrane fluidity can
change according to temperature. According to Gershfeld and Muramaya (1988) studies indicate
that a transformation of the membrane bilayer occurs when the normal ambient temperature of
the cell is exceeded. In the warm ethylene glycol treatment the cell membrane increases in
fluidity and allowes for the ethylene glycol to move more easily into the cell which causes the
time to hemolysis to be much faster than in room temperature. When the red blood cells were
placed in the cold treatment, the time to hemolysis was slowed down significantly due to the
structure of the bilayer which changes from a liquid to a more rigid, gel like (semi crystalline)
state. (Inouye 2012) Our results are in agreement with a similar experiment done by Gershfeld
and Muramaya (1988) which concluded that temperature is a dependent factor in hemolysis
4
Literature Cited:
Gershfeld N. L., & Murayama, M (1988) Thermal Instability of Red Blood Cell Membrane
Bilayers Temperature Dependence of Hemolysis. Journal of Membrane Biology, 101(1),67-72.
--7
.
Goodman, B.E. 2002. Transport of small molecules across cell membranes: water channels and
urea transporters. Advan. Physiol. Educ. 26: 146-157.
Inouye, C.Y. 2012. Water and solute movement through red blood cell membranes. Biology
3151 Laboratory Manual.
Manalis, R.S. 1996. Water and solute movement through red blood cell membranes. ABLE 17:
99-116.
Sherwood, L., H. Klandorf, and P.B. Yancey. 2013. Animal Physiology From Genes to
Organisms, 2ndedition. Thomson Brooks/Cole, California.
5
BIOL 3151-
Fall 2012
SCORING RUBRIC - PHASE II RBC OSMOSIS PAPER
Additional
Points
Earned
TITLE PAGE (up to 2 pts. for Phase I + Phase II)
Original, appropriately
scientific
descriptive
title of the study including
both common
names of the study species with the title page formatted
standards set for Phase I. Includes the suggested revisions.
and
according to the
uK
[up to 2 pts.]
RESULTS (up to 18 pts. for Phase I + Phase II)
Text of the Results Section
Thorough verbal, factual description of the results provided
for Phase I. Includes the appropriate revisions.
per the standards
/
e;
set
[up to 8 pts.]
!
Figures
Graphic depiction
the appropriate
of results provided
[up to 10 pts.]
DISCUSSION (up to 12 pts. for Phase I
Discussion including
+ Phase II)
accurate physiological
of relevantbackground
references
Includes the appropriate
-(- LF
per the standards set for Phase I. Includes
revisions.
explanation
provided
of results and integration
per the standards set for Phase I.
revisions.
-s
fLt-;
[up to 12 pts.]
LITERATURE CITED (up to 3 pts. for Phase I + Phase II)
All references
cited in the Discussion section listed in proper format.
include any references
Writing
Proficiency
not cited in the Discussion section.
Does not
[up to 3 pts.]
-1-
jt~
(up to 5 pts. for Phase I + Phase II)
Overall report demonstratesproper
and clarity and incorporates
grammar,
the appropriate
Level of Improvement/Demonstration
punctuation,
spelling, conciseness,
revisions.
+- J
[up to 5 pts.]
of Effort
[5 pts.]
+3~
-
Electronic
Upload to Blackboard/Turnitin
Past Deadline
(if report
not received
by 11:59 PM on Fri., 11/30/12
by due date, -1 pt./day)
I
¢
/
TOTAL ADDITIONAL
POINTS FOR PHASE II
+ Phase II)
',.
Comments:
I'Ll
/--r'
SCORE FROM PHASE I
TOTAL SCORE (45 pts. possible for Phase I
'
:.//
[2 pts.]
-:
(0