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Hemolytic Effect of Different Solutes

to
Red Blood Cells
Basco, Vince; Cruz, Ma. Lizette; Guilaran,
Diane; Masangkay, Anne and Perez, James Benedict
Department of Biology, College of Science, Polytechnic University of the Philippines

Abstract
A cells permeability to a solute is dependent to the size, polarity and
charge of the particular molecule. The hemolytic data on salt NaCl, urea
(CH4N2O) and fructose (C6H12O6) solutes in red blood cells (RBC) was
measured by light transmission of blood in a spectrophotometer at 510 nm
and 100% transmittance (T). Percent (%) T was used as an indirect measure
of RBC volume and changes in cell volume as used as an indicator of the
osmotic response of red blood cells to solutions with differing osmolarity. RBC
exposed to NaCl and fructose solutes at low concentrations are hemolyzed
due to their high %T, while RBC in urea exhibited outstanding %T regardless
of its concentration. NaCl has the lowest %T, with fructose having middling
%T and urea having high %T. Thus, RBCs membrane permeability is high in
urea, moderate in fructose and low in NaCl. The three solute solutions have
a negative or indirect relationship with the % Transmission. Each specific
molecule has its own way of entering the cell membrane; they differ on the
transport mechanism which enables them to enter the semi-permeable
membrane.
Keywords: red blood cell, cell membrane, spectrophotometer, hemolysis

INTRODUCTION
Red blood cell is the most
used model for membrane-solventsolute interactions (Scott, 1993)
where
the
cell
membrane
permeability is test in solute
transport understands how it
affects
the
cells
metabolic
processes,
and
physiological
systems (Langsdorf and Zydney,
1994). Haemolysis on the other
hand is a process where the red

blood cells shrink or crenate due to


the water efflux due to osmosis
(Scott, 1993). In the study of
Strand
(1983)
shows
that
hemolysis time can be used as an
index of the rate of osmosis.
Therefore, it can be use for
determining whether the RBC is
exposing in hypertonic, hypotonic
or isotonic solution.

Hemolysis is accompanied by
the changes of light absorbance of
cells suspension where the RBC
membrane burst and it settle to the
bottom causing the solution to be
clear (Hess et al., 2005). Light
absorbance can be measured
though
spectrophotometer
to
determine
the
percentage
of
transmitted
light
absorbance.
Spectrophotometer
indirectly
measures the amount of light in the
particular wavelength absorbed by
the substance (Abramoff and
Thomson, 1986), this is supported
by Beers Law, where the amount
of light which is absorbed or
transmitted is usually proportional
to the concentration of the
particular molecule in solution so
spectrophotometer is a proper tool
to measure the percentage of
transmitted light absorbance.
The
objective
of
this
experiment is to determine the
properties
of
the
cellular
membranes and the movement of
the water molecule across them;
understand what concentration of
solvent is viable to hemolysis or
crenation; and determine the rate
of osmosis of RBC in different
concentration of urea, fructose and
NaCl.

METHODOLOGY
Preparation of Stock Solution
Blood samples were collected
using a syringe, contained in
heparin coated tubes and stored in

a bucket with ice. Blood sample


and saline solution were mixed to
make a 10 mL blood solution (1 ml
blood and 9 ml saline) which will be
used throughout the experiment.
The blood solution was kept in cold
environment
to
prevent
coagulation of blood.
Preparation
Concentration

of

Solute

The NaCl, Urea and Fructose


solutes were prepared to make 25
ml with varying concentrations
(0.02M, 0.04M, 0.08M, 0.16M and
0.32M). A separate tube was
labeled
as
"blank
solution"
consisting of 1 ml of blood stock
solution and 4.5 ml of distilled
water. Each solute concentration
solution was placed 0.9 ml in a test
tube, adding 0.1 ml of blood stock
solution to make a test solute
solution which will be used in
spectrophotometer. The solution
were then mixed using a vortex.
Reading of Absorbance
Spectrophotometer

in

Spectrophotometer was set


in 510 nm and 100%T. Blank tube
solution was inserted and set at the
maximum
value
(100%
transmittance). The test solutions
were then placed immediately in a
cuvette and placed inside the
spectrophotometer. Each solution
was re-measured at 0, 10 and 15
minutes to observe the changes in
percentage transmission.

RESULTS AND DISCUSSION


The ability of an extracellular
solution to make water move into
or out of a cell by osmosis is known
as its tonicity. Solutions could be
classified into isotonic, hypertonic
and hypotonic solutions. In an
isotonic solution, the extracellular
fluid has the same osmolarity as
the cell, and there will be no net
movement of water into or out of
the cell, while in an hypertonic

solution, the extracellular fluid has


a higher osmolarity than the cells
cytoplasm, and water will move out
of the cell to the region of higher
solute concentration and on the
other hand a hypotonic solution
means that the extracellular fluid
has the same osmolarity as the
cell, and there will be no net
movement of water into or out of
the cell (Khan Academy 2016,
Scott, L.A. 1993).

highest
percent
transmission.
However, 0.32 M concentration of
NaCl showed the lowest percentage
100
transmission
among
the
80
concentrations. NaCl solution with
0.02 M concentration showed the
60
highest percent transmission and
% Transmission
as the time increases percentage
40
concentration decreases. This is
probably because the RBC in the
20
solution hemolysed due to low
concentration of NaCl. On the other
0
0.02 0.04 0.08 0.16 0.32 hand 0.04 to 0.32 M has lower
transmission but has
Concentrations of NaCl (M) percent
increasing trend.
120

0 mins

15 mins

30 mins

Figure 1. Change in % transmission of RBC


in varying concentrations of NaCl at a 15minute interval. Error bars represent SEM.

The percent transmission of


the red blood cells in NaCl solution
increases as the concentration of
the test solution decreases (Figure
1). The red blood cells in 0.02 M
concentration of NaCl showed the

When red blood cells are


exposed to a hypertonic NaCl
solution, osmosis occurs in which
water moves to the area of higher
solute concentration (Sherwood et
al., 2005). The movement is from
the
cell
going
through
the
aquaporins and to the outside of
the cell which results to crenation
of the cell. Crenation of the cell
causes the turbidity in the solution
which
results
to
decreased

percentage transmission. On the


other hands, when the red blood
cells are exposed to hypotonic NaCl
concentration, swelling of the cells
occurs faster which is known as
hemolysis. Hemolysis is directly
proportional to the concentration of
the NaCl solutions. When the cell is
in a hypotonic solution, the cell will
swell and eventually hemolyze
(Inouye, 2012) which causes the
transmission percentage to be
higher because there is no longer a
cell membrane present to block the
light
sensor
in
the
spectrophotometer.
These
phenomena are due to the
impermeability of NaCl and the
high permeability of water to the
cell membrane.
The rate of diffusion of NaCl
crossing the cellular membrane
seems fast especially when the
concentration gradient increases.
NaCl
is
a
nonpenetrating
electrolytes (Naccache, 1973) that
can cause hemolysis caused by
influx of water by osmosis at lower
molar concentrations (SowemimoCoker, 2002). And because NaCl is
an electrolyte it can dissociate into
two ions and every ion in the
dissociated solution exerts the
same
osmotic
pressure
as
produced by the entire NaCl
molecule. Based from the Figure 1,
there is a big difference in each
percentage transmission due to
varying concentration in which
varying in amount of electrolyte is
present as well.

120
100
80
60

% Transmission

40
20
0

0.02 0.04 0.08 0.16 0.32

Concentrations of Fructose (M)


0 mins

15 mins

30 mins

160
140
120
100

% Transmission

80
60
40
20
0

0.02 0.04 0.08 0.16 0.32

Concentrations of Urea (M)


0 mins

15 mins

30 mins

Figure 2. Change in % transmission of RBC


in varying concentrations of urea at a 15minute interval. Error bars represent SEM.

When the red blood cells are


exposed to varying concentrations

of urea, the transmission remains


at high percentage regardless of
the concentration (Figure 2) since
urea has high permeability across
most plasma membranes. Urea
(just like water) is highly permeable
to cell membrane because of the
presence
of
numerous
urea
transporters
(Inouye,
2012).
Hemolysis happens 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
(Goodman,
2002).
However, Figure 2 shows
that there are inconsistency in data
when it has increasing time
interval.
Urea has 0.1 m s -1
membrane permeability rate, its
molecular weight is small (MW =
60) and no charge (Scott, 1993).
This permeability rate can be
treated as rationale because it has
fast rate of osmosis in the red
blood cells solution. However,
results showed that researchers
inferred inconsistency of data is
due to different blood samples from
different individuals that were used
or
there
is
glitch
of
the
concentrations of urea used.
Figure 3. Change in % transmission of RBC
in varying concentrations of fructose at a
15-minute interval. Error bars represent
SEM.

Results above showed that


there is an decreasing percentage
transmission as the concentration
of fructose decreases. This is
similar to what happens in RBCs
exposed to NaCl concentrations.
The lower levels of sugar mean
that there is a higher water
potential, meaning there are more
freely moving water molecules
since less of them are bound to
sucrose so more have the potential
to move. Since there are more free
moving water particles out of the
cell, the water will move into the
red blood cell by osmosis. This
extra water will put pressure on the
membrane of the cell causing them
to swell and lyse (they swell and
the membrane ruptures) (Wingfield
& Wilbur, 1949).
Figure 3 showed the slow rate
of diffusion of fructose based on
the
difference
of
percent
transmission of RBC. Fructose is a
nonpenetrating
and
nonelectrolyte compound like glucose
(Cadwallader and Phillips, 1969). It
can cause hemolysis by influx of
water by osmosis with a same
molar concentration (Jung et al.,
1973). Fructose has big molecular
weight (MW=180) due to heavy
molecular weight it can slow down
the diffusion rate in the RBC
membrane so there is a little
difference within the time intervals.

Table 4. Correlation Values of %Transmission against Concentration and Time

Pearson r - values
% Transmission against Solute Concentration

NaCl
-0.75
Urea
-0.16
Fructose
-0.87
% Transmission against Time
NaCl
0.43
Urea
0.40
Fructose
-0.82
Table 4 shows the correlation
values of % Transmission against
concentration and time. Specifically
the table has shown negative
correlation/relationship between %
transmission and concentration in
all of the three solutes. The table
also shows that NaCl and Urea`s %
Transmission
have
positive
correlation with time while in
contrary,
Fructose`s
%
Transmission have a negative
correlation with time.
The results have shown an
indirect relationship between %
Transmission
and
solute
concentration
which
may
be
explained by the shared idea above
because as the concentration of
the solute increases, the attraction
of water to that solution also
increases which eventually lead
into the movement of water from
the cell into the solution resulting
into a decrease in %transmission or
low hemolysis rate.
Transports in and out the cell
happens most of the times but it
can only be done by specific
molecules along with their specific
transports. There are different
kinds of transport mechanisms
which can be observed in a cell

membrane, but different molecules


have also different ways of coming
in and out of the cell some may
just need diffusion caused by the
difference
in
gradient
concentration, some may use
specific signals in order to enter
the cell and some may enter the
cell with the use of enzymes or
other biomolecules (Reece et al.
2010). Due to the differences in
mechanism
and
molecular
structure,
different
molecules
consumes different amount of time
in order to enter the membrane.
NaCl and Urea may enter the
membrane
through
a
simple
diffusion (Freeman, W.H. et al.
2000), fructose in the other hand
requires specific binding enzymes
in order to enter the cell membrane
(Kielhorn
C.
2016),
hence
explaining the continuous gradual
increase of % Transmission of NaCl
and urea and an unstable gradient
concentration the fructose solution.
CONCLUSION
After
conducting
this
experiment the researchers were
able to come up with the following
conclusions: Solutions could be

classified into isotonic, hypertonic


and hypotonic solutions which
would be caused by difference in
solute concentration which would
also cause different effects on the
blood
cells.
The
percent
transmission of the red blood cells
in the NaCl and fructose solution
increases as the concentration of
the test solution decreases this was
also indicated by the correlation
values which show that the three
solutes solutions have a negative
or indirect relationship with the %
Transmission.
Each
specific
molecule has its own way of
entering the cell membrane; they
differ on the transport mechanism
which enables them to enter the
semi-permeable membrane.
RECOMMENDATION
In conducting this experiment
the researchers were able to come
up
with
the
following
recommendations: other factors
such as environment temperature,
pH, etc should have also been
considered in order to have a much
stronger basis for the conclusion.
More
concentration
gradients
would have given much better data
and a better conclusion. Other
solute solutions may have also
given
a
much
expounded
discussion and a much stronger
conclusion.
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