Malig-on, Kuven A.

BCH 2-2 Biochemistry of the Gene - Laboratory

BIOCHEMISTRY OF THE GENE 3. Erythrocytes provides a good model for toxicity

Laboratory Activity studies because of the following:

• few internal membrane structures and

PRE-LAB Answers
1. Red blood cells have three main functions:
A. Transportation - transports oxygen to the cells
for metabolism
B. Regulation
• helps to keep the body in balance for
important values
• maintains temperature through blood
plasma through absorption of heat while
blood vessel expansion causes heat loss
easy to be obtained
• plasma membrane is a multi-component
structure that keeps the cell morphology,
elasticity, flexibility and deformability
• can undergo eryptosis (programmed cell
death) in response to cellular injury in
which other mechanisms of toxicity
implicated by apoptosis of nucleated
cells can be studied
• vulnerable to peroxidation making it
useful for analyzing the oxidative stress
and lipid peroxidation of various
xenobiotics
(Farag, Alagawany, 2017)

C. Protection

• Protects the body evident as blood clots

for to stop bleeding.
• works with white blood cells for the
immune system

2. Cellular toxicity refers to the ability of

xenobiotics (foreign substances to the body), or
mediator cells to destroy living cells. With these,
cells could undergo necrosis, in which the cells
lose membrane integrity and die due to cell lysis,
or apoptosis, in which the cells undergoes a
process of cellular self-destruction.

The toxicity of a substance depends on three

factors:

• its chemical structure – determines its

mechanism on how it would affect or
deteriorate cells and its biological
functions
• body’s ability to absorb the substance
• body's ability to detoxify the substance
and eliminate it
Malig-on, Kuven A. BCH 2-2
POST LAB Answers
1. Hemolysis is the disruption of a red blood cells
membrane. When red blood cells burst,
hemoglobin, (the part that carries oxygen), is
released into the rest of the blood. This results to
a decrease in the amount of oxygen that the body
gets.
In vitro, hemolysis can be caused of the
administration of solutions of inappropriate
osmolarity, (osmoles of solute per liter), resulting
to rupturing and release of internal membrane
components of the cell. (Biomarkers in
Toxicology, 2014)
In vivo, hemolysis can occur intravascularly or
extravascularly:
• Intravascular hemolysis – occurs when
an extensive antibody/complement
activation damages and triggers an
immune reaction that causes
intravascular red blood cell destruction.
This type of intravascular hemolysis is
may be classified as either warm or cold
reacting. Hemolysis occurs when red
blood cells are coated with
immunoglobulins (warm reacting),
thereby induces cell-destruction.
• Extravascular hemolysis - occurs when
the antibody/complement adhering to the
red blood cell membrane induces
damage to the red blood cell only. The
spherocyte (damaged cell), is extremely
vulnerable to phagocytic cells in the
spleen, liver, or bone marrow. The
filtering system of the spleen allows for
prolonged contact between the sensitive
red blood cells then, removing the
damaged red blood cells from the
circulation.
(Silberstein, 2007)
Biochemistry of the Gene - Laboratory
2. Red blood cells are washed to remove excess
potassium, cytokines, and other allergen proteins
from the supernatant and/or to avoid storage
lesion (refers to changes in red cells during
storage). (Schimdt et. al., 2016). With the
unwanted components already washed out, this
will prevent any unwanted interactions between
the test substances and the sample which may
alter the results of the test to be employed i.e.
absorbance.
3. Hemolysis assay assesses, in vitro, the
hemolytic potential of test items in human blood.
Knowing that intravascular hemolysis results from
the rupture or lysis of red blood cells within the
circulation in vivo, the in vitro hemolysis assay
simulates the in vivo process and evaluates
hemoglobin release in the plasma (indicator of
red blood cell lysis) after exposure to the test
substances.
4. As described that cellular toxicity refers to the
ability of xenobiotics (foreign substances to the
body), or mediator cells to destroy living cells,
hemolytic activities of the test substances can be
related to its toxicity because as the red blood
cells membrane is ruptured, the release of
internal membrane components can be toxic to a
living organism and affect its biological
mechanisms at a certain intensity depending on
the dosage.
5. Triton X-100 is a non-ionic surfactant used for
permeabilizing cells wherein the polar head group
of TX100 disrupts the hydrogen bonding in lipid
bilayer as it interacts with the lipid bilayer and
destroying the integrity of the of the lipid
membrane of the red blood cell at the critical
micelle concentration. With this, it is proven to
induce significant hemolysis and is a viable
positive control for the assay.
Malig-on, Kuven A. BCH 2-2 Biochemistry of the Gene - Laboratory

Sample Data Calculations

0.9000
0.8000
Table 1 Absorbance and Percentage Hemolysis with Diethyl Ether as Solvent 0.7000
0.6000

% Hemolysis
Diethyl ether
Solvent 0.5000
Absorbance Percentage Hemolysis
Conc %(v/v) 0.4000
Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3
0.3000
0 0 0 0 0 0 0
20 0.5261 0.4682 0.5003 10.78% 9.00% 9.99% 0.2000
40 0.8391 0.8843 0.9491 20.42% 21.81% 23.80% 0.1000
60 1.207 1.935 1.572 31.74% 54.16% 42.98% 0.0000
80 2.21 2.096 2.105 62.62% 59.11% 59.39% 0 20 40 60 80 100 120
100 2.787 2.747 2.374 80.39% 79.16% 67.67% Solvent Concentration %(v/v)

Table 2 LC50 of diethyl ether at three Trial 1 Trial 2 Trial 3

different trials and their mean
ATriton X-100 = 3.424 Figure 1 Percentage Hemolysis against Diethyl Ether as Solvent
LC50
ANegativeCtrl = 0.1759 Trial 1 69.2910
Trial 2 65.4808
Trial 3 72.1854 Trial 1
Ave 69.08967 Eqn. of the Line: y = 0.8126x - 6.3029
R2 = 0.9519

Diethyl ether exhibited a mean LC50 value at 69.09% Trial 2

solvent concentration (v/v) which means that when Eqn. of the Line: y = 0.8264x - 4.1133
erythrocytes dissolved into diethyl ether to 69.09% R2 = 0.9669
concentration, half of the total red blood cell content will
lyse.
Trial 3
Eqn. of the Line: y = 0.7225x – 2.1512
R2 = 0.9887
Malig-on, Kuven A. BCH 2-2 Biochemistry of the Gene - Laboratory

Table 3 Absorbance and Percentage Hemolysis with Methanol as Solvent

0.8
Methanol 0.7
Solvent
Absorbance Percentage Hemolysis
Conc %(v/v) 0.6
Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3

% Hemolysis
0.5
0 0 0 0 0 0 0
20 0.5073 0.4712 0.4883 0.4
9.91% 8.79% 9.32%
40 0.8587 0.8383 0.9363 20.78% 20.15% 23.18% 0.3
60 1.527 1.555 1.651 41.46% 42.32% 45.30% 0.2
80 2.11 2.026 2.065 59.50% 56.90% 58.11% 0.1
100 2.597 2.577 2.545 74.57% 73.95% 72.96%
0
0 20 40 60 80 100 120
Table 4 LC50 of methanol at three Solvent Concentration %(v/v)
different trials and their mean
ATriton X-100 = 3.419
LC50 Trial 1 Trial 2 Trial 3
ANegativeCtrl = 0.1871 Trial 1 70.1787
Trial 2 71.2929 Figure 2 Percentage Hemolysis against Methanol as Solvent
Trial 3 69.9396
Ave 70.4704

Trial 1
Eqn. of the Line y = 0.7747x - 4.3659
Methanol exhibited a mean LC50 value at 70.47% solvent R2 = 0.9857
concentration (v/v) which means that when erythrocytes
dissolved into diethyl ether to 70.47% concentration, half of Trial 2
the total red blood cell content will lyse. Eqn. of the Line y = 0.7661x - 4.6175
R2 = 0.9846

Trial 3
Eqn. of the Line y = 0.7618x - 3.28
R2 = 0.9897