The analysis of lipid peroxidation in liver homogenate induced by FeSO4, H2O2 and ascorbic acid
by measuring TBARS
1. Introduction
In the last 200 years life expectancy doubled in most countries, due to a better lifestyle, water, nutrition
quality as well as improved medical care. The process of aging is inevitable with people now living
longer than before this is becoming more apparent. Aging is caused by many factors such as
Mitochondrial dysfunction, genomic instability, loss of proteolysis and stem cell exhaustion. Experts have
named these the Hallmarks of aging.
Another Hallmark of aging is mitochondrial damage, which causes impairment of biological
macromolecules in tissues or organs, such as lipids, proteins, and DNA, is caused by reactive oxygen
species and the acquisition of oxidative stress. Reactive oxygen species (ROS) are highly reactive radicals
that can attack various classes of biomolecules for example; DNA, proteins, lipids, etc. These ROS can be
produced both by enzymatic and by non-enzymatic chemical partial reduction of a molecular oxygen
(O2) to superoxide(O2-), hydrogen peroxide (H2O2), lipid peroxides (ROOH), or the corresponding
hydroxyl (HO+) and peroxyl radicals (ROO+). The phenomenon that ROS attacks biomolecules is called
oxidative stress, which can cause harm to people's health. Studies have shown that oxidative stress can
lead to aging and all kinds of diseases; including cancer, cardiovascular disease, neurodegenerative
diseases, diabetes, renal, pulmonary, skeletal muscle disorders, liver diseases, aging and many more.
In particular the reaction between ROS and lipids is called lipid peroxidation. Lipid peroxidation
preferentially oxidizes polyunsaturated fatty acids. In cells lipid peroxidation happens via Fenton-like
reactions. Originally the fenton reaction is the reaction between Fe(H2O)62+ and H2O2 but many other
fenton like reactions have been discovered, which change the metal ion, peroxide and water ligand.
However, in cells this fenton like reaction is mostly accomplished using iron ions.
2+¿ +O ¿
2
3+ ¿+∙ O−¿→ Fe ¿
¿
Fe 2
Equation 1: haber-weiss reaction
−¿+∙ OH.¿
3+¿+ O H ¿
F e 2+¿+ H 2 O2 → F e ¿
Equation 2: Fenton like reaction
Figure 1: The haber-weiss reaction and fenton like reaction that happens when Fe3+ reacts with oxygen
and is reduced to Fe2+. Fe2+ can further react with H2O2 to create an OH radical.
First the Fe3+ reacts with oxygen to produce Fe2+, also called the Haber-Weiss reaction and studies
proposed that OH radicals are the product of the reaction of Fe2+ with peroxides, because Fe2+ can
donate an electron. The end products of lipid peroxidation include malondialdehyde (MDA), 4-hydroxy-
2-nonenal (HNE) and other reaction products. These lipid peroxides can exert their toxic effects through
two general mechanisms. First of all, when lipid peroxidation takes place, the assembly, composition,
structure and dynamics of the cellular membrane is altered as lipids normally maintain the integrity of a
cellular membrane. Secondly, lipid peroxides are highly reactive so they are able to further generate ROS
and can crosslink DNA and proteins.
, The body has antioxidants to fight off the ROS and turn the radicals back to their normal form; they do
this by donating an electron to the radical. An imbalance of ROS and antioxidants can cause oxidative
stress. For example ascorbic acid (vitamin C) can donate an electron to vitamin E which will then act as
an antioxidant, reducing lipid peroxidation. Ascorbic acid has been thought to have a dual role because
ascorbic acid also reacts with Fe3+ to reduce it to Fe2+ (Haber-Weiss reaction), which means that Fe2+
can react with peroxides and enhance lipid peroxidation. This hypothesis has been further tested by using
ascorbic acid as an antioxidant in post-exercise supplementation to inhibit ROS production. Multiple
studies have shown that ingesting ascorbic acid can indeed increase antioxidant power in the muscles.
However, the supplementation did not prevent muscle damage in these studies, so further research is
needed on the exact effect of ascorbic acid on ROS production.
Metabolism of several compounds in the liver can cause formation of liver disease via ROS production.
Excessive alcohol consumption as well as leads to hepatic inflammation and lipid peroxidation.
Furthermore, liver cancer can be induced by lipid peroxidation initiated by ferroptosis, visceral obesity
and nonalcoholic fatty liver disease (NAFLD). Examination of FeSO4 and H2O2 concentration as well as
the antioxidant function of ascorbic acid, are important to evaluate, to find potential treatment for NAFLD
and consequently also for inhibiting cancer development. In our study we want to participate in these
investigations by also accessing the antioxidant function of ascorbic acid in lipid peroxidation.
In order to measure the intensity of lipid peroxidation, we can measure the MDA concentration using a
Thiobarbituric Acid Reactive Substances (TBARS) assay. The MDA will bind to two thiobarbituric acid
(TBA) to form a complex. This complex can be measured with an absorbance measurement, because it
has a pink color.
The aim of this study is to determine the impact of the factors FeSO 4, H2O2 and ascorbic acid on lipid
peroxidation. This will be studied by using a TBARS assay, which will measure the MDA concentration.
Our hypothesis is that H2O2 and FeSO4 will enhance lipid peroxidation, and ascorbic acid will reduce
lipid peroxidation. We will also research the interaction between FeSO4 and ascorbic acid. We
hypothesize that they will have an interaction and increase lipid peroxidation.
2. Materials and Methods
2.1. Sample:
In the study 225 liver homogeneous samples of pigs were used, the experiment was undertaken at
Maastricht University.
2.2. Method:
Sample preperation:
5 gram of liver were used to prepare tissue homogenate. The tissue was first washed in PBS, to
remove remaining blood and then cut into small pieces. Afterwards, those pieces are homogenized
in Phosphate buffer saline (PBS), which mimics the human body's pH of 7.4, osmolarity and ion
concentration. The homogenate was transferred to a clean tube and centrifuge for 10 miutes at
1800 rpm in a large centrifuge, in order to receive the supernatant and separated into another clean
tube. The liver homogenate was stored in the freezer at -20 °C until it was used.
by measuring TBARS
1. Introduction
In the last 200 years life expectancy doubled in most countries, due to a better lifestyle, water, nutrition
quality as well as improved medical care. The process of aging is inevitable with people now living
longer than before this is becoming more apparent. Aging is caused by many factors such as
Mitochondrial dysfunction, genomic instability, loss of proteolysis and stem cell exhaustion. Experts have
named these the Hallmarks of aging.
Another Hallmark of aging is mitochondrial damage, which causes impairment of biological
macromolecules in tissues or organs, such as lipids, proteins, and DNA, is caused by reactive oxygen
species and the acquisition of oxidative stress. Reactive oxygen species (ROS) are highly reactive radicals
that can attack various classes of biomolecules for example; DNA, proteins, lipids, etc. These ROS can be
produced both by enzymatic and by non-enzymatic chemical partial reduction of a molecular oxygen
(O2) to superoxide(O2-), hydrogen peroxide (H2O2), lipid peroxides (ROOH), or the corresponding
hydroxyl (HO+) and peroxyl radicals (ROO+). The phenomenon that ROS attacks biomolecules is called
oxidative stress, which can cause harm to people's health. Studies have shown that oxidative stress can
lead to aging and all kinds of diseases; including cancer, cardiovascular disease, neurodegenerative
diseases, diabetes, renal, pulmonary, skeletal muscle disorders, liver diseases, aging and many more.
In particular the reaction between ROS and lipids is called lipid peroxidation. Lipid peroxidation
preferentially oxidizes polyunsaturated fatty acids. In cells lipid peroxidation happens via Fenton-like
reactions. Originally the fenton reaction is the reaction between Fe(H2O)62+ and H2O2 but many other
fenton like reactions have been discovered, which change the metal ion, peroxide and water ligand.
However, in cells this fenton like reaction is mostly accomplished using iron ions.
2+¿ +O ¿
2
3+ ¿+∙ O−¿→ Fe ¿
¿
Fe 2
Equation 1: haber-weiss reaction
−¿+∙ OH.¿
3+¿+ O H ¿
F e 2+¿+ H 2 O2 → F e ¿
Equation 2: Fenton like reaction
Figure 1: The haber-weiss reaction and fenton like reaction that happens when Fe3+ reacts with oxygen
and is reduced to Fe2+. Fe2+ can further react with H2O2 to create an OH radical.
First the Fe3+ reacts with oxygen to produce Fe2+, also called the Haber-Weiss reaction and studies
proposed that OH radicals are the product of the reaction of Fe2+ with peroxides, because Fe2+ can
donate an electron. The end products of lipid peroxidation include malondialdehyde (MDA), 4-hydroxy-
2-nonenal (HNE) and other reaction products. These lipid peroxides can exert their toxic effects through
two general mechanisms. First of all, when lipid peroxidation takes place, the assembly, composition,
structure and dynamics of the cellular membrane is altered as lipids normally maintain the integrity of a
cellular membrane. Secondly, lipid peroxides are highly reactive so they are able to further generate ROS
and can crosslink DNA and proteins.
, The body has antioxidants to fight off the ROS and turn the radicals back to their normal form; they do
this by donating an electron to the radical. An imbalance of ROS and antioxidants can cause oxidative
stress. For example ascorbic acid (vitamin C) can donate an electron to vitamin E which will then act as
an antioxidant, reducing lipid peroxidation. Ascorbic acid has been thought to have a dual role because
ascorbic acid also reacts with Fe3+ to reduce it to Fe2+ (Haber-Weiss reaction), which means that Fe2+
can react with peroxides and enhance lipid peroxidation. This hypothesis has been further tested by using
ascorbic acid as an antioxidant in post-exercise supplementation to inhibit ROS production. Multiple
studies have shown that ingesting ascorbic acid can indeed increase antioxidant power in the muscles.
However, the supplementation did not prevent muscle damage in these studies, so further research is
needed on the exact effect of ascorbic acid on ROS production.
Metabolism of several compounds in the liver can cause formation of liver disease via ROS production.
Excessive alcohol consumption as well as leads to hepatic inflammation and lipid peroxidation.
Furthermore, liver cancer can be induced by lipid peroxidation initiated by ferroptosis, visceral obesity
and nonalcoholic fatty liver disease (NAFLD). Examination of FeSO4 and H2O2 concentration as well as
the antioxidant function of ascorbic acid, are important to evaluate, to find potential treatment for NAFLD
and consequently also for inhibiting cancer development. In our study we want to participate in these
investigations by also accessing the antioxidant function of ascorbic acid in lipid peroxidation.
In order to measure the intensity of lipid peroxidation, we can measure the MDA concentration using a
Thiobarbituric Acid Reactive Substances (TBARS) assay. The MDA will bind to two thiobarbituric acid
(TBA) to form a complex. This complex can be measured with an absorbance measurement, because it
has a pink color.
The aim of this study is to determine the impact of the factors FeSO 4, H2O2 and ascorbic acid on lipid
peroxidation. This will be studied by using a TBARS assay, which will measure the MDA concentration.
Our hypothesis is that H2O2 and FeSO4 will enhance lipid peroxidation, and ascorbic acid will reduce
lipid peroxidation. We will also research the interaction between FeSO4 and ascorbic acid. We
hypothesize that they will have an interaction and increase lipid peroxidation.
2. Materials and Methods
2.1. Sample:
In the study 225 liver homogeneous samples of pigs were used, the experiment was undertaken at
Maastricht University.
2.2. Method:
Sample preperation:
5 gram of liver were used to prepare tissue homogenate. The tissue was first washed in PBS, to
remove remaining blood and then cut into small pieces. Afterwards, those pieces are homogenized
in Phosphate buffer saline (PBS), which mimics the human body's pH of 7.4, osmolarity and ion
concentration. The homogenate was transferred to a clean tube and centrifuge for 10 miutes at
1800 rpm in a large centrifuge, in order to receive the supernatant and separated into another clean
tube. The liver homogenate was stored in the freezer at -20 °C until it was used.
