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SERUM ANTIOXIDANT LEVELS
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IN COLON CANCER
A research on surgery associated
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oxidative stress

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Submitted by:

Arka Sengupta
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MSc. Medical Biochemistry
Kasturba Medical College
Manipal
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 ACKNOWLEDGEMENT

I express my deepest gratitude to Dr.Shobha U.Kamath and Dr. Ullas

Kamath, Dept. of Biochemistry, Kasturba Medical College and Melaka
Manipal Medical College Manipal, for providing me with their invaluable
guidance, knowledge and support which helped me in successfully
completing this research.

I am thankful to the entire staff of the Departments of Biochemistry and

Oncology at Kasturba Medical College for support that they extended for
my research.

Finally, I take this opportunity to extend my deepest appreciation to my

family and friends, for being with me during the crucial times of the
completion of my project.

2
TABLE OF CONTENTS

TITLE PAGE NUMBER

ACKNOWLEDGEMENT 2
TABLE OF CONTENTS 3
ABSTRACT 4
LITERATURE REVIEW 5-15
INTRODUCTION TO PROTEIN 16
THIOLS
MAIN RESEARCH INTRODUCTION 17

AIM AND OBJECTIVES 18

METHODS AND MATERIALS 18-19
PROTOCOL AND PROCEDURE 20
CACULATION 20
RESULTS 21-23
ANALYSIS AND DISCUSSION 24-25
SCOPE 25-26
REFERENCES AND BIBLIOGRAPHY 27-29

3
 ABSTRACT

Reactive oxygen species (ROS) could be important causative

agents of a number of human diseases, including cancer. Thus,
antioxidants, which control the oxidative stress state, represent a
major line of defense regulating overall health. Human plasma
contains many different non-enzymatic antioxidants. Because of
their number, it is difficult to measure each of these different
antioxidants separately. In addition, the antioxidant status in
human plasma is dynamic and may be affected by many factors.
Thus, the relationship between non-enzymatic antioxidant
capacity of plasma and levels of well-known markers of oxidative
stress (oxidized proteins, lipid hydro-peroxides, decreases in thiol
groups) better reflects health status

Thiol compounds, such as glutathione (GSH), cysteine (CSH) and

homocysteine (HCSH) are a natural reservoir of the reductive
capacity of the cell. The most significant of the multifarious roles
played by thiols in vivo is their function as components of the
intracellular and extracellular redox buffer. A diminished cellular
GSH level accompanies such pathological states as diabetes,
alcoholism, AIDS, acute hemorrhagic gastric erosions, cataract,
neurological diseases, malnutrition and also has been observed
during aging.

The present study considers antioxidant capacity and oxidative

stress in human plasma of patients with colon cancer before and
after surgical removal of tumors. Healthy blood donors were used
as controls. Colon cancer patients demonstrated a significant
decrease in total thiol groups with respect to healthy controls. In
patients with precancerous lesions, the only unmodified
parameter was the thiol group level. After surgery, the levels of
total thiol groups were restored to those seen in healthy subjects.

4
 OXIDATIVE STRESS

Oxidative stress:
It is caused by an imbalance between the production of reactive oxygen and a
biological system's ability to readily detoxify the reactive intermediates or easily
repair the resulting damage. All forms of life maintain a reducing environment within
their cells. This reducing environment is preserved by enzymes that maintain the
reduced state through a constant input of metabolic energy. Disturbances in this
normal redox state can cause toxic effects through the production of peroxides and
free radicals that damage all components of the cell, including proteins, lipids, and
DNA.

In humans, oxidative stress is involved in many diseases, such as atherosclerosis,

Parkinson's disease and Alzheimer's disease and it may also be important in
ageing. However, reactive oxygen species can be beneficial, as they are used by
the immune system as a way to attack and kill pathogens. Reactive oxygen species
are also used in cell signaling. This is dubbed redox signaling.

Chemical and biological effects

In chemical terms, oxidative stress is a large increase (becoming less negative) in
the cellular reduction potential, or a large decrease in the reducing capacity of the
cellular redox couples, such as glutathione and related protein and non-protein
bound thiols. The effects of oxidative stress depend upon the size of these changes,
with a cell being able to overcome small perturbations and regain its original state.
However, more severe oxidative stress can cause cell death and even moderate
oxidation can trigger apoptosis, while more intense stresses may cause necrosis

5
REACTIVE OXYGEN SPECIES ( ROSs)

A particularly destructive aspect of oxidative stress is the production of reactive

oxygen species, which include free radicals and peroxides. Some of the less
reactive of these species (such as superoxide) can be converted by oxidoreduction
reactions with transition metals or other redox cycling compounds including
quinones into more aggressive radical species that can cause extensive cellular
damage. Most of these oxygen-derived species are produced at a low level by
normal aerobic metabolism and the damage they cause to cells is constantly
repaired. However, under the severe levels of oxidative stress that cause necrosis,
the damage causes ATP depletion, preventing controlled apoptotic death and
causing the cell to simply fall apart.

Oxidant Description

•O2-, superoxide One-electron reduction state of O2, formed in many autoxidation reactions and by the
anion electron transport chain. Rather unreactive but can release Fe2+ from iron-sulphur
proteins and ferritin. Undergoes dismutation to form H2O2 spontaneously or by
enzymatic catalysis and is a precursor for metal-catalyzed •OH formation.

H2O2, hydrogen Two-electron reduction state, formed by dismutation of •O2- or by direct reduction of
peroxide O2. Lipid soluble and thus able to diffuse across membranes.

•OH, hydroxyl Three-electron reduction state, formed by Fenton reaction and decomposition of
radical peroxynitrite. Extremely reactive, will attack most cellular components

ROOH, organic Formed by radical reactions with cellular components such as lipids and
hydroperoxide nucleobases.

RO•, alkoxy and Oxygen centred organic radicals. Lipid forms participate in lipid peroxidation
ROO•, peroxy reactions. Produced in the presence of oxygen by radical addition to double bonds or
radicals hydrogen abstraction.

HOCl, hypochlorous Formed from H2O2 by myeloperoxidase. Lipid soluble and highly reactive. Will readily
acid oxidize protein constituents, including thiol groups, amino groups and methionine.

OONO-, Formed in a rapid reaction between •O2- and NO•. Lipid soluble and similar in
peroxynitrite reactivity to hypochlorous acid. Protonation forms peroxynitrous acid, which can
undergo homolytic cleavage to form hydroxyl radical and nitrogen dioxide.

6
Production and consumption of oxidants

PRODUCTION: The most important source of reactive oxygen under normal

conditions in aerobic organisms is probably the leakage of activated oxygen from
mitochondria during normal oxidative respiration.

Other enzymes capable of producing superoxide are xanthine oxidase, NADPH

oxidases and cytochromes P450. Hydrogen peroxide is produced by a wide variety
of enzymes including monoxygenases and oxidases. Reactive oxygen species play
important roles in cell signalling, a process termed redox signaling. Thus, to
maintain proper cellular homeostasis, a balance must be struck between reactive
oxygen production and consumption.

CONSUMPTION: The best studied cellular antioxidants are the enzymes

superoxide dismutase (SOD), catalase, and glutathione peroxidase. Less well
studied (but probably just as important) enzymatic antioxidants are the
peroxiredoxins and the recently discovered sulfiredoxin. Other enzymes that have
antioxidant properties (though this is not their primary role) include paraoxonase,
glutathione-S transferases, and aldehyde dehydrogenases.

Oxidative stress contributes to tissue injury following irradiation and hyperoxia. It is

suspected (though not proven) to be important in neurodegenerative diseases
including Lou Gehrig's disease (aka MND or ALS), Parkinson's disease, Alzheimer's
disease, and Huntington's disease. Oxidative stress is thought to be linked to certain
cardiovascular disease, since oxidation of LDL in the vascular endothelium is a
precursor to plaque formation. Oxidative stress also plays a role in the ischemic
cascade due to oxygen reperfusion injury following hypoxia. This cascade includes
both strokes and heart attacks.

Antioxidants are molecules that slow or prevent the oxidation of other molecules.
Oxidation is a chemical reaction that transfers electrons from a substance to an
oxidizing agent. Oxidation reactions can produce free radicals, which start chain
reactions that damage cells. Antioxidants terminate these chain reactions by
removing radical intermediates, and inhibit other oxidation reactions by being

7
oxidized themselves. As a result, antioxidants are often reducing agents such as
thiols or polyphenols.

Although oxidation reactions are critical for life, they can also be damaging; hence,
plants and animals maintain complex systems of multiple types of antioxidants, such
as glutathione, vitamin C, and vitamin E as well as enzymes such as catalase,
superoxide dismutase and various peroxidases. Too low levels of antioxidants or
inhibition of the antioxidant enzymes causes oxidative stress and may damage or kill
cells.

Metabolites
Overview
Antioxidants are classified into two broad divisions, depending on whether they are
soluble in water (hydrophilic) or in lipids (hydrophobic). In general, water-soluble
antioxidants react with oxidants in the cell cytoplasm and the blood plasma, while
lipid-soluble antioxidants protect cell membranes from lipid peroxidation. These
compounds may be synthesized in the body or obtained from the diet. The different
antioxidants are present at a wide range of concentrations in body fluids and
tissues, with some such as glutathione or ubiquinone mostly present within cells,
while others such as uric acid are more evenly distributed throughout the body.

The relative importance and interactions between these different antioxidants is a

complex area, with the various metabolites and enzyme systems having synergistic
and interdependent effects on one another. The action of one antioxidant may
depend on the proper function of other members of the antioxidant system. The
amount of protection provided by any one antioxidant therefore depends on its
concentration, its reactivity towards the particular reactive oxygen species being
considered, and the status of the antioxidants with which it interacts.

Some compounds contribute to antioxidant defense by chelating transition metals

and preventing them from catalyzing the production of free radicals in the cell.
Particularly important is the ability to sequester iron, which is the function of iron-
binding proteins such as transferrin and ferritin. Selenium and zinc are commonly
referred to as antioxidant nutrients, but these chemical elements have no
antioxidant

8
action themselves and are instead required for the activity of some antioxidant
enzymes, as is discussed below.

Antioxidant Concentration in human serum Concentration in liver tissue

Solubility
metabolite (μM)] (μmol/kg)

Ascorbic acid (vitamin

Water 50 – 60 260 (human)
C)

Glutathione Water 325 – 650 6,400 (human)

Lipoic acid Water 0.1 – 0.7 4 – 5 (rat)

Uric acid Water 200 – 400 1,600 (human)

β-carotene: 0.5 – 1
5 (human, total carotenoids)
Carotenes Lipid
retinol (vitamin A): 1 – 3

α-tocopherol (vitamin
Lipid 10 – 40 50 (human)
E)

Ubiquinol (coenzyme
Lipid 5 200 (human)
Q)

9
Ascorbic acid
Ascorbic acid or "vitamin C" is a monosaccharide antioxidant found in both animals
and plants. As it cannot be synthesised in humans and must be obtained from the
diet, it is a vitamin. Most other animals are able to produce this compound in their
bodies and do not require it in their diets. In cells, it is maintained in its reduced form
by reaction with glutathione, which can be catalysed by protein disulfide isomerase
and glutaredoxins. Ascorbic acid is a reducing agent and can reduce and thereby
neutralize reactive oxygen species such as hydrogen peroxide. In addition to its
direct antioxidant effects, ascorbic acid is also a substrate for the antioxidant
enzyme ascorbate peroxidase, a function that is particularly important in stress
resistance in plants.

FIG: The Free Radical Mechanism of Lipid Peroxidation.

10
Glutathione
Glutathione is a cysteine-containing peptide found in most forms of aerobic life. It is
not required in the diet and is instead synthesized in cells from its constituent amino
acids. Glutathione has antioxidant properties since the thiol group in its cysteine
moiety is a reducing agent and can be reversibly oxidized and reduced. In cells,
glutathione is maintained in the reduced form by the enzyme glutathione reductase
and in turn reduces other metabolites and enzyme systems as well as reacting
directly with oxidants. Due to its high concentration and its central role in maintaining
the cell's redox state, glutathione is one of the most important cellular antioxidants.

Melatonin
Melatonin is a powerful antioxidant that can easily cross cell membranes and the
blood-brain barrier. Unlike other antioxidants, melatonin does not undergo redox
cycling, which is the ability of a molecule to undergo repeated reduction and
oxidation. Redox cycling may allow other antioxidants (such as vitamin C) to act as
pro-oxidants and promote free radical formation. Melatonin, once oxidized, cannot
be reduced to its former state because it forms several stable end-products upon
reacting with free radicals. Therefore, it has been referred to as a terminal (or
suicidal) antioxidant.

Tocopherols and tocotrienols (vitamin E)

Vitamin E is the collective name for a set of eight related tocopherols and
tocotrienols, which are fat-soluble antioxidant vitamins. Of these, α-tocopherol has
been most studied as it has the highest bioavailability, with the body preferentially
absorbing and metabolising this form. The α-tocopherol form is the most important
lipid-soluble antioxidant and protects cell membranes against oxidation by reacting
with the lipid radicals produced in the lipid peroxidation chain reaction .This removes
the free radical intermediates and prevents the propagation reaction from
continuing. The oxidised α-tocopheroxyl radicals produced in this process may be
recycled back to the active reduced form through reduction by ascorbate, retinol or
ubiquinol. The functions of the other forms of vitamin E are less well-studied,
although γ-tocopherol is a nucleophile that may react with electrophilic mutagens,
and tocotrienols may have a specialised role in neuroprotection.

11
Pro-oxidant activities
Antioxidants that are reducing agents can also act as pro-oxidants. For example,
vitamin C has antioxidant activity when it reduces oxidizing substances such as
hydrogen peroxide, however, it can also reduce metal ions which leads to the
generation of free radicals through the Fenton reaction.

2 Fe3+ + Ascorbate → 2 Fe2+ + Dehydroascorbate

2 Fe2+ + 2 H2O2 → 2 Fe3+ + 2 OH· + 2 OH−
The relative importance of the antioxidant and pro-oxidant activities of
antioxidants are an area of current research, but vitamin C, for example,
appears to have a mostly antioxidant action in the body. However, less data
is available for other dietary antioxidants, such as polyphenol antioxidants,
zinc, and vitamin E.

Enzyme systems

Enzymatic Pathway For Detoxification Of Reactive Oxygen Species.

Overview
As with the chemical antioxidants, cells are protected against oxidative
stress by an interacting network of antioxidant enzymes. Here, the
superoxide released by processes such as oxidative phosphorylation is first
converted to hydrogen peroxide and then further reduced to give water.
This detoxification pathway is the result of multiple enzymes, with
superoxide dismutases catalysing the first step and then catalases and
various peroxidases removing hydrogen peroxide. As with antioxidant
metabolites, the contributions of these enzymes can be hard to separate

12
from one another, but the generation of transgenic mice lacking just one
antioxidant enzyme can be informative.

Superoxide dismutase, catalase and peroxiredoxins

Superoxide dismutases (SODs) are a class of closely related enzymes that
catalyse the breakdown of the superoxide anion into oxygen and hydrogen
peroxide. SOD enzymes are present in almost all aerobic cells and in
extracellular fluids.] Superoxide dismutase enzymes contain metal ion
cofactors that, depending on the isozyme, can be copper, zinc, manganese
or iron. In humans, the copper/zinc SOD is present in the cytosol, while
manganese SOD is present in the mitochondrion. There also exists a third
form of SOD in extracellular fluids, which contains copper and zinc in its
active sites. The mitochondrial isozyme seems to be the most biologically
important of these three, since mice lacking this enzyme die soon after
birth. In contrast, the mice lacking copper/zinc SOD are viable but have
lowered fertility, while mice without the extracellular SOD have minimal
defects. In plants, SOD isozymes are present in the cytosol and
mitochondria, with an iron SOD found in chloroplasts that is absent from
vertebrates and yeast.

Catalases are enzymes that catalyse the conversion of hydrogen peroxide

to water and oxygen, using either an iron or manganese cofactor. This
protein is localized to peroxisomes in most eukaryotic cells. Catalase is an
unusual enzyme since, although hydrogen peroxide is its only substrate, it
follows a ping-pong mechanism. Here, its cofactor is oxidised by one
molecule of hydrogen peroxide and then regenerated by transferring the
bound oxygen to a second molecule of substrate. Despite its apparent
importance in hydrogen peroxide removal, humans with genetic deficiency
of catalase — "acatalasemia" — suffer few ill effects.

13
Peroxiredoxins are peroxidases that catalyze the reduction of hydrogen
peroxide, organic hydroperoxides, as well as peroxynitrite. They are divided
into three classes: typical 2-cysteine peroxiredoxins; atypical 2-cysteine
peroxiredoxins; and 1-cysteine peroxiredoxins. These enzymes share the
same basic catalytic mechanism, in which a redox-active cysteine (the
peroxidatic cysteine) in the active site is oxidized to a sulfenic acid by the
peroxide substrate. Peroxiredoxins seem to be important in antioxidant
metabolism, as mice lacking peroxiredoxin 1 or 2 have shortened lifespan
and suffer from hemolytic anaemia, while plants use peroxiredoxins to
remove hydrogen peroxide generated in chloroplasts.

Thioredoxin and glutathione systems

The thioredoxin system contains the 12-kDa protein thioredoxin and its
companion thioredoxin reductase. Proteins related to thioredoxin are
present in all sequenced organisms, with plants such as Arabidopsis
thaliana having a particularly great diversity of isoforms The active site of
thioredoxin consists of two neighboring cysteines, as part of a highly-
conserved CXXC motif, that can cycle between an active dithiol form
(reduced) and an oxidized disulfide form. In its active state, thioredoxin acts
as an efficient reducing agent, scavenging reactive oxygen species and
maintaining other proteins in their reduced state. After being oxidized, the
active thioredoxin is regenerated by the action of thioredoxin reductase,
using NADPH as an electron donor.

The glutathione system includes glutathione, glutathione reductase,

glutathione peroxidases and glutathione S-transferases. This system is
found in animals, plants and microorganisms. Glutathione peroxidase is an
enzyme containing four selenium-cofactors that catalyzes the breakdown of
hydrogen peroxide and organic hydroperoxides. There are at least four
different glutathione peroxidase isozymes in animals. Glutathione
peroxidase 1 is the most abundant and is a very efficient scavenger of
hydrogen peroxide, while glutathione peroxidase 4 is most active with lipid
hydroperoxides. Surprisingly, glutathione peroxidase 1 is dispensable, as
mice lacking this enzyme have normal lifespans, but they are
hypersensitive to induced oxidative stress. In addition, the glutathione S-
transferases are another class of glutathione-dependent antioxidant
enzymes that show high activity with lipid peroxides. These enzymes are at

14
 particularly high levels in the liver and also serve in detoxification
metabolism.

Oxidative stress in disease

Oxidative stress is thought to contribute to the development of a wide range of

diseases including Alzheimer's disease, Parkinson's disease, the pathologies
caused by diabetes, rheumatoid arthritis, and neurodegeneration in motor neurone
diseases. In many of these cases, it is unclear if oxidants trigger the disease, or if
they are produced as a consequence of the disease and cause the disease
symptoms; as a plausible alternative, a neurodegenerative disease might result
from defective axonal transport of mitochondria, which carry out oxidation
reactions. One case in which this link is particularly well-understood is the role of
oxidative stress in cardiovascular disease. Here, low density lipoprotein (LDL)
oxidation appears to trigger the process of atherogenesis, which results in
atherosclerosis, and finally cardiovascular disease.

PROTEIN THIOLS

15
 Thiol compounds, such as glutathione(GSH), cysteine (CSH)
and homocysteine(HCSH) are a natural reservoir of the reductive
capacity of the cell. The most significant of the multifarious roles
played by thiols in vivo is their function as components of the
intracellular and extracellular redox buffer. A diminished cellular
GSH level accompanies such pathological states as diabetes,
alcoholism, AIDS, acute hemorrhagic gastric erosions, cataract,
neurological diseases, malnutrition and has also been observed
during aging. The concept of plasma redox status postulates that
there are dynamic interactions between the different redox forms
of thiols realized through redox reactions, including thiol-disulfide
exchange .Formation and breakage of disulfide bonds depends
largely on the vailability of electron donors and acceptors, which
determines the redox potential of the environment. Therefore, a
change in the thiol : disulfide ratio, i.e. a change in the redox
status of thiols, significantly influences the structure and function
of cellular and extracellular proteins. Glutathione, cysteine and
homocysteine are present in plasma mostly in the form of
symmetrical and mixed disulfides, which belong to the free
fraction, called acid-soluble fraction, and also to the protein-
bound fraction. Since the plasma GSH concentration reflects its
levels in various tissues, it is believed that a lowered plasma GSH
level can be a diagnostic indicator of a pathological state. For this
reason, and also due to the atherosclerotic action of
homocysteine, thiols have become a focus of increasing interest.

16
 SERUM ANTIOXIDANT LEVELS IN
COLON CANCER

Reactive oxygen species (ROS) could be important causative

agents of a number of human diseases, including cancer. Thus,
antioxidants, which control the oxidative stress state, represent a
major line of defense regulating overall health. Human plasma
contains many different nonenzymatic antioxidants. Because of
their number, it is difficult to measure each of these different
antioxidants separately. In addition, the antioxidant status in
human plasma is dynamic and may be affected by many factors.
Thus, the relationship between nonenzymatic antioxidant
capacity of plasma and levels of well-known markers of oxidative
stress (oxidized proteins, lipid hydroperoxides, decreases in thiol
groups) better reflects health status. The present study considers
antioxidant capacity and oxidative stress in human plasma of
patients with colon cancer before and after surgical removal of
tumors . Healthy blood donors were used as controls. Colon
cancer patients demonstrated a significant decrease in total thiol
groups with respect to healthy controls. In patients with
precancerous lesions, the only unmodified parameter was the
thiol group level. After surgery, the levels of total thiol groups
were restored to those seen in healthy subjects.

17
AIM AND OBJECTIVE:
The antioxidant status of human plasma is dynamic and can be
affected by various factors, including diet, physical exercise,
injury, and disease. To verify the involvement of free radical
damage in tumor progression, the present study was directed at
evaluating total thiol groups in plasma of human subjects with
colon cancer. In these cancerous patients the same experimental
parameter was assayed before and after surgical removal of
tumor.

METHODS AND MATERIALS:

Patient Selection. Ten patients with colorectal cancer and 20

healthy subjects were randomly selected for this study.
Determinations of total thiols were performed in serum, before
and after the surgical removal of the tumor

Sample Preparation. Venous blood was collected and allowed to

stand at room temperature for 30 minutes. Plasma was separated
by centrifugation @ 2500 rpm for 10 min. Plasma samples were
immediately analyzed for total thiol groups.

Total Thiol Group Determination. Total thiol groups were

measured in 50μL of plasma using a spectrophotometric assay
based on the reaction of thiols with 2,2- dithio-bis-nitrobenzoic
acid ( DTNB) at [λ] = 412 nm . Results are expressed as μmol/L
plasma. PROTEIN THIOLS WERE ESTIMATED BY THE METHOD OF
A.P. MOTCHNIK et al.

18
 The method was standardized using standard 1mM reduced
Glutathione (GSH) which is prepared fresh and stored in cold .
Exposure to light is avoided.

REAGENTS:

1) DI SODIUM HYDROGEN PHOSPHATE ( Na2HPO4) ; 0.2 M

MW= 141.96
2) Di Sodium EDTA ( Na2 EDTA) ; 2mM
MW = 292.35

Always prepared fresh

3) Di Thio Nitro Benzene –DTNB (C14H8N208S2) ; 10mM

MW = 396.35

To be prepared fresh and stored in a dark bottle. However it can be

stored in cold for only a week.

4) Reduced Glutathione – GSH ( C10H17N3O6S) ; 1mM

MW = 307.32

Always prepared fresh and stored in cold in a dark bottle.

19
 PROTOCOL AND PROCEDURE:

TEST GSH (μL) Na2 EDTA Na2HPO4 DTNB

TUBES (μL) (μL) (μL)
1 mM
2 mM 0.2 M 10 mM

BLANK 0 900 100 20

in all test in all test
tubes tubes

S1 10 90

20 80

70,80.. 30, 20….

Absorbance measured at 412nm.

For test samples replace GSH with test. ( 50 μl of test is

taken)

20
 CALCULATION:

Thiols (μmol/L) = Value from graph x Dilution factor x

1000
Mol.wt of GSH
Dilution factor in this case = 20

RESULTS

Graph

700

600
Mean THIOLS ( PRE vs POST)

500

400

300

200
1 2

GROUPS

21
GROUP 1 -- PRE -- SURGERY THIOLS

GROUP 2 -- POST – SURGERY THIOLS

FIG: Results are mean ± SD of two determinations per patient.

Statistical analysis by Student t test. *P < 0.001 compared with
healthy subjects.

Means

Case Processing Summary

Cases
Included Excluded Total
N Percent N Percent N Percent
THIOLS ( PRE vs
20 100.0% 0 .0% 20 100.0%
POST) * GROUPS

Report

THIOLS ( PRE vs POST)

GROUPS Mean N Std. Deviation
1 275.4460 10 74.9304
2 639.0630 10 69.2682
Total 457.2545 20 199.3147

T-Test

Group Statistics

Std. Error
GROUPS N Mean Std. Deviation Mean
THIOLS ( PRE vs POST) 1 10 275.4460 74.9304 23.6951
2 10 639.0630 69.2682 21.9045

22
 Independent Samp les Test

Levene's Test for

Equality of Variances t-test for Equality of Means
95% Confidence
Interval of the
M ean Std. Error Difference
F Sig. t df Sig. (2-tailed) Difference Difference Lower Upper
THIOLS ( PRE vs POST)Equal variances
.027 .872 -11.268 18 .000 -363.6170 32.2686 -431.4109 -295.8231
assumed
Equal variances
-11.268 17.890 .000 -363.6170 32.2686 -431.4408 -295.7932
not assum ed

Protein Thiols Mean ± SEM Mean difference* 95% CI

Before surgery 275.5 ± 23.7 - 363.6 -431.4, -295.8

(N=10)

After surgery 639.1 ± 21.9 -363.6 -431.4, -295.8

(N=10)

* p<0.001

23
ANALYSIS AND DISCUSSION
Data obtained in this study demonstrated a significant decrease
in total thiol groups in subjects with colon cancer compared with
healthy subjects to determine the effect of surgical removal of
tumors in colon cancer patients, the same experimental
parameter were evaluated before and after treatment. As can be
seen in Figure 1, surgery restored levels of, total thiol groups to
those seen in healthy subjects.

Over recent years, researchers have focused on the pathologic

role of free radicals in a variety of diseases, among which the
most important are atherosclerosis and cancer (4, 13). It has
been proposed that oxygen free radicals mediate the detrimental
effects of malignancy and that removing them results in a
survival advantage (13–15). Colon cancer is a major health
problem, particularly because of the number of patients affected
each year. It has been demonstrated that the 5-year survival
period is increased when the disease is discovered early and the
tumor is not yet fully developed (16). It is, therefore, important to
find new, reliable markers enabling an early diagnosis of this
pathology It has been suggested that oxygen and organic free
radical intermediates are involved in the initiation, promotion,
and/or progression stages of carcinogenesis (17, 18). Increased
production of reactive species may result in a decrease in total
antioxidant capacity in vivo Results obtained here confirm that
oxidative stress is involved in carcinogenesis and neoplasia
results when antioxidant defenses are unable to counteract free
radicals. In fact, it has been demonstrated that inflammatory cells
are particularly effective in generating oxygen-derived oxidants
(19). The possibility that chronic inflammation poses a risk for
cancer in men is inferred from considerable clinical experience
24
indicating human malignancies often occur at sites of ongoing
chronic inflammation as well as from a number of recent
experimental observations (20). Our results also demonstrate
that changes in the plasma of patients are related to neoplasia. In
fact, the results reported in Figure 2 show that surgical tumor
removal restored levels of total thiol groups to those of healthy
subjects . Plasma thiol groups are critical endogenous
antioxidants that act concurrently in scavenging and/or reducing
free radicals, thus breaking the peroxidative chain and allowing
the repair of oxidatively damaged molecules. Thus, the reduction
of total thiol groups observed after surgical removal of tumor
confirms a mechanism of action of these treatments that might
evoke an adaptive response resulting in an increased antioxidant
capacity. Other evidence emerging from these data suggest that,
in addition to their positive effect on general health, antioxidants
may also exert specific beneficial effects on tumor progression
and may represent a valid therapeutic support during treatment.

SCOPE
Gastrointestinal and especially colon cancer remains today an
important cause of
death, especially in Western countries. Improvements in
screening programs and the encouraging results of surgery have
prolonged the lifespan of patients with this pathology, but
mortality is still very high. It has been demonstrated that the
factors able to influence the prognosis are: grading, staging,
nodal involvement, adjacent tissue involvement, and hepatic
recurrences (1, 2). Several studies have been undertaken to find
new oncological markers able to identify a tumor before its
macroscopic development. Evidence suggests the pathological
role of free radicals in a variety of diseases, among which the
most important are atherosclerosis, chronic inflammation, and
cancer (3, 4). Free radicals are inevitable byproducts of biological
redox reactions. In fact, reactive oxygen species, such as ·OH,
H2O2, and other chemical forms, are produced as part of many
normal and essential biological processes (4, 5). Plasma and other
biological fluids are rich in antioxidant molecules, which can be
subdivided into two major groups: those that prevent initiation
and those that slow down the progression of a peroxidative chain
reaction (6–8). The former includes primary antioxidants such as
25
ceruloplasmin and transferrin, which act by binding metal ions;
the latter group includes vitamins A, E, and C and reduced
glutathione, which act by reducing the propagation and
amplification chain. However, a great majority of antioxidants
have multiple antioxidant properties and can thus act by binding
metal ions, as well as by directly scavenging oxidizing species or
by regenerating other oxidized antioxidants. In addition, plasma
also contains noncharacterized antioxidants, which may
contribute to counteract oxidative stress. The multiform nature of
the primary antioxidant renders its quantitative analysis
extremely vague; thus, a “battery” of measurements is necessary
to adequately assess oxidative stress in biological systems.
Because the antioxidant status of human plasma is dynamic and
can be affected by various factors, including diet, physical
exercise, injury, and disease, the relationship between nonproteic
antioxidant capacity (NPAC) of plasma and oxidized protein, lipid
hydroperoxides and total thiol groups better reflects real
oxidative stress and health status. To verify the involvement of
free radical damage in tumor progression, a more comprehensive
study can be conducted that can be directed at evaluating
oxidized proteins, lipid hydroperoxide levels, total thiol groups,
and NPAC in plasma of human subjects

with colon cancer or precancerous lesions (ulcerative colitis,

polyposis). In addition, in some cancerous patients the same
experimental parameters can be assayed before and after
surgical removal of tumor and/or chemo/radiation therapy.

26
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