serum indicators of oxidative stress

8/9/2019 Serum Indicators of Oxidative Stress

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SERUM ANTIOXIDANT LEVELSIN COLON CANCER

A research on surgery associated

oxidative stress

Submitted by:

Arka SenguptaMSc. Medical BiochemistryKasturba 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 myfamily and friends, for being with me during the crucial times of the

completion of my project.

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TABLE OF CONTENTS

TITLE PAGE NUMBER

ACKNOWLEDGEMENT 2

TABLE OF CONTENTS 3

ABSTRACT 4

LITERATURE REVIEW 5-15

INTRODUCTION TO PROTEIN

THIOLS

16

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

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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 plasmacontains 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 observedduring 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.

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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

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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 andcausing the cell to simply fall apart.

Oxidant Description

O2-, superoxide

anion

One-electron reduction state of O2, formed in many autoxidation reactions and by the

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

peroxide

Two-electron reduction state, formed by dismutation of O2- or by direct reduction of

O2. Lipid soluble and thus able to diffuse across membranes.

OH, hydroxyl

radical

Three-electron reduction state, formed by Fenton reaction and decomposition of

peroxynitrite. Extremely reactive, will attack most cellular components

ROOH, organic

hydroperoxide

Formed by radical reactions with cellular components such as lipids and

nucleobases.

RO, alkoxy and

ROO, peroxy

radicals

Oxygen centred organic radicals. Lipid forms participate in lipid peroxidation

reactions. Produced in the presence of oxygen by radical addition to double bonds or

hydrogen abstraction.

HOCl, hypochlorousacid

Formed from H2O2 by myeloperoxidase. Lipid soluble and highly reactive. Will readilyoxidize protein constituents, including thiol groups, amino groups and methionine.

OONO-,

peroxynitrite

Formed in a rapid reaction between O2- and NO. Lipid soluble and similar in

reactivity to hypochlorous acid. Protonation forms peroxynitrous acid, which can

undergo homolytic cleavage to form hydroxyl radical and nitrogen dioxide.

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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'sdisease, 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

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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

OverviewAntioxidants 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 differentantioxidants 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

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Ascorbic acidAscorbic 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.

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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 reactingdirectly 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.

MelatoninMelatonin 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, cannotbe 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, -tocopherol has

been most studied as it has the highest bioavailability, with the body preferentially

absorbing and metabolising this form. The -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 -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 -tocopherol is a nucleophile that may react with electrophilic mutagens,

and tocotrienols may have a specialised role in neuroprotection.

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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 Fe

3+

+ Ascorbate 2 Fe

2+

+ Dehydroascorbate2 Fe2+ + 2 H2O2 2 Fe

3+ + 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.

OverviewAs 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

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from one another, but the generation of transgenic mice lacking just one

antioxidant enzyme can be informative.

Superoxide dismutase, catalase and peroxiredoxinsSuperoxide 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. Thisprotein 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.

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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 lifespanand suffer from hemolytic anaemia, while plants use peroxiredoxins to

remove hydrogen peroxide generated in chloroplasts.

Thioredoxin and glutathione systemsThe 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

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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 ofdiseases 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 inatherosclerosis, and finally cardiovascular disease.

PROTEIN THIOLS

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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 redoxstatus 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.

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SERUM ANTIOXIDANT LEVELS IN

COLON CANCER

Reactive oxygen species (ROS) could be important causativeagents of a number of human diseases, including cancer. Thus,antioxidants, which control the oxidative stress state, represent amajor line of defense regulating overall health. Human plasmacontains many different nonenzymatic antioxidants. Because oftheir number, it is difficult to measure each of these differentantioxidants separately. In addition, the antioxidant status inhuman plasma is dynamic and may be affected by many factors.

Thus, the relationship between nonenzymatic antioxidantcapacity of plasma and levels of well-known markers of oxidativestress (oxidized proteins, lipid hydroperoxides, decreases in thiolgroups) better reflects health status. The present study considersantioxidant capacity and oxidative stress in human plasma ofpatients with colon cancer before and after surgical removal oftumors . Healthy blood donors were used as controls. Coloncancer patients demonstrated a significant decrease in total thiolgroups with respect to healthy controls. In patients withprecancerous lesions, the only unmodified parameter was the

thiol group level. After surgery, the levels of total thiol groupswere restored to those seen in healthy subjects.

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AIM AND OBJECTIVE:

The antioxidant status of human plasma is dynamic and can beaffected by various factors, including diet, physical exercise,injury, and disease. To verify the involvement of free radicaldamage in tumor progression, the present study was directed atevaluating total thiol groups in plasma of human subjects withcolon cancer. In these cancerous patients the same experimentalparameter was assayed before and after surgical removal oftumor.

METHODS AND MATERIALS:

Patient Selection. Ten patients with colorectal cancer and 20healthy subjects were randomly selected for this study.Determinations of total thiols were performed in serum, beforeand after the surgical removal of the tumor

Sample Preparation. Venous blood was collected and allowed tostand at room temperature for 30 minutes. Plasma was separatedby centrifugation @ 2500 rpm for 10 min. Plasma samples wereimmediately analyzed for total thiol groups.

Total Thiol Group Determination. Total thiol groups were

measured in 50L of plasma using a spectrophotometric assaybased on the reaction of thiols with 2,2- dithio-bis-nitrobenzoic

acid ( DTNB) at [] = 412 nm . Results are expressed as mol/L

plasma.PROTEIN THIOLS WERE ESTIMATED BY THE METHOD OFA.P. MOTCHNIK et al.

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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.

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PROTOCOL AND PROCEDURE:

TEST

TUBES

GSH (L)

1 mM

Na2 EDTA

(L)

2 mM

Na2HPO4(L)

0.2 M

DTNB

(L)

10 mM

BLANK 0 900

in all test

tubes

100 20

in all test

tubes

S1

10 90

20

70,80..

80

30, 20.

Absorbance measured at 412nm.

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

taken)

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CALCULATION:

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

1000

Mol.wt of GSH

Dilution factor in this case = 20

RESULTS

Graph

GROUPS

21

MeanTHIOLS

(PRE

vsPOST)

700

600

500

400

300

200

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GROUP 1 -- PRE -- SURGERY THIOLS

GROUP 2 -- POST SURGERY THIOLS

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

Statistical analysis by Studentt

test. *P

< 0.001 compared withhealthy subjects.

Means

Case Processing Summary

20 100.0% 0 .0% 20 100.0%THIOLS ( PRE vs

POST) * GROUPS

N Percent N Percent N Percent

Included Excluded Total

Cases

Report

THIOLS ( PRE vs POST)

275.4460 10 74.9304

639.0630 10 69.2682

457.2545 20 199.3147

GROUPS

1

2

Total

Mean N Std. Deviation

T-Test

Group Statistics

10 275.4460 74.9304 23.6951

10 639.0630 69.2682 21.9045

GROUPS1

2

THIOLS ( PRE vs POST)N Mean Std. Deviation

Std. Error

Mean

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Independent Samples Test

.027 .872 -11.268 18 .000 -363.6170 32.2686 -431.4109 -295.8231

-11.268 17.890 .000 -363.6170 32.2686 -431.4408 -295.7932

Equal variances

assumedEqual variances

not assumed

THIOLS ( PRE vs POST)

F Sig.

Levene's Test for

Equality of Variances

t df Sig. (2-tailed)

Mean

Difference

Std. Error

Difference Lower Upper

95% Confidence

Interval of the

Difference

t-test for Equality of Means

23

Protein Thiols Mean SEM Mean difference* 95% CI

Before surgery(N=10)

275.5 23.7 - 363.6 -431.4, -295.8

After surgery(N=10)

639.1 21.9 -363.6 -431.4, -295.8

* p


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ANALYSIS AND DISCUSSION

Data obtained in this study demonstrated a significant decreasein total thiol groups in subjects with colon cancer compared withhealthy subjects to determine the effect of surgical removal oftumors in colon cancer patients, the same experimental

parameter were evaluated before and after treatment. As can beseen in Figure 1, surgery restored levels of, total thiol groups tothose seen in healthy subjects.

Over recent years, researchers have focused on the pathologicrole of free radicals in a variety of diseases, among which themost important are atherosclerosis and cancer (4, 13). It hasbeen proposed that oxygen free radicals mediate the detrimentaleffects of malignancy and that removing them results in asurvival advantage (1315). Colon cancer is a major health

problem, particularly because of the number of patients affectedeach year. It has been demonstrated that the 5-year survivalperiod is increased when the disease is discovered early and thetumor is not yet fully developed (16). It is, therefore, important tofind new, reliable markers enabling an early diagnosis of thispathology It has been suggested that oxygen and organic freeradical intermediates are involved in the initiation, promotion,and/or progression stages of carcinogenesis (17, 18). Increasedproduction of reactive species may result in a decrease in total

antioxidant capacity in vivo Results obtained here confirm thatoxidative stress is involved in carcinogenesis and neoplasiaresults when antioxidant defenses are unable to counteract freeradicals. In fact, it has been demonstrated that inflammatory cellsare particularly effective in generating oxygen-derived oxidants(19). The possibility that chronic inflammation poses a risk forcancer in men is inferred from considerable clinical experience

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indicating human malignancies often occur at sites of ongoingchronic inflammation as well as from a number of recentexperimental observations (20). Our results also demonstratethat changes in the plasma of patients are related to neoplasia. Infact, the results reported in Figure 2 show that surgical tumorremoval restored levels of total thiol groups to those of healthysubjects . Plasma thiol groups are critical endogenous

antioxidants that act concurrently in scavenging and/or reducingfree radicals, thus breaking the peroxidative chain and allowingthe repair of oxidatively damaged molecules. Thus, the reductionof total thiol groups observed after surgical removal of tumorconfirms a mechanism of action of these treatments that mightevoke an adaptive response resulting in an increased antioxidantcapacity. Other evidence emerging from these data suggest that,in addition to their positive effect on general health, antioxidantsmay 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 animportant cause ofdeath, especially in Western countries. Improvements inscreening programs and the encouraging results of surgery haveprolonged the lifespan of patients with this pathology, butmortality is still very high. It has been demonstrated that thefactors able to influence the prognosis are: grading, staging,nodal involvement, adjacent tissue involvement, and hepaticrecurrences (1, 2). Several studies have been undertaken to findnew oncological markers able to identify a tumor before itsmacroscopic development. Evidence suggests the pathologicalrole of free radicals in a variety of diseases, among which themost important are atherosclerosis, chronic inflammation, andcancer (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 manynormal and essential biological processes (4, 5). Plasma and otherbiological fluids are rich in antioxidant molecules, which can besubdivided into two major groups: those that prevent initiationand those that slow down the progression of a peroxidative chainreaction (68). The former includes primary antioxidants such as

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ceruloplasmin and transferrin, which act by binding metal ions;the latter group includes vitamins A, E, and C and reducedglutathione, which act by reducing the propagation andamplification chain. However, a great majority of antioxidantshave multiple antioxidant properties and can thus act by bindingmetal ions, as well as by directly scavenging oxidizing species orby regenerating other oxidized antioxidants. In addition, plasma

also contains noncharacterized antioxidants, which maycontribute to counteract oxidative stress. The multiform nature ofthe primary antioxidant renders its quantitative analysisextremely vague; thus, a battery of measurements is necessaryto adequately assess oxidative stress in biological systems.Because the antioxidant status of human plasma is dynamic andcan be affected by various factors, including diet, physicalexercise, injury, and disease, the relationship between nonproteicantioxidant capacity (NPAC) of plasma and oxidized protein, lipid

hydroperoxides and total thiol groups better reflects realoxidative stress and health status. To verify the involvement offree radical damage in tumor progression, a more comprehensivestudy can be conducted that can be directed at evaluatingoxidized 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 sameexperimental parameters can be assayed before and after

surgical removal of tumor and/or chemo/radiation therapy.

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serum indicators of oxidative stress

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