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Title: ANTIOXIDANTS in health and diseases


1
ANTIOXIDANTSin health and diseases
MUHAMMAD IQBAL CHOUDHARY
Dr. Panjwani Center for Molecular Medicine and
Drug Research International Center for Chemical
and Biological Sciences University of Karachi,
Karachi-75270
2
Oxidation and Human Health
  • One of the paradoxes of life on this planet is
    that the molecule that sustain aerobic life,
    oxygen, is not only fundamentally essential for
    energy metabolism and respiration, but implicated
    in many diseases and degenerative conditions.
  • Marx, Science, 235, 529-531 (1985).

3
Learning Objectives
  • Understanding the relationship between oxidative
    stress, and health and diseases
  • What are oxidants or ROS, types, sources, and
    activities?
  • Their role in normal physiological process
  • Their detrimental role in the onset and
    progression of diseases, chemical basis of
    oxidative damage!
  • Biomarkers of oxidative damage to the vital
    biomolecules and their analysis
  • What are antioxidants, types, sources and
    activities?
  • Perceived role of antioxidants in the
    preservation of health and prevention of diseases
  • Anti-oxidant drug development- challenges and
    opportunities

4
CONTENT
  • What is oxidation?
  • Oxidation in biological system
  • What are free radicals?
  • Sources of free radicals
  • Harmful effects of free radicals
  • Damage to proteins and associated diseases
  • Damage to DNA and associated diseases
  • Damage to lipids and associated diseases
  • Damage to carbohydrates and associated diseases
  • What are antioxidants?
  • Natures antioxidants system
  • Dietary sources of antioxidants
  • Oxidative Stress- Imbalance between oxidation and
    anti-oxidation
  • Types of antioxidants
  • Mechanism of anti-oxidation
  • Bioassays used to discover new antioxidants

5
Why This Topic?
  • If you search for Antioxidants
  • Sci-Finder 305,081 Articles (April 2013)
  • Pubmed 384,341 Hits (April 2013)
  • Google Search Over 7,000,000 Web pages (Jan.
    2014)
  • Chemical Abstracts 131,961 Publications
    (2003-2013)

6
What is Oxidation?
  • Combination of substrate with oxygen.
  • Reaction in which the atoms in a compound lose
    electrons.
  • Any compound, including oxygen, that can accept
    electrons is an oxidant or oxidizing agent
    (pro-oxidant), while a substance that donates
    electrons is a reductant or reducing agent
    (antioxidant).

7
Oxidation in Biological System
  • We live in an aerobic environment
  • Oxygen is the life sustaining element
  • We consume approximately 3.5 kilograms of oxygen
    every day
  • 2.8 percent of the oxygen is not properly used
    and forms free radicals
  • Several kilograms of peroxides (harmful oxidized
    lipids) are produced in our body every year

8
What are Free Radicals?
  • Free radicals (pro-oxidants) are any chemical
    species, capable of independent (although
    extremely short) existence with one or more
    unpaired electrons
  • Highly unstable and reactive
  • Looking for electrons from other sources to
    stabilize themselves. In this process they
    initiate a chain reaction of oxidation
  • Most commons are Reactive Oxygen Species (ROS)
  • Reactive Nitrogen Species (RNS) (NO., ONOO-, etc)
    or Reactive Sulfur Species (RSS)

9
What are Reactive Oxygen Species (ROS)?
  • ROS are
  • oxygen derived radicals (O2.-, .OH, ROO., 1O2,
    RO.)
  • oxygen-derived non radical species (O2, H2O2, O3,
    ROOH, HOCl)
  • They are now considered as major players in
    biochemical reactions, cellular response, and
    clinical outcome

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Common Free Radicals
Oxidant Description
O2-, superoxide anion One-electron reduced state of O2, formed in many autoxidation reactions and by the electron transport chain. Rather unreactive but can release Fe2 from iron-sulfur 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
13
Common Free Radicals
Oxidant Description
ROOH, organic hydroperoxide Formed by radical reactions with cellular components such as lipids and nucleobases.
RO, alkoxy and ROO, peroxy radicals Oxygen centered organic radicals. Lipid forms participate in lipid peroxidation reactions. Produced in the presence of oxygen by radical addition to double bonds or hydrogen abstraction.
ONOO-, 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. Source Wikipedia
14
What Free Radical does?
  • Free radicals are cellular renegades they wreak
    havoc by damaging DNA, altering biochemical
    compounds, corroding cell membranes and killing
    cells outright. Such molecular mayhem, scientists
    increasingly believe, plays a major role in the
    development of ailments like cancer, heart or
    lung diseases and cataracts. Many researchers are
    convinced that the cumulative effects of free
    radicals also underlie the gradual deterioration
    that is the hallmark of aging in all individuals,
    healthy as well as sick.
  • TIME, April 6, 1992 publications

15
Internal Sources of Free Radicals
  • Mitochondria
  • Phagocytes (macrophages)
  • Xanthine oxidase
  • Reaction involving iron and other transition
    metals
  • Arachidonate pathways
  • Peroxisomes
  • Exercise
  • Inflammation
  • Ischaemia/Reperfusion

16
External Sources of Free Radicals
  • Cigarette smoke
  • Environmental pollutants
  • Radiations
  • Ultraviolet radiations
  • Ozone
  • Certain drugs, pesticides, anesthetics and
    industrial solvents

17
Useful Functions of Free Radicals
  • Necessary in the maturation processes of cellular
    structures
  • Necessary in the antibacterial activity- White
    blood cells (phagocytes) releases free radicals
    to destroy invading pathogenic microbes as part
    of the bodys defense mechanism
  • Necessary in the immune system
  • Necessary in the prostaglandin biosynthesis
  • Some of them play an important role in cell
    signaling

18
History Harmful Effect of Free Radicals
  • 1775 Priestly- Toxicity of oxygen to the organism
    similar to burning of candle
  • 1954 Gilbert and Gersham- Free radicals are
    important player in biological environment and
    responsible for deleterious process in the cell
  • 1969 Mc Cord and Fridovich- Superoxide theory of
    toxicity

19
Harmful Effect of Free Radicals- Perception
  • Free radicals can damage all cellular
    macromolecules including proteins, carbohydrates,
    lipids and nucleic acids
  • Destructive effect play a role in the onset and
    progression of different diseases and in normal
    aging.

20
Oxidative Damage to Organs

21
Ageing is one of the major consequences of
oxidative damage
1956 Denham- Free Radical Theory of Ageing
22
Human Ailments Associated with Oxidative Damage
  • Neurological
  • Alzheimers Disease
  • Parkinsons Disease
  • Endocrine
  • Diabetes
  • Gastrointestinal
  • Acute Pancreatitis

23
Human Ailments Associated with Oxidative Damage
  • Others Conditions
  • Obesity
  • Loss of catalytic functions of proteins
  • Toxicity
  • Chronic Inflammation and arthritis

24
Diseases Related to Oxidative Damage

25
Exogenous v/s Endogenous Sources of Free Radicals
  • Exogenous ROS are extremely high
  • Exposure to endogenous oxidants is much more
    important and extensive, because it is a
    continued process during the entire life span
  • Mitochondria play an extremely important role in
    endogenous ROS production
  • Presence of metals (iron, copper, chromium,
    cobalt, vanadium) in un-complexed form
    significantly increase the level of oxidative
    stress.

26
Damage to Lipids
  • Lipids are highly prone to get oxidized.
  • Polyunsaturated fatty acid (PUFA)- major part of
    the low-density lipoprotein (LDL) in blood.

27
Damage to Lipids
  • Lipid peroxidation, if not terminated rapidly,
    can cause damage to cell membranes.
  • Removal of lipid peroxides is essential for
    mammalian life (Glutathione peroxidase IV
    knock-out mouse doesnt survive beyond embryonic
    state).
  • Malondialdehyde (MDA) is an important biomarker
    of oxidative stress. It reacts with DNA bases to
    for DNA-adduct

28
End Products of Lipid Peroxidation
These end products are the markers for lipid
peroxidation determination. For example
malondialdehyde (MDA) is detected in TBARS
(Thiobarb- ituric Acid Reactive Substance)
assay, specific for lipid peroxidation
determination. 13-HPODE 13- Hydroperxy- 9Z,11E-o
ctdecdienoic acid 4-HNE Hydroxynonenal
29
Damage to Lipids-Associated Diseases
  • Alterations in the structures of lipid molecules
    lead to change in their physical properties, such
    as permeability, surface adhesion, etc.
  • It cause damage to the cell membrane, made up of
    mainly lipids.
  • Risk of cardiovascular diseases (CVD), including
    atherosclerosis.

30
Damage to Lipids-Associated Diseases
  • End products of lipid peroxidation can also cause
    mutagenesis and carcinogenesis. For example
    malondialdehyde reacts with deoxyadenosine and
    deoxyguanosine in DNA to form a variety of DNA
    adducts.
  • Body has evolved a range of molecules, such as
    Vitamin E, and enzymes such as SOD, catalase, and
    peroxidase to control lipid peroxidation.
  • Knockout animals (which can not produce
    anti-oxidant enzymes), generally do not survive.

31
Atherosclerosis and Oxidative Stress
  • Compelling evidence points oxidative stress as an
    important trigger in the complex chain of events
    leading to atherosclerosis.
  • It involves accumulation of macrophages in the
    arterial wall. Which then promptly incorporate
    oxidized LDL to form foam cells.
  • ROS can lead to platelet activation and
    thrombosis formation.
  • Probucol has shown reduced progression of carotid
    atherosclerosis in clinical trials.

32
Oxidative Damage to Proteins
  • .OH and RO. and cause damage to proteins
  • Direct damage include peroxidation, damage to
    specific amino acid residues, change in tertiary
    structures, degradation and fragmentation
  • No efficient mechanism of repair of protein
    damage exists
  • Proteolytic enzymes play an important role in the
    removal of damaged proteins

33
Protein Oxidation Products
  • Aldehydes, keto and other carbonyl compounds
  • 3-Nitrotyrosine, produced by interaction of
    tyrosine and ONOO-, is a useful biomarker of
    oxidative protein damage
  • Ortho- and meta-tyrosines from phenylalanine.
  • Other damaged products include hydroxyproline,
    glutamyl semialdehyde, etc
  • Crossed linked proteins

34
Damage to Proteins- Associated Problems
  • Modified oxidized proteins are susceptible to
    many changes in their functions
  • This include chemical fragmentation, inactivation
    and increased proteolytic degradation
  • Oxidative changes in the structures of catalytic
    proteins lead to loss of enzyme activity
  • Altered cellular functions such as energy
    production, interference with the creation of
    membrane potential and change in the type and
    level of cellular proteins
  • Non- enzymatic glycation of proteins lead to
    multiple poteopathic disorders
  • Serum protein carbonyl concentration is directly
    related to muscle dysfunctions.

35
Mechanism of Glycation of Protein- Role of ROS
Catalyzed by transition metals (M) and the
superoxide radical generated are converted to the
hydroxyl radical via the Fenton reaction.
36
Oxidative Damage to DNA
  • DNA is stable, well protected molecules
  • ROS, specially .OH, can interact with it and
    cause several types of damage
  • Including modification of DNA bases, single- and
    double helical breaks, loss of purines, damage to
    deoxyribose sugar, DNA protein cross linkage and
    DNA repair system
  • Out of four bases, guanine is the most easily
    oxidizable nucleic acid base.

37
Oxidative Damage to DNA

38
Oxidative Damage to DNA
  • Oxidative products of guanosine serve as
    biomarkers of damage to DNA molecule.

39
Oxidative Damage to DNA
  • ROS in the cells lead to DNA damage, cause stable
    DNA lesions which are mutagenic, if un-repaired
  • Damaged DNA provide the wrong genetic code
    leading to unregulated protein synthesis and/or
    cell growth which results in cancer.
  • Presence of 8-oxo-2-deoxyguanosine (oxo8dG) in
    DNA is an important indicator of oxidative damage
    to DNA
  • Oxidative damage to DNA accumulate with ageing,
    increasing the possibilities of cancers and other
    disorders

40
Damage to DNA- Associated Problems
  • Number of oxidative hits to DNA per cell per day
    is about 100,000 in the rat and about 10,000 in
    the human (Reason????)
  • There is an inherent mechanism (specific repair
    glycosylases, etc.) to repair most of the DNA
    damage caused by ROS
  • Oxidative lesions in DNA accumulate with age and
    eventually lead to serious health challenges
    (well established relationship between onset of
    cancers and age)

41
Oxidative Stress Markers Oxidative stress end
products detection
  • Lipoperoxidation markers
  • malondialdehyde (MDA), conjugated dienes,
    isoprostanes
  • Oxidative damage to protein markers
  • protein hydroperoxides
  • Oxidative damage to DNA
  • modified nucleosides

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43
Potentialities of oxidative/nitrosative
stress-related biomarkers
Acta Medica Okayama, 61 (4), 181-189, 2007
44
What are Antioxidants?
  • Antioxidants (reductants or reducing agents) are
    compounds capable of preventing the pro-oxidation
    process or biological oxidative damage by
    scavenging or stabilizing reactive oxidative
    species.

45
An antioxidant is a molecule stable enough to
donate an electron to a rampaging free radical
and neutralize it, thus reducing its capacity to
damage.
46
What are Antioxidants?
  • Antioxdiants produced during normal metabolism
    include glutathione, ubiquinol and uric acid
  • Antioxidant enzymes include glutathione
    peroxidases, superoxide dismutases and catalase
  • Antioxidants from dietary sources such as
    Vitamins E and C and carotenoids
  • Antioxidants from non-dietary sources include
    phenolic or polyphenolic compounds as well as
    selenium

47
What Antioxidant Do???
48
WHY ARE ANTIOXIDANTS IMPORTANT ?
  • They inhibit the conversion of nitrites to
    nitrosamines (which are tumor promoters) and
    enhance the immune response.
  • Vitamins E, and C, ubiquinones, etc. remove free
    radicals from the epidermis of the skin and
    counteract their potentially damaging effect.
  • They terminate free radical- induced cellular
  • damage and functional degeneration (aging).
  • They trap and neutralize free radicals and
    protect our body tissues from environmental
    pollutants.















49
Sources of Antioxidants
  • More than 4,000 antioxidants are known
  • Endogenous- Antioxidant enzymes include
    glutatione peroxidases, superoxide dismutases and
    catalase
  • Antioxidants from dietary sources, such as
    Vitamin E, Vitamin C and carotenoids
  • Antioxidants from non-dietary sources include
    phenolic or polyphenolic compounds

50
Antioxidant Enzymes
  • Glutathione peroxidases (Seleno proteins)
    catalyze the reduction of lipid hydroperoxides to
    their corresponding alcohols
  • Superoxide dismutases, a family of
    metal-containing enzymes (Mn, Fe, Zn, Cu),
    catalyze the dismutation of superoxide into
    oxygen and hydrogen peroxide
  • Catalases catalyze the decomposition of hydrogen
    peroxide to water and oxygen

51
Major Antioxidants- Vitamin E
  • Vitamin E, fat-soluble vitamin which exists in
    eight different forms. a-Tocopherol is the most
    active form in humans.
  • Vitamin E protect cells through its ability to
    limit production of free radicals
  • Dietary sources include wheat, almonds, sunflower
    seeds, etc.

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Major Antioxidants- Vitamin C
  • Vitamin C, or L-ascorbate is an essential
    nutrient, water-soluble
  • Vitamin C scavenge aqueous peroxyl radicals

54
Major Antioxidants- Carotenoids
  • Organic pigments naturally occur in plants
  • 600 known carotenoids, and tetraterpenoids exist
    in nature
  • Most common are lycopene and vitamin A precursor
    b-carotene
  • They quench singlet oxygen primarily by a
    physical mechanism in which the excess energy of
    the singlet oxygen is transferred to the
    carotenoids electron rich structure

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Lycopene
57
Major Antioxidants- Plant Phenolics
  • A large number of plant phenolics, such as
    flavanoids, and catechins act as free radical
    scavengers
  • Tea is the richest source of plant phenolics
  • French paradox Flavanoids in red wine

58
Major Antioxidants- Plant Phenolics
  • Apigenin
  • Apigenin
    Catechin

59
Antioxidants at a glance
  • Nutrient RDI Dietary Sources Evidence
  • Vitamin E 30 IU Vegetable oils (soy, corn,
    olive, 100-800 IU may lower cotton-seed,
    safflower, sunflower), heart disease risk
    by nuts, sunflower seed, wheat germ 30-40
  • Vitamin C 60 mg Citrus, strawberries,
    tomatoes, no evidence that RDI
    cantaloupe, broccoli, asparagus, supplement
    form can peppers, spinach, potatoes prevent
    CHD or cancer
  • ß-Carotene NA Dark green, yellow, and orange may
    protect against vegetables spinach, collard
    green CHD and macular broccoli, carrots,
    peppers, sweet degeneration potatoes yellow
    fruits peaches
  • Selenium 70 ug Egg yolks, tuna, seafood,
    chicken, 150-200 ug may lower
  • 55 ug liver, whole grains, plant grown
    in prostate cancer risk selenium-rich soil.

60
Balance Between Oxidation and Antioxidation- Key
to Health











  • Balance between pro-oxidants and oxidants is
    tightly regulated and extremely important for
    maintaining vital cellular and biological
    functions
  • The objective of the antioxidant therapy is not
    to eliminate all the ROS, but to strike a healthy
    balance.



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What is Oxidative Stress?
  • If there are too many free radicals produced and
    too few anti-oxidants, a condition of oxidative
    stress develop which may cause chronic diseases.
  • An imbalance between the production of various
    reactive species and the ability of the
    organisms natural protective mechanism to cope
    with these reactive compounds and prevent adverse
    effects.
  • It has been proven to be related to degenerative
    diseases such as cancers, diabetes, premature
    ageing, Alzheimers disease, arthritis, etc.
  • Difficult to measure.

63
Oxidative Stress Markers
  • Free radical detection
  • Very difficult to analyze because of chemical and
    physical properties e.g. short half life
  • Oxidative stress end products detection (foot
    print measurement)
  • more simple, a wide range of techniques available

64
Some Common In Vitro Antioxidant Assays
  • NO ( Nitric Oxide) Radical Inhibition
  • Assay for DPPH Radical Scavenging Activity
  • Assay for xanthine oxidase inhibition Activity
  • Assay for superoxide anion scavenging Activity
  • a. Enzymatic generation of superoxide anion
    (through xanthine oxidase)
  • b. Non-Enzymatic generation of superoxide
    anion (through
  • NADH/Phenazine methosulphate system)

65
Some Common In Vivo Antioxidant Assays
  • In vivo CCl4 (Carbon tetrachloride) hepatoxicity
    assay.
  • Antioxidant testing kits are now available to
    detects free radical activity in body.

66
DILEMA OF REDOX IN LIVING SYSTEM
  • Difficult to quantify the oxidizing stress
  • Difficult to extrapolate in vitro and in vivo
    situations
  • Bioavailability is an issue
  • Interaction with other molecules
  • Whether antioxidants do help in the prevention of
    diseases?
  • More questions than answers!!!!

67
Antioxidant Drug Development- Challenges and
Opportunities
  • Clinical trials nightmare
  • Non-conclusive results
  • Requires new type of clinical studies than
    conventional double blind placebo trials.
  • Preventive v/s therapeutic
  • Raise questions to old believes?

68
THANK YOU VERY MUCH
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