Title: ANTIOXIDANTS in health and diseases
1ANTIOXIDANTSin 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).
3Learning 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
4CONTENT
- 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
5Why 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)
6What 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).
7Oxidation 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
8What 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)
9What 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
10(No Transcript)
11(No Transcript)
12Common 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
13Common 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
14What 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
15Internal Sources of Free Radicals
- Mitochondria
- Phagocytes (macrophages)
- Xanthine oxidase
- Reaction involving iron and other transition
metals - Arachidonate pathways
- Peroxisomes
- Exercise
- Inflammation
- Ischaemia/Reperfusion
16External Sources of Free Radicals
- Cigarette smoke
- Environmental pollutants
- Radiations
- Ultraviolet radiations
- Ozone
- Certain drugs, pesticides, anesthetics and
industrial solvents
17Useful 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
18History 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
19Harmful 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.
20Oxidative Damage to Organs
21Ageing is one of the major consequences of
oxidative damage
1956 Denham- Free Radical Theory of Ageing
22Human Ailments Associated with Oxidative Damage
- Neurological
- Alzheimers Disease
- Parkinsons Disease
- Endocrine
- Diabetes
- Gastrointestinal
- Acute Pancreatitis
23Human Ailments Associated with Oxidative Damage
- Others Conditions
- Obesity
- Loss of catalytic functions of proteins
- Toxicity
- Chronic Inflammation and arthritis
24Diseases Related to Oxidative Damage
25Exogenous 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.
26Damage to Lipids
- Lipids are highly prone to get oxidized.
- Polyunsaturated fatty acid (PUFA)- major part of
the low-density lipoprotein (LDL) in blood.
27Damage 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
28End 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
29Damage 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.
30Damage 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.
31Atherosclerosis 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.
32Oxidative 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
33Protein 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
34Damage 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.
36Oxidative 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.
37Oxidative Damage to DNA
38Oxidative Damage to DNA
- Oxidative products of guanosine serve as
biomarkers of damage to DNA molecule.
39Oxidative 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
40Damage 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)
41Oxidative 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
42(No Transcript)
43Potentialities of oxidative/nitrosative
stress-related biomarkers
Acta Medica Okayama, 61 (4), 181-189, 2007
44What 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.
45An antioxidant is a molecule stable enough to
donate an electron to a rampaging free radical
and neutralize it, thus reducing its capacity to
damage.
46What 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
47What Antioxidant Do???
48WHY 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.
49Sources 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
50Antioxidant 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
51Major 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.
52(No Transcript)
53Major Antioxidants- Vitamin C
- Vitamin C, or L-ascorbate is an essential
nutrient, water-soluble - Vitamin C scavenge aqueous peroxyl radicals
54Major 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
55(No Transcript)
56Lycopene
57Major 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
58Major Antioxidants- Plant Phenolics
59Antioxidants 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.
60Balance 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.
61(No Transcript)
62What 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.
63Oxidative 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
64Some 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)
-
65Some 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.
66DILEMA 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!!!!
-
67Antioxidant 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?
68THANK YOU VERY MUCH