1. ANTIOXIDANTS in 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, (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?
Nature’s 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. If you search for Antioxidants…
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 )
Chemical Abstracts 131,961 Publications ( )

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)
(RO

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

12. 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 body’s 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
Alzheimer’s Disease
Parkinson‘s 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 doesn’t 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-octdecdienoic 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

43. Potentialities of oxidative/nitrosative stress-related biomarkers
Acta Medica Okayama, 61 (4), , 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.

53. 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

56. 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, 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, 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.

62. 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 organism’s 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, Alzheimer’s 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