biochem

1. Oxidative stress and
Antioxidants

2. Introduction
• Oxidative stress is an imbalance of free
radicals and antioxidants in the body which
can damage different cells and tissues.

3. Free Radicals  Highly reactive chemical species with
an unpaired electron
General Representation:

4. Reactive Oxygen Species (ROS)
Molecules or Ions of Oxygen with Unpaired Electrons

5. Examples of ROS:
 Superoxide Anion Radical (O2⁻)˚
 Hydrogen Peroxide (H2O2)
 Hydroperoxy Radical (HOO)˚
 Hydroxyl Radical (OH)˚
 Lipid Peroxide Radical (ROO)˚
 Singlet Oxygen (1O₂)
Examples of RNS:
 Nitric Oxide (NO)˚
 Peroxy Nitrite (ONOO-)˚
Not classified as free
radicals, but due to their
Reactivity, they are included
in the group of ROS

6. Features of ROS
• Extreme Reactivity
• Short Half Life
• Generation of new ROS by Chain Reaction
• Damage to various tissues

7. Generation of free Radicals / ROS

8. ENDOGENOUS
• ETC
• Phagocytosis
• Reaction with metal iron/transition metal
• Xanthine oxidase
• Arachidonate pathway
• Lipoxygenase in platelets and WBC
• Peroxisomes
• Exercise
• Inflammation
• Ischemia

9. Exogenous
• Ionizing radiations
• Uvlight
• Cigarette smoke
• Carbon tetrachloride
• Pollutants
• Drugs
• Pesticides

10. Mitochondrial Respiratory Chain
During normal Oxidative Phosphorylation Molecular O₂
is reduced by the addition of 4H⁺+ 4e⁻ to produce
water (2H2O)
NADH
Complex-I
Complex-II
(FADH2)
Q
Complex-III
Cyt-c
Complex-IV
4H+
O2
2H2O
4e-

11. Oxygen Superoxide Anion Radical
Hydrogen Peroxide
Hydroxyl Radical

12. Mitochondrial Respiratory Chain
Leakage of Electrons form the ETC  Partial Reduction
of Oxygen  Generation of ROS
About 3-5% of O₂ intake is converted into these Free
Radicals [i.e.1.5mol of ROS produced] per day.

13. Cytosolic oxidation
In ER, Lysosomes and Peroxisomes O₂ is utilized for
Hydroxylation Reactions in which Superoxide is
generated as an Intermediate Product
Examples:
Hydroxylation of Steroid and Drugs

14. Some Normal metabolism
• In catabolism of purines by Xanthine oxidase (FAD,
Mo, Fe) Hydrogen peroxide is formed
Hypoxanthine Xanthine
Uric acid
H2O +O₂ H2O2
O₂ + H2O
H2O₂

15. By the PMN cells
2O₂ 2O₂˚ H₂O₂
HClO
NADPH Oxidase Superoxide Dismutase
Myeloperoxidase
Cl
NADPH2
HClO is powerful oxidant and highly Microbicidal
(Phygocytosed microorganism are killed)
Absence of enzyme NADPH oxidase causes Chronic
Granulomatous Disease

16. In Nitric oxide (NO) metabolism
NO is free radical having one unpaired electron.
Half life ≈ 0.1 sec.
Synthesized from Arginine by NOS, that requires NADPH
and Mol O₂
On exposure to superoxide, NO is converted to
Peroxynitrite (OONO) which is more toxic.

17. Role of Transition Metals (Cu, Fe)
In Fenton reaction:
H₂O₂ + Fe⁺⁺  OH˚ + OH⁻ + Fe⁺⁺⁺
H₂O₂ is rather less toxic but due to presence of Ferrous
form of Iron it becomes more toxic
Haber Weiss reaction:
O₂ + H₂O Fe/Cu  O₂ + OH˚ + OH⁻

18. Exogenous
Ionizing Radiation  can damage tissues by the
production of Hydroxyl, Hydrogen Peroxide and
Superoxide Anion radicals
UV-Rays, Gamma Rays  Can hydrolyze Water (H2O)
into Hydroxyl (OH
.) Free Radicals.
Light of appropriate Wavelength  Can cause
Photolysis of Oxygen to produce Singlet Oxygen

19. Cigarette Smoking; Chemical Pollutants
Cigarette smoke contains high concentrations
Superoxide
Hydroxyl
Nitric Oxide,
Inhalation of Air Pollutants increases the production of
Free Radicals such as Hydroxyl Radical

20. Effects of Free Radicals

21. Lipid peroxidation
1. Initiation Phase
• RH + OH' ––––––– R' + H2O (Reaction 1-A)
metal ion
• ROOH ––––––– ROO' + H+ (Reaction 1-B)

22. 2. Propagation Phase
• R' + O2 → ROO' (Reaction No.2)
• ROO' + RH → ROOH + R' (Reaction No.3)

23. 3. Termination Phase
• ROO' + ROO' → RO--OR + O2 (Reaction 4-A)
• R' + R' → R--R (Reaction 4-B)
• ROO' + R' → RO--OR (Reaction 4-C)

24. Clinical Significance
1. Chronic Inflammation
2. Acute Inflammation
3. Respiratory Diseases
4. Diseases of the Eye
5. Reperfusion Injury:
6. Atherosclerosis
7. Carcinogenesis:
8. Aging related Diseases:

25. 1. Chronic Inflammation: ROS induced tissue
damage Involved in the pathogenesis of,
Rheumatoid Arthritis
Chronic Ulcerative Colitis
Chronic Glomerulonephritis, etc
2. Acute Inflammation: Respiratory Burst and
increased activity of NADPH Oxidase, as seen in PMN

26. 3. Respiratory Diseases:
Breathing of 100% Oxygen for long Release of free
radicals by activated Neutrophils endothelial damage
and Pulmonary Edema
Bronchopulmonary Dysplasia (In premature Newborn
Infants): Due to prolonged exposure to high oxygen
concentration

27. • ARDS
Recruitment of Neutrophils to the lungs Subsequent
release of free radicals
• Cigarette Smoke Contains Free Radicals; Soot
Attracts Neutrophils More free radicals released
Lung damage

28. 4. Diseases of the Eye:
Retrolental Fibroplasia: Seen in Premature Infants
treated with pure Oxygen for a long time
Cataract formation (Related with aging): Partly due to
Photochemical generation of Free Radicals

29. 5. Reperfusion Injury:
After MI Caused by Free Radicals.
Ischemia increased activity of Xanthine
Oxidase;
When Myocardium is reperfused Availability
of O2 and subsequent formation of free
radicals Injury

30. 6. Atherosclerosis:
LDL deposited under the endothelial cells
Undergo Oxidation by Free Radicals
Chemoattraction of Macrophages Engulfment
of Oxidized LDL Formation of Foam Cells
Initiation of Atherosclerotic Plaque Formation

31. 7. Carcinogenesis:
Free Radicals DNA damage; Accumulated damages
Somatic Mutation and Malignancy
Radiotherapy of Cancer: Irradiation Production of
ROS in the Cells death of Cancer Cells
8. Aging related Diseases:
ROS role in the Degenerative Brain Disorders such as:
Parkinsonism, Alzheimer’s Disease, Multiple Sclerosis

32. Antioxidants
Compounds that dispose of the Reactive Oxygen
Species by
– Scavenging them,
– Suppressing their formation or
– Opposing their actions

33. Free Radical Scavengers
 Superoxide Dismutase
 Catalase
 Glutathione Peroxidase
 Glutathione Reductase
 Ferricytochrome
 Endogenous Ceruloplasmin

34. Superoxide Dismutase (SOD):
• Present in both Cytosol and Mitochondria
– Mitochondrial SOD  Manganese (Mn) dependent
– Cytosolic SOD  Copper-zinc (Cu-Zn) dependent
Reaction Catalyzed:
2(O2
-)o + 2 (H+)  H2O2 + O2

35. Glutathione Peroxidase and Glutathione reductase:

36. Catalase:
Liver peroxisomes
When H2O2 is generated in large amounts  catalase
destroys it to O2
Reaction catalyzed:
H2O2  2H2O + O2

37. Ferricytochrome:
Helps in the oxidation of Superoxide Anions
O2
- + Fe3+ (Cyt c)  O2 + Fe2+ (Cyt c)
Endogenous Ceruloplasmin:
Acts as ‘Feroxidase’  Can convert Fe2+ to Fe3+  can
halt the Haber Weiss reaction  Prevents further
formation of highly reactive hydroxyl free radicals
O₂ + H₂O Fe/Cu  O₂ + OH˚ + OH⁻

38. Antioxidants-Classification
In relation to Lipid Peroxidation:
• Preventive Antioxidants: inhibit the initial
production of free radicals. Eg: catalase, glutathione
peroxidase, and ethylene diamine tetra-acetate
(EDTA)
• Chain Breaking Antioxidants: inhibit propagative
phase. Eg: superoxide dismutase, uric acid and
vitamin E.

39. On the basis of Site of Action:
• Cell membrane: Vitamin E, β-Carotene
• Cytosol: Vitamin C, Enzymes
• Plasma: Uric Acid, Bilirubin, Ceruloplasmin,
Albumin, Transferrin

40. On the basis of Nature:
• Artificial: Propyl Gallate, Butylated Hydroxy Anisole
(BHA), Butylated Hydroxy Toluene (BHT)
• Natural:
• Lipid Soluble: Vitamin E (Tocopherol), β-Carotene
(An Antioxidant at low pO2), Lycopene
• Water Soluble: Vitamin C (Ascorbic Acid), Urates

41. Vitamin E: As an Antioxidant
The major Lipid-soluble Antioxidant in Cell
Membranes and Plasma Lipoproteins
Chain-breaking and Free-radical trapping Antioxidant
Reacts with the Lipid Peroxide Radicals (formed by
Peroxidation of Polyunsaturated Fatty Acids)

43. Vitamin C: As an Antioxidant
Ascorbate + O2
•−  H2O2 + Monodehydroascorbate
Ascorbate + OH• → H2O + Monodehydroascorbate
Catalase and Peroxidases catalyze the reaction:
2H2O2  2H2O + O2

44. Antioxidants as Pro-oxidants
Pro-oxidants: Compounds capable of generating Free
Radicals or ROS (particularly)
1. Vitamin C
2. Beta-Carotene (β-carotene):
3. Vitamin E

45. 1. Vitamin C: Can also be a source of Superoxide Radicals by
reaction with Oxygen, and Hydroxyl Radicals by reaction
with Cu2+ ions
Ascorbate + O2  O2
•− + Monodehydroascorbate
Ascorbate + Cu2+ Cu+ + Monodehydroascorbate
Cu+ + H2O2  Cu2+ + OH- + OH•

46. 2. Beta-Carotene (β-carotene):
A Radical-trapping Antioxidant under conditions of
Low Partial Pressure of Oxygen
In Tissues like lungs (with high pO2 and especially in
high concentrations)  Autocatalytic Pro-oxidant
Can initiate damage to Lipids and Proteins

47. 3. Vitamin E
Chain breaking antioxidants may have pro oxidant
role.
Some research has shown high dose vitamin E
supplement may have pro oxidant role.

48. Thank You