Antioxidants in health and disease

1. Role of Antioxidant in Health
and Disease
By-Dr Amit Gupta
PG-2
Deptt of Pharmacology

2. Contents
• Free radicals
• Antioxidant defense system
• Methods of Total antioxidant capacity assessment
• Conclusion

3. Free Radicals
• It is a molecular species having an unpaired electron
and thus is a highly reactive entity (being unstable)
• Free radicals are constantly produced in human
system during metabolism or deliberately during the
process of phagocytosis

4. • Apart from these, free radicals can also be generated
from toxic enviromental pollutants, ionizing
radiations, ozone, heavy metal poisoning, cigarette
smoking and chronic alcohol intake
• Free radicals being highly reactive can oxidise
biomolecules leading to tissue injury and cell death

5. • Now it is proved that free radicals on one hand have
key role in many fundamental cellular reactions and
on the other hand, they are important in the
pathophysiology of common diseases including
atherosclerosis, chronic renal failure, and diabetes
mellitus
• Thus free radicals have dual role

6. • To stabilize itself, a free radical may donate its
unpaired electron or may accept one from other
biomolecule transforming a non-radical to another
free radical to set up disastrous chain reaction
• Thus initiation, propagation, and termination of chain
reaction occurs

8. Types of Free Radicals
1.Endognous
2.Exogenous

10. • Free radicals can be negatively or positively charged
or may be electrically neutral
• H2O2, HOCl are neutral and such agents which are
not free radicals in true sense are called as Reactive
Oxygen Species ( ROS)

11. Endogenous free radicals
The most important free radicals in the body are-
1. singlet oxygen (O2), hydroxyl radical (OH-),
nitric oxide (NO), hypochlorous acid (HOCl),
hydrogen peroxide (H2O2) and the superoxide
radical (O2-)
2. Carbon-centered free radicals

12. 3. Sulfur-centered radical e.g thiyl radical

13. Superoxide (O2-)
• Superoxide (O2-) is produced by the addition of a single
electron to oxygen
• Major source of superoxide is from the electron transfer
chain of the mitochondria
• Also produced during metabolism of drugs by CYP 450 e.g
of paracetamol or alcohol
• Some enzymes also catalyzes superoxide formation e.g
superoxide and hydrogen peroxide are produced during
oxidation of hypoxanthine to xanthine and uric acid

14. Hydrogen peroxide(H2O2)
• Hydrogen peroxide(H2O2) is not a free radical but
falls in the category of reactive oxygen species
• It is a powerful oxidising agent
• It is the main source of hydroxyl (OH-) radicals
• It is also involved in the production of HOCl by
neutrophils.

15. • In biological systems hydrogen peroxide is generated
by the production of superoxide
O2 + O2⁻+ 2H⁺ = H2O2 + O2
• The above reaction is called a dismutation reaction as
the radical reactants produce non- radical products

16. Hydroxyl radical (OH-)
• Hydroxyl radical (OH-) is probably the final mediator
of most free radical induced tissue damage
• The reason for this is that the hydroxyl radical reacts,
with extremely high rate constants, with almost every
type of molecule found in living cells

17. • Hydroxyl radical formation in vivo mainly occur by
transition metal catalysed decomposition of
superoxide and hydrogen peroxide
Fe2+ + H2O2 = Fe3+ + OH + OH−
• This reaction is called as Fenton’s reaction described
in 1894

18. Singlet oxygen
• Singlet oxygen (O2) is an electronically excited and
mutagenic form of oxygen
• It is similar to normal oxygen but it has an extra
electron
• It is generated by input of energy like radiation or
sunlight
• This free radical is involved in joint diseases (like
arthritis) and eye diseases

19. Peroxy-nitrite
• Cytotoxicity of NO is due to formation of
peroxynitrite
• It is produced by the reaction of nitric oxide with
superoxide NO + O2- = ONOO-
• Because of its oxidizing properties, peroxy-nitrite can
damage a wide array of molecules in cells, including
DNA and proteins and results in cell apoptosis

20. Hypochlorous acid
• Activated polymorphonuclear cells produce HOCl as a
major bactericidal agent
• This reaction occurs in the neutrophilic lysosomal
vesicles and helps in the killing of bacteria and viruses
• HOCl may cross cell membrane so it may contribute to
tissue damage during the inflammatory process

21. Promoters of free radical
• Several transition metals have variable oxidation
numbers which accordingly can accept or donate
electrons e.g Fe, Cu
• As a result, these metals serve as excellent promoters
of free radical
Fe3+ + e- = Fe2+
Cu2+ + e- = Cu+

22. EXOGENOUS FREE RADICALS
• Drugs: A number of drugs can increase the production of free
radicals e.g nitrofurantoin, antineoplastic agents as
bleomycin, anthracyclines (adriamycin) and methotrexate
• Radiation
• Tobacco smoking
• Inorganic particles e.g asbestos, silica, quartz
• Gases e.g ozone
• Pesticides, exhaust fumes

23. Role of Free radicals
• Body’s immune system’s cells purposefully create them to
neutralize viruses and bacteria
• In absence of free radicals body’s defense system will become
weak
• Normally, the body can handle free radicals, but if antioxidants
are unavailable, or if the free-radical production becomes
excessive, damage to tissues can occur
• Of particular importance is that free radical damage
accumulates with age.

24. • Free radicals are imlicated in many diseases e.g
autoimmune diseases, RA, carcinogenesis
• CNS- Parkinson’s disease, Alzheimer’s disease,
Huntington’s disease, MS
• CVS- MI, Ischaemic reperfusion injury,
atherosclerosis

25. • Endocrine- DM
• GIT- Peptic ulcer, cirrhosis, pancreatitis
• Renal- Nephrotoxicity due to aminoglycosides and
heavy metals
• RS- Toxicity due to cigarette smoke, asbestos, silia
• Eyes- Cataract, Retinopathy

26. Antioxidant Defense system
• An antioxidant can be defined as: “any substance that,
when present in low concentrations compared to that
of an oxidisable substrate, significantly delays or
inhibits the oxidation of that substrate”.
• They are substances that protect other chemicals of
the body from damaging oxidation reactions by
reacting with free radicals

27. • During this reaction the antioxidant sacrifices itself
by becoming oxidized
• However, antioxidant supply is not unlimited
• Therefore, there is a constant need to replenish
antioxidant resources, whether endogenously or
exogenously

28. • Antioxidant system is divided into three main groups:
1.Antioxidant enzymes
2.Chain breaking antioxidants
3.Transition metal binding proteins

29. Antioxidant Enzymes
Catalase
• First antioxidant enzyme to be characterized
• It catalyses the two stage conversion of hydrogen
peroxide to water and oxygen:
catalase–Fe(III) + H2O2 = compound I
compound I + H2O2 = catalase–Fe(III) +2H2O + O2

30. • Catalase consists of a haem group and a molecule of
NADPH
• Catalase is largely located within cells in peroxisomes,
which also contain most of the enzymes capable of
generating hydrogen peroxide
• Greatest activity is present in liver and erythrocytes

31. Glutathione peroxidase and glutathione reductase
• Glutathione peroxidase catalyze the oxidation of
glutathione at the expense of a hydroperoxide,
ROOH + 2GSH = GSSG + H2O + ROH
• Glutathione peroxidases requires selenium for its
activity
• Predominant subcellular distribution is in the cytosol
and mitochondria

32. • Highest availability is in liver
• Main scavenger of hydrogen
• Activity of the enzyme is dependent on the constant
availability of reduced glutathione. This is made
possible by glutathione reductase
GSSG + NADPH + H+ = 2GSH + NADP+
• NADPH is supplied by pentose phosphate pathway

33. • Any competing pathway that utilises NADPH (such
as the aldose reductase pathway) might lead to a
deficiency of reduced glutathione and hence impair
the action of glutathione peroxidase

34. Superoxide dismutase
• Superoxide dismutase catalyze the dismutation
of superoxide to hydrogen peroxide:
O2− + O2− + 2H+ = H2O2 + O2
• The hydrogen peroxide must then be removed by
catalase or glutathione peroxidase
• There are three forms of superoxide dismutase in
mammalian tissues:

35. (1) Copper zinc superoxide dismutase (CuZnSOD):
• It is found in the cytoplasm of all cells
• It contains catalytically active copper and zinc atom
(2) Manganese superoxide dismutase
(3) Extracellular superoxide dismutase (ECSOD):
• EC-SOD is a secretory copper and zinc containing
SOD distinct from the CuZnSOD described above

36. • EC-SOD is synthesised by fibroblasts and endothelial
cells
• EC-SOD might play a role in the regulation of
vascular tone, because endothelial derived relaxing
factor (nitric oxide or a closely related compound) is
is neutralized in the plasma by superoxide

37. Chain Breaking Antioxidants
• Such antioxidants can be conveniently divided into
lipid phase and aqueous phase antioxidants
1. Lipid phase antioxidants
• These antioxidants scavenge radicals in membranes
and lipoprotein particles and are crucial in preventing
lipid peroxidation
• Most important of these is Vit E

38. • They react rapidly with peroxyl radicals and hence
act to break the chain reaction of lipid peroxidation
• Besides, Vit.E also stabilizes cell membrane so its
deficiency may cause hemolysis and peripheral
neuropathy
• Vitamin E also inhibits the conversion of nitrites in
smoked and pickled foods to nitrosamines in the
stomach
• Nitrosamines are strong tumor promoters

39. Beta Carotene
Fontbonne A, Charles MA, Juhan-Vague I et al. The effect of metformin on the metabolic
abnormalities associated with upper-body fat distribution. BIGPRO Study Group. Diabetes
Care. 1996;19(9):920.
• Carotenoids are pigmented micronutrients present in
fruits and vegetables
• Carotenoids are precursors of vitamin A and also have
antioxidant effects
• Beta-carotene is the most widely studied
• It is composed of two molecules of vitamin A
(retinol) joined together

40. • Dietary beta-carotene is converted to retinol at the
level of the intestinal mucosa.
• Beta-carotene scavenges singlet oxygen, free radicals
and inhibits lipid peroxidation
• Carotenoids also have been reported to have a
number of other biologic actions, including immuno-
enhancement, inhibition of mutagenesis and
regression of premalignant lesions

41. Flavanoids
• Flavonoids are a large group of polyphenolic
antioxidants found in many fruits, vegetables, and
beverages such as tea and wine e.g quercetin
• Epidemiological studies suggest an inverse relation
between flavonoid intake and incidence of chronic
diseases such as coronary heart disease (CHD)

42. Ubiquinol-10
• Ubiquinol-10 (reduced coenzyme Q10) is an effective
chain breaking antioxidant
• Whenever plasma or isolated low density lipoprotein
(LDL) cholesterol is exposed to radicals, ubiquinol-
10 is the first antioxidant to be consumed, suggesting
its important role in preventing the propagation of
lipid peroxidation

43. Aqueous phase chain breaking antioxidants
• These antioxidants will directly scavenge radicals
present in the aqueous compartment
• Most important antioxidant of this type is vitamin C
(ascorbate)
• Its best known role is as a cofactor for prolyl and
lysyl oxidases in the synthesis of collagen
• Ascorbate has been shown to scavenge superoxide,
hydrogen peroxide, the hydroxyl radical

44. Uric acid
• Uric acid efficiently scavenges free radicals
• Urate is important in providing protection against
certain oxidizing agents such as Ozone
• It has been suggested that the increase in life span
occurred during human evolution is partly explained
by the protective action provided by uric acid in
human plasma

45. Albumin
• Albumin, predominant plasma protein has several
sulphydryl groups and a single cysteine residue
• This chemical structure is responsible for the
antioxidant effect of albumin
• Due to this, albumin plays important role in
transporting free fatty acids in the blood
• In addition, albumin has the capacity to bind copper
ions and will inhibit copper dependent lipid
peroxidation and hydroxyl radical formation

46. Interactions between chain breaking
antioxidants
• It is vital to remember that in vivo, complex
interactions between antioxidants occur e.g ascorbate
helps in regenerating alpha-tocopherol and glutahione
helps in regenerating ascorbate
• Therefore, it becomes difficult to predict how
antioxidants will function in vivo and which
antioxidant is more important than other

47. Transition metal binding proteins
• Transition metal binding proteins (ferritin, transferrin,
lactoferrin, and caeruloplasmin) act as a crucial
component of the antioxidant defence system
• By sequestering iron and copper, they inhibit the
formation of the hydroxyl radical

48. Melatonin
• Melatonin is a powerful antioxidant
• Melatonin easily crosses cell membranes and the
blood-brain barrier
• Melatonin, once oxidized, cannot be reduced to its
former state. Therefore, it has been referred as
terminal (or suicidal) antioxidant

49. Agents augmenting endogenous antioxidants
• N-acetylcysteine is a glutathione precursor while
ebselen is a congener of glutathione peroxidase
• Both augments endogenous glutathione peroxidase
activity
• Former is used as antioxiant in treating paracetamol
toxicity

50. Exogenous(Pharmacological antioxidants)
• Several pharmaceutical agents have been found to
exert an antioxidant effect
1. Xanthine oxidase inhibitors: e.g. allopurinol
2. NADPH inhibitors: e.g. adenosine
3. Albumin
4.Inhibitors of iron redox cycling: deferoxamine,
apotransferrin
5. Statins

51. Plant Sources
• Garlic, grape fruit juice, soyabean, turmeric
(cucurminoids), tomato (lycopene) contains
bioflavinoids which possess good antooxidant
properties
• These are claimed to reduce the risk of
atherosclerosis, MI and various cancers

52. • Spirulina is a blue-green algae with excellent
antioxidant properties
• It is a good source of SOD, beta-carotene and B-
complex vitamins

53. Are antioxidants really beneficial?
• Large clinical trials with a limited number of
antioxidants detected no benefit and even suggested
that excess supplementation with certain antioxidants
may be harmful
• Antioxidant supplements have no clear effect on the
risk of chronic diseases such as cancer and heart
disease in the long run

54. • Because 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 will also reduce metal ions that
generate free radicals
• Other example of pro-oxidants are vit E, uric acid

55. Methods of Total Antioxidant Capacity
Assessment

56. Antioxidant
capacity assay
Principle of the method End-product
determination
Spectrometry
DPPH(2,2-diphenyl-1-
Picrylhydrazyl)
Antioxidant reaction
with an organic
radical
Colorimetry
FRAP (ferric reducing
antioxidant power)
Antioxidant reaction
with a Fe(III) complex
Colorimetry
PFRAP(potassium
ferricyanide reducing
power)
Potassium ferricyanide
reduction by
antioxidants and
subsequent reaction
of potassium
ferrocyanide with Fe3+
Colorimetry
Spectrometric Techniques

57. Antioxidant
capacity assay
Principle of the method End-product
determination
CUPRAC(cupric
reducing antioxidant
power)
Cu (II) reduction to Cu
(I) by antioxidants
Colorimetry
TRAP(total peroxyl
radical trapping
antioxidant parameter)
Antioxidant capacity to
scavenge luminol-
derived peroxyl radicals
Chemiluminescence
quenching

60. Conclusion

61. THANK YOU

62. REFERENCES
• HL sharma, KK sharma. Principles of Pharmacology.
2nd ed.2011;p 901
• Katzung BG, Trevor AJ. Basics and clinical
pharmacology. 13th ed.McGraw Hill
education:2015;p664-5
• For various trial details (https://clinicaltrials.gov)