Moderator: Dr.Shantha

Dept. of Biochemistry

Presenter: Dr.Hariharan

(proteins, lipids, carbohydrates and nucleic acids)

High energy

Fe

Hb

Other cells

Cyt P 450

superoxideMet Hb

H2O2

NADPH oxidase

SOD

Neutro phils

Glu perRBC

Habe

r-wei

ss

catalaseH2O

OH. H2O

xanthine oxidase

bacteria

5

Physiological Stimuli that Form Free radicals

• Normal respiration – O. , H2O2, HOCL, NO

• Transition metals present inside our body when are in free form behave as free radicals. Fe2+, Cu+

• Body cells-– Endothelium (NO,NO2) – Macrophages (NO2)

• Ageing• Phagocytosis or biogenetics • Oxidation of foods and endogenous compounds. • Transportation of substances for energy production.• Breakdown of drugs by body-CCl3

6

Pathological Stimuli that Form Free radicals

Radiation, Ultraviolet rays, X-raysBreaks the water inside our body: H2O =H+ + OH-

Emotional stress Sunlight Stress Toxins Pollution Exercise Food additives Environmental Chemicals Synthetic materials Household cleaners Solvents Pesticides,Herbicides

SmokingSmoking 10 Quad Trillion free radicals 10 Quad Trillion free radicals

per cigarette!per cigarette!

Free radicalsSuperoxide, O2

· -

Hydroxyl radical, OH ·

Peroxyl, ROO ·Alkoxyl, RO ·

Hydroperoxyl, HO2 ·

Nitric oxide, NO .

Nitrous oxide, NO2 .

Free radicals - physiological function

• NADPH-oxidase of leukocytes against bacteria (ROS/RNS)

• Signal molecules-first messenger second messenger information net-This info net function is affected by the redox state of cells

9

Actions of Free radicals

• Lipid peroxidation.• Alteration of the enzyme and

receptor proteins. • Fracturing on the cell nucleus

resulting in single strand DNA damage.• Damages LDL

Lipid Peroxidation

• Initiation

ROOH + metal (n)+ ROO. + metal(n-1) + H+

X . +RH R . +XH• Propagation

R . + O2 ROOH + R.

• Termination

ROO. + ROO. ROOR + O2

ROO. + R . ROOR

R . + R . RR

R.

ROO.

ROOH

Fe2+

O2

Free radical

Fe3+

RO.

OROO.

O2

12

• This chain reaction may produce diseases like: –Carcinogenesis.–Myocardial reperfusion injury.– Shock related injury.–Arteriosclerosis.–Rheumatoid arthritis.–Adult respiratory diseases.–Diabetes.–Obesity.– Lipid abnormality. Etc.

ANTI-OX

PRO-OX

SODGSH

Vit C, ECarotenoidO-

2OH NOH2O2

An imbalance between the prooxidants and the antioxidants of the organism, in favour of the former lead to disease

Ideal Antioxidants

SafetyAntioxidant effectivenessOff-odor, Off-colorStability to pH and food processingAvailability, CostNon-adsorbableEffective in low concentrationFat-solubleMechanism of Antioxidants•Hydrogen donation •Formation of a complex between the lipid radical and the antioxidant radical (free radical acceptor).

Antioxidants-enzymatic

• Glutathione peroxidase(for hydroperoxides) H2O2 + 2GSH GSSG + 2H2O

• Catalase2H2O2 H2O + O2

• Superoxide dismutase (SOD) for superoxide O2

- + O2- + 2H+ H2O2 + O2

Antioxidants-non enzymatic- fixed in membranes ( -tocopherol, -carotene, coenzyme Q 10)

- out of membranes (ascorbate, transferrin, bilirubin, dihydrolipoic acid (DHLA), Flavonoids)

• carotenoids, -carotene, vitamin A-removing the radicals from lipids

• glutathione (GSH, GSSG)-in all mammalian cells (1-10 mmol/l)-important redox buffer

• 2 GSH GSSG + 2e- + 2H+

ROS elimination, stabilisation in reduction form ( SH- groups, tocopheryl and ascorbate regeneration)-substrate of glutathione peroxidases

• Lipoic acid (lipoate)-tocopheryl and ascorbate regeneration• melatonin-lipophilic ; hydroxyl radicals scavenger• Selenium:influence to vitamin E resorption, part of selenoproteins• Zinc-cell membrane stabilisation, Fe antagonist• thioredoxin

selenium

Zinc copper

Superoxide dismutaseUrateVitamin EVitamin CQuercetin

Anthocyanin

R.

ROO.

ROOH

Fe2+

O2

Free radical

Fe3+

RO.

OROO.

O2

Donates H+

CatalaseGlutathione peroxidaseSinglet oxygen quencher-Transition metal chelators (Phosphoric acid, Citric acid, Ascorbic acid, Ethylene-Diamine-Tetra-Acetate (EDTA), haptoglobin, hemopexin, albuminDiethylene diamine tetra acetate(DTPA)

Transferin for Fe, Ceruloplasmin for Cu

Vitamin C (ascorbic acid)• Ascorbic acid can easily give up two hydrogen

with their electrons, thereby becoming dehydroascorbic acid.

• Molecules with unpaired electrons (free radicals) combine vitamin C to form . Gives up electrons very easily when they are needed-recycle themselves over and over again.

• Helps to reactivate Vitamin E• Protects oxygen and iron from oxidation.

AscAscH

O

OH

OHO

O

OH

R+ RH+

O

O

HO

O

OH

O

Glutathione/Selenium Redox Cycleto Regenerate Vitamin C

Reduced Glutathione

(GSSG)Oxidized Glutathione

Oxidized Vitamin C(Ascorbate)

Vitamin C(Ascorbic Acid)

Selenium

Thioredoxin ReductaseGlutathione Reductase

Vitamin E- Membrane bound

alpha tocopherol radical

alpha-tocopherol + free radical

Membrane bound

alpha-tocopherol

H+

Vit C

Flavanoids as “Super Antioxidants.” • antiviral, antiallergic, anti-inflammatory,

antithrombogenic and anticarcinogenic effects.• Scavenge for free radicals associated with oxygen and

iron; or by inhibiting oxidative enzymes.• Over 4000 flavanoids have been found, fall in four

different groups: flavones, flavanones, catechins, and anthocyanins.

• Found in: certain fruits, flowers, roots, stems, tea, wine, grains and vegetables.

Phyto chemicals

Food Phytochemical(s)

Allium vegetables(garlic, onions, chives, leeks) Allyl sulfides

Cruciferous vegetables(broccoli, cauliflower,cabbage, Brussels sprouts,kale, turnips, bok choy,kohlrabi)

Indoles/glucosinolates-Sulfaforaphane, Isothiocyanates/thiocyanates,Thiols

Solanaceous vegetables(tomatoes, peppers) Lycopene

Umbelliferous vegetables(carrots, celery, cilantro,parsley, parsnips)

Carotenoids-PhthalidesPolyacetylenes

Compositae plants (artichoke) Silymarin

Citrus fruits(oranges, lemons, grapefruit)Glucarates

Monoterpenes (limonene)Carotenoids

Other fruits (grapes, berries,cherries, apples, cantaloupe,watermelon, pomegranate)

Ellagic acid-Phenols Flavonoids (quercetin)

Beans, grains, seeds-(soybeans, oats, barley, brownrice, whole wheat, flax seed)Protease inhibitors

Flavonoids (isoflavones) Phytic acid, Saponins

Herbs, spices (ginger, mint,rosemary, thyme, oregano,sage, basil, tumeric, caraway,fennel)

Gingerols –Flavonoids,Monoterpenes (limonene)

Licorice root-Green tea,Polyphenols Glycyrrhizin Catechins

•Tomato-against cancer by protecting DNA from oxidative damage

•Broccoli sprouts -phytochemical –sulforaphane

•Garlic -allicin

•Apples-flavonoids

•Soybeans-flavonoids

•resveratrol

• Citrus fruits(vit C) Don’t throw it. The wine bottle is full of antioxidants

• 20 times stronger antioxidant then Vitamin C and 50 times stronger then Vitamin E.

• Found in: Grape seed, Red Wine, Pine Bark (Pycnogenol), grape skin, peanut skin, and some white wines and fruits.

Oligomeric Proanthocyanidins(Bioflavanoids)

“You’re not getting older, just your DNA”

Aging

Lung Cancer

Prostate Cancer

Macular Degeneration & Cataracts

Osteoarthritis

Rheumatoid Arthritis

Skin Cancer & Melanoma

Photo-aging (wrinkles)

Stroke, Alzheimer’sParkinson’s

PeriodontitisEmphysema Coronary Heart Disease

Alcohol &Viral Liver Diseases

Diabetes

Hypertension &Chronic Kidney Disease

Colo-rectal CancerPeripheral Vascular Disease

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Role of Antioxidants in Carcinogenesis

Reactive O2+

O2-Derived Free Radicals

Oxidized basesin DNA

Normal cell

Lipid peroxidationin cell membranes

Cell repairExposure of DNACell DeathLipid Peroxides

DNA Repair

Normal Cell

Mutations Cell DeathNo Repair

CompensatoryCellularHypoproliferation

Less regulation of cellgrowth & differentiation

Cell Hyperproliferation

Exposure of DNA

Further Mutations

Antioxidant Scavengers &

Enzyme Systems

Assessment of free radical damage and antioxidant

status• Protein oxidation-fluorescence spectroscopy• Lipid peroxidation-Diene conjugates : early marker of

lipid peroxidation 235 – 255 mm• Thiobarbituric acid - Absorbance measured at 532 nm

or 553 nm• Measurement of enzymes: Superoxide Dismutase,

catalase, Glutathione peroxidase in bloodOXYGEN RADICAL ABSORBANCE CAPACITY Standard assay for measuring antioxidant capacity of

foods and supplements. Foods with high ORAC is said to have high antioxidant

capacity.

Oxidative stress markers

Lipoperoxidation markers:malondialdehyde (MDA), conjugated diens,

isoprostanesOxidative damage to protein markers :protein hydroperoxidesOxidative damage to DNA :modified nucleosidesAntioxidant determination:ascorbate,tocopheryl,glutathion

•Harper’s- Biochemistry

References