Free radicals and

antioxidants in health and disease

• Electrons in an atom or molecule occupy orbits

• The outer most orbital contains two electrons, each

spinning in opposite directions

• The chemical covalent bond consists of a pair of electrons,

each component of the bond donating one electron each

• A free radical is a molecule or molecular fragment that

contains one or more unpaired electrons in its outer orbital

• Free radical is conventionally represented by a superscript

dot (R●)

Introduction

• Aerobic organisms produce a number of reactive free radicals continuously in cells during respiration, metabolism and phagocytosis.

• Out of these, the most important source of free radicals being the respiratory chain where ~ 1 to 2% oxygen is converted into superoxide radicals (O2•−).

• Oxidation reactions ensure that molecular oxygen is completely reduced to water

• Products of partial reduction of oxygen are highly reactive and make havoc in the living systems

• Hence they are also called Reactive oxygen species (ROS)

Introduction

Members of ROS group

• Superoxide anion radical (O2-)

• Hydroperoxyl radical (HOO●)

• Hydrogen peroxide (H2O2)

• Hydroxyl radical (OH●)

• Lipid peroxide radical (ROO●)

• Singlet oxygen (1O2)

• Nitric oxide (NO●)

• Peroxy nitrite (ONOO--●)

Univalent reduction steps of oxygen

Important characteristics

• Extreme reactivity

• Short life span

• Generation of new ROS by chain reaction

• Damage to various tissues

Generation of free radicals

• Constantly produced during the normal oxidation of foodstuffs,

due to leaks in the electron transport chain in mitochondria

• Some enzymes such as xanthine oxidase and aldehyde oxidase

form superoxide anion radical or hydrogen peroxide

• NADPH oxidase in the inflammatory cells produces superoxide

anion by a process of respiratory burst during phagocytosis

• Macrophages also produce NO from arginine by the enzyme

nitric oxide synthase

• Peroxidation is also catalysed by lipo-oxygenase in platelets and

leukocytes

Generation of free radicals

• Ionising radiation damages tissues by producing hydroxyl radicals, hydrogen peroxide and superoxide anion

• Light of appropriate wavelengths can cause photolysis of oxygen to produce singlet oxygen

• In the presence of free iron, H2O2 can generate OH● which is

highly reactive

• Cigarette smoke contains high concentrations of various free radicals

• Toxic compounds such as carbon tetrachloride, drugs and inhalation of air pollutants will increase the production of free radicals

Respiratory burst

• NADPH oxidase in the inflammatory cells produces superoxide anion

• The superoxide is converted to hydrogen peroxide and then to hypochlorous acid (HClO) with the help of superoxide dismutase (SOD) and myeloperoxidase (MPO)

• The superoxide and hypochlorous ions are the final effectors of bactericidal action

• This is a deliberate production of free radicals by the body

• About 10% of the oxygen uptake by macrophage is used for free radical generation

• Along with the activation of macrophages, the consumption of oxygen is increased drastically; this is called respiratory burst

Damage produced by ROS

• Free radicals are extremely reactive

• Their mean effective radius of action is only 30Å

• Their half life is only a few milliseconds

• When a free radical reacts with a normal compound, other free radicals are generated

• Peroxidation of PUFA in plasma membrane leads to loss of membrane functions

• Lipid peroxidation and consequent degradation products such as malondialdehyde are seen in biological fluids

• Their estimation in serum is often employed to assess the oxidative stress

Damage produced by ROS

• Almost all biological macromolecules are damaged by the

free radicals

• Oxidation of sulfhydryl containing enzymes, modification

of amino acids, loss of function and fragmentation of

proteins are noticed

• Polysaccharides undergo degradation

• DNA is damaged by strand breaks

• The DNA damage may directly cause inhibition of protein

and enzyme synthesis and indirectly cause cell death or

mutation and carcinogenesis

Clinical significance

1. Chronic inflammation

• Chronic inflammatory diseases such as rheumatoid

arthritis is self-perpetuated by the free radicals released

by neutrophills

• Both corticosteroids and non-steroid anti-inflammatory

drugs interfere with formation of free radicals and

interrupt the disease process

• ROS induced tissue damage appears to be involved in

pathogenesis of chronic ulcerative colitis, chronic

glomerulonephritis etc.

Clinical significance

2. Acute inflammation

• At the inflammatory site, activated macrophages produce

free radicals

• Respiratory burst and increased activity of NADPH

oxidase are seen in macrophages and neutrophils

Clinical significance

3. Respiratory diseases

• Breathing of 100% oxygen for more than 24 hrs produces destruction of endothelium and lung edema

• This is due to release of free radicals by activated neutrophils

• In premature newborn infants, prolonged exposure to high oxygen concentration is responsible for broncho-pulmonary dysplasia

• ARDS is produced when neutrophils are recruited to lungs which subsequently release free radicals

• Cigarette smoke contains free radicals. Soot attracts neutrophils to the site which releases free radicals

Clinical significance

4. Diseases of the eye

• Retrolental fibroplasia or retinopathy of prematurity is a condition seen in premature infants treated with pure oxygen for a long time

• It is caused by free radicals, causing thromboxane release, sustained vascular contracture and cellular injury

• Cataract is partly due to photochemical generation of free radicals

• Tissues of the eye, including the lens, has high concentration of free radical scavenging enzymes

Clinical significance

5. Reperfusion injury

• Reperfusion injury after myocardial ischemia is caused by free

radicals

• During ischemia, the concentration of xanthine oxidase is

increased and when reperfused this causes conversion of

hypoxanthine to xanthine and superoxide anion

• At the same time, the availability of scavenging enzymes is

decreased, leading to aggravation of myocardial injury

• Allopurinol, a xanthine oxidase inhibitor, reduces the severity

of reperfusion injury

Clinical significance

6. Shock related injury

• Release of free radicals from phagocytes damage membranes

by lipid peroxidation

• They release leucotrienes from platelets and proteases from

macrophages

• All these factors cause increased vascular permeability,

resulting in tissue edema

• Antioxidants have a protective effect

Clinical significance

7. Atherosclerosis and myocardial infarction

• Low density lipoproteins (LDL) promote atherosclerosis

• They are deposited under the endothelial cell, which undergo

oxidation by free radicals released from endothelial cells

• This attracts macrophages which are then converted to foam

cells

• This initiates the atherosclerotic plaque formation

• Alpha tocopherol offers some protective effect

Clinical significance

8. Peptic ulcer

• Peptic ulcer is produced by erosion of gastric mucosa by

HCl

• It is shown that superoxide anions are involved in the

formation of ulcer

• Helicobacter pylori infection perpetuates the disease

• This infection potentiates the macrophage oxidative burst

leading to tissue destruction

Clinical significance

9. Skin diseases

• Due to inborn defects, porphyrins accumulate in the skin

• Exposure to sunlight will lead to erythema and eruptions in the

patients

• Sunlight acting on porphyrins produces singlet oxygen, which

trigger inflammatory reaction, leading to the above symptoms

• Certain plant products, called psoralens are administered in the

treatment of psoriasis and leukoderma

Clinical significance

10. Cancer treatment

• Free radicals contribute to cancer development because of their mutagenic property

• Free radicals produce DNA damage, and accumulated damages lead to somatic mutation and malignancy

• Cancer is treated by radiotherapy

• Irradiation produces reactive oxygen species in the cells which trigger the cell death

• To increase the therapeutic effect of radiation, radio-sensitisers are administered, which increase the production of ROS

Lipid peroxidation

• Polyunsaturated fatty acids (PUFA) are normal constituents of

cellular and subcellular membranes

• The integrity of the cell membranes is affected by peroxidation

of PUFA

• Damage to cell membrane will cause increased permeability to

sodium ions, rapid influx of calcium, osmotic entrance of water

into the cell, leading to cell damage

Lipid peroxidation

• The auto-oxidation of membrane lipids proceeds as a chain

reaction and it occurs in 3 steps

– Initiation phase

– Propagation phase

– Termination phase

Initiation phase

• During this phase, the primary event is the production of R●

(PUFA radical) or ROO● (lipid peroxide radical) by the

interaction of a PUFA with free radicals generated by other

means

RH + OH● R● + H2O

ROOH ROO● + H+

• The R● and ROO●, in turn, are degraded to malondialdehyde

which is estimated as an indicator of fatty acid breakdown by

free radicals

Propagation phase• The R● rapidly reacts with molecular oxygen forming ROO●

which can attack another polyunsaturated lipid molecule

R● + O2 ROO●

ROO● + RH ROOH + R●

• The net result of two reactions is conversion of R● to ROOH

• But there is simultaneous conversion of a R● to ROO●. This would lead to continuous production of hydroperoxide with consumption of equimolecular quantities of PUFA

• The progression of this chain of events will destroy PUFA present in the membrane lipids

• Accumulation of such lipid damages lead to the destruction of fine architecture and integrity of the membranes

Termination phase

• The reaction would proceed unchecked till a peroxyl

radical reacts with another peroxy radical to form inactive

products

ROO● + ROO● RO-OR + O2

R● + R● R-R

ROO● + R● RO-OR

Role of anti-oxidants

• The damage produced by ROS may be prevented by

antioxidants

• There are two types of antioxidants

a) Preventive antioxidants – will inhibit the initial production

of free radicals. They are catalase, glutathione peroxidase,

EDTA (ethylene diamine tetra acetate)

b) Chain breaking antioxidants – once the peroxyl radicals are

generated, the chain breaking antioxidants can inhibit the

propagation phase. They include superoxide dismutase, uric

acid and vitamin E

Superoxide dismutase

• SOD is a non-heme protein

• The gene coding SOD is on chromosome 21

• Different iso-enzymes of SOD are described.

• The mitochondrial enzyme is manganese dependent;

cytoplasmic enzyme is copper-zinc dependent

• A defect in SOD gene is seen in some patients with

amylotrophic lateral sclerosis

Glutathione peroxidase

• The H2O2 generated is

removed by glutathione

peroxidase (POD)

• It is a selenium containing

enzyme

Glutathione reductase

• The oxidised glutathione is reduced by glutathione

reductase (GR) in presence of NADPH

• This NADPH is generated with the help of glucose-6-

phosphate dehydrogenase (G6PD) in HMP shunt pathway

• Therefore in G6PD deficiency the RBCs are liable to lysis,

especially when oxidising agents are administered (drug

induced hemolytic anemia)

Catalase

• When H2O2 is generated in

large quantities, the

enzyme catalase is also

used for its removal

Alpha-tocopherol

• Alpha tocopherol (T-OH) would intercept the peroxyl free

radical and inactivate it before a PUFA can be attacked

T-OH + ROO● TO● + ROOH

• The phenolic hydrogen of the alpha tocopherol reacts with the

peroxyl radical, converting it to a hydroperoxide product

• The tocopherol radical thus formed is stable and will not

propagate the cycle any further

• The tocopherol radical can react with another peroxyl radical

getting converted to inactive products

TO● + ROO● inactive products

Alpha tocopherol

• Vitamin E acts as the most effective naturally occurring chain

breaking antioxidant in tissues

• Only traces of tocopherol is required to protect considerable

amounts of polyunsaturated fat

• But in this process vitamin E is consumed

• Hence it has to be replenished to continue its activity

• This is achieved by daily dietary supply

Vitamin C

• Vitamin C, or ascorbic acid, is a water-soluble vitamin.

• This vitamin is a free radical scavenger, it is considered

to be one of the most important antioxidants in extra

cellular fluids.

• Its protective effects extend to cancer, coronary artery

disease, arthritis and aging.

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