Role of free radicals in health and disease

Introduction
Free radical is defined as an atom or molecule that contains one or more unpaired electrons. Mitochondria are the major source of intracellular free radicals. Free radicals can be anionic , cationic or neutral. The first organic free radical identified was triphenylmethyl radical, by Moses Gomberg in 1900 at the University of Michigan.

Formation of Free Radical

Free radicals are produced during the homolytic fission of a covalent bond.

Excessive free radical formation can be caused by environmental radiation, cigarette smoking, myocardial reperfusion, infection, hyperglycemia, hypoxia, chemical pollutants and certain drugs.

Reactive Oxygen Species

Molecular oxygen is completely reduced to water. The partial reduction of oxygen are highly reactive and create havoc in the living systems. Hence they are called Reactive oxygen species or ROS. ROS are ions or very small molecules that include oxygen ions, free radicals and peroxides, both inorganic and organic. ROSs form a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling.

 Reactive oxygen species are formed by several different mechanisms. The interaction of ionizing radiation with biological molecules. Byproduct of cellular respiration. Some electrons passing down the electron transport chain leak away from the main path and go directly to reduce oxygen molecules to the superoxide anion Synthesized by an enzymes in phagocytic cells like neutrophils and macrophages NADPH oxidase (in both type of phagocytes) Myeloperoxidase (in neutrophils only)

ROS (reactive oxygen species)

Free radicals Superoxide, O2 Hydroxyl radical, OH Peroxyl, ROO Alkoxyl, RO Hydroperoxyl, HO2 Particals, which are not free radicals Hydrogen peroxide, H2O2 Hypochlorous acid, HClO ozone, O3 Singlet oxygen, 1O2

RNS (reactive nitrogen species)

Particals, which are not free Free radicals radicals Nitrogen(II) oxide, NO . Nitrosyl, NO+ . Nitrogen(IV) oxide, NO2 Nitrous acid, HONO Nitogen(III) oxide, N2O3 peroxynitrite, ONOO Alkylperoxinitrite, ROONO

Main sources of free radicals

Membranes enzymes and/or coenzymes with flavine structures, hem coenzymes, enzymes containing Cu atom in an active site. 1. Respiratory chain mitochondria : Mainly superoxide and then H2O2 Approx 1- 4% O2 enters into respiratory chain (mainly complexes I and III)

2. Endoplasmic reticulum Superoxide creation by cytochrome P- 450 3. Special cells (leukocytes) Superoxide creation by NADP-oxidase 4. Hemoglobin to methemoglobin oxidation Erytrocyte is full of antioxidants

Free Radical Scavenger System

Superoxide Dismutase (SOD) to get rid of superoxide produced from electron transport chain, the product is hydrogen peroxide.
MnSOD (mitochondria). CuZn SOD (cytosol). Non-heme protein

2O2 + 2H

H 2 O 2 + O2

Glutathione
GSH is a tripeptide, -glutamylcysteinyl-glycine The sulfur atom of the cysteine moiety is the reactive site which provides electrons GSH is stable because the bond in glutamyl-cysteine (not the a peptide bond) is resistant to cellular peptidases

Glutathione
GSH is the most abundant non-protein thiol in mammalian cells GSH is a substrate for two enzymes that are responsible for detoxification and antioxidation. Other physiological roles including cysteine storage and transport, prostaglandin metabolism, immune function, cell proliferation and redox balance

Glutathione Synthesis
Protein
Methionine

Cysteine
Glutamate

g-Glutamylcysteine
Glycine

Glutathione Peroxidase (GSH PX) To get rid of hydrogen peroxide (H2O2) and some lipid peroxide. - It requires reduced glutathione (GSH) as substrate and produces oxidized glutathione (GSSG) as product. A cytosolic enzyme.
H2O2 + 2 G-SH G-S-S-G + 2H2O

Glutathione reductase (GSR or GR) Reduces glutathione disulfide (GSSG) to the sulfhydryl form GSH, which is an important cellular antioxidant. GSSG +NADPH +H+ 2GSH +NADP The activity of glutathione reductase is used as indicator for oxidative stress The activity can be monitored by the NADPH consumption In the case of erythrocytes, if the PPP is non-functional, then the oxidative stress in the cell will lead to cell lysis and anemia.

Catalase To get rid of hydrogen peroxide produced in peroxisome. 2 H2O2 2 H2O + O2 Tetramer with Fe, needs NADPH

Oxidative stress
Oxidative stress represents an imbalance between the production and manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Be carefull - this equilibrium can be disbalance in both sides

Oxidative damage to lipids

Damage Unsaturated bonds loss Arising of reactive metabolites (aldehydes) Sequel Changes in fluidity and permeability of membranes Membranes integral enzymes are influenced

Lipid Peroxidation
A free radical prefers to steal electrons from the lipid membrane of a cell, initiating a free radical attack on the cell known as lipid peroxidation. A deficiency in glucose 6-phosphate dehydrogenase produces hemolytic anemia. This enzyme is the only source of NADPH in the red blood cells (they lack mitochondria). With no NADPH, glutathione in the red blood cells cannot be maintained in its reduced form.

Stages of Lipid Peroxidation

Oxidative damage to proteins

Damage
Agregation, fragmentation and cleveage Reaction with hem iron ion Functional group modification

Sequel
Changes in: enzymes activity, ions transport Proteolysis

Oxidative damage to DNA

Damage Saccharide ring cleveage Bases modification Chain breakeage Sequal Mutation Translation mistakes Proteosynthesis inhibition

Oxidative stress markers

Lipoperoxidation markers: Malondialdehyde (MDA), conjugated diens, isoprostanes Oxidative damage to protein markers : Protein hydroperoxides Oxidative damage to DNA : Modified nucleosides

Various Diseases
Lungs Asthma Chronic bronchitis Joints Arthritis Rheumatism Kidney Glomerulonephritis Chronic Renal Failure Fetus Pre-eclampsia IU growth restriction Eyes Cataract Retinal diseases

Brain Alzheimers Parkinsons Memory loss Depression Stroke

Multi Organ Cancer Aging Diabetes Inflammation Infection

Heart-vessels Artherosclerosis Hypertension Ischemia Cardiomyopathy Heart failure

Biochemistry of Aging
Reactive oxygen species play a vitol role in the degenerative brain disorders such as Parkinsonism, Alzheimers dementia and multiple sclerosis The increasing accumulation of lipofuscin in cells, like heart, muscle, nerve, ganglia and nerve cells demarcates aging.

 Brown pigment granules compound of lipid containing residues of lysosomal digestion. It is one of the aging wear and tear pigments. Age- dependent accumulation of lipofuscin in brain cells is one of the most consistent features of aging.

 Centrophenoxine cleans out LF in the cells in the body and brain, improving their function and their longevity. Concentration of carnosine in muscles correlates with maximum life span, a fact that makes it a promising bio-marker of aging. It prevents lipid peroxidation within the cell membrane.

Antioxidants
Is a substance neutralizes free radicals by donating an electron to an unpaired electron of free radical before it causes damage to the cell. Prevents the transfer of electron from O2 to organic molecules. Stabilizes free radicals. Terminates free radical reactions.

High-molecular endogennous antioxidants

Transferrin- iron transport protein Ferritin -iron storage protein Haptoglobin Hemopexin Albumin

Low-molecule endogennous antioxidants

Ascorbate (vitamin C) Alfa-tocopherol a vitamin E Collagen synthesis Localise in membranes Dopamine to epinephrine Produces hydroperoxides, conversion which are changes by GSHPx Reduction agent Fe absorption Antioxidant = reduction O2 - OH , ROO, HO 2 Prooxidant

Low-molecule endogennous antioxidants

Ubiquinone (coenzyme Q) Electron carrier in respisratory chain Co-operates with tocopheryl Carotenoides, -carotene Removing the radicals from lipids

Low-molecule endogennous antioxidants

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

Low-molecule endogennous antioxidats

Lipoic acid (lipoate) PDH cofactor tocopheryl and ascorbate regeneration Melatonin Lipophilic ; hydroxyl radicals scavenger Uric acid (urates) Bilirubin Flavonoids

Trace elements influence to Free Radical metabolism

Selenium Influence to vitamin E resorption, part of selenoproteins of Se = insufficient immune response, erythrocytes hemolysis, methemoglobin synthesis Zinc Cell membrane stabilisation Fe antagonist

Defending Against Cancer

Cancers occur when cellular DNA is damage causing mutations.
Sometimes this damage is caused by free-radical attacks.

Antioxidants may reduce cancer risk by protecting DNA from oxidative damage. Lower cancer rates among people who consume abundant fruits and vegetables rich in antioxidants.

Defending Against Cancer

Diets rich in vitamin C correlated with lower cancer rates, especially cancer of the mouth, larynx, esophagus, and stomach. Such correlations reflect the benefit of consuming a diet rich in fruits and vegetables and low in fat, but it does not necessarily support taking vitamin C supplements to treat or prevent cancer.

Defending Against Cancer

Protective effect of vitamin E against cancer is less consistent than that for vitamin C. However, people with low vitamin E blood levels have higher rates of certain types of cancer. Several studies also report a cancer protective effect of fruits and vegetables rich in betacarotene and the other carotenoids.

Defending Against Heart Disease

High LDL cholesterol is a major risk factor for cardiovascular disease. One way LDL cholesterol increases the risk of cardiovascular disease is that free radicals in the arteries oxidize LDL cholesterol. Oxidized LDL cholesterol accelerates the formation of artery-clogging plaques.

Defending Against Heart Disease

Free radicals also oxidize polyunsaturated fatty acids in cell membranes, initiating additional changes in artery walls which decrease blood flow. Oxidative damage in artery walls is increased by a diet high in saturated fat or cigarette smoke.

Defending Against Heart Disease

On the other hand, diets high in fruits and vegetables, especially in combination with low saturated fat, increase antioxidant action against LDL cholesterol oxidation which can help decreases the development of atherosclerosis.

Defending Against Heart Disease

Antioxidants, especially vitamin E, may protect against the development of cardiovascular disease. Epidemiological studies have reported that people who consume diets rich in vitamin E have lower rates of death from cardiovascular disease.

Defending Against Heart Disease

Some studies also suggest that vitamin C protects against LDL cholesterol oxidation, raises HDL, lowers total cholesterol, and improves blood pressure. Vitamin C may also decrease free radical oxidation in the artery wall that typically follows a high-fat meal.

Carotenoids
Carotenoids are a group of red, orange and yellow pigments found in plant foods, particularly fruits and vegetables. Some carotenoids like bcarotene act as a precursor of vitamin A; others do not.

Rheumatoid Arthrities
Diseases like rheumatoid arthrities is selfpropagated by the free radicals released by the neutrophils. Drugs like corticosteroids and NSAIDs interfere with the formation of free radicals and thus provides relief

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