Free Radicals and Antioxidants The Price for Living – Free Radical Generation and Defense Mechanism. By Dhandapani Ganesan

Ten Leading Causes of Death in the United States (%) 1. Heart diseases (32)* 6. Pneumonia & Influenza (4) 2. Cancers (23)* 7. Diabetes Mellitus (3)* 3. Strokes (7)* 8. AIDS (2) 4. Lung Diseases (5) 9. Suicide (1) 5. Accidents (4) 10. Liver Disease (1) indicates diet-related diseases (65%) >90% of disease incidence involves free radical damage

Questions asked What are free radicals? Types of free radicals Sources of free radicals Oxygen metabolism

What are free radicals? Any molecule containing one or more unpaired electrons. These unpaired electrons readily form free radical molecules which are chemically reactive and highly unstable.

Types of free radicals 1. Superoxide, O Hydrogen peroxide, H 2 O 2 3. Hydroxyl radical, OH - 4. Singlet oxygen, 1 O 2 5. Hydroperoxy radical, HOO - 6. Lipid peroxide radical, ROO - 7. Nitric oxide, NO - 8. peroxynitrite, ONOO -

O2O2 e-e- O2-O2- e -, 2H + H2O2H2O2 e -, H + OH - e -, H + H2OH2O H2OH2O Superoxide Hydrogen peroxide Hydroxyl radical

Properties of free radicals 1. Highly reactive 2. Very short half-life 3. Generate new radicals by chain reaction 4. Cause damage to biomolecules, cells and tissues Most free radicals in biological systems are derivatives of oxygen (Reactive Oxygen Species, ROS), but there are also derivatives of nitrogen (Reactive Nitrogen Species, RNS), Reactive Metabolites or Intermediates.

Reactive Oxygen Species (ROS) Superoxide (O 2. - ) Hydrogen Peroxide (H 2 O 2 ) Hydroxyl Radical (OH. ) Singlet oxygen, 1 O 2 Reactive Oxygen Species is used in a broad sense to collectively free radicals (O 2.-, OH. ) and non-free radicals (H 2 O 2, 1 O 2, which are extremely reactive) of the biological system.

Generation of Free Radical 1.Cellular metabolism About 1-4% of oxygen taken up in the body is converted to free radicals. They are constantly produced during the normal oxidation of foodstuffs. a) due to leaks in the electron transport chain in mitochondria. b) Some enzymes such as xanthine oxidase and aldehyde oxidase form superoxide anion radical or hydrogen peroxide. c) Macrophage also produces NO from arginine by the enzyme nitric oxide synthase. This is also an important anti-bacterial mechanism.

Sources of oxygen free radicals In mitochondria: - generation of energy - ATP - glucose, fatty acids, amino acids - O 2 2H 2 O 4e - +4H + - leakage of O 2 -. (superoxide) H 2 O 2 (hydrogen peroxide)

Generation of Free Radical 2. Environmental effects: a) due to drug metabolism. b) due to damages caused by UV or X-rays c) cigarette or alcohol.

FREE RADICAL FORMATION FREE RADICALS : THE CAUSE OF VIRTUALLY ALL DISEASES Industrial pollution Pesticides & herbicides High fat foods Environmental pollution Excessive Alcohol & smoking

Harmful effects of free radicals A. Free Radical and biomolecules 1.Proteins Cause oxidation of sulfhydryl groups, and modification of AA. ROS may damage protein by fragmentation, aggregation results in the loss of biological activity of proteins. 2. Lipids The polyunsaturated lipid molecules of cell membranes are particularly susceptible to damaging free radicals process and contribute to the uncontrolled chain reaction (lipid peroxidation).

Lipid peroxidation Lipid peroxidation refers to the oxidative degradation of lipids. It is the process whereby free radicals "steal" electrons from the lipids in cell membranes, resulting in cell damage. This process proceeds by a free radical chain reaction mechanism. It most often affects polyunsaturated fatty acids(PUFA). In addition, end products of lipid peroxidation may be mutagenic and carcinogenic

Harmful effects of free radicals A. Free Radical and biomolecules 3. Carbohydrates Glycation increases the susceptibility of proteins to the attack by free radicals. 4. Nucleic acid cause DNA strand breaks, fragmentation of bases and deoxyribose results in cytotoxicity and mutations.

Oxidative Damage Free Radicals Proteins Lipids DNA/RNA (-SH)(R-OO. ) (-OH. )

Harmful effects of free radicals B. Diseases 1. Cardiovascular diseases (CHD): ox-LDL, formed by the action of free radicals, promote CHD and atherosclerosis (AS). 2. Cancers: damage DNA and cause mutation and cytotoxicity, play a key role in carcinogenesis. 3. Inflammatory diseases: damage on the extracellular components such as collagen and hyaluronic acid, promote glomerulonephritis and ulcerative colitis. 4. Respiratory diseases: destroy endothelium and cause lung edema. Cigarette smoke contains free radicals and promotes the production of more free radicals.

Macrophage take up oxidized LDL, when overload with lipid, become “foam cells”. Conglomerate of foam cells form fatty streaks or yellow patches visible in the arterial wall. Dying foam cells release lipid that form lipid pool within the arterial wall.

Harmful effects of free radicals B. Free Radical and diseases 5. Diabetes mellitus: Destruction of islets results in pathogenesis. 6. Cataract 7. Male infertility: reduce sperm motility and viability. 8. Aging process 9. Others: such as Parkingson’s disease, Alzheimer’s disease, multiple sclerosis, liver cirrhosis, muscular dystrophy.

Severity of Oxidative Stress & Biological Consequences Severity of Oxidative Stress Biological Consequences A. Low level & gradual Aging B. Medium level & rapid Carcinogenesis Mutagenesis C. Large level & rapidDeath, Stroke, Trauma, Ionizing irradiation

Bacteria Anti-biotic Free Radicals Anti-oxidants (Oxidants)

Antioxidant The substance present in low concentrations relative to the oxidizable substrate that significantly delays or reduces oxidation of the substrate. They reduce the effect of dangerous oxidants by binding together with these harmful molecules, decreasing their destructive power. They can also help repair damage already sustained by cells. They may be considered as the scavengers of free radicals.

During this reaction the antioxidant sacrifices itself by becoming oxidized. However, antioxidant supply is not unlimited as one antioxidant molecule can only react with a single free radical. Therefore, there is a constant need to replenish antioxidant resources.

Antioxidants Prevents the transfer of electron from O 2 to organic molecules Stabilizes free radicals Terminates free radical reactions

Classification of antioxidant Ⅰ. According to their location a)Plasma antioxidants: –a–ascorbic acid (Vitamin C), bilirubin, uric acid, transferrin, ceruloplasmin, β-carotene; b) Cell membrane antioxidants: –α–α-tocopherol (Vitamin E) c) Intracellular antioxidants: –s–superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx)

Classification of antioxidant Ⅱ. According to their nature and action a)Enzymatic antioxidants: –S–SOD, catalase, GPx, glutathione reductase b) Non-enzymatic antioxidants: –N–Nutrient antioxidants: β-carotene, α-tocopherol, ascorbic acid, –M–Metabolic antioxidants: bilirubin, uric acid, ceruloplasmin, ferritin, transferrin, albumin, glutathione

Enzymatic antioxidant 1. superoxide dismutase (SOD) SOD is present in essentially every cell in the body which actually represented by a group of metalloenzymes with various prosthetic groups. SOD appears in three forms: a) Cu-Zn SOD: in the cytoplasm with two subunits b) Mn-SOD: in the mitochondrion c) Cu-SOD: extracellular SOD 2O 2 · ⁻ + 2H + H 2 O 2 + O 2 SOD This is the first line of defence to protect cells from the injurious effects of superoxide.

Oxygen Radical Defense Enzyme O2¯O2¯H2O2H2O2 H 2 O + O 2 Mn SOD Catalase GSH Peroxidase CuZnSOD OH Fe 2+

Enzymatic antioxidant 2. catalase, CAT 2H 2 O 2 2H 2 O + O 2 catalase Catalase, iron dependent enzyme, is present in all body organs being especially concentrated in the liver and erythrocytes. The brain, heart and skeletal muscle contains only low amounts.

Enzymatic antioxidant 3. glutathione peroxidase, GPx GPx is a selenium-dependent enzyme. The entire process is driven by energy production at the cellular level, which involves proper thyroid hormone levels, healthy mitochondrial function, and an active pentose-phosphate metabolic pathway.

Nutrient antioxidant 1. α-tocopherol (vitamin E) The most important lipid-soluble antioxidant Present in all cellular membranes. Protect against lipid peroxidation.

1.Vitamin E was shown to be stored in adipose tissue. 2.Vitamin E prevents the peroxidation of membrane phospholipids and avoids cell membrane damage through its antioxidant action.

Nutrient antioxidant 2. ascorbic acid (vitamin C) It is a water-soluble, antioxidant present in citrus fruits, potatoes, tomatoes and green leafy vegetables. It is a chain breaking antioxidant as a reducing agent or electron donor. It scavenges free radicals and inhibits lipid peroxidation. It also promotes the regeneration of α- tocopherol. + 2O 2 · ⁻ + 2H + H 2 O 2 + Dehydroascorbate, DHA

Nutrient antioxidant 3. carotenoids Carotenoids consist of C40 chains with conjugated double bonds, they show strong light absorption and often are brightly colored (red, orange). They occur as pigments in bacteria, algae and higher plants.

β-carotene is the most important. It is composed of two molecules of vitamin A (retinol) joined together. Dietary β-carotene is converted to retinol at the level of the intestinal mucosa. It can quench singlet oxygen. – Quenching of singlet oxygen is the basis for it's well known therapeutic efficacy in erythropoietic protoporphyria (a photosensitivity disorder). Lycopene is responsible for color of certain fruits and vegetables like tomato. It also possesses antioxidant property.

Nutrient antioxidant 4. α-lipoic acids It is vitamin-like compound, produced in the body, besides the supply from plant and animal sources. It plays a key role in recycling other important antioxidants such as ascorbic acid, α- tocopherol and glutathione.

Antioxidant Source 1.Coenzyme Q 10 organ meats (best heart), beef, chicken 2. Selenium sea foods, meats, whole grains 3. Proanthocyanidins grape seeds 4. Catechins green tea 5. Quercetin onions, red wine, green tea 6. Ellagic acid berries, walnuts, pomegranates Other important nutrient antioxidants

Metabolic antioxidant 1. glutathione, GSH In addition to its role as a substrate in GSH redox cycle, GSH is also a scavenger of hydroxyl radicals and singlet oxygen. GSH also has an important role in xenobiotic metabolism.

1. Uric acid scavenge singlet oxygen and hydroxy radical 2. Ceruloplasmin inhibit iron and copper dependent lipid peroxidation 3. Transferrin prevents iron-catalyzed radical formation 4. Albumin scavenge radicals on its surface 5. Bilirubin protects albumin bound FFA from peroxidation 6. Haptoglobin bind to free Hb and prevent the acceleration of lipid peroxidation Metabolic antioxidant

Normally, cellular homeostasis is a delicate balance between the production of free radicals and our antioxidant defenses.

Chinese Saying -- 药补不如食补 “ Supplementation with drugs is never as good as supplemention with foods …. ”

GREEN TEA – A WONDERFUL ANTIOXIDANT 200 times more powerful than Vitamin E Scavenges free radicals, high rate Reduce the risk of heart diseases Lowers LDL oxidation Prevents Red blood Cell breakdown Protects against digestive & respiratory infections Prevents cancers of colon, pancreas & stomach Primarily consumed in China, Japan, Middle East, North Africa & North America - Rich in polyphenol – an antioxidant

FOR A HEALTHY TOMORROW… What should you do?

Notice: Countering the Harmful Effects of Free Radicals Don’t smoke – if you do then make it a point to quit Don't overdo your exposure to the sun Don’t over consume alcohol Don’t consume foods containing trans fats or hydrogenated oils Get your cardio exercise from sprinting or interval training Do consume antioxidant rich foods and use a good antioxidant formula Keep your stress levels down

Points Free radicals –Definition, types and Properties –ROS, RNS –Generation of Free Radical –Harmful effects of free radicals Antioxidant –Enzymatic antioxidants SOD, catalase, GPx, glutathione reductase –Non-enzymatic antioxidants Nutrient antioxidants: β-carotene, α-tocopherol, ascorbic acid, Metabolic antioxidants: glutathione (GSH)