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Kuliah Biokimia Geriatri
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Free Radicals and Antioxidants
Dep.of BiochemistryFaculty of Medicine.Hasanuddin Univ.
Rosdiana Natzir.,MD.,Ph.D.,Prof.Sp.Biok.
•SYSTEM GERIATRIC
TOPICS
• THE FREE RADICAL THEORY. • MITOCHONDRIA AND AGING. • THE GLYCATION THEORY. • PROTEINS DAMAGE AND MAINTENANCE IN AGING. • DNA DAMAGE AND DNA REPAIR. • TELOMERES THEORY.
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Introduction There was an era when people were worried about the disease processes and its treatment.
But off late, tremendous efforts are being made to find out the ways of prevention of important diseases.
The important research studies are rightly to be repeated with other populations, but nevertheless give an opportunity to consider the integrated actions of the known reducing (Antioxidant) systems.
Inadequacy of certain internally produced reducing systems has been postulated as being at the core of some disease processes.
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It is believed that life has originated from basic chemicals.
By free radical reaction, largely initialled by ionising radiation from sun.
Paradoxically the same reactions creating life may also be responsible for many diseases, ageing and death.
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Free Radicals
• These are highly reactive chemical entities that have a single unpaired electron in their outer most orbit.
• Under certain conditions can be highly toxic to the cells.
• Generally unstable and try to become stable, either by accepting or donating an electron.
• Therefore if two FRs react, they neutralise each other.• However, if the FRs react with stable molecules, there
is generation of more free radicals.
• This characteristic enables the FRs to participate in auto catalytic chain reactions,
– Molecules with which they react are themselves converted to free radicals to propagate the chain of damages.
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Reactive Oxygen Species (ROS): -
These are free radicals derived initially from oxygen. But as they do not contain unpaired electrons in their outermost orbit, they do not qualify as free radicals and so are referred to separately as ROS.
Eg. - H2O2, HOCL, NO.
Free radicals are formed in side our body by both PHYSIOLOGICAL (Natural) and PATHOLOGICAL stimuli : -
They are two conditions of stimuli to form FRs :
1.Physiological stimuli.2.Pathological stimuli.
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Physiological Stimuli that Form FRs
• Normal respiration –
– O2 – Superoxide, – H2O2 – Hydrogen Peroxide– HOCL – Hypochlorous acid– NO – Nitric Oxide
• Transition metals present inside our body when are in free form behave as free radicals. Fe2+, Cu+
• Body cells-
– Endothelium (NO3 – Nitric Oxide, NO2 – Nitrous Oxide), – Macrophages (NO2) – Neurones (ONOOH – Peroxy nitrite).
• Ageing
• Phagocytosis or biogenetics
• Oxidation of foods and endogenous compounds. • Transportation of substances for energy production.
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Pathological Stimuli that Form FRs
• Radiation Breaks the water inside our body: H2O =H+ + OH-
• Metabolism of drugs CCl3• Transition Metals Cu+, Fe2+• Ultraviolet rays• Emotional stress
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Actions of FRs
Mechanism of Action: • They act on the cell membranes and
membranes of different organelles of cells and cause cell injury and death by oxidative reactions.
• So FRs are also called OXIDANTS.
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Actions of FRs
• FRs cause lipid peroxidation.– The PUFA of cell membrane are more vulnerable
for this injury. By lipid peroxidation FR increases the permeability of cells, leading to calcium influx and altered PH of the cell.
• FRs alter the enzyme and receptor proteins.– FRs cause – cross-linking of proteins and
fragmentation of protein strands, oxidation of amino acids like cysteine, Methionine.
– These alterations in the enzymes and receptors inside the cell lead to abnormal cell behaviour.
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Actions of FRs
• FRs cause fracturing on the cell nucleus resulting in single strand DNA damage.
• This oxidative injury may be.– Lethal – Leading to cell death and ultimately
removed by phagocytosis.– Sub lethal - which may result in
• Increased cell permeability.• Toxicity.• Mutation of cells.
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FRs Induce Chain Reaction
• During the process of oxidant damage resulting in tissue destruction & degeneration, some electrons may escape oxidation and become FRs
• 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.
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Antioxidants (AOs)
• These are substances, which protect us against potentially harmful free radicals derived from oxygen.
• To combat the injurious effects of FRs our body has its own system of ‘in vivo’ Antioxidants’.
• In normal healthy state a balance is maintained between FRs & AOs
• The ‘AO’ activity of serum is measured as - % inhibition of lipid peroxidation in a standardised brain homogenate.
• Moreover we can as well supplement these from outside (in vitro Antioxidants).
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In Vivo Antioxidants: -
I.EnzymesI.Enzymes
Name Acts against Present in
SOD (Superoxide dismutase)
Super oxide cytosol mitochondria.
CATALASE H2 O2 Blood, bone marrow, Mucus membrane Kidney; Liver.
GOP (Glutathion peroxidase)
H2 O2, lipid
peroxidation
membranes of lipids, Haemoglobin and erythrocytes
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In Vivo Antioxidants: -
These are binding proteins. They keep the free ions of plasma in a binding form, so prevent oxidation injury. Eg.-Transferin for Fe, Ceruloplasmin for Cu
II. II. Preventive AOPreventive AOss
III. III. Scavenger AOScavenger AOss
Also called chain breaking enzymes, they break the catalytic chain propagated by FRs.
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In Vivo Antioxidants: - Source
Vitamin A, C & E,
Cystine, Glutathion, Melthionine,
Bioflavines, Se, Zn.
I. In the form of Medicines:
II. Food sources:
Green & yellow vegetables
Herbs: -Turmeric/kunyit, Garlic, Grape, Tea, Berries, Carrot, Spinach, Broccoli,
Red Meat, Kidney, Liver & Lipoic Acid
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Oxidative Stress: -
• Under normal conditions body maintains an equilibrium between its own FR’s and Antioxidants.
• When this equilibrium breaks, a state called oxidation stress arises with in, due to FR formation or AO system.
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Example:
AO Status in Normal Pregnancy: -
• There is increased need for AOs as there is increased production of FRs due to
– Pregnancy being a stressful condition. – Because of the rapidly growing foetus there is increased
cellular activity.
• Thus AO activity during normal pregnancy progressively increases as demonstrated by
Serum tocopherol. Activity of GOP. Serum ceruloplasmin & transferring level.
• However, there is no evidence to suggest the need for administration of ‘AOs’ during normal pregnancy but drugs that lead to ‘FR’ formation must be avoided.
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AOs & Diabetic Embryopathy: -
• Oxidative stress has been suggested to contribute to the increased risk of foetal malformations in poorly controlled diabetics. – There are reports of lipid peroxidation in cell
membranes in diabetic pregnancies – Periods of maternal hyperglycaemia & hypoglycemia may
cause marked changes in the availability of glucose to the foetus.
– Also conc. of lipids, notably the ketone bodies and branched chain amino acids in the maternal circulation contribute to altered nutrition for the embryo
Leading to FR conc. & foetal malformation in embryo.
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AOs & Diabetic Embryopathy: -
• During later part of pregnancy
load of glucose in the mitochondria may accelerate the flow of electrons through respiratory chain including mitochondrial leakage of free radicals.
– This leads to production of FR in embryonic tissues to cause congenital malformations.
• Thus maintenance of normal concentration of metabolites of all nutrient class may be important for prevention of adverse foetal outcome.
• But the question is “ will dietary supplements alone hold the key to the future in diabetic embryopathy”?
• However, maintenance of blood glucose level at euglycemic level is always important for prevention of Diabetic embryopathy .
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AOs & Down Syndrome: -
• Free radicals being the hallmark of aging, are greatly increased with maternal age.
• So FRs play a role in pathogenesis of Down’s syndrome.
• Administration of AOs may help in preventing this disease.
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FRs & AOs in PIH : -
• Increased activity of free radicals promote maternal uterine vascular malformations.
– FRs are promoters of maternal vasoconstriction.
• O2 , H2 O2 & NO2 in combinations
– Inactivate the NO (a vasorelaxant) – Causes PG synthatase activity.– Produce peroxynitrate, a potent oxidant
PIH –pituitary inhibiting hormone
• leading to the subsequent development of PIH • Evidence-.
– Lipid peroxide in pre-eclamptic placenta is about 1.8 times higher in comparison to normal placenta.
– Vit. E is less in serum of PIH patients.
– Severity of hypertension has been found to be inversely proportional to concentration of Vit. E.
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FRs & AOs in Preterm Delivery: -
• Sub clinical chorioamnionitis leads to liberation of bacterial antitoxins (polysaccharide in nature) and inflammatory cytokines.
• These cause stimulation of inflammatory cells and cells of CX. which in turn produce NO2.
• Evidence: There is N2O in vaginal samples of females having preterm delivery.
• So antioxidants may play a part in the treatment of preterm delivery.
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FRs & AOs in New Born Babies: -
• In recent years experimental and clinical data have provided compelling evidences for involvement of oxygen derived ‘FRs’ in disorders of prematurity.– Chronic lung disease– Retinopathy– Intra ventricular haemorrhages.– Necrotising Enterocolitis: - FRs cause
microvascular injury and cell permeability leading to necrotising enterocolitis.
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FRs & AOs in New Born Babies: -
• PDA: - Hypoxanthine radical in NBB leads to formation
of PGE2 and causes PDA.• Kwashiorkor: -
– Studies suggest that kwashiorkor babies have FRs in their body .
‘AO’ in Kwashiorkor babies are due to deficiency of Vit. A, E, C, Inositol & Selinium.
• Ideally the future treatment of Neonates with all these disease should include AO therapy.
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Carcinogenesis: -
• Basics of cancer formation: -– A normal cell can undergo malignant
transformation in presence of procarcinogens and carcinogens.
– However, in early stages, it can revert back to normal cell when detected and corrected by our body immune system.
– Without immune system detection a normal cell is converted to malignant cell in stages –
• Initiation Promotion Progression
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FRs in Carcinogenesis: -
• Free radicals are formed from stimulants like – Radiation - Xenobiotics - Inflammatory cells - Respiration etc, which act on cellular targets to cause oxidant
DNA damage in form of mutagenesis & clostogenesis,
- which cause: - initiation of carcinogenesis by-
• Activation of protooncogens.• Inactivation or loss of tumour suppresser genes.• Normal cell becomes initiated cell.
– Procarcinogens are metabolically activated by FRs, which cause promotion and progression of these initiated cells to cancer
– Ultimately carcinogenesis sets in
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FRs & AOs in Carcinogenesis: -Normal cell
Repair by AOsInitiation
Initiated Cell
Premalignant Cell
Malignant Cell
Repair by AOs
Repair by AOs
FRs Activation of procarcinogens & Carcinogens Genetic Damage
Promotion
Progression
FRs
- Tumour promoters
- Spontaneous
FRs
- Dysregulation
- Greater Cell autonomy
- Reduced Growth Factor dependence
CLINICAL CANCERCLINICAL CANCER
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AOs in Cancer Prevention: -
• DEFENCE: - enzymes with antioxidant property cause first line of defence by: -– Protecting the lipids and enzymes against oxidation
Retarding the generation of free radicals Creating a balance of ‘AOs’ against FRs in the body PREVENTS : -
• Cell pathology.• Metabolic disturbances.• Changes in cell permeability.• Eormation of toxic products.
Prevention of initiation of Carcinogenesis
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AOs in Cancer Prevention: -
• INTERVENTION at promotion & progression stages: – Local deactivation of genotoxins responsible for
further nuclear mutations– Inactivation of tumour promoters eg.- activation of
granulocytes. – Simulate oxygen.– Maintenance of proper function of gap junction
communication.– Maintenance of physical stability of membrane &
also within cells.
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AOs in Cancer Prevention: -
• Majority of epidemiological data suggest supplementation with antioxidants
- Vit.- A, E, C; - beta carotene & selenium, decreases the
incidence of various cancers.
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RECOMMENDED FOR CARCINOMA PREVENTION(Federation of Obstetric & Gynaecological Societies of India)
AOs in Cancer Prevention: -
Antioxidant RDA Recommended Dose
Possible Toxicity Level
Features Causing Req.
Vitamin ‘A’ 5000 IU 12,500 IU Chronic intake of 125,000 IU
Smoking
Vitamin ‘E’ 10-20 IU 200-800 IU >1,200 IU High PUFA intake, Smoking
Vitamin ‘C’ 60 mg 1000 mg Negligible / 1-2Gms
Stress, OCP,Smoking
Selenium None 50-200 mg >200mg Aging, High PUFA intake, Smoking, Heavy metals
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FRs & AOs in Infertility - Male
• Various studies have suggested Conc. of Malon –di-Aldehyde (MDA) is inversely proportional to fertility in case of males.
• In asthenospermic and oligoasthenospermic males there is increased serum concentration of MDA.
• Even in normospermic males if there is concentration of MDA there is reduced fertility.
• Addition of vitamin E causes decreased concentration of MDA and improves fertility.
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FRs & AOs in Infertility - Female
• FRs cause -– Short luteal phase and LPDS.– In IVF arrest the cell growth (div) at 2, 4, 8 cell
stages– Hamper the regulation of corpus luteum.
• Addition of ‘AOs’ improves the results.
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CONCLUSION
Oxidant generation is a part of human life.
Every O2 atom we breath in ,is converted to water
inside our body by addition of four electrons
sequentially.
When four electrons are added three ‘FRs’- O2,
H2 O2 & OH are formed along with water.
So where there is life there are oxidants.
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CONCLUSION
But when produced in excess, they can cause
any disease.
So our concern should be to FRs in systemic
circulation.
However, careful use of ‘AOs’ and newer and
more accurate methods to measure oxidant
generation in humans , will go a long way to find
out the exact contribution of oxidants in disease
processes and the role of ‘AOs’ to prevent it.
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THANK YOUTHANK YOU