Introduction
- Free radicals are highly reactive chemical species that possess one or more unpaired electrons in their outer orbital.
- The presence of unpaired electrons makes them unstable and highly reactive, enabling them to interact with nearby molecules to gain stability.
- These reactions may damage cellular structures and macromolecules such as lipids, proteins, carbohydrates, and nucleic acids.
- Free radicals are continuously produced in the body during normal metabolic processes and participate in essential physiological functions including immune defense, intracellular signaling, and regulation of vascular tone. However, excessive production or inadequate antioxidant defenses results in oxidative stress, which contributes to cellular injury and numerous pathological conditions.
- The study of free radicals has become increasingly important in biochemistry because they are implicated in aging, diabetes mellitus, cardiovascular diseases, cancer, inflammatory disorders, and neurodegenerative diseases.
Definition of Free Radical
A free radical is defined as:
“An atom, molecule, or ion capable of independent existence that contains one or more unpaired electrons in its outer orbital.”
Characteristics:
- Possess unpaired electrons
- Highly unstable
- Highly reactive
- Short half-life
- Capable of initiating chain reactions
- Can damage biological molecules
Historical
The concept of free radicals in biology was introduced by:
Denham Harman
He proposed the Free Radical Theory of Aging in 1956, suggesting that accumulation of oxidative damage contributes to aging.
Properties of Free Radicals
Physical properties
- Exist independently
- Short-lived
- Extremely reactive
Chemical properties
- Undergo oxidation-reduction reactions
- Extract electrons from nearby molecules
- Initiate chain reactions
Biological properties
- Damage cell membranes
- Modify proteins
- Alter DNA structure
- Influence cellular signaling pathways
Types of Free Radicals
Free radicals may be classified according to their chemical origin.
1. Oxygen-Centered Free Radicals
Reactive oxygen species (ROS) are oxygen-containing reactive molecules.
Radical ROS
Examples: Superoxide anion
O₂•⁻
Formation: O₂ + e⁻ → O₂•⁻
Characteristics:
- Initial ROS formed in cells
- Less reactive than hydroxyl radical
- Generates secondary radicals
Hydroxyl radical
OH•
Hydroxyl radical is considered the most destructive free radical.
Formation: Fe2++H2O2→Fe3++OH−+OH∙
Effects:
- Lipid peroxidation
- DNA damage
- Protein oxidation
Peroxyl radical
ROO•
Role:
- Important in lipid peroxidation
Alkoxyl radical
RO•
Role:
- Damages membranes and proteins
Non-radical ROS
Examples:
- Hydrogen peroxide (H₂O₂)
- Singlet oxygen
- Hypochlorous acid
- Ozone
Although these species lack unpaired electrons, they generate radicals.
2. Nitrogen-Centered Free Radicals
Reactive nitrogen species include: Nitric oxide radical
NO•
Functions:
- Vasodilation
- Neurotransmission
- Immune regulation
Nitrogen dioxide radical
NO₂•
Effects:
- Oxidative tissue injury
Peroxynitrite
Formation: NO∙+O2∙−→ONOO−
Effects:
- Protein nitration
- DNA damage
3. Carbon-Centered Free Radicals
Examples:
- CH₃•
- CCl₃•
Role:
Produced during drug metabolism.
Clinical significance:
Associated with liver toxicity.
4. Sulfur-Centered Free Radicals
Examples:
- Thiyl radical (RS•)
Functions:
- Protein oxidation
- Redox signaling
Sources of Free Radicals
Sources are classified into endogenous and exogenous sources.
A. Endogenous Sources
1. Mitochondrial Electron Transport Chain
Major source of ROS.
Mechanism:
Electron leakage from complexes I and III reduces oxygen.
Reaction: O₂ + e⁻ → O₂•⁻
2. Respiratory Burst in Phagocytes
Activated neutrophils and macrophages generate ROS.
Enzyme: NADPH oxidase
Reaction: NADPH + 2O₂ → NADP⁺ + H⁺ + 2O₂•⁻
Purpose:
- Destruction of microorganisms
3. Xanthine Oxidase System
Converts hypoxanthine to uric acid.
Generates:
- Superoxide radicals
- Hydrogen peroxide
4. Peroxisomal Oxidation
Produces hydrogen peroxide during fatty acid metabolism.
5. Cytochrome P450 System
Produces ROS during detoxification reactions.
B. Exogenous Sources
Physical agents
- UV radiation
- X-rays
- Gamma rays
- Ionizing radiation
Chemical agents
- Cigarette smoke
- Air pollutants
- Heavy metals
- Pesticides
Drugs
- Alcohol
- Chemotherapeutic agents
- Carbon tetrachloride
Biological agents
- Viral infections
- Bacterial infections
Mechanism of Free Radical Formation
Free radicals exert their harmful effects primarily by reacting with cellular macromolecules such as lipids, proteins, nucleic acids, and carbohydrates. Because free radicals are highly unstable, they attempt to achieve stability by capturing electrons from neighboring molecules. This initiates chain reactions that progressively damage cells and tissues.
Major targets of free radical injury include:
- Cell membrane lipids
- Proteins
- DNA and nucleic acids
- Carbohydrates
- Mitochondria
1. Lipid Peroxidation
Lipid peroxidation is considered one of the most important mechanisms of free radical-mediated cellular injury.
Cell membranes contain large amounts of polyunsaturated fatty acids (PUFAs), which are highly susceptible to oxidation by free radicals.
Steps of Lipid Peroxidation
A. Initiation Phase
A free radical (usually hydroxyl radical) removes hydrogen from membrane lipids.
Reaction:
RH + OH• → R• + H₂O
Where:
- RH = Polyunsaturated fatty acid
- R• = Lipid radical
Effect:
Formation of unstable lipid radical.
B. Propagation Phase
The lipid radical reacts with molecular oxygen.
Reaction:
R• + O₂ → ROO•
ROO• + RH → ROOH + R•
Where:
- ROO• = Lipid peroxyl radical
- ROOH = Lipid hydroperoxide
Effect:
- Continuous chain reaction
- Amplification of membrane damage
C. Termination Phase
Two radicals combine to form stable products.
Reaction:
R• + R• → Stable molecule
ROO• + ROO• → Stable product
Effect:
Chain reaction stops.
Consequences of Lipid Peroxidation
- Loss of membrane fluidity
- Increased membrane permeability
- Membrane rupture
- Leakage of intracellular enzymes
- Cell death
Clinical marker:
Malondialdehyde (MDA)
2. Protein Oxidation
Free radicals attack amino acid residues and alter protein structure.
Mechanisms:
A. Oxidation of Amino Acids
Amino acids such as:
- Methionine
- Cysteine
- Tyrosine
- Histidine
undergo oxidation.
Effects:
- Structural alteration
- Reduced biological activity
B. Protein Cross-linking
Free radicals induce abnormal bonds between proteins.
Effects:
- Protein aggregation
- Reduced solubility
C. Enzyme Inactivation
Oxidative modification of active sites causes:
- Loss of catalytic activity
- Reduced metabolic function
Examples:
- Membrane enzymes
- Mitochondrial enzymes
Consequences of Protein Oxidation
- Loss of enzymatic function
- Structural instability
- Altered receptor activity
- Impaired transport systems
3. DNA Damage
DNA is highly vulnerable to oxidative damage.
Major sites affected:
- Nitrogenous bases
- Sugar-phosphate backbone
Mechanisms:
A. Base Modification
Hydroxyl radicals attack DNA bases.
Examples:
- Guanine → 8-hydroxyguanine
Effects:
- Mutation formation
B. DNA Strand Breaks
Free radicals break phosphodiester bonds.
Types:
- Single-strand breaks
- Double-strand breaks
Effects:
- Loss of genetic information
C. Cross-Link Formation
Abnormal linkage may occur between:
- DNA-DNA
- DNA-protein
Effects:
- Impaired replication
- Altered transcription
Consequences of DNA Damage
- Gene mutations
- Defective protein synthesis
- Carcinogenesis
- Apoptosis
- Cell death
4. Carbohydrate Oxidation
Free radicals oxidize carbohydrates and polysaccharides.
Effects:
- Fragmentation of carbohydrate molecules
- Altered membrane glycoproteins
- Disturbed cell signaling
Consequences:
- Cellular dysfunction
- Impaired membrane recognition
5. Mitochondrial Damage
Mitochondria are both producers and targets of free radicals.
Mechanism:
- ROS damage mitochondrial DNA
- Membrane proteins become oxidized
- ATP production decreases
- Cytochrome c is released
Effects:
- Reduced energy production
- Initiation of apoptosis
Consequences:
- Cell injury
- Cell death
6. Calcium Homeostasis Disturbance
Oxidative damage to membranes affects calcium channels.
Effects:
- Increased intracellular calcium concentration
- Activation of destructive enzymes
Examples:
- Phospholipases
- Proteases
- Endonucleases
Consequences:
- Membrane degradation
- DNA fragmentation
- Cell death
Oxidative Stress
- Oxidative stress is an important biochemical concept that refers to a condition in which the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) exceeds the capacity of antioxidant defense mechanisms.
- This imbalance results in damage to cellular structures and contributes to the pathogenesis of many diseases.
Oxidative stress is defined as:
“A condition resulting from an imbalance between oxidant production and antioxidant defense systems in favor of oxidants, leading to potential cellular and tissue damage.”
Relationship:
Reactive oxygen species (ROS) > Antioxidant defense system
↓
Oxidative stress develops
Concept of Oxidative Stress
Normal physiological conditions:
- Small amounts of free radicals are continuously produced
- Antioxidant systems neutralize these radicals
- Cellular homeostasis is maintained
Pathological conditions:
- Excessive ROS generation occurs
- Antioxidant defenses become inadequate
- Oxidative injury develops
Flow diagram:
Normal metabolism
↓
Generation of ROS
↓
Antioxidants neutralize ROS
↓
Normal cellular function
However:
Excess ROS production
↓
Reduced antioxidant capacity
↓
Oxidative stress
↓
Cellular damage
↓
Disease development
Causes of Oxidative Stress
The causes can be classified into endogenous and exogenous factors.
A. Endogenous Causes
1. Mitochondrial Electron Transport Chain
- Major source of ROS
- Electron leakage from respiratory chain generates superoxide radicals
2. Inflammation
Activated inflammatory cells produce:
- Superoxide radicals
- Hydrogen peroxide
- Nitric oxide
Examples:
- Chronic infections
- Autoimmune disorders
3. Ischemia-Reperfusion Injury
During restoration of blood supply:
- Sudden oxygen influx occurs
- Massive ROS production develops
Examples:
- Myocardial infarction
- Stroke
- Organ transplantation
4. Enzyme Systems
Enzymes producing ROS:
- Xanthine oxidase
- NADPH oxidase
- Cytochrome P450
5. Aging
Aging causes:
- Accumulation of oxidative injury
- Reduced antioxidant efficiency
B. Exogenous Causes
Environmental factors
- Air pollution
- Heavy metals
- Industrial chemicals
Radiation
- Ultraviolet rays
- X-rays
- Gamma rays
Lifestyle factors
- Cigarette smoking
- Alcohol consumption
- Stress
- Poor nutrition
Drugs and toxins
- Chemotherapeutic agents
- Carbon tetrachloride
- Certain antibiotics
Infections
- Viral infections
- Bacterial infections
Mechanisms of Oxidative Stress-Induced Cellular Damage
Excessive ROS attack multiple cellular components.
1. Lipid Peroxidation
Free radicals oxidize polyunsaturated fatty acids of membranes.
Effects:
- Membrane instability
- Increased permeability
- Cell lysis
Marker:
Malondialdehyde (MDA)
2. Protein Oxidation
Effects:
- Enzyme inactivation
- Protein denaturation
- Structural abnormalities
3. DNA Damage
Effects:
- Base modification
- DNA strand breaks
- Mutation formation
Consequences:
- Carcinogenesis
- Apoptosis
4. Mitochondrial Dysfunction
Effects:
- Reduced ATP synthesis
- Release of cytochrome c
- Cell death
Biological Effects of Oxidative Stress
Cellular effects
- Membrane injury
- Protein damage
- DNA mutations
- Cellular aging
Tissue effects
- Inflammation
- Fibrosis
- Necrosis
Organ effects
- Cardiac dysfunction
- Neurological disorders
- Renal damage
Antioxidant Defense System Against Oxidative Stress
The body possesses enzymatic and non-enzymatic antioxidant systems.
A. Enzymatic Antioxidants
Superoxide Dismutase (SOD)
Function: Converts superoxide radical into hydrogen peroxide.
2O2∙−+2H+→H2O2+O2
Catalase
Function: Converts hydrogen peroxide into water and oxygen.
2H2O2→2H2O+O2
Glutathione Peroxidase
Function: Reduces hydrogen peroxide using glutathione.
2GSH+H2O2→GSSG+2H2O
B. Non-Enzymatic Antioxidants
Vitamins
- Vitamin C
- Vitamin E
- Vitamin A
- β-carotene
Minerals
- Selenium
- Zinc
- Copper
Endogenous antioxidants
- Reduced glutathione
- Uric acid
- Bilirubin
- Albumin
Biomarkers of Oxidative Stress
Several biochemical markers are used to assess oxidative stress.
Lipid oxidation markers
- Malondialdehyde (MDA)
- Thiobarbituric acid reactive substances (TBARS)
DNA damage markers
- 8-hydroxydeoxyguanosine (8-OHdG)
Antioxidant markers
- Reduced glutathione
- Catalase activity
- Superoxide dismutase activity
Global assessment
- Total antioxidant capacity (TAC)
Diseases Associated with Oxidative Stress
Cardiovascular diseases
Mechanism: Oxidized LDL contributes to plaque formation.
Examples:
- Atherosclerosis
- Hypertension
- Myocardial infarction
Diabetes Mellitus
Effects:
- Endothelial dysfunction
- Diabetic nephropathy
- Retinopathy
Neurodegenerative diseases
Examples:
- Parkinson disease
- Alzheimer disease
- Huntington disease
Cancer
Mechanism:
- DNA mutation
- Activation of oncogenes
- Inactivation of tumor suppressor genes
Pulmonary diseases
Examples:
- Asthma
- COPD
- ARDS
Eye disorders
Examples:
- Cataract
- Macular degeneration
Aging
The free radical theory suggests that cumulative oxidative damage contributes to aging.
Antioxidants
- Antioxidants are substances that protect cells and tissues from oxidative damage by neutralizing free radicals and reactive oxygen species (ROS).
- They inhibit or delay oxidation reactions by donating electrons to unstable molecules without themselves becoming harmful.
- Antioxidants are essential components of the body’s defense system because they maintain the balance between oxidants and antioxidants, thereby preventing oxidative stress and cellular injury.
Antioxidants are defined as:
“Substances that delay, prevent, or remove oxidative damage to target molecules by neutralizing free radicals or inhibiting their formation.”
Concept of Antioxidant Action
Normal cellular metabolism continuously generates free radicals.
Mechanism:
Normal metabolism
↓
Free radical generation
↓
Antioxidants neutralize free radicals
↓
Cellular protection
When antioxidant defense decreases:
Excess free radicals
↓
Oxidative stress
↓
Cell damage
Functions of Antioxidants
Major functions include:
1. Neutralization of free radicals
Antioxidants donate electrons to unstable free radicals.
Result:
- Stable molecules formed
- Chain reactions terminated
2. Prevention of lipid peroxidation
Antioxidants inhibit oxidation of membrane lipids.
Effects:
- Protection of cell membranes
- Maintenance of membrane integrity
3. Protection of proteins
Functions:
- Prevent enzyme inactivation
- Prevent protein denaturation
4. Protection of nucleic acids
Functions:
- Prevent DNA strand breaks
- Reduce mutation formation
5. Maintenance of cellular homeostasis
Functions:
- Regulate redox balance
- Maintain normal physiological function
Classification of Antioxidants
Antioxidants are broadly classified into:
- Enzymatic antioxidants
- Non-enzymatic antioxidants
- Preventive antioxidants
- Chain-breaking antioxidants
A. Enzymatic Antioxidants
These are intracellular enzymes that convert toxic free radicals into harmless products.
1. Superoxide Dismutase (SOD)
Function: Converts superoxide radicals into hydrogen peroxide and oxygen.
2O2∙−+2H+→H2O2+O2
Types:
Cu-Zn SOD
Location:
- Cytoplasm
Mn-SOD
Location:
- Mitochondria
Extracellular SOD
Location:
- Extracellular fluids
Functions:
- First line of antioxidant defense
- Reduces superoxide toxicity
2. Catalase
Function: Catalyzes decomposition of hydrogen peroxide.
2H2O2→2H2O+O2
Location:
- Peroxisomes
- Erythrocytes
- Liver cells
Functions:
- Prevents accumulation of hydrogen peroxide
- Protects tissues against oxidative damage
3. Glutathione Peroxidase (GPx)
Function: Reduces hydrogen peroxide and lipid hydroperoxides.
2GSH+H2O2→GSSG+2H2O
Cofactor:
- Selenium
Functions:
- Prevents membrane lipid oxidation
- Protects mitochondria
4. Glutathione Reductase
Function: Regenerates reduced glutathione from oxidized glutathione.
Reaction:
GSSG + NADPH → 2GSH + NADP⁺
Importance:
Maintains intracellular glutathione levels.
B. Non-Enzymatic Antioxidants
These antioxidants directly scavenge free radicals.
Vitamin E (α-Tocopherol)
Characteristics:
- Fat-soluble antioxidant
- Major membrane antioxidant
Functions:
- Prevents lipid peroxidation
- Stabilizes cell membranes
Sources:
- Vegetable oils
- Nuts
- Green vegetables
Deficiency:
- Hemolytic anemia
- Neurological dysfunction
Vitamin C (Ascorbic Acid)
Characteristics:
- Water-soluble antioxidant
Functions:
- Scavenges ROS
- Regenerates vitamin E
- Protects plasma proteins
Sources:
- Citrus fruits
- Tomatoes
- Green vegetables
Deficiency:
- Scurvy
Vitamin A and β-Carotene
Functions:
- Scavenge singlet oxygen
- Protect epithelial tissues
Sources:
- Carrots
- Spinach
- Mangoes
Reduced Glutathione (GSH)
Structure:
Tripeptide composed of:
- Glutamate
- Cysteine
- Glycine
Functions:
- Detoxification of ROS
- Maintains sulfhydryl groups
- Protects cell membranes
Uric Acid
Functions:
- Scavenges singlet oxygen
- Neutralizes hydroxyl radicals
Bilirubin
Functions:
- Prevents membrane oxidation
Albumin
Functions:
- Binds transition metals
- Prevents metal-mediated oxidation
C. Preventive Antioxidants
Preventive antioxidants reduce free radical generation before damage occurs.
Examples:
Metal-binding proteins
- Transferrin
- Ferritin
- Ceruloplasmin
- Lactoferrin
Mechanism:
Bind iron and copper, preventing: Fe2++H2O2→Fe3++OH−+OH∙
D. Chain-Breaking Antioxidants
These interrupt propagation of free radical reactions.
Examples:
- Vitamin E
- Vitamin C
- β-carotene
- Glutathione
Mechanism:
Donate electrons to radicals.
Mechanism of Antioxidant Action
Antioxidants protect cells through multiple mechanisms:
1. Radical scavenging
Free radical + Antioxidant
↓
Stable molecule
2. Metal ion chelation
Functions:
- Bind iron
- Bind copper
Effects:
- Prevent hydroxyl radical generation
3. Repair of damaged molecules
Functions:
- Restore oxidized proteins
- Regenerate antioxidants
4. Breaking chain reactions
Effects:
- Stops propagation of lipid peroxidation
Synergistic Action of Antioxidants
Antioxidants work together as a network.
Example:
Vitamin E neutralizes lipid radicals
↓
Vitamin E becomes oxidized
↓
Vitamin C regenerates Vitamin E
↓
Glutathione regenerates Vitamin C
Antioxidant Defense System in Cells
First line defense
Prevention of radical generation:
- Catalase
- SOD
- Glutathione peroxidase
Second line defense
Scavenging radicals:
- Vitamin E
- Vitamin C
- Glutathione
Third line defense
Repair systems:
- DNA repair enzymes
- Proteases
- Lipases
