Free Radicals & Antioxidants
Free radicals are molecules with one or more unpaired electrons. They are highly reactive and can damage DNA, proteins, and lipids. Antioxidants protect cells by neutralizing free radicals.
Reactive Oxygen Species (ROS)
- Superoxide (O₂•⁻): Formed in ETC (Complex I, III leakage), NADPH oxidase (phagocytes), xanthine oxidase. First free radical generated.
- Hydrogen Peroxide (H₂O₂): Formed from superoxide via superoxide dismutase (SOD). Less reactive but generates hydroxyl radical in Fenton reaction.
- Hydroxyl Radical (•OH): Most reactive and damaging. Formed by Fenton reaction: H₂O₂ + Fe²+ → •OH + OH⁻ + Fe³+. Attacks DNA (strand breaks), lipids (peroxidation), proteins.
- Peroxynitrite (ONOO⁻): Formed from O₂•⁻ + NO•; highly toxic; damages proteins and DNA.
Sources of ROS
- Normal: ETC leakage (~1–2%), peroxisomal β-oxidation, CYP450
- Pathological: Ischemia-reperfusion injury, inflammation, radiation
- Phagocytic burst: NADPH oxidase generates O₂•⁻ → kills bacteria (MPO system)
Antioxidant Defenses
Enzymatic:
- Superoxide Dismutase (SOD): 2 O₂•⁻ + 2H+ → H₂O₂ + O₂ (Cu/Zn-SOD in cytosol; Mn-SOD in mitochondria)
- Catalase: 2 H₂O₂ → 2 H₂O + O₂ (in peroxisomes). Very efficient; activated Fe-containing enzyme.
- Glutathione Peroxidase (GPx): H₂O₂ + 2 GSH → GSSG + 2 H₂O. Requires Selenium as cofactor. Glutathione Reductase regenerates GSH from GSSG (requires NADPH from HMP shunt).
Non-enzymatic:
- Vitamin E (Tocopherol): Fat-soluble; scavenges peroxyl radicals; major lipid-phase antioxidant; protects PUFA in membranes
- Vitamin C (Ascorbate): Water-soluble; reduces superoxide; also regenerates Vitamin E from Vit E radical
- β-Carotene: Quenches singlet oxygen (excited O₂)
- Uric Acid: Important plasma antioxidant (in primates); accounts for ~50% of plasma antioxidant capacity
- Glutathione (GSH): Tripeptide (Glu-Cys-Gly); most important intracellular antioxidant
- Thioredoxin: Reduces protein disulfide bonds; part of antioxidant network
Oxidative Stress & Disease
When ROS production exceeds antioxidant capacity → oxidative stress → damage:
- Lipid peroxidation (membrane disruption) — atherosclerosis (oxLDL)
- DNA damage → mutations → cancer
- Protein oxidation → enzyme inactivation, neurodegeneration (Parkinson's, Alzheimer's)
- Ischemia-reperfusion injury (heart, brain during stroke) — major oxidative stress burst on reperfusion
Respiratory Burst (Phagocytes)
Neutrophils and macrophages use controlled ROS generation to kill pathogens:
- NADPH oxidase: 2 O₂ + NADPH → 2 O₂•⁻ + NADP+ + H+ (consumes O₂ — hence "respiratory" despite not producing ATP)
- SOD: O₂•⁻ → H₂O₂
- Myeloperoxidase (MPO): H₂O₂ + Cl⁻ → HOCl (hypochlorous acid — bleach; most bactericidal) → kill bacteria
Chronic Granulomatous Disease (CGD): NADPH oxidase defect → cannot kill catalase-positive organisms (Staph. aureus, Aspergillus) → recurrent, severe infections.