Free radicals and reperfusion injury
Reactive oxygen species come from normal metabolism, inflammation, radiation, metals and drug metabolism. They attack lipids, proteins and DNA unless the cell's defenses keep up, and they are why restoring flow can harm already injured tissue.
Radicals and reactive species
L1 p.19–20A free radical has a single unpaired electron in its outer orbital. It is unstable and grabs an electron from a neighbouring molecule, which becomes a radical in turn, so one radical can start a chain reaction. Not every damaging species is a radical, so the broader term is reactive oxygen species (ROS).
| Species | Radical? | Formed by | Note |
|---|---|---|---|
| Superoxide (O₂•⁻) | Yes | Electron leak from the respiratory chain; NADPH oxidase in phagocytes | Starting point for most ROS |
| Hydrogen peroxide (H₂O₂) | No, but reactive | Superoxide dismutase | Crosses membranes; source of hydroxyl radical |
| Hydroxyl radical (•OH) | Yes | Fenton reaction with Fe²⁺ (or Cu⁺); radiolysis of water | Most reactive and most damaging; no enzyme removes it |
| Nitric oxide (NO•) | Yes | NO synthase in endothelium and macrophages | A signalling molecule as well as a toxin |
| Peroxynitrite (ONOO⁻) | No, but a strong oxidant | NO + superoxide | Potent, bactericidal |
Superoxide (O₂•⁻)
- Radical?
- Yes
- Formed by
- Electron leak from the respiratory chain; NADPH oxidase in phagocytes
- Note
- Starting point for most ROS
Hydrogen peroxide (H₂O₂)
- Radical?
- No, but reactive
- Formed by
- Superoxide dismutase
- Note
- Crosses membranes; source of hydroxyl radical
Hydroxyl radical (•OH)
- Radical?
- Yes
- Formed by
- Fenton reaction with Fe²⁺ (or Cu⁺); radiolysis of water
- Note
- Most reactive and most damaging; no enzyme removes it
Nitric oxide (NO•)
- Radical?
- Yes
- Formed by
- NO synthase in endothelium and macrophages
- Note
- A signalling molecule as well as a toxin
Peroxynitrite (ONOO⁻)
- Radical?
- No, but a strong oxidant
- Formed by
- NO + superoxide
- Note
- Potent, bactericidal
Where they come from
L1 p.20–21Pathway schematic
- Normal metabolism: a small fraction of electrons leak from respiratory complexes I and III and partly reduce oxygen to superoxide.
- Inflammation: neutrophils and macrophages use NADPH oxidase to make superoxide in the oxidative burst that kills microbes.
- Ionizing radiation splits water into hydroxyl radicals.
- Metals: free iron and copper drive the Fenton reaction, Fe²⁺ + H₂O₂ → Fe³⁺ + •OH + OH⁻.
- Drugs and chemicals: cytochrome P450 in the liver turns acetaminophen into the reactive metabolite NAPQI and carbon tetrachloride into the trichloromethyl radical (CCl₃•).
- Nitric oxide from NO synthase combines with superoxide to form peroxynitrite.
What they damage
L1 p.19Lipids: peroxidation
- Radicals attack double bonds of membrane fatty acids
- Peroxyl radicals propagate a chain reaction
- Membranes become leaky; ion pumps and organelle membranes are damaged
Proteins
- Oxidation of side chains and sulfhydryl groups
- Cross-linking and fragmentation
- Loss of enzyme activity; misfolded proteins are degraded
DNA
- Base oxidation and strand breaks
- Mutations that accumulate with age and can contribute to cancer
- Severe damage activates p53 and apoptosis (1.7)
Defenses
L1 p.22–23| Defense | What it does | Where |
|---|---|---|
| Superoxide dismutase (SOD) | Converts superoxide to hydrogen peroxide | Mitochondria and cytosol |
| Catalase | Breaks hydrogen peroxide into water and oxygen | Peroxisomes |
| Glutathione peroxidase | Reduces hydrogen peroxide and lipid peroxides to water and alcohols, oxidizing 2 GSH to GSSG | Cytosol and mitochondria |
| Glutathione reductase | Regenerates GSH from GSSG using NADPH | Cytosol |
| G6PD (pentose phosphate pathway) | Produces the NADPH that keeps glutathione reduced | Cytosol, essential in red cells |
| Vitamin E | Fat-soluble; sits in membranes and stops lipid peroxidation chains; protects LDL from oxidation | Membranes, lipoproteins |
| Vitamin C | Water-soluble scavenger, effective against hydroxyl radical; regenerates vitamin E | Cytosol, plasma |
| Iron- and copper-binding proteins | Transferrin, ferritin and ceruloplasmin keep metals from driving the Fenton reaction | Plasma and cells |
Superoxide dismutase (SOD)
- What it does
- Converts superoxide to hydrogen peroxide
- Where
- Mitochondria and cytosol
Catalase
- What it does
- Breaks hydrogen peroxide into water and oxygen
- Where
- Peroxisomes
Glutathione peroxidase
- What it does
- Reduces hydrogen peroxide and lipid peroxides to water and alcohols, oxidizing 2 GSH to GSSG
- Where
- Cytosol and mitochondria
Glutathione reductase
- What it does
- Regenerates GSH from GSSG using NADPH
- Where
- Cytosol
G6PD (pentose phosphate pathway)
- What it does
- Produces the NADPH that keeps glutathione reduced
- Where
- Cytosol, essential in red cells
Vitamin E
- What it does
- Fat-soluble; sits in membranes and stops lipid peroxidation chains; protects LDL from oxidation
- Where
- Membranes, lipoproteins
Vitamin C
- What it does
- Water-soluble scavenger, effective against hydroxyl radical; regenerates vitamin E
- Where
- Cytosol, plasma
Iron- and copper-binding proteins
- What it does
- Transferrin, ferritin and ceruloplasmin keep metals from driving the Fenton reaction
- Where
- Plasma and cells
Mechanisms in disease
L1 p.19–22| Setting | Radical mechanism | Consequence |
|---|---|---|
| Atherosclerosis | LDL oxidized in the arterial wall is taken up by macrophages through scavenger receptors | Foam cells and fatty streaks, the earliest lesions of atherosclerosis |
| Aging | Damage to lipids, proteins and DNA accumulates as repair declines | Lipofuscin, mutations, declining function |
| Acetaminophen overdose | Glucuronidation and sulfation are saturated; P450 makes excess NAPQI, which uses up glutathione, then binds cell proteins and causes oxidative stress | Centrilobular (zone 3) hepatocyte necrosis; N-acetylcysteine supplies cysteine to rebuild glutathione |
| Carbon tetrachloride | P450 makes CCl₃•, which peroxidizes ER membranes | Fatty liver and centrilobular necrosis |
| Reperfusion | Restored oxygen meets damaged mitochondria and incoming neutrophils | Reperfusion injury (next subtopic) |
Atherosclerosis
- Radical mechanism
- LDL oxidized in the arterial wall is taken up by macrophages through scavenger receptors
- Consequence
- Foam cells and fatty streaks, the earliest lesions of atherosclerosis
Aging
- Radical mechanism
- Damage to lipids, proteins and DNA accumulates as repair declines
- Consequence
- Lipofuscin, mutations, declining function
Acetaminophen overdose
- Radical mechanism
- Glucuronidation and sulfation are saturated; P450 makes excess NAPQI, which uses up glutathione, then binds cell proteins and causes oxidative stress
- Consequence
- Centrilobular (zone 3) hepatocyte necrosis; N-acetylcysteine supplies cysteine to rebuild glutathione
Carbon tetrachloride
- Radical mechanism
- P450 makes CCl₃•, which peroxidizes ER membranes
- Consequence
- Fatty liver and centrilobular necrosis
Reperfusion
- Radical mechanism
- Restored oxygen meets damaged mitochondria and incoming neutrophils
- Consequence
- Reperfusion injury (next subtopic)
Two outcomes of reflow
L1 p.19Cells still viable (reversible injury)
- Mitochondria can use the returning oxygen
- ATP regenerates; pumps restart; water leaves
- Antioxidant defenses handle the small radical load
- Outcome: recovery
Cells already badly injured
- Damaged mitochondria partly reduce oxygen to superoxide
- Neutrophils carried in by the flow add superoxide (NADPH oxidase) and inflammation
- Lipid peroxidation and leaky membranes let calcium flood in
- Outcome: death, sometimes hastened; reperfused infarcts can become hemorrhagic, and myocardium shows contraction bands
The lecture's example is reperfusion injury after fibrinolytic therapy for acute myocardial infarction. Compare the two outcomes yourself in Cell on a Clock by switching between reflow while reversible and reflow after sustained injury.
Core activity · The Missing Arrow
The Missing Arrow
A coronary artery is reopened after the myocardium in its territory has been ischemic for hours. Complete the chain for the already-injured cells.
- Blood flow returns to injured myocardium
- Membranes leak; ion pumps are damaged
- calcium floods in through damaged membranes
Cards
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Knowledge check · 1.5
Answer, read why, and try again whenever you like. Answers are kept in both study modes.
- Q1Fenton reactionL1 p.20–21
In a patient with iron overload, which reaction converts hydrogen peroxide into the most damaging radical?
- Q2Acetaminophen and glutathioneL1 p.20, 22
A 19-year-old takes a large overdose of acetaminophen.
Why is N-acetylcysteine given?
- Q3Reperfusion injuryL1 p.19Midterm sim. p.1
A 62-year-old man with an anterior myocardial infarction receives thrombolysis 5 hours after his pain began. Flow is restored, but troponin rises steeply over the next hour, and at autopsy days later the infarct is hemorrhagic with contraction bands in the dead myocytes.
What best explains these findings?
- Q4Antioxidant defensesL1 p.23
Why do red cells of a person with G6PD deficiency hemolyze after an oxidant drug?
- Q5Vitamins E and CL1 p.22
Which antioxidant acts inside membranes to stop lipid peroxidation chain reactions?