Metabolism 5.2 Oxidative Stress and Antioxidant Defence Mechanisms

Oxidative Stress

• Definition: Condition where generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) exceeds the body’s ability to protect itself.

  • Consequences include increased oxidative damage to macromolecules (proteins, lipids, DNA) leading to cell/tissue damage.

  • Imbalance between oxidants and antioxidants drives cellular injury and disease.

Redox Reactions (Foundational Concept)

• Redox reactions: Involve simultaneous oxidation and reduction.

  • OIL RIG: Oxidation Is Loss, Reduction Is Gain of electrons.

  • Oxidizing agent (oxidant): Oxidises another substance and becomes reduced.

  • Reducing agent (reductant): Reduces another substance and is oxidised.

  • Practical implication: In biology, redox couples regulate signaling and oxidative damage.

Reactive Oxygen and Nitrogen Species (ROS & RNS)

• Reactive Oxygen Species (ROS): Includes superoxide ( ext{O}2^{ullet-}), hydrogen peroxide ($ext{H}</em>2 ext{O}*2$), hydroxyl radical ( ext{OH}^{ullet}).

• Reactive Nitrogen Species (RNS): Includes nitric oxide ( ext{NO}^{ullet}) and peroxynitrite ($ext{ONOO}^-$).

• Free radical definition: Atom/molecule with one or more unpaired electrons; highly reactive ( ext{OH}^{ullet}).

  • Readily diffusible and react with many cellular targets.

• Key interaction: Superoxide reacts with nitric oxide to form peroxynitrite ($ext{ONOO}^-$), a powerful oxidant.

Formation of ROS in Cells

• Electron Transport Chain (ETC) as a source: Electrons occasionally escape, reducing $ext{O}<em>2$ to form superoxide ( ext{O}2^{ullet-}).

  • Superoxide-derived ROS give rise to other ROS.

• Other endogenous sources: Peroxidases, nitric oxide synthases, NADPH oxidases, xanthine oxidase.

• Exogenous sources: Radiation, pollutants, drugs (e.g., paracetamol), toxins.

Key ROS/RNS Reactions and Consequences

• Superoxide to hydrogen peroxide: Superoxide dismutase (SOD) converts ext{O}2^{ullet-} to $ext{H}</em>2 ext{O}_2$.

  • Reaction: $2 \ \mathrm{O}<em>2^{\bullet-} + 2 \ \mathrm{H}^+ \rightarrow \mathrm{H}</em>2\mathrm{O}<em>2 + \mathrm{O}</em>2$

• Hydroxyl radical formation (Fenton chemistry): $\mathrm{Fe}^{2+} + \mathrm{H}<em>2\mathrm{O}</em>2 \rightarrow \mathrm{Fe}^{3+} + \mathrm{OH}^- + \mathrm{OH}^{\bullet}$.

• Peroxynitrite formation: $\mathrm{NO}^{\bullet} + \mathrm{O}_2^{\bullet-} \rightarrow \mathrm{ONOO}^-$.

• Nitric oxide synthases (NOS): Generate ext{NO}^{ullet} from arginine (iNOS, eNOS, nNOS).

Nitric Oxide Synthase (NOS) and Nitric Oxide (NO)

• NOS catalysis: Oxidation of arginine to citrulline and ext{NO}^{ullet}.

• NOS cofactors: FMN, FAD, heme, tetrahydrobiopterin (BH4).

• NO interactions: Reacts readily with $ext{O}*2$, metals, nucleic acids, and proteins.

• Low NO levels: Can inhibit cytochrome oxidase and support cell survival.

• High NO levels: Damage mitochondrial complexes, cause post-translational modifications (nitrosylation), and lead to cell death.

• Roles of NOS isoforms:

  • iNOS: Inducible, high ext{NO}^{ullet} production in phagocytes for antimicrobial effects.

  • eNOS: Endothelial signaling; vasodilation.

  • nNOS: Neuronal signaling.

DNA Damage from ROS

• Two main ROS-induced DNA damages:

  • Base damage: Leading to mispairing and mutations (oxidized bases).

  • Sugar/phosphodiester damage: Causing strand breaks during repair.

• 8-Oxo-2′-deoxyguanosine (8-oxo-dG): Marker for oxidative DNA damage.

  • Accumulation correlates with cancer risk when repair is compromised.

  • Used as an oxidative damage biomarker.

DNA Damage from ROS (Overview of Consequences)

• Consequences: ROS interactions with DNA drive mutations and genomic instability.

  • Failure to repair ROS-induced DNA lesions increases cancer risk.

Protein Damage from ROS

• ROS attack on proteins leads to:

  • Backbone fragmentation.

  • Side-chain modifications: carbonylation, hydroxylation.

  • Formation of protein cross-links (e.g., di-tyrosine dimers).

  • Loss or gain of function due to structural changes (e.g., disulfide bonds in insulin).

Lipid Peroxidation and Membrane Damage

• Mechanism: Unsaturated lipids react with ROS to form lipid peroxides.

• Consequences: Damages cell membranes, alters fluidity, and generates reactive aldehydes.

  • Implicated in early cardiovascular disease processes.

Nitric Oxide Synthase (NOS)–NO in Cells (Detailed)

• NOS reaction: $\text{Arginine} + \ \mathrm{O}<em>2 + \mathrm{NADPH} \rightarrow \text{Citrulline} + \mathrm{NO}^{\bullet} + \mathrm{NADP}^+ + \mathrm{H}</em>2\mathrm{O}$.

• NO interactions: Broaden signaling, but high NO can damage mitochondria and proteins.

  • Can participate in nitrosylation of thiol groups and other post-translational modifications.

Cellular Defence Mechanisms Against ROS/RNS

• Major defenses: Enzymatic and non-enzymatic systems to maintain non-damaging ROS/RNS levels.

• Non-enzymatic pool: Glutathione (GSH) in reduced (GSH) and oxidized (GSSG) forms.

• Key enzymes:

  • Glutathione peroxidase (GPx): Reduces $ext{H}<em>2 ext{O}</em>2$ and organic peroxides using GSH.

  • Glutathione reductase (GR): Regenerates GSH from GSSG using NADPH.

  • NADPH oxidase (NOX): Generates ROS for signaling/defense; tightly regulated.

  • Nicotinamide adenine dinucleotide phosphate (NADPH): Reducing power for GSSG-to-GSH regeneration.

• Pentose phosphate pathway (PPP): Generates NADPH, vital for maintaining GSH:GSSG ratio.

• GSH regeneration cycle: $\text{GSSG} + \mathrm{NADPH} + \mathrm{H}^+ \rightarrow 2\text{GSH} + \mathrm{NADP}^+$.

  • GSH donates electrons (via cysteine thiol) to reduce oxidants, becoming GSSG.

  • GR regenerates GSH from GSSG using NADPH.

  • Maintains cellular redox homeostasis and detoxifies peroxides.

Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency

• Definition: X-linked recessive enzyme deficiency, affects RBC protection against oxidative stress.

  • Common in African, Mediterranean, and Middle Eastern populations ($\sim$400 million people).

  • Provides some protection against malaria.

• Clinical presentation:

  • Most individuals asymptomatic.

  • Occasional severe haemolytic anaemia during oxidative stress (haemolytic crisis).

• Pathophysiology:

  • G6PD deficiency lowers PPP NADPH production.

  • Reduced NADPH limits regeneration of GSH from GSSG.

  • Decreased GSH leads to vulnerability to ROS, causing damage.

• Red cell consequences in oxidative crises: Heinz bodies, haemolysis.

G6PD Deficiency – Mechanistic View

• Pathway disruption: $\downarrow$G6PDH $\rightarrow \downarrow$NADPH $\rightarrow \downarrow$GSH reduction.

• Consequences:

  • Oxidative stress–related damage to lipids, proteins, and DNA.

  • Heinz bodies formation and hemolytic anemia if exposed to oxidants.

  • Higher sensitivity to infection, certain drugs, and fava beans as triggers.

Heinz Bodies and Related RBC Pathology (Context of G6PD)

• Heinz bodies: Dark inclusions due to precipitated hemoglobin (Hb) inside RBCs.

  • Bind to cell membrane, increasing rigidity and causing mechanical stress.

  • Spleen removes Heinz bodies, leading to blister cells.

  • Clinical sign of G6PD deficiency-related hemolysis.

Galactosaemia and Related Pathways (Oxygen/NADPH Context)

• Galactosaemia: Defects in galactose metabolism enzymes (Galactokinase, Uridyltransferase, UDP-galactose epimerase).

  • Consequence: Accumulation of galactitol via aldose reductase (polyol pathway) consuming NADPH.

  • NADPH depletion: Compromises antioxidant defenses, contributing to cataracts, hepatic, and renal complications.

  • Metabolic flow: Dietary lactose $<br>ightarrow$ galactose + glucose; galactose involved in polyol pathway.

Oxygen Toxicity (Clinical Implications of Excess Oxygen)

• Mechanism: Excessive oxygen exposure increases ROS formation.

• Detrimental effects: Damage to bronchioles/alveoli, retina, and other tissues.

  • Occurs in both infants and adults under prolonged respiratory support.

Diseases and Conditions Linked to Free Radical Damage (Broad Overview)

• Multifactorial associations: ROS/RNS contribute but are not sole causes.

• Linked conditions include:

  • Atheroma (atherosclerosis).

  • Cancers.

  • Autoimmune and inflammatory diseases (e.g., rheumatoid arthritis).

  • Neurodegenerative diseases (including dementia).

  • Cataracts and macular degeneration.

  • Aging processes.

Progression of Atheroma (Conceptual Model)

• Progression: Healthy artery $ightarrow$ atheroma plaque formation $ightarrow$ plaque rupture $ightarrow$ thrombosis $ightarrow$ impaired blood flow.

  • Oxidative stress contributes to lipid oxidation and plaque instability.

Cellular Defences – Antioxidants (Non-enzymatic Defenders)

• Vitamin E ($\alpha$-tocopherol): Lipid-soluble antioxidant; protects against lipid peroxidation.

• Vitamin C (ascorbic acid): Water-soluble antioxidant; regenerates reduced Vitamin E.

  • Vitamin E scavenges lipids and becomes oxidized; Vitamin C regenerates reduced Vitamin E.

• Other non-enzymatic antioxidants: Carotenoids, flavonoids, selenium (cofactor for GPx), uric acid, melatonin, zinc.

• General mechanism: Free radical scavengers donate a hydrogen atom (and its electron) to neutralize free radicals.

Antioxidant Enzyme Systems (Key Players)

• SOD: Converts superoxide to hydrogen peroxide.

• CAT (catalase) and GPX (glutathione peroxidase): Convert hydrogen peroxide to water.

• GSR (glutathione reductase): Regenerates GSH from GSSG using NADPH.

• GST (glutathione-S-transferase): Involved in detoxification pathways.

• Metal cofactors: Mn, Cu, Zn.

Antioxidant Foods and Supplements – Practical Perspective

• Dietary antioxidants: Vitamins A, C, E; minerals Mn, Se, Zn; carotenoids; plant-based foods (fruits, vegetables, coffee, dark chocolate).

  • Caveat: Not miracle cures; claims should be interpreted cautiously.

• Supplements: Can be helpful but not a panacea; effectiveness varies.

Respiratory Burst (Phagocytic Defense) and CGD

• Respiratory Burst: Phagocytes (e.g., neutrophils) rapidly release ROS/$\text{H}<em>2\text{O}</em>2$ to destroy invading bacteria.

• Chronic Granulomatous Disease (CGD): Genetic defects in the NADPH oxidase complex.

  • Leads to increased susceptibility to infections (atypical infections, pneumonia, abscesses, cellulitis).

  • Respiratory burst is a controlled ROS burst for immunity; defects heighten infection risk.

Summary of Key Concepts (Cohesive View)

• Oxidative stress: ROS/RNS production exceeding defenses, causing damage to DNA, proteins, lipids.

• ROS/RNS formation: Mitochondrial leakage, NO synthases, NADPH oxidases, other sources.

• ROS damages: DNA base/sugar damage (e.g., 8-oxo-dG), protein oxidation, lipid peroxidation, contributing to various diseases.

• Antioxidant defenses: Enzymatic systems (SOD, CAT, GPX, GR, GST) and non-enzymatic antioxidants (Vitamins E/C, carotenoids, GSH/GSSG cycle, NADPH via PPP).

• NADPH central role: Regenerating reduced antioxidants (GSH) and enzymatic defenses; PPP is primary source.

• G6PD deficiency: Limits NADPH supply, compromising oxidative defense, leading to hemolysis and Heinz bodies.

• NOS/NO biology: Dual roles ranging from physiological signaling to cytotoxicity at high levels or with ONOO$^-$.

• Oxygen toxicity: Excessive ROS from prolonged high oxygen exposure clinically.

• Pathophysiological links: Atheroma progression, cancer, neurodegeneration.

• Clinical implications: Cautious use of oxidant drugs, awareness of G6PD status (e.g., with nitrofurantoin, primaquine), judicious oxygen therapy.

Notable Ancillary Content (contextual and non-core items from the transcript)

• Product advertisement: Mothballs, medication panel (Page 23).

• Educational inserts: Oxygen toxicity in clinical care (Page 26–27).

• Galactosaemia section: Metabolic pathway details and NADPH consumption via aldose reductase (Page 23–25).

• Summary citation list: References to antioxidant studies (Page 35).

Connections to Foundational Principles and Real-World Relevance

• Redox biology: Unifying framework for understanding aging, cancer, neurodegeneration, cardiovascular disease.

• Balance: Between ROS production and antioxidant capacity is a dynamic determinant of cell fate.

• Real-world relevance: Clinical strategies (CGD diagnosis/management, NADPH-dependent antioxidant support, oxygen therapy) and public health (dietary antioxidants, malaria resistance in G6PD deficiency).

Equations and Key Reactions (LaTeX)

• Superoxide formation: $\text{O}<em>2 + e^- \rightarrow \text{O}</em>2^{\bullet-}$.

• Dismutation of superoxide by SOD: $2\,\text{O}<em>2^{\bullet-} + 2\,\text{H}^+ \rightarrow \text{H}</em>2\text{O}<em>2 + \text{O}</em>2$

• Fenton reaction: $\text{Fe}^{2+} + \text{H}<em>2\text{O}</em>2 \rightarrow \text{Fe}^{3+} + \text{OH}^- + \text{OH}^{\bullet}$.

• Peroxynitrite formation: $\text{NO}^{\bullet} + \text{O}_2^{\bullet-} \rightarrow \text{ONOO}^-$.

• NOS-catalyzed NO production: $\text{Arginine} + \text{O}<em>2 + \text{NADPH} \rightarrow \text{Citrulline} + \text{NO}^{\bullet} + \text{NADP}^+ + \text{H}</em>2\text{O}$.

• GSSG reduction by NADPH: $\text{GSSG} + \text{NADPH} + \text{H}^+ \rightarrow 2\text{GSH} + \text{NADP}^+$.

• GPx-catalyzed reduction of $ext{H}<em>2 ext{O}</em>2$: $2\text{GSH} + \text{H}<em>2\text{O}</em>2 \rightarrow \text{GSSG} + 2\text{H}_2\text{O}$.

• PPP-derived NADPH support: Conceptual role in sustaining GSH/GSSG balance.

Key Takeaways for Exam Preparation

• Define oxidative stress: Explain ROS/RNS balance and cellular outcomes.

• List major ROS/RNS: Their interconversions ($\text{O}<em>2$, $ext{O}</em>2^{\bullet-}$, $ext{H}<em>2 ext{O}</em>2$, $ext{OH}^{\bullet}$, $ext{NO}^{\bullet}$, $ext{ONOO}^-$).

• Describe ETC as ROS source: How ROS are formed inadvertently.

• Enumerate oxidant sources: Endogenous and exogenous.

• Explain ROS damage: DNA (8-oxo-dG), proteins (carbonylation), lipids (lipid peroxidation).

• Outline NOS/NO biology: iNOS/eNOS/nNOS roles and dual nature of NO.

• Describe GSH/GSSG cycle: Role of GPx, GR, and importance of NADPH/PPP.

• Explain G6PD deficiency: Inheritance, relevance, clinical consequences, Heinz bodies.

• Recognize oxygen toxicity: Clinical implications of oxygen therapy.

• Recall respiratory burst and CGD: Model of defense and infection vulnerability.

• Understand broad connections: Oxidative stress and diseases (atheroma, cancer, neurodegeneration, aging).