Oxidative Stress

Question 1

What diseases does oxidative stress have a role in?

Answer 1

Multiple Sclerosis

Cardiovascular disease

Alzheimer’s

Rheumatoid arthritis

Crohn’s disease

COPD

Ischaemia / reperfusion injury

Cancer

Pancreatitis

Parkinson’s disease

Question 2

What is a free radical?

Answer 2

A free radical is an atom or a molecule that contain one or more unpaired e^-.

They are very reactive and tend to acquire electrons from other atoms, molecules or ions.

Reactions of a radical with a molecule typically generates a second radical thereby propagating the damage.

Question 3

What Oxygen free radical can be produced? How are they produced?

Answer 3

Oxygen combines with an electrol to produce superoxide (O₂^.)

Superoxide then combines with 2H⁺ and e^- to produce Hydrogen Peroxide.

Hydrogen peroxide then combines with an e^- and a H⁺ to produce a hydroxyl radical and water. This hydroxyl radical is the most reactive and damaging.

Question 4

What are the reactive nitrogen species?

Answer 4

Nitric oxide (NO^•) which then combine with superoxide to produce peroxynitrite (ONOO-).

Peroxynitrite is not itself a free radical but, is a powerful oxidant that can damage cells.

Question 5

What damage can reactive oxygen species cause?

Answer 5

ROS damage to DNA…

react with base. - This modified base can lead to mispairing and mutation.

ROS react with sugar. (Ribose or deoxyribose). This can cause a strand break and mutations on repair.

ROS damage with proteins.

ROS damage lipids.

Question 6

What happens when ROS reacts with proteins?

Answer 6

ROS can damage the backbone or the sidechain of the DNA.

Damaging the backbone can lead to fragmentatin which leads to protein degredation.

Damaging the side chains can lead to modified amino acids eg: Carbonyls, Hydroxylated adducts, Ring opened species, dimers and disulphide bonds.

This then leads to a change in protein structure.

Question 7

What are the importance of disulphide bonds? How can innapropriate formation occur?

Answer 7

They play an important role in folding and stability of some proteins.

Disulphide bonds form between two thiol groups of cysteine residues.

Inappropriate disulphide bond formation can occur is ROS takes electrons from cysteines. This causes misfolding, crosslinking and disruption of function (e.g. enzymes)

Question 8

How do reactive oxygen species cause damage to lipids?

Answer 8

Free radicals (eg OH.) extracts H atom from a polyunsaturated fatty acid in membrane lipid.

Lipid radicals formed which can react with oxygen to form a lipid peroxyl radical.

This results in a chain reactin as lipid peroxyl radical extracts hydrogem from mearby fatty acids.

Hydrophobic environments of the bilayer are disrupted and the membrane integrity fails.

This is mportant in athlerosclerosis

Question 9

WHat are some endogenous sources of biological oxidants?

Answer 9

• Electron Transport Chain
• Peroxidases
• Nitric Oxide synthases
• Lipooxygenases
• NADPH oxidases
• Xanthine oxidase
• Monoamine oxidase

Question 10

What are some exogenous sources of biological oxidants?

Answer 10

• Radiation inc. cosmic rays, UV light and X rays.
• Pollutants
• Drugs eg Primaquine (anti-malerial)
• Toxins eg Paraquat (herbicide)

Question 11

How is the ETC a source of Reactive Oxygen Species?

Answer 11

Occasionally, electron can accidentally escape the ETC and react with dissolves O₂ to form superoxide.

Question 12

How are nitric oxide synthases (NOS) sources of reactive oxygen species?

Answer 12

iNOS: inducible nitric oxide synthase. Produces high NO conc. in phagocytes for direct toxic effect.

eNOS: Endothelial nitric oxide synthase (signalling).

nNOS: Neuronal nitric oxide synthase (signalling)

They all produce NO.

This has toxic effects at high levels. (iNOS) It is a signalling molecule that causes Vasodilation, Neurotransmission S-Nitrosylation

Question 13

How do respiration bursts cause ROS?

Answer 13

Rapid release of superoxide and H₂O₂ from phagocytic cellls (eg neutrophils and monocytes)

ROS and peroxynitrite destroy and invade bacteria.

Part of antimicrobiral defence system/

Question 14

WHat disease makes sufferers more susceptible to bacterial infection? Why?

Answer 14

Chronic granulomatose disease.

This is a genetic defect in NADPH oxidase complex which causes enhansed suseptibility to bacterial infections. e.g. Atypical infections, pneumonia, absesses, Impetigo, cellulitis

Question 15

What cellular defenses does the body have against reactive oxygen species?

Answer 15

• Superoxide dismutase (SOD)
• Catalase
• Glutatione
• Free radical scavengers (Vitamin E, Vitamin C ect..)

Question 16

How does SOD work?

Answer 16

Superoxide dismutate converts superoxide to H₂O₂ and oxygen.

This is a primary defence because superoxide is strong initiator of chain reactions.

There are three isoenzymes:

• Cu⁺-Zn²⁺ Cystolic
• Cu⁺-Zn²⁺ Extracellular
• Mn²⁺ Mitochondria

Question 17

How does catalase work?

Answer 17

Catalase converts H₂O₂ to water and oxygen.

Widespread enzyme. Important in immune cells to protect against axidative bursts.

You get grey hair because, as you age, there are declining levels of catalses in the hair folicles. This means you have more hydrogen peroxide which bleaches the hair and turns it grey.

Question 18

How does Glutathione work?

Answer 18

Glutathione is a tripeptide synthesised by the body to protect against oxidative damage.

The thiol group of cysteine donates e- to ROS. GSH (the reduced form of glutathione) then reacts with another GSH to form disulphide (GSSG which is the oxidised form of glutathione).

Glutathione peroxidase (the enzyme that catalyses disulphide bind formtion) requires Selenium.

GSSH is reduced back to GSH by glutathione reductase. This enzyme catalyses the transfer of electrons from NADPH to the disuphide bond.

NADPH from the pentose phosphate pathway is therefore essential for protection against free radical damage.

Question 19

Why is the pentose phosphate pathway useful?

Answer 19

Important source of NADH which is required for:

• Reducing power for biosynthesis
• Maintainance of GSH levels (reduced glutathione)
• Detoxification reactions

Produces C5-sugar ribose required for the synthesis of:

• Nucleotides
• DNA and RNA

This pathways starts from Glucose-6-phosphate.

There is no ATP synthesied or CO₂ produced.

The rate limiting enzyme is Glucose-6-phosphate dehydrogenase

Question 20

What are free radical scavengers?

Answer 20

Free radical scavengers reduce free radical damage by donating a hydrogen atom (and its electron) to free radicals in a nonenzymatic reaction. Examples include:

Vitamin E (a-tocopherol)

• This is a lipid soluble antioxidant
• It is important for the protection agaisnt lipid peroxidation.

Vitamin C

• This is a water soluble antioxidant
• Important role in regenerating reduced form of vitamin E

Others inc:

Carotenoids, Flavoniods, Uric acid, Melatonin

Question 21

When does oxidative stress occur?

Answer 21

When there is more oxidants formed than our defences can cope with.

Question 22

What is galactosaemia?

Answer 22

Galactosaemia is caused by a deficiency in one of three enzymes:

• Galactokinase
• Uridyl Transferase
• UDP-galactose epimerase

This caused the concentration of galactose to build up.

This galactose is metabolised by Aldose reductase into galacticol.

The increased activity of aldose reductase uses NADPH which is needed to combat ROS. This compromises our ROS defense system which means that the crystallin protein in the lense of our eye is denatured and causes cateracts.

Symptoms:

• Cataracts
• Vomitting
• Renal failiure
• Hepatomegally + cirrhosis
• Seizures and brain damage
• Hypoglycaemia

Question 23

What effect does G6PDH deficiency have?

Answer 23

G6PDH is the first enzyme in the pentose phosphate pathway.

Therefore, decreased G6PDH activity limits the amount of NADPH.

NADPH is required for the reduction of oxidised glutathione (GSSH) back to reduced glutathione (GSH).

Lower GSH means there there is less protection against damage from oxidative stress.

Oxidative sress leads to Lipid peroxidation inc cell membrane damage. This lack of deformity leads to mechanical stress.

It also leads to protein damage which results in aggregates of cross-linked haemoglobin (heinz bodies).

All these things lead to Haemolysis.

Question 24

Where G6DPH deficiency most effect?

Answer 24

G6PHD deficiencies mostly affect red blood cells because the pentose phosphate pathway is their only source of NADPH.

Question 25

WHat are Heinz bodies?

Answer 25

Dark staining within red blood cells reslting from precipitated haemoglobin.

They bind to the cell membrane and alter rigidity.

They increase mechanical stress when cells squeeze through small capillaries.

The spleen removes bound Heinz bodies resulting in “blister cells”

They are a clinical sign of G6PDH deficiency.

Question 26

How is paracetamol metabolised?

Answer 26

Paracetamol is normally metabolised in liver the to Glucuronide and sulphate.

Question 27

What does paracetamol metabolism produce if you overdose?

Answer 27

No longer convert all paracetamol to sulphide gluronide so, produce NAPQI which is a toxic metabolite of paracetamol.

Question 28

WHat does NAPQI cause?

Answer 28

NAPQI combines with glutathione.

This results in glutathione depletion so, can result in oxidative damage to liver cells.

Question 29

How is a paracetamol overdose treated?

Answer 29

Give the patient Acetylcysteine. This is the substrate for glutathione so, it will replenish the bodies reserves and prevent glutathione depletion and therefore liver cell damage due to oxidative stress.