Alcohol Metabolism And Oxidative Stress Flashcards
What is alcohol metabolised by?
- Most (>90%) alcohol is metabolised by liver
- Remainder excreted passively in urine and on breath.
- Alcohol oxidised by alcohol dehydrogenase to acetaldehyde and then to acetate by aldehyde dehydrogenase.
- Acetate converted to acetyl~CoA and used in TCA cycle or for fatty acid synthesis
- Smaller amounts of alcohol can also be oxidized by the cytochrome P450 2E1 enzyme (CYP2E1), or by catalase in brain.
What is the recommended limits for alcohol consumption
14 units a week spread over at east 3 days
What is the rate of alcohol metabolsim?
Rate of alcohol metabolism
• One unit of alcohol = 8 g
• Half pint of normal strength beer, small glass of wine
• Eliminated at rate of ~7g per hour
What are the steps in alcohol metabolism?
Ethanol -> Acetaldehyde
(NAD+ ->NADH)
Catalysed by alcohol dehydrogenase
Acetaldehyde -> Acetate
(NAD+ -> NADH)
Aldehyde dehydrogenase
What is Acetaldehyde and what are the effects of its build up
Intermediate between ethanol and acetate
Acetaldehyde is a toxic metabolite. Accumulation causes “Hangover”
What happens to acetate?
Conjugated to coenzyme A to form acetyl-CoA and metabolised in TCA cycle or utilised for fatty acid synthesis
What causes liver damage?
- Acetaldehyde toxicity normally kept to a minimum by aldehyde dehydrogenase (low Km for acetaldehyde)
- Prolonged and excessive alcohol consumption can cause sufficient acetaldehyde accumulation to cause liver damage
- Excess NADH and Acetyl-CoA lead to changes in liver metabolism
- “Fatty liver”
- Alcoholic hepatitis
- Alcoholic cirrhosis
What is the metabolic response to chronic alcohol consumption
See slide
What is disulfiram used for
- Disulfiram can be used as an adjunct in the treatment of chronic alcohol dependence.
- It is an inhibitor of aldehyde dehydrogenase
- If patient drinks alcohol acetaldehyde will accumulate causing symptoms of a ‘hangover
What are ROS and RNS?
Reactive oxygen species and reactive nitrogen species
Cause cell damage
What are defences against oxidative damage
Antioxidant enzymes
Small molecule antioxidants
What is oxidative stress
When cell damage > cell defences
What are free radicals?
- Electrons of atoms, molecules & ions usually associate in pairs. Each pair moves within a defined region of space (an orbital).
- A free radical is an atom or molecule that contains one or more unpaired electrons and is capable of independent (“free”) existence
- A superscript dot used to denote free radical (e.g. OH )
- Free radicals (usually) very reactive and tend to acquire electrons from other atoms, molecules or ions
- Reaction of a radical with a molecule typically generates a second radical thereby propagating damage.
Name some reactive oxygen species
O2 - Molecular oxygen is a biradical. It has 2 unpaired electrons in different orbitals
(+e-……)
Superoxide O2• - Produced by adding electron to molecular
oxygen. Also Important source of other ROS
(+2H+, e-…….)
Hydrogen peroxide - Not a free radical but can react e.g. with Fe2+ to produce free radicals. Readily diffusible.
(+H+, e- gives water and……..)
Hydroxyl radical OH• - Most reactive and damaging free radical. Reacts with anything!
What are reactive oxygen species?
Nitric oxide NO •
Peroxynitrite ONOO-
O2• + NO• -> ONOO-
- Superoxide can react with nitric oxide to produce peroxynitrite
- Peroxynitrite is not itself a free radical, but is a powerful oxidant that can damage cells
What are the 2 main types of ROS damage to DNA?
Two main types of damage
• ROS reacts with base
Modified base can lead to mispairing and mutation
• ROS reacts with sugar (ribose or deoxyribose)
Can cause strand break and mutation on repair
How can ROS damage to DNA lead to cancer?
ROS reacts with DNA -> DNA damage -> failure to repair can end to mutation -> can lead to cancer
Describe ROS damage to proteins
ROS interacts with protein -> backbone fragmentation -> protein degradation
ROS interacts with protein -> sidechain modified* -> change in protein structure -> protein degradation, loss of function (-> degradation) or gain of function
* Modified amino acid (e.g.) • Carbonyls • Hydroxylated adducts • Ring opened species • Dimers (e.g. di-tyrosine) • Disulphide bond (Cys)
How do disulfide bonds affect protein stability
- Play important role in folding and stability of some proteins (usually secreted proteins or in extracellular domains of membrane proteins)
- Formed between thiol groups of cysteine residues
- Inappropriate disulphide bond formation can occur if ROS takes electrons from cysteines causing misfolding, crosslinking and disruption of function (e.g. enzyme)
Describe ROS damage to lipids
- Free radical (e.g. •OH) extracts hydrogen atom from a polyunsaturated fatty acid in membrane lipid
- Lipid radical formed which can react with oxygen to form a lipid peroxyl radical
- Chain reaction formed as lipid peroxyl radical extracts hydrogen form nearby fatty acid
- Hydrophobic environment of bilayer disrupted and membrane integrity fails
What are sources of biological oxidants
Endogenous: • Electron transport chain • Peroxidases • Nitric oxide synthases • Lipooxygenases • NADPH oxidases • Xanthine oxidase • Monoamine oxidase
Exogenous • Radiation - Cosmic rays - UV light - X-rays • Pollutants • Drugs - Primaquine (anti-malarial) • Toxins - Paraquat (herbicide)
How can the electron transport chain be a source of ROS?
- NADH and FADH2 supply electrons (e−) from metabolic substrates
- e − pass through ETC and reduce oxygen to form H2O at Complex IV
- Occasionally electrons can accidently escape chain and react with dissolved O2 to form superoxide
Which is nitric oxide synthase?
- iNOS: Inducible nitric oxide synthase. Produces high NO concentrations in phagocytes for direct toxic effect.
- eNOS: Endothelial nitric oxide synthase (Signalling)
- nNOS: Neuronal nitric oxide sytnthase (Signalling)
NO• - toxis effects at high level
Signalling molecule - vasodilation, neurtransmission, s-nitrosylation
What is respiratory burst?
- Rapid release of superoxide & H2O2 from phagocytic cells (e.g. neutrophils and monocytes)
- ROS and peroxynitrite destroy invading bacteria
- Part of antimicrobial defence system
What is chronic granulomatous disease?
Genetic defect in NADPH oxidase complex causes
enhanced susceptibility to bacterial infections
• Atypical infections
• Pneumonia
• Abscesses
• Impetigo
• Cellulitis
What is superoxide dismutase?
Cellular defence Superoxide dismutase (SOD) • Converts superoxide to H2O2 and oxygen • Primary defence because superoxide is strong initiator of chain reactions • 3 isoenzymes: • Cu+-Zn2+ Cytosolic • Cu+-Zn2+ Extracellular • Mn2+ Mitochondria
What is catalase
Cellular defence
• Converts H2O2 to water and oxygen
• Widespread enzyme. Important in immune cells to protect against oxidative burst
• Declining levels in hair follicles with age may explain grey hair!
What is glutathione?
Cellular defence
• Tripeptide synthesised by body to protects against oxidative damage
• Thiol group of Cys donates e− to ROS. GSH then reacts with another GSH to form disulphide (GSSG).
• Glutathione peroxidase requires Selenium
• GSSG reduced back to GSH by glutathione reductase which catalyses the transfer of electrons from NADPH to disulphide bond
• NADPH from pentose phosphate pathway is therefore essential for protection against free radical damage
Major source of NADPH
SEE SLIDE
What is the pentose phosphate pathway?
- starts from glucose-6-phosphate
- important source if NADPH required for
- reducing power for biosynthesis
- maintenance of GSH levels
- Detoxification reactions
- produces C5 sugar ribose required for synthesis of
- nucleotides
- DNA and RNA
- No ATP synthesised
- CO2 produced
- rate limiting enzyme is Glucose 6-phosphate dehydrogenase
What are free radical scavengers?
Cellular defence
Free radical scavengers reduce free radical damage by donating hydrogen atom (and its electron) to free radicals in a nonenzymatic reaction
Vitamin E (α-tocopherol)
• Lipid soluble antioxidant
• Important for protection against lipid peroxidation
Vitamin C (Ascorbic acid)
• Water soluble antioxidant
• Important role in regenerating reduced form of Vitamin E
Others • Uric acid • Melatonin • Carotenoids • Flavonoids
What is galactosaemia?
Increased activity of aldose reductase consumes excess NADPH
Compromised defences against ROS damage
Crystallin protein in lens of eye denatured -> cataract
What are 3 key enzymes of which deficiency leads to galactosaemia?
Galactokinase
Uridyl transferase
UDP-galactose epimerase
What are the symptoms of galactosaemia?
Symptoms Hepatomegaly + Cirrhosis Renal failure Vomiting Seizure + Brain damage Cataracts Hypoglycaemia
What does G6PDH deficiency lead to?
Decreased G6PDH activity limits amount of NADPH
NADPH required for reduction of oxidised glutathione (GSSG) back to reduced glutathione (GSH)
Lower GSH means less protection against damage from oxidative stress
Oxidative stress then leads to
Lipid peroxidation
• Cell membrane damage
• Lack of deformity leads to mechanical stress Protein damage
• Aggregates of cross- linked haemoglobin (Heinz bodies)
Leads to haemolysis
What are Heinz bodies
- Dark staining within red blood cells resulting from precipitated haemoglobin
- Bind to cell membrane altering rigidity
- Increased mechanical stress when cells squeeze through small capillaries
- Spleen removes bound Heinz bodies resulting in “blister cells”
- Clinical sign of G6PDH deficiency
Describe the metabolism of paracetamol
At prescribed dosage paracetamol can safely be metabolised by conjugation with glucronide to sulphate
With high levels of paracetamol the toxic metabolite NAPQI accumulates.
NAPQI can have direct toxic effects:
Oxidative damage to liver cell
• Lipid peroxidation • Damage to proteins • Damage to DNA
NAPQI can be converted to glutathione but glutathione depletion can lead to oxidative damage to liver cells
Antidote acetylcysteine works by replenishing glutathione levels
