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Detoxification by the
Liver
Phase I and II reactions
Xenobiotics
• Foreign chemical substance
• Can be absorbed across lungs, skin or ingested
• Drugs are considered xenobiotics
• Excreted in bile, urine, sweat, breath
Pharmacologically Active
Compounds
• Lipophilic
• To pass through plasma membranes to reach
metabolising enzymes
• Non-ionised at pH7.4
• Bound to plasma proteins
• To be transported in the blood
Enzymes
• Microsomal and Non-microsomal enzymes
• BOTH be involved in phase I and II reactions
• Microsomal enzymes mainly Phase I
• Non-microsomal mainly Phase II
Microsomal enzymes
• Located on smooth endoplasmic reticulum
• Phase I reactions – biotransform substances
• Phase II – glucuronidation
• Activity can be induced or inhibited
• Drugs, food, age, bacteria, alcohol
• Examples: Cytochrome P450 (CYPs), Flavin monooxygenase (FMOs), UDPglucuronosyltransferase (UGT)
Non-microsomal enzymes
• Located in cytoplasm and mitochondria
• Non-specific so Phase I and Phase II reactions
• All conjugation reactions EXCEPT
GLUCURONIDATION
• Non-inducible
• Genetic polymorphic – affects metabolism
• Examples: protein oxidases, esterases, amidases, conjugases
(transferases), alcohol dehydrogenase, aldehyde dehydrogenase
Drug Metabolism
• Why?
• Most drugs excreted by kidneys
• Lipophilic drugs not effectively removed
• AIM: To make drugs more polar
• Mostly occur in liver
• 2 mechanisms – phase I and II reactions
• Usually sequentially
Phase I
• Non-synthetic catabolic reactions
• Oxidation, Reduction, Hydrolysis
Phase I reactions
• Oxidation
•
•
•
•
Hydroxylation (add –OH)
Dealkylation (remove –CH side chains)
Deamination (remove –NH)
Hydrogen removal
• Reduction
• Add hydrogen (saturate unsaturated bonds)
• Hydrolysis
• Split amide and ester bonds
Phase I
• Non-synthetic catabolic reactions
• Oxidation, Reduction, Hydrolysis
• Known as ‘functionalisation’
•
•
•
•
Introduces reactive group to drug
Includes adding or exposing –OH, -SH, -NH2, -COOH
Product usually more reactive
Small increase in hydrophilicity
• Mainly occur in the liver
• Mainly catalysed by Cytochrome P450
• Drug has to get into cell – more lipophilic
Cytochrome P450 Enzymes
• Type of microsomal enzyme
• Phase I reaction
• Haem group to oxidise substances
• Products more water soluble
Cytochrome P450
• Large family with prefix CYP – known as isoforms/
isozymes
• 1st number – indicates the family the enzyme belongs to
• Letter – to indicate subfamilies
• 2nd number – individual genes involved
• Isoforms catalyse different reactions (specificity)
• Some important isozymes – CYP1A2, CYP2C9,
CYP2C19, CYP2D6,CYP2E1, CYP3A4
Cytochrome P450 Reductase
• Flavoprotein
• Contains both Flavin adenine dinucleotide (FAD) and
Flavin mononucleotide (FMN)
• FAD – accepts electrons from NADPH
• FMN – electron donor to CYPs
Cytochrome P450 Reactions (oxidation)
Drug
NADPH
H
NADP+
P450[Fe3+]
Flavoprotein
(oxidised)
TIME TODrug
PUTHIT
NADPH +
H+
Flavoprotein
(reduced)
+ O2
TOGETHER!
+ RH  NADP+ + H O + R-OH
2
Drug
OH
e-
P450[Fe3+]
O2
P450[Fe2+]
Drug
P450[Fe3+]
H
eO2
Drug
P450[Fe2+]
Drug
H
H2O
2H+
OH
Remember!
• Non-microsomal enzymes
•
•
•
•
Alcohol dehydrogenase
Aldehyde dehydrogenase
Reduction
Hydrolysis
• Phase I reactions can:
•
•
•
•
Inactivate drug
Further activate drug
Activate drug from pro-drug (inactive form)
Make a drug into a reactive intermediate (could be
carcinogenic or toxic)
Phase II
• Synthetic anabolic reactions
• Glucuronidation, sulfation, Glutathione conjugation,
amino acid conjugation, acetylation, methylation, water
conjugation
• Known as ‘conjugation’ reactions
• Attachment of substituent groups (endogenous
molecules)
• Usually inactivate products
• Catalysed by transferases
• Significantly increase hydrophilicity for renal
excretion
• Also mainly in the liver
Glucuronidation
• Glucuronosyltransferase (UGT) – microsomal
enzyme, phase II reaction.
• Uridine diphospho-glucuronic acid (UDPGA)
needed to conjugate glucuronic acid.
• Pathway for bilirubin conjugation and drugs
including corticosteroids & paracetamol.
Glucuronidation Reaction
UDPGA
Drug
UGT
Uridine
diphosphate
Drug
Glucuronide
MORE HYDROPHILIC!
Remember!
• Most phase II reactions involved non-microsomal
enzymes
• Mostly found in the cytoplasm or mitochondria
Elimination (usually polar drug, excreted
unchanged)
DRUG
Phase II
Elimination
(functionalised without
Phase I)
Phase I
Phase II
Elimination
Aspirin
• Analgesic
• NSAID
• Non steroidal anti-inflammatory drug
• Antiplatelet
• Irreversibly inhibits cyclooxygenase (COX)
Aspirin Phase 1 metabolism
• Prodrug so it is activated upon metabolization
• Hydrolysis reaction
• Aspirin (+H20) → Salcylic acid + Ethanoic acid
• Salcyclic acid is the active anti-inflammatory and
analgesic
Aspirin Phase 2 metabolism
• Conjugated with glycine or glucuronic acid
• Forms a range of ionised metabolites
• Excreted in the urine
Paracetamol
• Also known as Acetaminophen
• Analgesic
• Antipyretic agent
Paracetamol Metabolism
• Predominantly PHASE 2 metabolism
• Conjugation with glucuronic acid and sulphate
Paracetamol toxicity
• If stores of glucuronic acid and sulphate are running
low…
• Undergoes PHASE 1 metabolism (oxidation) to
produce toxic NAPQI
• This is removed by conjugation with glutathione
• In overdose stores of glutathione can run low
leading to toxicity
• Treated with N-Acetyl Cysteine
Alcohol metabolism
• Ethanol
→
Acetaldehyde →
Acetate
(ADH)
(ALDH)
• Acetate  CO2 + H2O
• ADH – Alcohol Dehydrogenase
• ALDH – Aldehyde Dehydrogenase
• Operate at different speeds in different people
• Acetaldehyde
• Carcinogenic
• High levels: Facial flushing, rapid heartbeat, nausea
Any Questions?