:) The Liver Flashcards
1
Q
Healthy Liver Function: Bile & Bile salts - 5 examples
A
- Synthesis & secretion of bile for digestion & absorption of fats.
- Secretes bile into bicarbonate rich solution to neutralize acid in the duodenum.
- Secretes cholesterol into bile.
- Covers plasma cholesterol into bile salts.
- Bile salts – for absorption of fat-soluble vitamin K (clotting factors).
2
Q
Healthy Liver Function: Metabolism - 3 examples
A
- Coverts plasma glucose to glycogen and triglycerides.
- Produces glucose from glycogen (gluconeogenesis).
- Produces urea – product of amino acid (protein) catabolism releases into the blood.
3
Q
Healthy Liver Function: Metabolism of Fats - 5 examples
A
- Converts plasma amino acids to fatty acids
- Synthesizes triglycerides and secretes them as lipoproteins.
- Converts fatty acids to ketones during fasting.
- Cholesterol metabolism.
- Synthesizes cholesterol & releases into the blood.
4
Q
Healthy Liver Function: Plasma proteins & clotting factors: - 2 examples
A
- Synthesizes & secretes proteins including plasma albumin, acute phase proteins, binding proteins for hormones & lipoproteins.
- Produces many of the plasma clotting factors including prothrombin & fibrinogen.
5
Q
Healthy Liver Function: Storage - Examples
A
Storage
Vitamin B12, Iron, Kupffer cells
6
Q
Healthy Liver Function: Endocrine Functions - Example
A
Endocrine functions
e.g. Secretes insulin-like growth factor 1 (IGF-1) in response to growth hormone. This promotes cell division in a number of tissues including bone
7
Q
Healthy Liver Function: Excretory Functions - Example
A
Removal of toxins from circulation
e.g. break down of alcohol
8
Q
7 Causes of Liver Impairement
A
- Alcohol consumption
- Non-alcoholic fatty liver disease – obesity
- Metabolic Disorders
- Viral Infection
- Autoimmune Hepatitis
- Cholestatic Disorders
- Toxins
9
Q
Liver 4 health stages
A
- Chronic damage to the liver
- Cell damage causes matrix deposition, resulting in cell death, causing angiogenesis to occur.
- Early fibrosis occurs, the disrupted structure causes loss of function & aberrant hepatocyte regen.
- Cirrhosis occurs, requiring a liver transplant.
10
Q
4 causes of liver health change
A
Liver health may change due to:
1. Genetic polymorphisms
2. Epigenetic marks
3. Cofactors (e.g. obesity & alcohol)
4. Injury
11
Q
NAFLD (Non-alcoholic Fatty Liver Disease): 3 Stages
A
NAFLD (Non-alcoholic Fatty Liver Disease):
1. Non-alcohol related fatty liver – fatty infiltration
2. Non-alcohol related steatohepatitis (NASH) +/- fibrosis – inflammation & fibrosis
3. Non-alcohol related steatohepatitis (NASH) - cirrhosis
12
Q
Alcoholic liver disease: 3 Stages
A
Alcoholic liver disease:
1. Excess alcohol leads to build up of fatty deposits – Fatty infiltration
2. Alcoholic hepatitis – alcoholic hepatitis
3. Alcoholic hepatitis - cirrhosis
13
Q
7 Signs of Liver impairment
A
Non-specific:
1. Generally feeling unwell & tired all the time
2. Loss of appetite, weight & muscle
3. Feeling sick (nausea) & vomiting
4. Tenderness/pain in the liver area
5. Spider-like small blood capillaries on the skin above waist level (spider angiomas)
6. Blotchy red palms
7. Disturbed sleep pattern
14
Q
Ascites cause: Reduced aldosterone pathway, 7 steps
A
Cirrhosis:
1. Reduced aldosterone metabolism
2. Increased aldosterone
3. Activation of renin-angiotensin system
4. Underfilling of circulation
5. Reduced renal blood flow
6. Salt & water retention
7. Ascites (fluid retention)
15
Q
Ascites cause: Portal hypertension 1 step, Reduced albumin 5 steps
A
Cirrhosis:
1. Portal hypertension causing ascites (fluid retention)
Cirrhosis:
1. Reduced albumin
2. Plasma protein production, reduced albumin
3. Decreased oncotic pressure
4. Transudation of fluid
5. Ascites
16
Q
Hepatic circulation & Cirrhosis’ effects
A
Hepatic vein to inferior vena cava.
Aorta to Hepatic artery.
Hepatic portal vein delivers nutrients to liver.
Cirrhosis: the liver shrinks & is scarred making it blood flow harder, increasing pressure in portal vein, causing portal hypertension
17
Q
9 Symptoms relating to loss of function &/or portal hypertension:
A
- Pruritis (itch)
- Jaundice
- Bleeding &/or bruising
- In men: enlarged breasts and shrunken testes
- In women: irregular or lack of menstrual periods
- Confusion, memory difficulties – hepatic encephalopathy
- Increased sensitivity to alcohol & medications
- Trembling hands & ‘liver flap’
- Staggering gait
18
Q
Liver Function Tests (LFTs): Impaired Biliary Secretion or Cholestasis: GGT
A
GGT (Gamma-glutamyl Transferase):
Elevated in cholestasis, and acute toxic damage including following excessive alcohol consumption. Not used for liver disease but liver mortality. Can help stage disease.
19
Q
Liver Function Tests (LFTs): Impaired Biliary Secretion or Cholestasis: Bilirubin
A
Bilirubin: Elevated in cholestasis (reduced flow of bile) & jaundice. Breakdown of haem, excreted in bile.
Breakdown product of haem, excreted in bile.
Elevated in cholestasis & jaundice.
20
Q
Liver Function Tests (LFTs): Impaired Biliary Secretion or Cholestasis: ALP
A
ALP (alkaline phosphate): Slightly elevated in acute hepatitis & drug induced liver toxicity
ALP isoenzymes may present in bones, kidneys, intestine & placenta.
Elevated in cholestasis (reduced bile flow), hepatic infiltration.
21
Q
Liver Function Tests (LFTs): Markers of Parenchymal Liver Disease: ALT (alanine aminotransferase):
A
ALT (alanine aminotransferase):
Elevated values may be caused by acute hepatitis and drug induced liver toxicity (eg. Paracetamol overdose)
Non-liver related elevated uncommon due to presence of low concentration in non-hepatic tissue
Parenchymal – functional liver disease
Enzymes released into the blood stream in response to hepatocyte injury.
22
Q
Liver Function Tests (LFTs): Markers of Parenchymal Liver Disease : AST (aspartate aminotransferase):
A
AST (aspartate aminotransferase):
If significantly elevated may be related to acute hepatitis, alcoholic hepatitis or drug induced liver toxicity – interpret in the context of the history and other LFTs
Parenchymal – functional liver disease
Enzymes released into the blood stream in response to hepatocyte injury.
23
Q
Liver Function Tests (LFTs): Impaired synthetic & detoxifying functions: Albumin
A
Albumin: plasma protein produced by the liver, reduced levels if liver function is impaired
24
Q
Liver Function Tests (LFTs): Impaired synthetic & detoxifying functions: Ammonia
A
Ammonia: Reduced ability to detoxify the blood then ammonia levels rise & may cause encephalopathy
25
Liver Function Tests (LFTs): Impaired synthetic & detoxifying functions: INR &/or Prothrombin time
INR &/or Prothrombin time: Vitamin K dependent. Elevated values suggest reduced production of clotting factors.
26
9 Causes of Liver disease
1. Family history
2. High cholesterol
3. Obesity
4. Age
5. Medication
6. Smoking
7. T2Diabetes
8. Blood pressure
9. Ethnicity
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4 medications to counteract liver disease
1. Obeticholic acid – PBC, bile acid agonist, FDA – close monitoring due to deaths, pruritis.
2. Elafibrinor – reduces circulating triglycerides, increases insulin sensitivity, increases HDL, reduces inflammation.
3. Vitamin E – anti-oxidant, reduces inflammation but poor evidence & risks (cancers, strokes).
4. Pioglitazone – increased insulin sensitivity takes fat from liver to adipose but ALT reverts quickly & weight gain, unlikely to continue use long-term
28
6 ways to manage Pruitis (itch)
1. Treat obstruction (endoscopy)
2. Antihistamines
3. Topical therapies – emollients or menthol in aqueous
4. Ondansetron
5. Rifampicin
6. Opioid antagonists
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Managing Encephalopathy: Wernicke's
Wernicke’s – mostly associated with alcoholic liver disease.
1. Lack of Vitamin B1 – Thiamine breaks down carbohydrate & in detox process, less thiamine absorbed.
2. Less Vit B storage, malnourishment, may have lost weight recently.
3. Leads to Korsakoff’s syndrome (dementia) – lack of Vit B & leads to brain swelling (encephalopathy). Long term development is Korsakoff’s syndrome.
Treatment: Thamine & Vit B Co strong
30
qManaging Encephalopathy: Hepatic encephalopathy
Hepatic encephalopathy:
1. Reduced ability to clear toxins, increasing ammonia circulating, causing increasing confusion & encephalopathy.
2. Increased permeability of the BBB allowing other neurotoxins to enter the brain & alter function.
3. Treatment focuses on increasing clearance of ammonia – laxatives, primarily lactulose.
4. Not absorbed, increases acid in gut ionizing nitrogenous products reducing ammonia production & coliform bacteria. Also clears gut.
Treatment: Lactulose & Rifaximin minimises ammonia circulation.
31
Ascites
Build up of fluid in the abdomen due to increased portal vein pressure (portal hypertension), reduced oncotic pressure in the blood (low alb), & activation of the renin-angiotensin system leading to increased salt & fluid reabsorption.
32
Ascites: 5 methods of treatment
Treatment:
1. Paracentesis (liquid draining) – every 2-4 weeks as an outpatient.
2. Albumin cover is given to reduce the risk of Acute Kidney Injury.
3. Salt restriction and diuretics (e.g. furosemide) – help reduce swelling
4. Liver transplant
5. TIPPS (transjugular intrahepatic portosystemic shunting): new connection between portal vein & hepatic vein
33
Managing Oesophageal Varices
If patient has symptoms of GI bleeding investigate urgently.
Endoscopic management:
Telipressin & antibiotics
To prevent re-bleeding use a Non-selective Beta blocker.
34
6 Key considerations of Drug Handling in Liver Impairment
Key Considerations:
1. Reduced hepatic function = reduced ability to metabolise drugs
2. Reduced plasma proteins = increased free drug levels of protein bound drugs
3. Reduced first-pass metabolism in cirrhosis, increased bioavailability
4. Avoid hepatotoxic drugs
5. Avoid sedative drugs
6. Be alert for drug side effects – dose reductions may be necessary
35
Phase 1 metabolism: 2 facts
Phase 1 metabolism:
1. Functionalization/activation of drugs (often oxidative):
2. Most important enzymes are cytochrome P450 enzymes (a.k.a. CYPs).
35
Phase 2 metabolism: 4 facts
Phase 2 metabolism:
1. Enzymes involved in conjugation for detoxification &/or excretion;
2. Important enzymes: UDP-glucuronic acid transferases (UDPGTs) & glutathione-S-transferases (GSTs).
3. Several metabolic pathways for same drug & minor pathways:
4. Enzymes usually metabolises several drugs, often with no structural similarity (important for drug-drug interactions & tolerance).
35
Cytochrome P450 enzymes: 4 facts
1. Microsomes (enzymes in endoplasmic reticulum)
2. Membrane bound – metabolism of hydrophilic drugs
3. Reaction needs NADPH, O2, NADPH-cytochrome P450 reductase (2ndry enzyme)
4. More selective oxygenases for steroids in mitochondria
36
6 Phase 1 reaction methods
1. Oxidations (especially by cytochrome P450s, mixed function oxygenases):
2. Hydrolysis (important for prodrug activation)
3. Hydration:
4. Dethioacetylation:
5. Isomerisation:
6. Reduction:
37
Metabolism of Chiral drugs
Enzymes (& other biological materials) are built from chiral molecules;
Different stereoisomers of drugs are different drugs;
Often metabolised to different products with different kinetics.
38
Mechanisms of cytochrome P450 enzymes
Enzymes possess a prosthetic haem group (which contains iron). Reactivity depends on iron ligand.
Reaction requires O2 (from air), NADPH, & auxiliary enzyme (a reductase)
Enzymes catalyse their reactions by negative catalysis (enzyme generates highly reactive chemical species & suppresses unrequired reactions).
39
The 5 step cytochrome P450 catalytic cycle
1. D (Drug) with Fe3+D, gains an electron & becomes Fe2+D. O2 is gained to make Fe2+O2D.
2. Next, Cytochrome b5 & NADPH-P450 reductase donates a proton & an electron, forming Fe2+OOHD.
3. The NADPH-P450 reductase donates an electron to Fe3+D earlier in the reaction.
4. The Fe2+OOHD undergoes a hydration reaction, producing (FeO)3+D, which becomes Fe3+DOH.
5. The DOH is the desired product.
40
P450 2nd Catalytic Mechanism
Type 1 substrates do not coordinate to iron.
Substrate binding to protein causes iron low-spin (6 ligand) to high spin (5 ligand) change (vacant site for O2 binding).
Reduction (P450 reductase) is required before dioxygen binds.
A 2nd reduction step occurs following O2 binding, before hydroxylation.
More than one hydroxylation mechanism (may depend on substrate).
41
Uncoupled reaction cycles
O2 & NADPH are consumed but substrate is not oxidised.
Under most conditions uncoupled reactions are small proportion of catalytic cycles.
Product is reduced oxygen (hydrogen peroxide).
Source of H2O2 is unclear, but results from an abortive catalytic cycle.
P450 enzymes can also function as organic peroxidases.
42
Define: Enzyme super family, give 3 examples
Enzyme super-family: The whole group of enzymes that catalyse the same or a similar type of reaction using a similar mechanism & are related by primary sequence homology or identity.
There are 3:
1. CYP enzymes (various oxidative reactions);
2. UDP-glucuronosyl transferases (‘glucuronidation’);
3. Glutathione-S-transferases (‘glutathione conjugation’).
43
Define: Homology & Identity
Homology: when sequences are aligned an amino acid in a particular position is conserved by type e.g., leucine could be substituted for isoleucine or valine, but not arginine;
Identity: when sequences are aligned an amino acid in a particular position is always the same, i.e., leucine must be leucine.
44
Identity and homology in a sequence
These enzymes are inducible (increased expression) by small molecules/drugs – results in faster metabolism of all drugs processed by the same enzyme.
Classification of enzymes based on amino acid primary sequence identity.
45
6 Important members of the super-family
Important members of super-family for drug metabolism include:
1. CYP3A4
2. CYP3A5
3. CYP3A7
4. CYP2D6
5. CYP2E1
6. CYP3A4
46
Drugs can be metabolised by more than one CYP enzyme
Different expression in children and adults – therefore different metabolism;
Drugs with very different structures can be metabolised by the same enzyme (basis of drug-drug interactions).
47
Hydroxylation mechanisms: Radical rebound mechanism - 6 steps
Hydroxylation mechanisms: Radical rebound mechanism
1. Substrate binds to P450 enzyme. The haem group activates O2.
2. Haem reduced Fe(III) to Fe(II) through electron transfer from NADPH via cytochrome P450 reductases. Once reduced, P450 binds O2 to Fe(II), forming (Fe(II)-O₂).
3. O-O bond in O-Fe complex is cleaved, facilitated by electron transfer, forms intermediate.
4. The intermediate abstracts Hydrogen from the substrate, forming a C-centred radical where hydroxylation occurs.
5. The Oxygen rebounds from the intermediate, onto the C-centred radical, attaching to the substrate, forming a hydroxylated product.
6. After rebound, product is release & P450 returns to original state.
48
Hydroxylation mechanisms: Concerted mechanism - 4 steps
Hydroxylation mechanisms: Concerted mechanism
1. The iron centre of the haem group is reduced, Fe(III) to Fe(II), & binds O₂, forming a Fe(II)-O₂ complex. Electrons transfer to form the intermediate.
2. The oxygen atom (from the intermediate) directly attacks the substrate in a concerted manner, oxygen transfer & C-H bond breaking occurs simultaneously.
3. The oxygen atom is transferred directly to the substrate, & the C-H bond is cleaved in a single, concerted motion. The substrate undergoes hydroxylation without generating a C-centred radical on the substrate, & the -OH is added to the substrate.
4. The result of the concerted reaction is the direct hydroxylation of the substrate. The hydroxylation happens without the intermediary radical step. This is a key difference from the radical rebound mechanism, where the oxygen species temporarily forms a radical before transferring to the substrate.
49
What 3 types of Cytochrome P450 reactions results from decomposition of unstable hydroxylation products?
Results from decomposition of unstable hydroxylation product:
1. Aliphatic dealkylation after hydroxylation.
2. Oxidative deamination.
3. Alcohol oxidation.
50
Cytochrome P450 reaction: Aromatic hydroxylation - 3 facts
1. This is the most common reaction because many drugs and xenobiotics contain aromatic rings.
2. Position of hydroxyl group depends on specific enzyme and drug.
3. -OH bonds to an aromatic ring.
51
Cytochrome P450 reaction: Aliphatic hydroxylation - 3 facts
1. Hydroxylation is usually stereospecific when a new chiral centre is formed (i.e., get R- or S- product).
2. Position & chirality of hydroxyl group depends on specific CYP & drug.
3. -OH replaces a H, in CH2
52
Cytochrome P450 reaction: Aliphatic hydroxylation and dealkylation - 4 facts
1. Reactions can occur for O-Me, S-Me, N-Me, etc.
2. Dealkylation of ethyl, & other alkyl groups can also occur.
3. Unstable hydroxylated intermediate formed.
4. Carbonyl compound eliminated (methanal in demethylations).
53
Cytochrome P450 reaction: Oxidative deamination - 2 facts
1. Alcohol causes deamination, leaving group is an NH4+
2. Produces an intermediate.
54
Cytochrome P450 reaction: Alcohol hydroxylation - 3 facts
1. Alcohol is converted into an Aldehyde by P450 enzyme
2. Water is eliminated from hydrated Aldehyde.
3. Ethyl-1,1-diol becomes ethanoic acid + water.
55
Cytochrome P450 reaction: Epoxidation - 4 facts
1. Epoxides can be hydrolysed to diol by epoxide hydrolase.
2. Epoxides are electrophiles.
3. Epoxide metabolites are often highly reactive and cause toxicities.
4. Epoxides react with epoxide hydrolase into diol, then CYP forms the “ultimate carcinogen.”
56
Cytochrome P450 reaction: Heteroatom oxidation - 2 facts
1. heteroatom (an atom other than carbon or hydrogen) is oxidised.
2. e.g. S oxidised into double oxygen bond, or -OH
57
Cytochrome P450 reaction: Dehalogenation - 3 facts
1. Alcohol displaces halogen.
2. Forms an intermediate with alcohol and halogen.
3. Produces a Product and H-halogen.
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Cytochrome P450 reaction: Reduction - 2 facts
1. Reductive reactions by P450 enzymes are less common, and often involve heteroatoms (azo and nitro-reduction).
2. e.g. N=N bond reduced to NH2 by breaking into two separate molecules.
59
6 Intrinsic factors affecting drug metabolism by CYPs
1. Age (neonates/elderly – different expression of CYP enzymes).
2. Hormonal & gender differences.
3. Genetic variability of CYP enzymes (race, populations, individuals)
4. Due to polymorphisms (substitution of 1/< amino acids with the primary sequence that affects the reaction product or rate for at least one drug).
5. Can lead to differences in metabolic rates of up to a factor of 100.
6. Can increase/decrease risk of getting some diseases.
60
6 External factors affecting drug metabolism:
1. Diet & nutritional status.
2. Method of cooking (induction of enzymes).
3. Alcohol & smoking (induction of enzymes, drug interactions).
4. Co-administration of drugs (Drug/Drug Interactions).
5. Exposure to drugs, solvents, xenobiotics, pollutants (induction of enzymes).
6. Diseases (especially of liver e.g., alcoholism).
Effects can be divided into short term (typically hours), medium term (days/weeks/months) or long term (years).
61
Drug-drug interactions (short term) - list 5
1. Drug/drug interactions (DDIs) occur when 2 drugs are used together & metabolised by the same enzyme (due to super-families).
2. Acute administration of both drugs usually leads to one drug being metabolised (‘the perpetrator’) & one drug (‘the victim’) not been metabolised.
3. The ‘victim’ drug can rise to toxic levels.
4. Can have harmful consequences e.g., paracetamol / alcohol combination can lead to paracetamol poisoning.
5. Can be used therapeutically e.g., alcohol / ethylene glycol poisoning.
Effects can be divided into short term (typically hours), medium term (days/weeks/months) or long term (years).
62
Induction of enzymes (medium term) - list 5
1. Levels of enzyme increase in presence of drug.
2. Results in tolerance (larger doses required for same effect).
3. Tolerance for all drugs metabolised by same enzyme.
4. Levels return to basal levels once drug is removed.
5. Repeat exposure gives even higher levels.
Effects can be divided into short term (typically hours), medium term (days/weeks/months) or long term (years).
63
Long term effects - list 5
1. Reduction of drug metabolism due to age
2. anatomical change in liver
3. Extreme exposure to drugs or poisons can cause loss of metabolism (affects all drugs)
4. Often due to generalised tissue damage
5. Exposure or liver disease
Effects can be divided into short term (typically hours), medium term (days/weeks/months) or long term (years).
64
7 Phase 2 metabolisms
Phase 2 metabolism should detoxify the drug or drug metabolite &/or allow excretion by adding a water-solubilising group:
Types of reaction include:
1. Glucuronidation (nucleophiles)
2. Glutathione conjugation (electrophiles)
3. Sulfation and phosphorylation
4. Methylation
5. Amino acid conjugation
6. Acetylation and acylation
7. Acyl-CoA formation
65
Glucucordination - 4 facts
1. Glucuronidation: the addition of glucuronic acid (electrophile) to nucleophiles.
(Nucleophiles must possess lone pair of electrons or - charge)
2. Common nucleophiles: OH, phenols, SH groups, carboxylate groups
3. Protonated amines are non-nucleophilic because the lone pair of electrons is unavailable
4. Amides are non-nucleophilic because their lone pair of electrons is resonance stabilised with the carbonyl group.
66
6 Nucleophiles
Nucleophiles:
1. Alcohol pKa 16
2. Phenol pKa 10
3. Thiol pKa 8.5
4. Carboxylate pKa 4-5
5. Amine (conjugate acid) pKa 9
6. Hydroxamic acid pKa 9
67
Glucuronidation reaction steps - 3
1. Glucuronic Acid is attached to UDP, forming UDP-glucuronic acid (UDP-GlcA), which is the donor molecule in the reaction.
2. UDP-Glucuronosyltransferase catalyses the transfer of glucuronic acid to a drug.
3. The enzyme uses UDP-glucuronic acid as a cofactor & transfers the glucuronic acid to a functional group (e.g. hydroxyl, amine, carboxyl, or thiol) on the substrate.
68
5 Factors determining dose relating to UDP Glucuronsyl transferase
1. UDPGTs found in membrane of ER – water solubilisation of hydrophobic CYP products.
2. UDPGTs are found in many tissues, especially liver, skin, intestine, kidney, lung, adrenals & spleen.
3. Drugs Mw <200 excreted in urine, >200 excreted in bile.
4. Low expression/enzyme activity in babies & neonates
5. Decrease first pass metabolism of drugs
69
Enterohepatic Circulation - 5 facts
1. Drugs MW >200 Da excreted in bile – possibility of recycling due to presence of gut β-D-glucuronidase (entero-hepatic circulation).
2. Used to salvage important biochemical (such as steroid hormones).
3. Entero-hepatic circulation increases apparent half-life of drug due to removal of glucuronic acid & re-absorption of drug.
4. Possible to get extended therapeutic effects of drugs.
5. Can get increased toxicity e.g., in cancer chemotherapy.
70
Classifications of UDPGT super-family
Important UDPGT enzymes in drug metabolism:
Liver: 1A1, 1A3, 1A4, 1A6, 2B4, 2B7, 2B10, 2B11, 2B15
Other tissues, GI tract: 1A10 – broad substrate selectivity.
Also important for metabolism of steroids derived from cholesterol.
71
4 Causes in variations UDPGT
1. UDPGT is made from several coding regions within the gene
2. UDPGT share exons 2-5, have variable exon 1
3. Polymorphisms affect drug metabolism, & can affect rate of transcription & translation or enzyme stability (amount of enzyme), & or catalytic activity
4. Varied drug metabolism rate due to polymorphisms
72
Gilberts’ syndrome: Common UDPGT Deficiency: - 3 facts
Gilberts’ syndrome: Common UDPGT Deficiency:
1. Most common mutation in Caucasians (UDPGT1A1*28) in promoter region of the gene – affects enzyme expression.
2. Most common mutation in Asians is missense (replaces an amino acid) – reduces enzyme activity.
3. Limits/prevents formation of Glucuronic acid-conjugate.
73
Gilberts’ Syndrome: Consequences: 4 Consequence
1. Bilirubin levels are increased in the blood (>17 μM).
2. Rises tend to be cyclical – can be promoted by stress, heavy exercise, dehydration etc.
3. Generally mild symptoms & Periodic jaundice
4. Affects metabolism of a few drugs (e.g. Atazanavir, Paracetamol )
74
Drug Interactions with UDPGTs - 4 facts
1. Drug interactions can occur due to two drugs been metabolised by the same enzyme (as for CYP enzymes).
2. Depletion of UDP-glucuronic acid causes reduced metabolism: e.g. paracetamol causes toxicity in neonates & babies due to bilibrun accumulation (degradation product of haem)
3. Possible increased metabolism (enzyme induction from drugs, e.g. cigarettes)
4. Complex behaviour with chiral drugs & drugs with multiples glucuronidation site
75
Chiral selectivity of UDPGT - 4 facts
1. Different enantiomers (or diastereoisomers or epimers) can react with different UDPGTs.
2. This is because enzymes are chiral (diastereomeric situation is produced).
3. UDPGT1B7 is non-specific with respect to the chiral configuration.
4. UDPGT1A10 & 1A9 specific for particular configuration.
76
Different Reactions Catalysed By Different UDPGTs - 3 facts
1. Some drugs have multiple nucleophilic groups e.g., salicylic acid.
2. Multiple products can result.
3. Which group gets glucuronidated depends on the nucleophilicity of the groups and the availability and activity of the enzyme (tissue).
4. Produces minor & major products based on nucleophilicity
77
Structures of Glutathione - 5 facts
1. Glutathione needed for maintaining reducing environment (protection against radicals, reactive oxygen species etc.). High [in] cells (mM).
2. Oxidation occurs to form the disulfide species.
3. Reduction to the sulfhydral form is mediated by a NADPH-dependent reductase.
4. Glutathione reacts with electrophiles (cf. UDP-glucuronic acid which reacts with nucleophiles).
5. Disulfide bond forms due to ROS, Glutathione reductase breaks this bond, producing 2 separate molecules.
78
Enzymes subunit - 4 facts
1. Three super-families of enzymes in humans (cytosolic, microsomal & mitochondrial)
2. Cytosolic & microsomal enzymes important for drug metabolism
3. Cytosolic enzymes are dimers (2 subunits)
4. Microsomal enzymes are trimers (3 subunits) & mostly metabolise arachidonic acid (inflammatory mediators)
79
Reaction of Glutathione with Epoxides - 4 facts (do flashcard)
1. Glutathione: detoxifies electrophiles (electron deficient species).
2. -SH group is usually deprotonated by enzyme to increase reactivity.
3. Important for metabolism of derivatives of polycyclic aromatic compounds.
4. Negative S binds to epoxide to produce an alcohol on a ring, and the S joins two molecules.
80
Reaction of Glutathione by 1,4-Conjugate Addition - 4 facts
(do flashcard)
1. Conjugate addition (a.k.a. Michael addition) occurs with electron-deficient double bonds.
2. Requires electron-withdrawing (usually carbonyl) group.
3. Reaction proceeds via an enolate intermediate.
4. Negative S binds to C=C bond, causes it to move, and produce a double bond.
81
Reaction of Glutathione in Paracetamol Metabolism - 3 facts
(do flashcard)
1. Reactive intermediate produced by cytochrome P450 enzyme followed by dehydration.
2.Reaction with glutathione is a 1,4-conjugate addition.
3. In acute toxicity glutathione pools can be depleted.
82
Reaction Of Glutathione In 1,6-Conjugate Addition - 3 facts
(do flashcard)
1. Oxidation by cytochrome P450 enzyme produces quinone intermediate.
2. Glutathione reacts with 1,6-conjugate addition.
3. Reaction restores aromaticity (shifts double bonds).
83
Conversion of Conjugates to Mercapturates - 5 facts
(do flashcard)
1. Processing occurs in liver & kidneys.
2. Results in negatively charged mercapturate.
3. Mercapturate excreted in urine (or bile if Mw > 400 Da.)
4. Pathway involves sequential removal of glutamyl & glycyl residues.
5. Acetyl group is transferred from acetyl-CoA
84
Conversion of Mercapturates by Cysteine Lyases - 4 facts
(do flashcard)
1. Glutathione displaces chloride in a SN2 reaction.
2. Removal of glutamyl- & glycyl residues.
3. Pyridoxal phosphate lyase activity via imine intermediate.
4. Further metabolism of reactive products (can lead to toxicities).
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Isoforms Of Cytosolic GSTs
1. Enzymes exist as dimers (two subunits associated non-covalently).
2. Enzymes can be homo- or hetero-dimers with monomer subunits of 20-25 kDa.
3. 20 isoforms of GST found in humans.
4. Subfamilies arise due to different combinations of monomers.
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Cytosolic GSTs - 8 sub-families
Sub-families are:
alpha (α)
mu (μ)
pi (π)
sigma (σ)
tau (τ)
zeta (ζ)
omega (ο)
kappa (κ)
e.g., GSTA1-1 is a homodimer of α subunits. Some dimers may have 2 different.
87
Induction of GST Enzymes - 6 steps
1. Expression is tissue specific expression
2. Wide variability in basal levels between individuals.
3. Induction of cytosolic & microsomal enzymes by physiological (oxidative) stress & xenobiotics (including drugs) can occur.
4. Some drugs cause induction by stimulating receptors which regulate fatty acid metabolism e.g., Peroxisomal Proliferation Activating Receptor-γ (PPAR-γ).
5. Leads to higher metabolism rate due to higher enzyme levels & activity.
6. Over-expression of enzymes associated with treatment resistance in several cancers.
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Polymorphisms Of GSTs - 4 facts
1. Can be in protein sequence, usually reduces protein levels.
2. Polymorphisms in the promoter region are also known.
3. Polymorphisms can have difference levels of effect e.g., GST M1 polymorphisms found in >20 different cancers, whilst polymorphisms in GSTA1 are only associated with colorectal cancer.
4. Decreased expression of GSTM1 due to polymorphisms is associated with decreased risk of prostate cancer.
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Polymorphisms of GSTs & Cancer chemotherapy - 4 facts
1. Cancer chemotherapy often uses cytotoxic drugs as therapeutic agents. Often highly electrophilic e.g., mustard gases.
2. Cancer drugs often react with DNA but also react with other nucleophiles.
3. Metabolism of these drugs is via the GST enzymes.
4. Polymorphisms in GSTA1 & P1 predict survival following chemotherapy – due to different metabolic rates of the drugs.
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Hydrolysis - 4 facts
1. Hydrolases can hydrolyse a variety of substrates: e.g. oxygen esters, thioesters, amides & carbamates.
2. Hydrolysis is a minor pathway of phase 1 metabolism.
3. Esters often used as pro-drugs to promote drug distribution, including uptake through membranes by passive diffusion (requires a lipophilic drug).
4. Carboxyl-ester hydrolases (CEH) catalyse hydrolysis of oxygen esters, hence conversion of the pro-drug to the drug.
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Ester Hydrolases - 5 facts
1. Enzymes unselective respective to substrates, but one generally predominates.
2. Substrates with large acyl groups & small -OH hydrolysed by hCE1 (in liver).
3.Substrates with small acyl groups & large -OH hydrolysed by hCE2 (small intestine & liver). Kidney & lungs of 2ndry importance.
4. Enzymes important for activation of ester pro-drugs to their carboxylate drugs.
5. Can be important in phase 1 metabolism to eliminate drug e.g., aspirin (drug is eliminated or carboxylate glucuronidated).
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Variation in Hydrolase Activity - 5 facts
1. hCE1 varies greatly: 6-30 fold
2. Hce2 varies by 3 fold
3. Unknown reason why, possibly polymorphisms, enzyme induction & inhibition, changes due to liver disease
4. e.g. G-A polymorphism in Hce1 can result in 100% loss of catalytic activity
5. Incidence varies on race, dose change may be needed
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Sulfotransferases - 3 facts
1. Sulfation done by sulfotransferases. Sulphates endogenous substrates & xenobiotics (drug sulphate derivatives)
2. Found in superfamilies related by sequence
3. Enzymes have distinct but overlapping substrate profiles:
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SULT family profiles (4)
SULT1 family – phenol substrates.
SULT2 family – aliphatic alcohol substrates including steroid hormones.
SULT3 family – amine substrates (positively charged compounds to negatively charged).
SULT4 family – unknown substrate preference.
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Sulphation reaction: 4 facts
1, Part of phase 2 metabolism. Occurs in liver.
2. Requires 3’-phosphoadenosine 5’-phosphosulfate (PAPS) as cosubstrate.
3. Drug or metabolite requires OH, SH or another nucleophilic group.
4. Product is – charged sulphate ester.
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Competition between sulfation and glucuronidation - 5 facts
1. Most drugs with nucleophilic groups can be glucuronidated and sulphated. These pathways can compete.
2. Sulphation predominates at [low] & glucuronidation at high [drug].
3. Because PAPS is limiting (UDP-glucuronic acid is not)
4. Sulfation product from a carboxylate group likely unstable & rapidly hydrolyse (because it’s an acid anhydride)
5. Ergo, glucuronidation predominates carboxylate groups.
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Phosphorylation - 4 facts
1. Requires nucleophilic group to react with (typically aliphatic alcohol)
2. Cosubstrate is ATP
3. Important for anticancer & antiviral agents where nucleoside analogue pro-drugs are activated to their corresponding nucleotide drugs.
4. Drugs can have multiple phosphate groups, but separate enzymes are required for each phosphate group
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O-Methylation - 6 facts
1. Part of phase 2 metabolism
2. Needs nucleophilic group to react with (OH, NH, SH) to make methyl ether, methyl amine, or methyl thioether
3. Reaction catalysed by O-,N- or S-Me transferase enzyme.
4. Cosubstrate is S-adenosyl-L-methionine, which is demethylated to S-adenosyl-L-homocysteine.
5. O-Methylation of drugs mostly carried out by catechol-O-methyl transferase (COMT) in the cytosol.
6. Opposite reaction to demethylation by cytochrome P450 enzymes.
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N-Methylation - 5 facts
1. Part of phase 2 metabolism. Catalysed by N-methyl transferase (HNMT, selective for substrates) nicotinamide N-methyl transferase (NNMT, non-selective).
2. NNMT metabolises most drugs due to lower substrate selectivity. Can produce + charged quaternary amines, with increased renal clearance.
3. Both enzymes have polymorphisms affecting metabolic rates.
4. Non-specific amine methyl transferase widely distributed & important in drug metabolism. Two isoenzymes with broad and overlapping substrate selectivity.
5. Phenylethanolamine N-methyl transferase found in adrenals & methylates catecholamine hormones (noradrenalin etc.)
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S-Methylation - 6 facts
1. Part of phase 2 metabolism.
2. Thiopurine methyl transferase (TPMT) metabolises purine derivatives.
3. Thiol methyl transferase broad selectivity
4. Polymorphisms exist resulting in variation in response & toxicity.
5. Can also get methylation of trivalent Arsenic compounds, found as environmental pollutants.
6. Pathways are complex with redox chemistry. Methylation is catalysed by arsenic (III) methyl transferase (where III represents the arsenic oxidation state).
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Amino acid conjugation - 3 (flashcard)
1. Part of phase 2 metabolism. Converts carboxylate drug to carboxylate.
2. Drug activated with ATP to give AMP mixed anhydride.
3. Conjugate has higher Mw & ergo may be excreted in the bile.
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Coenzyme A formation - 5 (flashcard)
1.Part of phase 2 metabolism. COOH activated as AMP mixed anhydride
2. CoA-SH used to form acyl-CoA ester
3. Acyl-CoA esters good electrophiles & react with nucleophiles e.g. OH(form esters), amines (form amines)
4. Sig. toxicities (allergies) result because of reaction of acyl-CoA ester with proteins & other biomolecules
5. Acyl-CoA formation for pharmacological activation of ibuprofen & similar drugs.
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Acetylation and Acylation - 5 (flashcard)
1. Part of phase 2 metabolism. Acetylation adds acetyl to amino, OH, or sulfhydryl to make an amide. ester, or thioester.
2. Product heavier (increased biliary excretion) & less polar. Drug may cause renal toxicity by precipitation.
3. Reaction uses N-acetyl transferase.
4. Important source of chloramphenicol resist (used treating eye infections)
5. Can also add fatty acids to steroid drugs using corresponding acyl-CoA ester & acyl transferase
