Biochem Flashcards
What are the urinary and digestive system effective at removing and what aren’t they good at?
The urinary system and digestive system are good at removing soluble compounds, such as urea, but not good for hydrophobic compounds
What are hydrophobic toxins called?
Xenobiotics
What is phase I detoxification?
CYPs catalyse a range of reactions: hydroxylation, epoxidation, dealkylation& oxidation
=> makes toxins more soluble
What does a typical cytochrome p450 catalysed reaction look like?
RH + NADPH + H+ + O2 → ROH + H2O + NADP+
What is phase II detoxification?
Addition of a sugar (egglucuronate) to the –OH group makes the foreign molecule even more soluble
How many CYPs do humans have?
How many different families are these from?
49
17
What is the most important CYP in humans - what does it do?
CYP3A4 - metabolism
These are inducible upon exposure to appropriate substances
What is the most important CYP in terms of polymorphisms - what does that mean?
CYP2D6
There is considerable variation in the efficiency and amount of CYP2D6 enzyme produced between individuals. Hence for drugs that are metabolized by CYP2D6 will show variation in processing time
What is the main organ for CYP mediated detox?
Liver
Which organelle plays the most important detox role?
ER
From which Microsomes are derived
What is common to all cytochrome P450s?
An active site that is specificfor a particular class of substrate
A haem prosthetic group with iron that alternates between Fe2+ and Fe3+ as electrons are donated to oxygen.
A cysteineanchor (-CH2-SH) from the protein that forms a ligand to the Fe in haem (the side chain sulphurcoordinates to Fe
A hydrophobic protein “foot”that anchors the P450 to the ER membrane
What are the steps involved in a P450 reaction?
Step 1 -NADPH is oxidised by cytochrome P450 reductase (releases H+and 2 e-)
Step 2 -The H+and 2 e-are used by CYP to reduce one of the two atoms of molecular oxygen to water.
Step 3 -The other oxygen atom is retained in a highly reactive form, and used to force a reaction (eg hydroxylation) on the substrate.
What are the major P450 reactions?
1) hydroxylation (MAIN!!!)
2) Aromatic hydroxylation
3) Epoxidation - Has double bonds which can then react with DNA
4) De alkyation,
What are the minor P450 reactions?
1] N-oxidation,
2] Sulfoxidation,
3] Dehalogenation
Name an example of a toxin created by CYP450
Aspergillusfungus on corn & peanuts makes aflatoxin
→ Converted to aflatoxin B1 epoxide by P450
→ Reacts with DNA
→ Mutagen
Define a poor metaboliser
Homozygous for one deficient allele or heterozygous for two different deficient alleles (4% of caucasians)
Define an intermediate metaboliser
Heterozygous for one deficient allele or carry two alleles that cause reduced activity (32%)
Define an extensive metaboliser
Two wild-type alleles (55%)
Define an ultra rapid metaboliser
Multiple gene copies (9 %)
What percentage of the population have inactive CYP2D6 - what is the consequence?
5-10%
Codeine has no analgesic action unless some is converted to morphine by the action of CYP 2D6. => Poor metabolisers get no relief from codeine
CYP2D6 deficient patients also fail to metabolise about 25 other drugs
What problem do ultra rapid metabolisers have?
They can rapidly destroy some drugs, making them ineffective due to too low concentration
- This is especially true with antidepressants and antipsychotics
What is a side effect of SSRIs with regard to CYPs
Some Selective Serotonin Reuptake Inhibitors (SSRI antidepressants) such as Prozac inhibit many CYPs (espCYP2D6)
This makes “extensive metabolisers” resemble “poor metabolisers”
=> contraindicated with other CYP2D6-metabolised drugs such as tricyclic antidepressants and antipsychotics
Why is grapefruit contraindicated when taking a number of drugs?
Grapefruit contains furanocoumarin, which inhibits CYP 3A4
What do cruciferous vegetables (brussels sprouts, Kale, cabbage etc) induce?
Induce the expression of CYP1A2
=> May decrease bioavailability of drugs such as haloperidol (an antipsychotic)
What are some other functions of CYPs?
CYPs are involved in vascular autoregulation, particularly in the brain.
CYP are involved in the formation of cholesterol, steroidsand arachidonic acid metabolites (in mitochondria, especially in the adrenals)
What is erythropoietin?
It is a hormone which is secreted by the kidney.
Signals via JAK2, required for survival, proliferation + differentiation of committed erythroid progenitors; major function is to promote erythroid differentiation + initiate Hb synthesis
What is erythropoiesis?
Broduction of RBCs Stem cell (pluripotent blast cell, not multipotent) -> RBC (7 days) = 2 million/ day
What are the stages of haemapoeitic stem cell development?
Blast cell (stem cell) -> proerythroblast (large cell) -> polychromatic erythroblast (slightly smaller) -> matures by expelling its nucleus + digesting its nucleus -> reticulocyte (immature red cell) -> 1 day later -> RBC (7-10um)
What is MetHaemoglobin?
It is a heam molecule with a Fe3+ iron. This has no value in carrying oxygen and is thus recycled
What are the regions of the spleen called?
White pulp
Red Pulp
What is the role of white pulp?
Contains T cells, B cells and accessory cells -> similar to LN but captures blood-borne antigens b
What is the role of red pulp?
Red pulp = system of blood vessels arranged to facilitate removal of old or damaged RBC from the circulation (this is affected by macrophages)
What are the cells responsible for RBC phagocytosis?
Primarily monocytes and macrophages, in LN and the spleen,
KUPFFER CELLS in liver
What is the mononuclear phagocyte system?
It is the system which regulates the blood
What are the functions of the mononuclear phagocyte system?
- Formation of new RBCs and WBCs (in embryo).
- Destruction of old RBCs and WBCs.
- Formation of antibody
- Formation of plasma proteins.
- Formation of bile pigments.
How do B and T lymphocytes assist in the destruction of old RBCs?
B and T lymphocytes may assist in recognition of old RBCs by coating with antibodies (opsonisation). Surface changes in the RBCs can also be directly recognised by phagocyticcells.
What are the properties of RBC that macrophages respond to in order to stimulate phagocytosis?
Reduced Neuramic acid
More phosphatidyl serine flips to the outer side of the membrane
Oxidative damage products
What aspect of neuramic acid do macrophages respond to?
As the RBC ages it encounters nueraminidase, which cuts off sialic acid (Neuramic acid). Over time enough is cut off that the macrophage will detect and destroy the cell
What are the oxidative products of the RBC that are detected from the macrophage?
Heinz bodies
Thallasaemia and Sickling also change the RBC surface to activate macrophages
What are the causes of haemolysis?
External attack on RBC
Congenital/genetic factors
What are some external attacks on the RBC which causes haemolysis?
Streptococcus, enterococcus (many species of bacteria) parasitic haemolysis eg. malaria
What are some congenital or genetic factors which cause haemolysis?
Sickle cell
Haemolytic disease of new born due to Ab (i.e. from mother’s antibodies crossing the placenta to the foetus)
What is the definition of haemolysis?
Shortens survival of circulating RBCs to less than 100 days (reducing haemtocrit)
What is hepatoblobin?
It is a plasma protein that binds Hb released from haemolysed RBCs at sites remote from the spleen.
What percentage of RBCs are haemolysed as sites remote from the spleen?
10%
Why does Hb need to be mopped up?
Hb contains Fe and haem = toxic
How do we measure haemolysis?
The hapatoglobin assay is used to monitor intravascular haemolytic anaemia.
High haemolysis decreases Hp levels.
When Hp is depleted free Hb can be excreted in the urine (giving it a red/reddish brown tinge)
What is the role of hemopexin?
Secondary system that transports haem to liver, where the iron can be recycled.
What can low hemopexin levels indicate?
Haemolytic anaemia
What are the consequences of increased haemolysis?
If excessive haemolysis -> conversion of Hb to bilirubin > capacity of liver to conjugate and excrete bilirubin -> unconjugated (indirect)hyperbilirubinaemia and jaundice.
Bilirubin catabolism causes ↑ stercobilin in the stool and ↑ in the urine and sometimes cholelithiasis (gall stones from bile acids)
Bone marrow increases RBC production (in response to haemolysis) -> reticulocytosis
What are the symptoms of increased haemolysis?
1) Pallor, fatigue, dizziness, possibly hypotension.
2) Haemolytic crisis (severe, acute haemolysis) is uncommon: accompanied by chills, fever, body pain, shock.
3) Severe haemolysis splenomegaly and jaundice.
4) Haemoglobinuria red-brown urine.
What are the steps in haem breakdown?
- Globin chain is released and hydrolysed to amino acids
- Iron which is found inside the haem molecule is then carried by transferrin
- Iron may be stored in bone marrow or liver.
3a. It may also be used for erthropoiesis in bone where it is added to globin using VitB12
How much Hb is bound to CO?
At any given time, around 1% of our haem carries CO (because they are being broken down); 1% burden of haem with CO that is non-functional
In smokers, 5% burden of haem-CO (less functional haem).
Describe the process of bruise formation.
1) Initially red as blood trapped in interstitium.
2) 1-2 days bluish-purple or blackish (deoxy and met-Hb)
3) 5-10 days: greenish/yellowish (biliverdin)
4) 10-14 days: yellow-light brown (bilirubin)
How is biliverdin produced from Heme?
Heme + NADPH + H+ + 3O2 → biliverdin + Fe2+ + CO + NADP+ + H2O
What the overall steps in haem breakdown?
1) HgB -CO → Red
2) Biliverdin → Green and very Hydrophobic
3) Bilirubin → Yellow
What is the role of serum albumin, why?
Serum albumin is responsible for carrying fats and hydrophobic molecules in the blood. In order to allow this it has crevices which allow hydrophobic molecules to bury their structure.
Bilirubin is highly hydrophobic and thus needs to be transported via serum albumin.
Osmotic control (as well)
How is bilirubin conjugated?
1) Bilirubin is released into the plasma and recirculated to the liver bound to albumin. (bilirubin-Albumin = here, it is unconjugated bilirubin)
2) Once it enters in to the liver via the fenestrated sinusoids
3) → into the space of Disse and transported across and into hepatocyte membrane
4) UGT1A1 (UDP glucuronosyltransferase 1) in liver hepatocyte conjugates 2 glucuronic acid moclecules onto bilirubin →bilirubin diglucuronide
5) This makes it water soluble, conjugated bilirubin
How does bilirubin get out of the liver?
cMOAT (canalicular multi specific organic anion transporter) actively pumps bilirubin diglucuronide into bile canaliculi and into the bile.
What is Dubin-Johnson syndrome?
Autosomal recessive defect in cMOAT = can’t pump conjugated bilirubin into bile canaliculi.
What happens in Dubin-Johnson syndrome?
Conjugated (water-soluble) bilirubin diglucuronides accumulation = conjugated hyperbilirubinaemia.
However, this is usually asymptomatic because diglucuronide can readily and alternatively be excreted by the kidneys.
what is Crigler-Najjar syndrome?
Genetic deficiency leading to total/partial lack of UGT1A1
What happens in Crigler-Najjar syndrome?
Bilirubin can’t be conjugated → can’t be excreted via bile nor can it be excreted by the kidneys
→high levels of unconjugated biliruibin in blood/plasma
What are the symptoms of Crigler-Najjar syndrome?
1) (unconjugated hyperbilirubinaemia) → persistent jaundice.
2) kernicterus → bilirubin encephalopathy
3) brain damage
What is the treatment for Crigler-Najjar syndrome?
Light therapy (10-12 hours a day to reduce bilirubin levels reducing kernicterus risk)
Patient’s survival is dependent on the indefinite continuation of this therapy [INCONVENIENT]
ii. Effective in children but efficacy reduces due to↑ thickness of skin and body surface/weight ratio
iii. Liver transplantation is efficacious, but limited donors + life-long immune suppressio
Why does light therapy help individuals with Crigler-Najjar syndrome?
Blue fluorescent tubes are optimum for converting free bilirubin to a more soluble isomer that can be excreted unconjugated in bile
The Z,Z isomer flips to E,Z and E, E-isomers and bilirubin becomes more soluble and is excreted by the kidney
What are the components of Bile?
1) Water (Mostly)
2) Bile pigments (breakdown products of Hb; in bile, this is mostly bilirubin diglucuronides)
3) Bile salts → detergents/emulsifiers, amphipathic.
4) Lecitihin→Fatty acids,
5) Cholesterol.
How much bile is produced bet pay?
About 1L
Where is bile reasbsorbed?
What percentage/
Terminal ileum
95%
How concentrated is the bile from the the gall bladder?
5x
What happens to bilirubin once it enters into the gut?
Excreted in the bile into the duodenum
Conjugated bilirubin is acted on by bacterial proteases;
Bacterial proteases cleave off the 2 glucuronic acid molecules →deconjugates bilirubin.
Bilirubin converted to → urobilinogen → stercobilin and urobilin.
What is the fate of stercobilin?
It is excreted in the feces and give it the brown colour
What happens to urobilinogen?
90% is excreted in the feces
10% is reabsorbed
Portal vein
Heaptocytes
9% - exits via cMOAT into the bile again
1% enters into the blood and is eventually excreted by the kidneys
What is urobilinogen oxidised to? What is the result?
It is oxidised to urobilin
Urobilin is yellow hence yellow pee
What do increased levels of urobilinogen in the urine indicate?
Increased RBC breakdown, heamtoma, poisionging and liver cirrhosis
What do low levels of urobilinogen in the urine indicate?
Low levels indicate a blockage in bile ducts and bile production failure.
Could be due to a gallstone
What is kernicterus?
Bilirubin is neurotoxic at high levels and thus can cause encephalopathy (kernicterus)
What is the enterohepatic urobilinogen cycle?
Urobilinogen is a colourless product of bilirubin reduction. It is formed in the intestines by bacterial action. Some urobilinogen is reabsorbed from the gut and excreted by the kidney. This constitutes the normal “enterohepatic urobilinogen cycle”.
