HIS02 Iron Metabolism And Haem Biosynthesis Flashcards
Why do we need Fe and issues with Fe metabolism
Fe:
- abundant metal
- multiple oxidation states —> useful to **relay electrons from one molecule to another —> **Oxygen transport
- can only acquire from external environment
Important issues:
- How to reduce toxicity of Fe
- Availability / Storage but without overloading —> Fe homeostasis
***Where is iron found in human body
- Does not exist as free metal ion in body (∵ very toxic)
- Must be bound to molecule (protein / organic compound e.g. **Heme OR in form of a structure: **Iron-sulphur cluster found in proteins in mitochondrial respiration)
- Intracellularly: Fe usually exist in 2+ oxidation state (**Fe2+) —> excess Fe bound to **Ferritin (storing Fe)
- Extracellularly (e.g. plasma): ***Fe3+
Problems with Fe homeostasis
- Main issue: ***Uptake is controlled, Loss is not
- Mechanisms exist for uptake of Fe but NO mechanism for excreting Fe
- Fe is lost but not excreted from body
- Loss of Fe cannot be controlled (natural slothing of cells from GI tract / menstrual cycle)
- Fe homeostasis cannot be achieved by usual principle of balancing acquisition with excretion
- Clinical significance: Fe overloading in blood transfusion
How to achieve Fe homeostasis
- Recycle Fe within body with 0% loss
- but not possible due to inevitable loss for various reasons
- also growth and development of body have ↑ demand for Fe - Placing ***intake of Fe under control of the “uncontrollable loss” —> achieve Fe homeostasis
—> Better option
Example of Hb:
Physiological / Pathological events (e.g. Hb biosynthesis, blood loss)
—> Loss of Fe
—> Reduction of total Fe pool
—> Stimulate uptake of Fe from dietary sources
—> Repletion of total Fe pool
Summary: Uncontrollable loss of Fe —> Depletion of Fe —> Repletion of Fe from dietary sources (Under Control)
Heme
- Organic molecule of Fe
- Fe confers Hb with ability to ***carry oxygen
- Fe react with ***O2 chemically rather than remaining as stable partners without any chemical interactions —> ∴ free Fe is toxic
- Fe complex with ***Porphyrin (a cage) —> Heme —> reactivity of Fe stabilised (stay in 2+ oxidation state) —> ∵ orbitals of Fe are now partially engaged
- However, Heme in ***free state is still significantly reactive / toxic to cells
Toxicity of heme
Free heme react with organic hydroperoxide, unsaturated fatty acids, alcoholic compounds
—> ***Free radicals, Superoxide anions
—> Readily damaging other biological compounds
Toxicity of heme is much reduced when interact with **a.a residues of **Globin chains
—> Fe in Hb now only “active” enough to hold oxygen in ***molecular state without forming Fe/O2 compound
Heme synthesis
Synthesis of Porphyrin begins in mitochondria matrix:
**Glycine + **Succinyl-CoA (intermediate in TCA cycle)
—(condensation, by ALA synthase)—> 5-aminolevulinic acid (ALA)
—(transported out of mitochondria into cytoplasm, acted on by enzymes in stepwise manner)—> Coproporphyrinogen III
—(transported back to mitochondria matrix, at the same time further transformation)—> Porphyrin acquire Fe2+ ion on inner membrane of mitochondria
—> Heme
Problem: **Balance between Fe supply and Porphyrin synthesis
- too much Porphyrin / free Fe —> toxic
—> **Biosynthesis of Porphyrin has to match with availability of Fe
Fe availability and Porphyrin synthesis
Erythropoietin (Epo)
—> secreted by Kidney
—> bind to Epo-receptor in **Erythroblast
—> stimulate Erythroblast-specific transcription factors responsive to Epo
—> activate transcription of **ALAS-2 gene
—> ALAS-2 mRNA
—> ***an Iron-responsive-element (stem loop structures, cannot be translated, act as regulator) is present in the 5’ untranslated region of ALAS-2 mRNA
Absence of Fe / Fe depletion: —> ***Iron Regulatory Protein (IRP) bind to Stem loop structure —> ***block ribosome attachment —> ***block translation of ALAS-2 mRNA —> no enzyme to synthesise Porphyrin
Presence of Fe-S cluster / Presence of Fe2+:
—> bind to Fe-S cluster (S atoms from cysteine)
—> conformational change of IRP
—> Dissociation of IRP from Stem loop
—> allow translation of ALAS-2 mRNA
—> **ALAS-2 protein (*enzyme synthesising Porphyrin)
—> Porphyrin + Fe2+
—> Heme
Heme release and translation of ALAS-2 mRNA + Globin mRNA
Recruitment of initiator tRNA can be blocked by intracellular protein in erythroid cells know as **HRI (Heme-regulated inhibitor, a protein kinase)
—> HRI **phosphorylate ribosome
—> ***block binding of initiator tRNA to ribosome
Presence of **Heme
—> **block HRI action by binding to HRI (inactivated protein kinase)
—> allow binding of initiator tRNA to ribosome
—> translation of **ALAS-2 mRNA + **Globin mRNA
—> **Porphyrin + **Globin
—> Haemoglobin
***Overall: Erythropoietin —> Transcription of ALAS-2 gene —> ALAS-2 mRNA —> Presence of Fe2+ —> IRP dissociate from ALAS-2 mRNA —> Translation of ALAS-2 mRNA —> ↑ ALAS-2 protein —> ↑ Porphyrin —> ↑ Heme —> block HRI —> further translation of ALAS-2 mRNA + Globin mRNA —> further ↑ Porphyrin + ↑ Globin (consume Heme —> prevent accumulation of free Heme (toxic)) —> ↑ Haemoglobin (↑ Haemoglobin formation to consume Fe2+ to avoid Fe overload)
***Whole process of haemoglobin formation
Erythropoietin —> Transcription of ALAS-2 gene —> ALAS-2 mRNA —> ***Presence of Fe2+ —> ***IRP dissociate from ALAS-2 mRNA —> Translation of ALAS-2 mRNA —> ↑ ALAS-2 protein —> ↑ Porphyrin —> ***↑ Heme —> ***block HRI —> further translation of ALAS-2 mRNA + Globin mRNA —> further ↑ Porphyrin + ↑ Globin (consume Heme —> prevent accumulation of free Heme (toxic)) —> ↑ Haemoglobin (↑ Haemoglobin formation to consume Fe2+ to avoid Fe overload)
Negative feedback pathway (Heme-mediated):
Heme: May inhibit ***unloading of Fe from transferrin (in form of Transferrin-bound Fe3+)
—> prevent release of Fe2+
—> make less heme
—> balance globin synthesis from time to time
***Transfer of Fe into RBC precursor and Regulation by heme
Transferrin-bound Fe3+
—> bind to Transferrin receptor
—> Transferrin + Fe3+ + Transferrin receptor
—> Endocytosis
-
Within endosome:
1. Fe3+ is reduced by **Ferrireductase to Fe2+ —> transported out of endosome by **DMT1 (Divalent metal ion transporter 1)
2. Transferrin dissociate from Fe3+ —> ***Apotransferrin —> Apotransferrin + Transferrin receptor —> Recycled to cell surface
Heme:
- May inhibit unloading of Fe from transferrin —> prevent release of Fe2+
- actual mechanism unknown
Heme regulation in termination of Hb synthesis during maturation of red cells
Postulated negative feedback model: Fe2+ —> ↑ Heme synthesis —> inhibition of unloading of Fe from Fe-loaded transferrin —> ↓ Fe uptake —> ↓ ALAS-2 expression —> ↓ Heme synthesis —> Termination of Hb synthesis
***Haemoglobin protein
- Largest consumer of Fe in body
- Fe mainly lost in form of Fe-containing protein
- Most abundant protein in haematological system
- Critical for aerobic respiration
- Synthesised by erythroid cells in their late stage of differentiation
- Mature Hb protein composed of multiple components:
1. Globin polypeptides (α + β chains)
2. Porphyrin
3. Fe
—> ALL components must be in balance (NO components should be in excess)
—> Biosynthesis of Hb is a highly coordinated process
—> Communication (***via Heme) between Fe availability, Porphyrin synthesis, Globin synthesis
Fe in excess: **Toxic
Porphyrin in excess: **Porphyria (hereditary disorder)
Globin in excess: ***Protein stress in cells
Recycling of Fe in human body
Theoretically Fe is self-sufficient:
Aging of RBC
—> Phagocytised in Liver and Spleen (+ Bone marrow)
—> Degradation of senescent RBC
—> Release of Fe into blood (+ Turnover of Fe-containing proteins in non-erythroid cells e.g. CYP450)
—> Fe taken up by erythroid cells for Heme synthesis
—> Maturation of erythroid cells
—> RBC
—> However, there is inevitable loss of Fe from body due to sweating, blood, exfoliation of epithelial cells from body
—> Repletion of Fe from diet
***Degradation of Haemoglobin
Haemoglobin
—> Heme (+ Globin)
—(**Heme oxygenase)—> Porphyrin **break open
—> Biliverdin + Free Fe
—(Biliverdin reductase)—> ***Bilirubin (transported in blood in association with serum albumin)
—> Urobilinogen
—> Urobilin / Stercobilin
Fe:
Fe2+ released from lysosome-related vesicles via **NRAMP1 (~ DMT1) into cytosol
—> exit cell via **Ferroportin (at the same time oxidised by ***Ferroxidase / Hephaestin)
—> Fe3+
—> Transferrin-bound Fe3+ (transport in blood)
Enzymes involved:
- ***Heme oxygenase —> catalyse cleavage of one of Methine bridges of protoporphyrin
- ***Cytochrome P450 reductase —> maintain Fe as Fe2+ (NADPH provide electrons)
- ***Biliverdin reductase —> converts Biliverdin to Bilirubin (NADH, NADPH provide electrons)
***Recovery of Fe from Heme in macrophage
Phagocytosis of RBC
—> RBC broken down
—> Fe2+ released from lysosome-related vesicles via ***NRAMP1 (~ DMT1) into cytosol
Within cytosol:
- Released to extracellular space via **Ferroportin (at the same time oxidised by **Ferroxidase / Hephaestin)
—> Fe3+
—> transported in blood as Transferrin-bound Fe3+ (more stable form, Fe2+ is unstable as it will release electron —> toxic) - Stored as protein-bound Fe3+
Transport of Fe in blood
Major (High affinity) (20-30% occupation):
***Apotransferrin
—(+ Fe3+)—> Monoferric transferrin
—(+ Fe3+)—> Diferric transferrin (2 Fe)
Minor (Low affinity) / Non-transferrin bound Iron (NTBI):
- Serum albumin —(+ Fe3+)—> Iron-bound serum albumin
- Low molecular weight compound (e.g. a.a., glucose)—(+ Fe3+)—> complex
Prevention of Fe overload in blood
- Large buffer / reserve of free Transferrin in blood
- High affinity of Transferrin for Fe (Kd: 10^-23)
- Further Fe-binding capacity provided by non-proteinaceous compounds
- ∴ Very low concentration of free Fe found in plasma —> important for minimising toxic effects of Fe
- Excess Fe in blood can also be taken up by cells
Dietary Fe to compensate for loss of Fe
2 types of Fe sources:
-
**Non-heme Fe (Fe in a.a. / sugars, mostly Fe3+, low solubility)
—(Gastric HCl)—> **Soluble Fe3+ (bioavailable)
—> mostly absorbed in duodenum, jejunum -
**Heme Fe (e.g. in meat)
—(digestion)—> **Isolated form (available for absorption)
—> mostly absorbed in duodenum, jejunum (but by a different mechanism from that of Non-heme Fe)
Absorption of Heme Fe by enterocyte
- Heme **transporter (apical)
—> Heme directly transported out of cell (basolateral)
—> Heme bound to **Hemopexin
—> Heme-Hemopexin complex transported in blood and picked up by other cells
OR
—> Degradation to Fe2+
—> Fe2+ stored as **Ferritin in enterocyte / transported out via **Ferroportin (at the same time oxidised by **Ferroxidase / Hephaestin)
—> Fe3+ transported in blood as **Transferrin-bound Fe3+ - Heme **receptor (apical)
—> **endocytosis
—> degradation of Heme within endosome by **Heme oxygenase
—> Fe2+ stored as **Ferritin in enterocyte / transported out via **Ferroportin (at the same time oxidised by **Ferroxidase / Hephaestin)
—> Fe3+ transported in blood as ***Transferrin-bound Fe3+
Fe-Ferritin: storage form of Fe, when there is no immediate demand for Fe
Absorption of non-Heme Fe by enterocyte
Non-Heme Fe (mostly Fe3+)
—(Ferrireductase / Duodenal cytochrome b at apical surface)—> Fe2+ (in GI lumen)
—> transported into cell via **DMT1
—> Fe2+ stored as **Ferritin in enterocyte / transported out via **Ferroportin (at the same time oxidised by **Ferroxidase / Hephaestin)
—> Fe3+ transported in blood as ***Transferrin-bound Fe3+
***Major factors influencing absorption of Fe
- Iron stores in body
- Rate of erythropoiesis
- Hypoxia
- Inflammation (***↑ Hepcidin!!!)
—> ALL induce liver to ↓ level of Hepcidin (a hormone)
—> ↓ Degradation of Ferroportin
—> ↑ Fe3+ transported out of enterocyte into blood
Fe controls synthesis of Hepcidin
Major cellular Fe reservoir —> ↑ Demand for heme —> Depletion of Fe reservoir —> ***↓ in Plasma Fe-Transferrin —> ***Plasma Fe level sensed by liver —> Hepcidin level changes —> Change in uptake of Fe —> Restore Fe in cellular Fe reservoir
Regulation of Ferroportin by Hepcidin
Hepcidin bind to Ferroportin
—> ***Internalisation of Ferroportin-Hepcidin complex into cell by Endocytosis
—> Endosome
—> Degradation within cell
Adjustment of Fe absorption in response to ↑ plasma Fe
↑ Plasma Transferrin-bound Fe3+ (Fe-Tf)
—> ↑ Hepcidin synthesis in liver
—> Hepcidin bind to Ferroportin
—> ↑ ***Internalisation of Ferroportin-Hepcidin complex into cell by Endocytosis
—> ↑ Degradation of Ferroportin
—> ↓ Release of Fe from basolateral side of enterocyte
Model for regulation of expression of Hepcidin gene
Body Fe status —> reflected by ***plasma Fe-Tf level
**HFE-TfR1, **TfR2, **BMP receptor (all on hepatocyte surface, detect Fe-Tf level in blood)
—> down-stream signals
—> control **SMAD family of proteins in cytosol
—> migrate to nucleus
—> Activate / Inhibit ***transcription of Hepcidin gene
BMP6 (a binding protein derived from non-parenchymal cells in liver)
—> bind to Fe-Tf
—> BMP6-Fe-Tf complex bind to BMP receptor
Stress erythropoiesis
e.g. Hypoxia
—> ↑ Erythropoietin from Kidneys
—> ↑ **Erythroferrone (ERFE) from **bone marrow
—> ↓ Hepcidin from liver
—> ↓ Degradation of Ferroportin on enterocyte
—> ↑ Fe availability
—> ↑ Porphyrin (i.e. Heme)
—> ↑ Globin synthesis
—> ↑ Haemoglobin synthesis
—> ↑ Erythrocyte synthesis in bone marrow
Ferritin vs Transferrin
Ferritin: Fe storage
Transferrin: Fe transport
DMT1: transport of Fe2+ into cell (from endosome / outside cell)
NRAMP1: transport of Fe2+ into cell (from lysosome)
Ferroportin (Macrophage + Enterocyte): transport of Fe2+ out of cell (ferroxidase convert Fe2+ into Fe3+)
Ferrireductase: convert Fe3+ to Fe2+ (uptake of Fe3+ by Erythroblast + absorption of Non-heme Fe)
Ferroxidase: convert Fe2+ to Fe3+ (transport of Fe3+ in blood)
