HIS04 Red Cell Metabolism Flashcards
Major functions of mature RBC
Carrier of oxygen —> effectively deliver oxygen to tissues and cells
Oxidative damage to RBC
- Hb(Fe2+) —(+ O2, H+ (from hydroxonium ion (H3O+)))—> Hb(Fe3+) + ***Superoxide
—> Superoxide
—> H2O2 (non-specific oxygen species)
—> Oxidative damage in RBC
(Hb(Fe2+): Deoxyhaemoglobin; Hb(Fe3+): Methaemoglobin —> cannot bind oxygen)
- Neutrophils, Macrophage, Endothelial cells
—> produce Reactive oxygen species (probably H2O2) (to kill pathogens)
—> diffuse freely into plasma membrane of RBC
—> Oxidative damage in RBC
***2 major approaches to suppress formation of Methaemoglobin Hb(Fe3+)
- Chelation of Fe2+ by Histadine residue in Hb (i.e. binding in Porphyrin ring)
—> ***Histadine residue of Globin blocks access of H+ to participate in Fe2+ oxidation (Steric hindrance) - RBC can protect itself against oxidative damage (**PPP + **Glutathione antioxidant system)
—> Glucose as source of electrons
—> Oxidation of G6P by G6PD during Pentose phosphate pathway (PPP)
—> produces NADPH
—> **NADPH serves as donor of electrons
—> Transfer of electrons to **GSSG (glutathione disulfide, oxidised form) by **Glutathione reductase (NADPH —> NADH to be recycled again)
—> **GSH (reduced form, carrying electrons)
—> Reduction of ROS (e.g. H2O2) by antioxidant enzymes (e.g. ***Glutathione peroxidase) (electrons from GSH)
AND
—> Inhibit damaging effect of Hydroxyl free radical to lipids, proteins, DNA etc.
簡單而言:
PPP —> NADPH —> donate electron —>
GSSG —(Glutathione reductase)—> GSH (contain electron) —(Glutathione peroxidase)—> Reduction of ROS
(- RBC can produce energy via glycolysis / PPP
- PPP uses G6P dehydrogenase to oxidise G6P
- PPP: Glucose —> G6P —> Ribose (for glycolysis / synthesis of nucleotides, unimportant in RBC))
Glutathione antioxidant system
Glutathione: Tripeptide
- Glutamate + ***Cysteine (most important due to S atom) + Glycine
Oxidised Glutathione (GSSG): Oxidised S atom —> 2 Glutathione dimerised via Disulfide bond —> 2GSH Reduced Glutathione (GSH): Reduced S atom —> Donate electron for reduction of ROS
Adaptation of RBC antioxidant system to high and low oxygen states
Lung: RBC under high O2 partial pressure —> Hb loaded with O2 —> ↑ chance of oxidation to Hb(Fe3+) —> ↑ chance of oxidative damage to RBC —> need more antioxidant capacity
Peripheral tissue: RBC under low O2 partial pressure —> ↓ Hb loaded with O2 —> ↓ chance of oxidation to Hb(Fe3+) —> ↓ chance of oxidative damage to RBC —> but need to prepare for meeting varying demands for O2 (prepare to unload O2 to tissue)
***Expand antioxidant capacity in response to high risk of oxidative damage
Spectrin skeleton (cytoskeleton in RBC)
—> Protein framework that gives shape to RBC
—> Anchored to plasma membrane by cluster of ***Band 3 protein
C-terminal region of Band 3 protein
—> contains binding site for **Glycolytic enzymes + **Deoxyhaemoglobin
—> Glycolytic enzymes and Deoxyhaemoglobin compete for same binding sites on Band 3 protein
—> Glycolytic enzymes ***inhibited when bound to Band 3 protein
Competition between Glycolytic enzymes and Deoxyhaemoglobin
—> mediates coupling between O2 level to Glycolytic activity
High oxygen tension (e.g. Lung) —> ↓ Deoxyhaemoglobin —> ↑ Glycolytic enzyme bound to Band 3 —> ***↓ Glycolytic activity —> ***↑ G6P to PPP pathway —> ↑ NADPH produced —> Adapts RBC to an environment with high oxidative stress
Low oxygen tension (e.g. Peripheral tissue)
—> ↑ Deoxyhaemoglobin
—> ↓ Glycolytic enzyme bound to Band 3 (displacement of glycolytic enzymes from Band 3)
—> **↑ Glycolytic activity
—> **↓ G6P to PPP pathway
—> ↓ NADPH produced, ↑ ATP produced
—> enable RBC to respond to demand of O2 by nearby cells + favour unloading of O2
Coordinated changes in Glycolytic and PPP activity at High, Low O2 level
Glucose —(independent of O2 level)—> G6P
—> High O2 —> ↓ G6P to glycolysis —> ↑ G6P to PPP pathway
—> Low O2 —> ↑ G6P to glycolysis —> ↓ G6P to PPP pathway
Summary
- Sequestration of Glycolytic enzymes as a mean to regulate glycolysis in RBC
- Relative rates of glycolysis and PPP are ultimately connected to changes in oxygen level in surrounding environment of RBC
- Coordinated changes in rates of glycolysis and PPP —> Basis of oxidative defence in RBC
***3 mechanisms of adaptation to high demand for O2 in low O2 environment
- Unloading more oxygen from oxyHb by ***2,3-BPG
- Evidence that Glycolysis can affect O2-transport function of RBC (apart from cooperative binding of O2 to Hb)
- Pyruvate kinase deficient RBC
—> accumulation of special glycolytic intermediate (Later found to be 2,3-BPG)
—> lower O2 affinity of Hb - ***Increase blood flow to hypoxic region in response to local hypoxia
-
**Stress erythropoiesis (in lecture 2)
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
Formation of 2,3-BPG
Normal glycolysis: Glyceraldehyde 3-phosphate —> 1,3-Bisphosphoglycerate (1,3-BPG) —> 3-Phosphoglycerate (3-PG) —> 2-Phosphoglycerate
1,3-BPG (支線)
—(Bisphosphoglycerate mutase)—> 2,3-BPG
—(2,3-BPG phosphatase)—> 3-PG
Bisphosphoglycerate mutase:
- not a regular enzyme in glycolytic pathway
- shift phosphate position from 1 to 2
- activated by ***phosphorylation by AMPK (AMP-activated protein kinase)
2,3-BPG phosphatase:
- not a regular enzyme in glycolytic pathway
- removal of phosphate from 2,3-BPG
Balance between **Bisphosphoglycerate mutase and **2,3-BPG phosphatase
—> determines [2,3-BPG] in RBC
***Interaction between 2,3-BPG and Haemoglobin
2,3-BPG preferentially binds to T-state of Hb (∵ central cavity that accommodates 2,3-BPG is reduced in size in R-state Hb)
—> 2,3-BPG ***stabilise T-state Hb
—> ↓ Affinity of Hb for O2
—> shifts O2-binding curve to right (extent of shifting depend on [2,3-BPG])
—> encourage unloading of O2 from Hb
2,3-BPG:
- ***allosteric regulator for Hb (by changing affinity of Hb for O2)
- control ***interconversion of Hb between T-state and R-state
***2,3-BPG level and Hypoxic conditions
Low O2 level stimulate Glycolysis + BPG mutase
High-altitude / Hypoxic conditions:
—> Body Hypoxia
- ↑ Glycolytic activity (Glycolytic enzyme: Aldolase)
—> ↑ Precursor supply (Glyceraldehyde 3-phosphate) + ↑ ATP production - ↑ **sCD73 (ecto 5’-nucleotidase) in plasma
—> sCD73 catalyse **breakdown of ATP
—> ATP —> Adenosine
—> **Adenosine bind to ADORA2B receptor on RBC membrane
—> **Stimulate AMPK
—> Stimulate / **Phosphorylate Bisphosphoglycerate mutase
—> **↑ 2,3-BPG production in RBC
—> ↓ Affinity of Hb for O2 (indicated by ↑ P50 (pressure required to saturate 50% of Hb))
—> Shifting O2-binding curve of Hb to right hand side
—> Favour ***unloading of O2 from Hb
—> Resolve body hypoxia
***Involvement of membrane lipids in O2 release from OxyHb under low O2 condition
Sphingomyelin
—(sphingomyelinase)—> Ceramide
—(ceramidase)—> Sphingosine
—(sphingosine kinase)—> ***Sphingosine-1-phosphate (S1P)
High-altitude / Hypoxic conditions
—> ↑ S1P formation
—> S1P **bind to Deoxyhaemoglobin and recruit it to plasma membrane
—> S1P **facilitate interaction between Deoxyhaemoglobin and Band 3
—> ***Liberate more glycolytic enzymes
—> ↑ Glycolytic activity
—> ↑ 2,3-BPG
***Responding to local hypoxia
Local hypoxia
—> Local demand of O2 > total O2 carrying capacity of locally present RBC
—> ↑ Glycolysis
—> Secretion of **ATP by RBC
—> Stimulation of **purinergic receptors on endothelium
—> Signals generated and propagates to upstream arterioles
—> ***Dilatation of arterioles
—> ↑ Blood flow
—> ↑ RBC in local area
—> ↑ O2 delivery to hypoxia regions
Overall summary
High oxygen tension: 1. Chelation of Fe2+ by Histadine residue in Hb 2. ↓ Deoxyhaemoglobin —> ↑ Glycolytic enzyme bound to Band 3 —> ↓ Glycolysis, ↑ PPP —> ↑ NADPH —> Glutathione antioxidant system
Low oxygen tension: 1. ↑ Deoxyhaemoglobin + ↑ S1P formation + ↑ sCD73 (ATP breakdown) —> ↓ Glycolytic enzyme bound to Band 3 —> ↑ Glycolysis + ATP breakdown —> Stimulate AMPK —> Phosphorylate Bisphosphoglycerate mutase —> ↑ 2,3-BPG production in RBC —> Unloading more oxygen
- Increase blood flow to hypoxic region in response to local hypoxia
- Stress erythropoiesis
