L04 - RBC metabolism

Question 1

2 sources of oxidative damage to RBCs?

Answer 1

1. Neutrophils, macrophages, endothelial cells release reactive oxygen species (probably H2O2): diffuse into red blood cell
2. In red blood cell: deoxyhemoglobin (Hb)(Fe2+)
   Fe2+&raquo_space; Fe3+ generates H202 from O2

Question 2

Explain how superxoides are formed inside the RBC?

Answer 2

Increase O2 inside RBC

e- from Fe2+ is transferred to O2 to form superoxide as Fe2+ is oxidised to Fe3+

Superoxide > H2O2 > Oxidative damage

Question 3

2 major approaches to suppress formation of Hb (Fe3+)/ prevent oxidative damage ?

Answer 3

1. Chelation of Fe2+ to His. residue in Hb&raquo_space; Steric hinderance blocks access of H+ to initiate Fe2+ oxidation
2. RBC protect itself via glycolysis and pentose phosphate pathway

Question 4

Pentose phosphate pathway sequence?

Answer 4

Glucose 
>> Glucose 6-phosphate 
>> [G6PD + make NADP]
>> 6 - phosphogluconate + NADPH
>> [NADP]
>> Ribulose 5- phosphate + NADPH
>> Ribose 5-phosphate

Question 5

What is the fate of R5P in RBC?

Answer 5

R 5-P not used for nucleotide synthesis in mature RN which is anuclear

Recycled for glycolysis

Question 6

Explain how the pentose phosphate pathway is used to overcome oxidative stress in RBC?

Answer 6

Oxidative phase of pentose phosphate pathway (PPP) produces 2 NADPH:

> > serves as a donor of electrons to make GSSG into Glutathione(GSH) via Glutathione reductase enzyme

1) GSH turns H2O2 into water via GSH peroxidase
2) GSH reduces hydroxyl free radicals

Question 7

Why is GSH such a good anti-oxidant?

Answer 7

GSH = glutamate + cysteine + glycine

Cysteine sulphur has many oxidation states available for oxidation&raquo_space; used in reduction of ROS

GSH is dimerized to GSSH after reducing ROS

Question 8

RBC has higher chance of oxidation to Hb (Fe3+) in the lungs than in peripheral tissue. T or F? Why?

Answer 8

True

PO2 in lungs is high and amount of Hb loaded with O2 is also high

Question 9

What protein is used to protect against increased risk of oxidative damage in RBC in the lungs?

Answer 9

Band 3 protein cluster

i) anchors spectrin skeleton to the plasma membrane
ii) C- terminus is a competitive binding site for DeoxyHb and several glycolytic enzymes

Question 10

Explain the Band 3 mechanism in RBC in increased or decreased O2 levels?

Answer 10

C- terminus is a competitive binding site for DeoxyHb and several glycolytic enzymes

• Increasing O2 level&raquo_space; more oxyHb formed&raquo_space; incresae glycolytic enzyme binding to Band 3&raquo_space; reduce glycolysis and more pentose phosphate pathway&raquo_space; make more NADPH, GSH
• Decreasing O2 level&raquo_space; more deoxyHb formed&raquo_space; detach glycolytic enzymes from band 3&raquo_space; more glycolysis and less PPP&raquo_space; less NADPH, GSH, more ATP

Question 11

Why does the level of NADPH and ATP made at various O2 levels important for oxidative stress?

Answer 11

High O2&raquo_space; More NADPH made&raquo_space; adapt to high oxidative stress

Low O2&raquo_space; Less NADPH made, More ATP made for responding to O2 demand of nearby cells/ Unload O2 ATP- Purigenic receptor pathway at endothelium

Question 12

Production of G 6-P is dependent on the O2 level to cope with various oxidative stress. T or F?

Answer 12

False

Glucose&raquo_space; G 6-P is independent of O2 level, conversion is constant

Depends on the [glycolytic enzymes] released from Band 3 protein to determine what to make from the G6P

Question 13

Pyruvate kinase deficiency can cause a shift in the O2 dissociation curve in which direction?

Answer 13

To the right = Decrease affinity of Hb to O2 in addition to co-operative binding

due to Accumulation of glycolytic intermediates

Question 14

How does glycolysis relate to O2 transport by erythrocytes?

Answer 14

Via A special glycolytic metabolite in erythrocyte

2,3- Bisphosphoglycerate (BPG)

Question 15

Pathway of forming 2,3 BPG?

Answer 15

1,3- Bisphosphoglycerate 
--[bisphosphoglycerate mutase]-->
2,3 Bisphosphoglycerate 
--[2,3- bisphosphoglycerate phosphatase]-->
3- Phosphoglycerate

Question 16

What determines the concentration of 2,3 - BPG in RBC?

Answer 16

Balance between bisphosphoglycerate mutase enzyme activity vs 2,3- bisphosphoglycerate phosphatase enzyme activity

Question 17

Mechanism of 2,3 - BPG in shifting the O2 dissociation curve? *

Answer 17

Act as an allosteric regulator for Hb, changing the affinity of Hb for oxygen

2,3-BPG preferentially binds to stabilize T-state of haemoglobin (Hb) in erythrocytes

(R-state Hb central cavity is smaller and cannot bind 2,3 BPG)

> > reduces the affinity of Hb for Oxygen and encourage unloading

Question 18

The extent of O2 dissociation curve shift cause by 2,3 BPG depends on what?

Answer 18

Extent of shifting depends on [BPG]

Question 19

Which shifts the O2 dissociation curve more to the right: Hb + CO2, or Hb + BPG, or Hb + CO2 + BPG?

Answer 19

Increasing shift:

Hb + CO2
> Hb + BPG
> Hb + CO2 + BPG

Question 20

How does high altitude shift the O2 dissociation curve to the right via glycolytic intermediate? (Note: Not asking about EPO and more erythropoiesis) exam

Answer 20

High altitude/ hypoxic

1) Low O2 = More deoxyHb displace ALDOLASE enzyme from Band 3
ALDOLASE = F-1,6-BP&raquo_space;> Glyceraldehyde 3-Phosphate (precursor for 2,3 BPG)

2) AMP-activated protein kinase (AMPK) increase
» phosphorylates and activates BPG Mutase

> > > Increase 2,3 - BPG level = T-state stabilized, decrease affinity of Hb to O2

Question 21

List the sequence of reactions involved with 2,3 BPG, starting with Glyceraldehyde 3-phosphate?

Answer 21

Glyceraldehyde 3- Phosphate 
--[GAPDH]-->
1,3- Bisphosphoglycerate 
--[BPG mutase]-->
2,3 BPG 
--[2,3-BPG phosphatase]-->
3- Phosphoglycerate 
--[PGM]-->
2 - Phosphoglycerate 

*1,3 - BPG can bypass synthesis of 2,3- BPG by directly going to 3-phosphoglycerate via PGK enzyme

Question 22

Mechanism of increasing supply of precursors to 2,3- BPG in hypoxia?

Answer 22

Hypoxia

> > More deoxyHb formed
More glycolytic enzyme liberated from Band 3 i.e. Aldolase
Increase catalysis of F 1,6-BP
Increase supply of precursor Gylceraldehyde 3- phosphate
GAPDH + BPG mutase = 2,3- BPG

Question 23

Mechanism of lipid involvement in RBC under hypoxic conditions?

Answer 23

Hypoxia

> more deoxyHb made
Increase production of sphinogosin 1- phopshate (S1P)
S1P binds to deoxyHb and recruits it to plasma membrane to facilitate interaction with Band 3
more deoxyHb bind to Band 3, displace more glycolytic enzymes
More glycolysis = more precursor for 2,3 BPG

Question 24

Mechanism of vasodilation of arterioles to local hypoxic tissue?

Answer 24

Local hypoxia

> Local demand of O2 exceed total O2 carrying capacity of local RBC
Secretion of ATP by RBC **
Stimulation of PURINERGIC RECEPTOR **on endothelium
Signals generated, dilatation of arterioles upstream to increase blood flow
Increase O2 delivery