Heme 06: Heme Catabolism

Front 1

What quantity of hemoglobin is synthesized each day? Why?

Back 1

6-7g / day to replace heme lost through the normal turnover of RBCs

Front 2

Where is most iron in the human body? How much?

Back 2

~70% of total iron is present as heme iron in RBCs

Front 3

What concerns does the body have to deal with as it breaks down heme?

Back 3

- Handling the hydrophobic products of the porphyrin ring cleavage
- Retention, safe mobilization, and re-utilization of iron

Front 4

What form of iron can generate reactive oxygen species (ROS)?

Back 4

Ferrous iron (Fe2+) can generate ROS by the Fenton reaction → promotes oxidative stress

Front 5

What structure can free heme and heme iron negatively affect?

Back 5

Lipid membranes

Front 6

How does free heme and heme iron negatively affect lipid membranes?

Back 6

- Heme is hydrophobic and can intercalate into lipid bilayers
- Heme iron can generate ROS by Fenton Reaction
- ROS participate in oxidation of lipid membrane components and cause lipid peroxidation

Front 7

What effect do structural changes to lipid membranes caused by ROS produced by heme iron have?

Back 7

- Alters fluidity and channels
- Alters membrane-bound signaling proteins
- Increases ion permeability

Front 8

What is the effect of lipid peroxidation caused by ROS produced by heme iron?

Back 8

Lipid peroxidation products form adducts and cross-links with non-lipids (eg, proteins and DNA)

Front 9

What ROS does ferrous iron (Fe2+) help form?

Back 9

1) Fe2+ + O2 → Fe3+ + O2˙-
2) 2 O2˙- + 2H+ → H2O2 + O2
3) H2O2 + Fe2+ → Fe3+ + HO˙ + OH-

O2˙- = Superoxide radical
HO˙ = Hydroxyl radical

Front 10

What type of endothelium is affected by free plasma hemoglobin and heme?

Back 10

Toxic to vascular endothelium

Front 11

What effect does HEME IRON have on vascular endothelium?

Back 11

- Heme iron promotes oxidative stress
- Can cause endothelial activation responsible for vaso-occlusive events and thrombus formation

Front 12

What effect does FREE HEMOGLOBIN have on vascular endothelium?

Back 12

- Scavenges NO (NO binds to heme iron)
- Reduces NO bioavailability
- This prevents smooth muscle relaxation (usually achieved via GC→ cGMP)
- Dysregulation of endothelium vasodilator-vasoconstrictor balance can lead to severe VASOCONSTRICTION and HTN, a common feature of pathologic conditions associated with hemolysis

Front 13

What pathologic conditions are commonly associated with hemolysis? Why?

Back 13

- Severe vasoconstriction and HTN
- Caused by free hemoglobin scavenging NO (which leads to dysregulation of the endothelial vasodilator - vasoconstrictor balance)

Front 14

Which of the following statements is incorrect?
a) Heme can intercalate into a lipid bilayer
b) Heme iron can generate ROS
c) Hemoglobin cannot bind NO
d) Lipid peroxidation can alter fluidity of membranes
e) NO triggers vasodilation

Back 14

Hemoglobin cannot bind NO is incorrect

Front 15

What happens to old RBCs?

Back 15

- RBCs become smaller in size and cell surface is modified with aging
- They become senescent → Extravascular Hemolysis → phagocytosed by macrophage

Front 16

What are the two ways that RBCs can be lysed? Which type predominates?

Back 16

- Extravascular hemolysis (80-90%)
- Intravascular hemolysis (10-20%)

Front 17

What happens to RBCs undergoing Intravascular Hemolysis?

Back 17

1) RBC membrane is perturbed releasing Hb and sometimes heme from Hb
2) Haptoglobin binds free Hb and Hemopexin binds free heme
3) Haptoglobin-Hb and Hemopexin-Heme can each be taken up by scavenger receptors via Receptor-Mediated Endocytosis into macrophages (CD163 for Hb and CD91 for Heme)
4) Heme is degraded to produce bilirubin, which is excreted into bile by liver

Front 18

Which molecule binds free Hemoglobin? How is this complex taken up by receptor mediated endocytosis?

Back 18

- Haptoglobin
- CD163

Front 19

Which molecule binds free Heme? How is this complex taken up by receptor mediated endocytosis?

Back 19

- Hemopexin
- CD91

Front 20

What happens if there are reduced Haptoglobin levels when there is acute intravascular hemolysis?

Back 20

- Hb remains unbound
- Leads to hemoglobinuria
- Renal tubules reabsorb → Hemosiderinuria
- Can also excrete excess Hb → Possible iron deficiency

Front 21

Where does Extravascular Hemolysis take place?

Back 21

Within macrophages of spleen and liver ("tissue macrophages")
- Red pulp macrophages of spleen
- Kupffer cells of liver

Front 22

What does Intravascular Hemolysis require?

Back 22

CD163 positive macrophages to scavenge hemoglobin-haptoglobin complex

Front 23

How can you get an idea of how much Intravascular Hemolysis is happening?

Back 23

Measure Haptoglobin levels (the less haptoglobin there is the more intravascular hemolysis is happening)

Front 24

What is Haptoglobin? What does it bind? Where is it made?

Back 24

- Acute phase glycoprotein that binds free Hemoglobin (Hb)
- Nearly irreversible binding to Hb
- Produced mostly in liver by hepatocytes and secreted into blood circulation

Front 25

When is there an increase in Haptoglobin levels?

Back 25

During acute phase of inflammation and in neoplastic disease in response to inflammatory cytokines

Front 26

What is Haptoglobin a marker of?

Back 26

- Useful marker for inflammation-related diseases (levels increase)
- Clinical marker for hemolysis (levels decrease)

Front 27

What is the function of Haptoglobin?

Back 27

Antioxidant:
- Following its release into plasma, Hb dissociates from tetramer into αβ dimers
- Oxy-Hb dimers bind Haptoglobin tightly
- Haptoglobin sequesters the dimers, preventing release of free heme and oxidative damage of heme iron

Mechanism to conserve iron:
- Haptoglobin-Hb complex can't pass through glomerular filter

Front 28

How are Haptoglobin-Hb complexes cleared from circulation if they cannot pass through the glomerular filter?

Back 28

Hb scavenger receptor CD163 that is expressed on circulating monocytes and tissue macrophages

Front 29

What are the structures of the phenotypes of Haptoglobin?

Back 29

3 phenotypes of Haptoglobin:
- Hp1-1
- Hp2-1
- Hp2-2

Each contains 2 α-chains and 2 β-chains
- All have the same β-chains
- Two types of α-chains (1-83aa and 2-142aa) which is denoted in their names

Front 30

Which phenotype of Haptoglobin is found to be over-represented in auto-immune and inflammatory diseases?

Back 30

Hp2-2

Front 31

How do the different phenotypes of Haptoglobin affect HDL?

Back 31

Hp1-1:
- Preserves HDL function

Hp2-2:
- Defective HDL function

Front 32

How do the different phenotypes of Haptoglobin differ in size and antioxidant capacity?

Back 32

Hp1-1:
- Small dimer
- Highest antioxidant capacity

Hp2-2:
- Large cyclic polymer
- Lowest antioxidant capacity

Front 33

What type of Haptoglobin preserves HDL function? Implications?

Back 33

Hp1-1 genotype:
- ↑ reverse cholesterol transport
- ↑ antioxidant activity

Front 34

What type of Haptoglobin causes defective HDL function? Implications?

Back 34

Hp2-2 genotype:
- ↓ reverse cholesterol transport
- ↓ antioxidant activity

Front 35

What is the mechanism of Receptor-Mediated Endocytosis for Haptoglobin-Hb?

Back 35

1) Haptoglobin-Hb complex binds CD163 receptor on macrophages
2) Membrane is coated with clathrin
3) Clathrin-coated vesicle pinches off and clathrin dissociates to return to membrane
4) Vesicle fuses with early endosome which acidifies via V-type ATPase
5) CD163 receptor releases Haptoglobin-Hb complex and is recycled back to cell membrane
6) Haptoglobin-Hb complex is digested in lysosome

Front 36

What is CD163? What does it bind? Where is it expressed?

Back 36

- Type I Transmembrane Glycoprotein that contains 9 scavenger receptor cysteine rich (SRCR) domains
- Binds to Haptoglobin-Hb complex
- Expression restricted to monocyte / macrophage lineage; recycles between early endosomes and plasma membrane

Front 37

Which of the following is not required for CD163 scavenger receptor expression on the cell surface?
a) signal recognition particle (SRP)
b) golgi
c) endoplasmic reticulum
d) ubiquitination
e) signal sequence

Back 37

Ubiquitination

Front 38

What happens when Haptoglobin's buffering capacity is overwhelmed?

Back 38

Hb undergoes a rapid conversion to metHb, liberating heme

Front 39

What happens to the heme that is liberated when the haptoglobin buffering system for Hb is overwhelmed?

Back 39

Ferriheme binds to albumin and other plasma components including lipoproteins and is subsequently transferred to Hemopexin

Front 40

What is Hemopexin? What does it bind? Where is it made?

Back 40

- Acute phase glycoprotein that binds free heme
- High binding affinity for heme (higher than albumin) and coordinates heme via its conserved clustering of histidine residues
- Produced mostly in liver by hepatocytes and secreted into blood circulation

Front 41

When are Hemopexin levels elevated?

Back 41

- During acute phase of inflammation in response to inflammatory cytokines
- During heme overload

Front 42

What is CD91? What does it bind? Where is it expressed?

Back 42

- Type I Transmembrane Glycoprotein that is also known as the LDL receptor-related protein (LRP1)
- Binds to Hemopexin-Heme complex
- Expression is found in numerous cell types (NOT restricted to macrophages); recycles between early endosomes and plasma membrane

Front 43

What happens to hemoglobin components in the lysosome?

Back 43

- Globin proteins are degraded to amino acids in lysosome
- Heme is transferred to cytosol where it is catabolized by Heme Oxygenase-1 (HO-1) that is associated w/ ER membrane w/ activate site facing cytoplasm

Front 44

What does heme degradation produce?

Back 44

Bilirubin which is normally excreted into bile by liver

Front 45

What can result if there are elevated levels of bilirubin int he skin and sclera?

Back 45

Jaundice or icterus - imparts a yellow color to these tissues

Front 46

What can cause hyperbilirubinemia?

Back 46

Inherited disorders of abnormal bilirubin metabolism

Front 47

Where does heme catabolism occur?

Back 47

~80% occurs in senescent erythrocytes
~20% comes from turnover of immature RBCs and various cytochromes in non-erythroid tissues

Front 48

What happens to senescent RBCs?

Back 48

They are taken up by macrophages (extravascular hemolysis) of the reticuloendothelial system of the liver and spleen

Front 49

What is bilirubin? What color?

Back 49

- Orange pigment derived from degradation of heme proteins
- Potentially toxic waste product that is generally harmless because of binding to serum albumin

Front 50

Why is bilirubin generally harmless despite having toxic potential?

Back 50

It binds to serum albumin

Front 51

How does the heme ring open mechanistically?

Back 51

1) Ferroprotoporphyrin IX ring is selectively cleaved at α-methene bridge
2) Non-enzymatic oxidation by molecular O2 w/ elimination of CO
3) Iron is released after addition of e-, releases green pigment biliverdin
4) Conversion of biliverdin (green) to bilirubin (orange) in macrophage
5) Binding of bilirubin to albumin
6) Uptake, storage, conjugation, and excretion of bilirubin in liver

Front 52

What happens during the first step of heme catabolism?

Back 52

- Ferroprotoporphyrin IX ring is selectively cleaved at α-methene bridge
- Heme Oxygenase (HO-1) catalyzes reaction and requires e- from NADPH cytochrome P450 Oxidoreductase (CYPOR)

Front 53

What happens during the second step of heme catabolism, after opening of α-methene bridge in Ferroprotoporphyrin IX ring?

Back 53

- Non-enzymatic oxidation by molecular O2 w/ elimination of CO (CO is bound by hemoglobin)
- Only known reaction in human tissues and cells that produces CO as byproduct

Front 54

What happens during the third step of heme catabolism, after the non-enzymatic oxidation by molecular O2, which eliminates CO?

Back 54

- Iron is released after addition of e- (Fe2+/3+ is bound by Ferritin)
- Releases green pigment biliverdin

Front 55

What happens during the fourth step of heme catabolism, after Biliverdin is released?

Back 55

- Conversion of biliverdin (green) to bilirubin (orange) in macrophage
- Catalyzed by Biliverdin Reductase, which uses either NADH or NADPH for activity
- Bilirubin is less polar than biliverdin and crosses membranes more readily
- Bilirubin is an anti-oxidant and appears to be particularly important during neonatal period, when concentrations of other antioxidants are low in body fluids

Front 56

What happens during the fifth step of heme catabolism, after Biliverdin is reduced to Bilirubin?

Back 56

- Binding of bilirubin to albumin (in blood) tightly but reversibly
- Albumin keeps bilirubin in solution and transports it from primary sites of production (spleen and BM) to primary site of excretion (liver)
- Uptake, storage, conjugation, and excretion of bilirubin in liver

Front 57

What is the significance of the changing of colors in heme catabolism?

Back 57

Biliverdin (green) converted to Bilirubin (orange)

Front 58

Which of the following is NOT a component that serves a protective function during intravascular hemolysis?
a) antibody
b) haptoglobin
c) hemopexin
d) CD91
e) CD163

Back 58

Antibody (part of extravascular hemolysis)

Front 59

Which enzyme converts heme to a linear molecule?

Back 59

Heme Oxygenase (HO-1) which requires NADPH Cytochrome P450 Oxidoreductase (CYPOR)

Front 60

What enzyme removes iron during heme catabolism?

Back 60

Heme Oxygenase (HO-1) which requires NADPH Cytochrome P450 Oxidoreductase (CYPOR)

Front 61

What enzyme produces carbon monoxide (CO)?

Back 61

Heme Oxygenase (HO-1) which requires NADPH Cytochrome P450 Oxidoreductase (CYPOR)