Hemoglobin metabolism

Patarabutr Masaratana

major protein in erythrocytes

large complex protein: heme (3%) + globin (97%)

tetramer: heme จบกบ globin chain (1:1)

heme: porphyrin ring with iron in the center

globins: 2 pairs of globin chains

(α-chains, non α-chains)

twisted together to expose heme

on the exterior of the molecule

Hemoglobin (Hb)

NH

pyrrole ring

side chains

M = methyl -CH3

V = vinyl -CH = CH2

P = propionyl -CH -CH2 -COOH

1 2

HC 1

2

3

4

CH

N

NH

HC CH

N

HN

3

4

5 6

7

8

a

b g

d

porphyrin = 4 pyrrole rings เชอมกนดวย methine bridges (=CH-)

Porphyrin (Greek) = purple

Fe-porphyrin prosthetic group: hydrophobic

heme ใน hemoglobin, myoglobin:

protoporphyrin IX + ferrous (Fe2+) at the center

heme อยใน hydrophobic

pocket ของ globin โดยอาศย

hydrophobic interaction

Heme

เพม aqueous solubility ของ heme

exterior: polar amino acids high solubility

interior: hydrophobic residue hydrophobic pocket ส าหรบ

heme

folded globin polypeptide: ลดการเกด oxidation ของ Fe2+ เปน Fe3+ ใน

สภาวะทม O2

ชนดของ Hb ขนอย กบ globin chain ทเปนสวนประกอบ

Globin

globin chain ชนดของ Hb

α2β2

α2δ2

α2γ2

A

A2

F

97%

6

ชนดของ Hb ปกต % at birth % in adult

HbF (α2γ2) 75 (50-90) <1 (หลงอาย 2 ป)

HbA (α2 β2) ~20 ~97

HbA2 (α2 δ2) ~1 ~2.5

คาปกตของ Hb ใน ชาย 15 g/dL และ Hct 45%

หญง 13 g/dL และ Hct 40%

Globin synthesis

Hemoglobin synthesis

เกดขนใน immature erythrocytes ในไขกระดก

(65% ใน nucleated RBCs, 35% ใน reticulocytes)

immature erythrocytes ไดรบ Fe ในรป transferrin

เขาสเซลล mitochondria

การสงเคราะห heme เกดใน mitochondria และ cytosol

การสงเคราะห globin เกดใน cytosol

heme + globin Hb ท cytosol

การสงเคราะห heme เกดขนในเนอเยอเกอบทกชนด

Puy H, et al. Lancet. 2010;375(9718):924–937.

Heme synthesis

rate-limiting enzyme: δ-aminolevulinic acid synthase (ALA

synthase) ควบคมปฏกรยาแรกในการสงเคราะห heme

glycine + succinyl Co A

δ-aminolevulinic acid

ALA synthase

pyridoxal phosphate

Heme synthesis

glycine +

succinyl CoA

ALA

mitochondria cytosol

N H

porphobilinogen

N H

N

H N

H

N H Fe2+

condensation of 2 ALAs

ALA dehydratase

N H

N

H N

H

N H

N H

N

H N

H

N H

Uroporphyrinogen III Coproporphyrinogen III

Protoporphyrin IX

U I

C I

P

A

P

A

P

A

A P

P A

N H

N

H N

H

N H

P

M

P

M

M P

P M

Protoporphyrinogen IX

M

N H

N

H N

H

N H

P

M

V

M

P M

V

Fe2+

globin

Heme synthesis

glycine +

succinyl CoA

ALA

mitochondria cytosol

N H

porphobilinogen

N H

N

H N

H

N H Fe2+

condensation of 2 ALAs

ALA dehydratase

N H

N

H N

H

N H

N H

N

H N

H

N H

Uroporphyrinogen III Coproporphyrinogen III

Protoporphyrin IX

U I

C I

P

A

P

A

P

A

A P

P A

N H

N

H N

H

N H

P

M

P

M

M P

P M

Protoporphyrinogen IX

M

N H

N

H N

H

N H

P

M

V

M

P M

V

Fe2+

globin

Regulation of heme synthesis

ALA synthase

- rate-limiting enzyme of heme biosynthetic pathway

- 2 forms encoded by different genes

- ALAS1: ubiquitously expressed throughout the body

-ALAS2: erythroid-specific form (red blood cell precursor cells)

mRNA contains 5’-IRE

- different mechanisms of regulation

rate-limiting enzyme: δ-aminolevulinic acid synthase (ALA

synthase) ควบคมปฏกรยาแรกในการสงเคราะห heme

Regulation of heme synthesis in non-erythroid cells

glycine + succinyl Co A

δ-aminolevulinic acid

ALA synthase

pyridoxal phosphate

heme glucose,

Heme: inhibits both the synthesis, transport of ALA synthase Glucose: inhibits transcription of gene encoding ALA synthase

Puy H, et al. Lancet. 2010;375(9718):924–937.

Puy H, et al. Lancet. 2010;375(9718):924–937.

Regulation of heme synthesis

Puy H, et al. Lancet. 2010;375(9718):924–937.

Regulation of heme synthesis

Heme synthesis

glycine +

succinyl CoA

ALA

mitochondria cytosol

N H

porphobilinogen

N H

N

H N

H

N H Fe2+

condensation of 2 ALAs

ALA dehydratase

N H

N

H N

H

N H

N H

N

H N

H

N H

Uroporphyrinogen III Coproporphyrinogen III

Protoporphyrin IX

U I

C I

P

A

P

A

P

A

A P

P A

N H

N

H N

H

N H

P

M

P

M

M P

P M

Protoporphyrinogen IX

M

N H

N

H N

H

N H

P

M

V

M

P M

V

Fe2+

globin

Heme synthesis

ALA dehydratase (porphobilinogen synthase)

- cytosolic enzyme

- very sensitive to heavy metals esp Pb

Ferrochelatase

- เตม Fe2+ เขาไปใน protoporphyrin IX heme

- sensitive to heavy metals, iron deprivation

- iron deprivation zinc-protoprophyrin IX

(Zn ถกเตมแทน Fe)

Transferrin-bound iron

Duodenum

Spleen Liver

Erythrocytes

Bo

ne

mar

row

Absorption Mucosal sloughing

Hemoglobin degradation

Extravascular pathway

- 90% ของ Hb ในเลอดถกสลายโดยวธน

- reticuloendothelial (mononuclear phagocyte) system

Hb

Hemoglobin degradation

Intravascular pathway

- <10% ของ Hb ในเลอดจะถกปลอยเขาสกระแสเลอดโดยตรง

- Hb แยกตวออกเปน α-dimer และ β-dimer จบกบ haptoglobin

- stabilize heme-globin bond, ปองกนการเสย Hb ทางปสสาวะ

- ถกก าจดทตบ โดยวธการเดยวกบ extravascular pathway

- Hb บางสวนอาจถก oxidise ได methemoglobin ซงจะปลดปลอย heme ออกมา

และจบกบ hemopexin ตอไป

Hemoglobin degradation

Intravascular pathway

- หากม α-dimer และ β-dimer มาก จนเกนความสามารถในการจบของ

haptoglobin และการดดกลบของทอไต

- Hb dimer ถกขบออกมาทางปสสาวะ

- hemoglobinuria

- heme ทมมากเกนความสามารถในการจบของ hemopexin จะจบกบ albumin

เปน

methemalbumin เพอ

สงตอ heme ใหกบ

hemopexin อณใหม

Hemoglobin derivatives

altered form of hemoglobin

Methemoglobin

Carboxyhemoglobin

Sulfhemoglobin

Glycosylated hemoglobin

Glycosylated hemoglobin (HbA1c)

4-6% of HbA

nonenzymatic attachment of D-glucose to amino groups

of globin polypeptide

HbA1c levels: increased with mean blood glucose

concentration in the previous 3-6 weeks

parameter of blood sugar control in DM patients

Conditions associated with abnormal Hb synthesis

1. Heme synthesis abnormalites

- porphyrias

2. Globin synthesis abnormalities

- hemoglobinopathies: qualitative defects

- thalassemias: qauntitative defects

Porphyrias

congenital or acquired deficiencies of heme biosynthetic

enzymes

retention of heme precursors toxicities

excess early precursors (ALA, PBG)

- neuropsychiatric symptoms

excess later intermediates (Uro-, Copro-, Proto-)

- cutaneous symptoms

Porphyrins accumulation

later intermediates (Uro-, Copro-, Proto-)

porphyrins: light absorptivity photosensitivity

porphyrin deposition in dentin teeth

discoloration/fluorescent teeth (under UV)

porphyrin excretion in urine/feces florescent

urine/feces under UV!

may require precipitating factors

31

Porphyrin-induced photosensitivity

32

33

porphyrinurias

acquired disorders

toxin/drugs interfere heme biosynthesis

lead (Pb) inhibits ALA dehydratase, ferrochelatase

Secondary porphyrias

Conditions associated with abnormal Hb synthesis

1. Heme synthesis abnormalites

- porphyrias

2. Globin synthesis abnormalities

- hemoglobinopathies: qualitative defects

- thalassemias: qauntitative defects

Hemoglobinopathies

mutations of α/β–globin genes: mostly point mutations

abnormal globin abnormal Hb altered Hb properties

Hb E

common in SE Asia

beta 26, GluLys Hb S

(homozygote sickle cell disease)

common in African Americans

beta 6, GluVal Hb CS (Constant Spring)

alpha 142, TermGln extra 31-amino acids

37

beta 6, GluVal

surface-localized charged amino acid hydrophobic amino acid

deoxy Hb S becomes less soluble

form filamentous polymers

precipitate

distorted RBCs

Thalassemias

reduced rate of normal globin synthesis

gene deletions or point mutations

α-thalassemia: reduced α-globin production

β-thalassemia: reduced β-globin production

Hemoglobin (Hb)

• Globin synthesis: developmental changes

a2g2 : HbF a2b2 : HbA a2d2 : HbA2

reduced α- or β-globin synthesis

accumulation of unmatched chains in developing RBCs

precipitation

cell destruction

anemia

b-thalassemia

a b

precipitate

Rbc destruction (spleen)

Excess a-chains

Erythroblast destruction (bone marrow)

Hemolysis Ineffective erythropoiesis

Anemia

b-thalassemia

Bone marrow

expansion

Iron overload

Tissue hypoxia

Iron

absorption

Anemia

Erythropoietin

Blood transfusion

Bone deformity

Hyperuricemia

Folate deficiency

Adapted from Weatherall DJ, Clegg JB. The Thalassaemia Syndromes, 4th ed. Blackwell Science.

α-thalassemia

mostly caused by deletion of α–globin gene

humans: 2 α–globin genes/chromosome 4 genes

α-thalassemia

1. Silent carriers 1 gene deleted (-α/αα)

- no clinical abnormality

2. α-thalassemia trait 2 genes deleted (--/αα)

- mild anemia (-α/-α)

3. Hb H disease 3 genes deleted (--/-α)

- β-chain accumulation β4 (Hb H)

- precipitation Hb H inclusions RBCs life span

- moderate anemia

α-thalassaemia

4. Hydrops fetalis 4 genes deleted (--/--)

- most severe form total absence of α-globin

- γ4 (Hb Bart’s): extremely high O2 affinity

- almost no O2 transport

- stillbirth or die shortly after birth

β-thalassemia

usually caused by point mutations of β–globin gene

humans: 1 β–globin genes/chromosome 2 genes

β+: reduced synthesis (5-30%), β0: no synthesis

homozygous/heterozygous β-thalasemia

thalassemia minor/intermedia/major

a2 b2 = A

a2 d2 = A2

a2 g2 = F

a b

d g

a b

d g

β0/β0 homozygote???

Hb E syndrome

Asymptomatic

- Hb E heterozygote/homozygote

Symptomatic

- β-thalassaemia/Hb E compound heterozygote

- Hb E A Bart’s disease (Hb E + Hb H disease)

- Hb E F Bart’s disease

(Hb H disease + β-thalassemia/Hb E or homozygous Hb E)

Laboratory diagnosis

complete blood count

blood smear

solubility test

Hb electrophoresis

DNA analysis