Hematology Block- Medical Biochemistry and Molecular Genetics

Metabolism of Hemoglobin
Porphyrins & Porphyrias

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▪ Describe the structure, synthesis, degradation and types
of haemoglobin.
Specific Objectives:
▪ Describe the pathway and rate-limiting steps by which
heme is synthesized.
▪ List the normal hemoglobins found in fetal and adult
blood.
▪ Describe the pathway for the degradation of heme.
▪ List the clinical conditions associated with abnormal
metabolism of heme.

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Metabolism of Hemoglobin
porphyrins & porphyrias

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 Haemoglobin
➢Haemoglobins is globular composed of haem
and globin.
➢Globin is a simple protein
➢ “globin” in hemoglobin refers to the individual
protein subunits
➢Haem is composed of Fe++ & Porphyrins

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 Typical Hemoglobin Values

Hemoglobin levels are measured by the amount of

hemoglobin in grams (gm) per deciliter (dl) of blood. The
normal ranges for hemoglobin values are dependent on the
age and sex. Normal ranges are:

▪ Newborns: 17-22 gm/dl

▪ One (1) week of age: 15-20 gm/dl
▪ One (1) month of age: 11-15gm/dl
▪ Children: 11-13 gm/dl
▪ Adult women: 12-16 gm/dl
▪ Adult males: 14-18 gm/dl
▪ Women after middle age: 11.7-13.8 gm/dl
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▪ Men after middle age: 12.4-14.9 gm/dl
 Hemoglobin
▪ 65% of the Hb is synthesized in the erythroblasts, and
35% at the reticulocyte.

▪ Haem synthesis occurs largely

in the mitochondria.

▪ Globin synthesis occurs in the

polyribosomes.

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 Haemoglobin
▪ Each molecule of normal adult haemoglobin (Hb-A)
consists of four polypeptide chains a2b2, each with its
own haem group.

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 Globin synthesis
▪ polypeptide subunits: two alpha chains and two beta chains,
each with 141 and 146 amino acids respectively.
▪ The synthesis of these globins is under genetic control.
▪ Humans normally carry functional globin chains, arranged
in two, duplicated gene clusters:
▪ b-like cluster (b, g, d and e globin genes) on the short arm
of chromosome 11 and
▪ a-like cluster (a and z globin genes) on the short arm of
chromosome 16.

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α Alpha β Beta γ Gamma δ Delta ε Epsilon
ζ Zeta and η Eta 9
 Hb in adult blood
Hb A Hb A2 Hb F

Structure a2b2 a2d2 a2g2

Normal % 96-98 % 1.5-3.2 % 0.5-0.8 %

The major switch from fetal to adult haemoglobin occurs 3-6 months after birth.

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 STRUCTURE OF HEME

▪ Ferrous iron (Fe2+)

▪ Protoporphyrin IX: comprised of 4 pyrrole rings linked together by
methenyl bridges

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 Iron Metabolism

• Low pH of stomach solubilizes Fe-containing ionic compounds.

Gut

• Fe transporters facilitate absorption into blood stream

• Fe3+ ions are bound and chelated by Transferrin (Tf).

• Transferrin transports Fe to tissues
Blood

•Maintains solubility
•Keeps Fe ions unreactive

• Transferrin endocytosis is receptor-mediated (TfR)

• Endocytosis results in Fe3+ release
• Fe is distributed to distinct regions of the cell via Fe transporter
Cells

and/or by the help of channels

• Function of iron in the cell: Protein components (Heme)
• Storage: Ferritin (Fe2+)
• Toxicity
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 Iron transport to the Cell

Fe(III)2-Tf Tf

Transferrin
Receptors (TfR)

Proteins: Catalysis
Electron, oxygen transport
[Fe] Structural stabilization
Formation of protein-bound radicals

[Fe] Storage and Sequestration: Ferritin

[Fe] Toxicity: Oxidative stress

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 Heme Synthesis
85% of total heme synthesis occurs in red blood cells (RBC)

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Succinyl CoA

8 Glycine**

Heme

** Amino acid (building blocks of protein) synthesized in your body

Dr Gihan Gawish 15
 Heme
synthesis

Dr Gihan Gawish 16
Heme Synthesis

Dr Gihan Gawish 17
 Summary
IRON

Dr Gihan Gawish 18
 Regulation Of Heme And Globin Synthesis:

Heme:

➢ Inhibits activity of pre-existing d-ALA synthase

➢ diminishes the transport of d-ALA synthase from cytoplasm
to mitochondria.
➢ Represses the production of d-ALA synthase by regulating
gene transcription.
➢ Stimulates globin synthesis to ensure that levels of free heme
remain low in concentration.

➢Inhibition of the synthase and stimulation of globin synthesis are the

most important aspects in balancing hemoglobin production.
➢d-ALA synthase Requires pyridoxal phosphate (Vitamin B6) as a
coenzyme
Dr Gihan Gawish 19
 Disorders of Heme Synthesis

▪ Acquired: Lead poisoning

▪ Congenital: Porphyrias

▪ Deficiency of heme has serious effects in relation to

hemoglobin and cytochromes.

Dr Gihan Gawish 20
 LEAD TOXICITY
Symptoms
• Irritibility • Poor appetite
• Lethargy • Abdominal pain (with or without vomiting)
• Sleeplessness
• Headaches • Constipation

LEAD
➢Binds to any compound with a sulfhydryl group
➢Inhibits multiple enzyme reactions including those involved in
heme biosynthesis (PBG synthase &ferrochelatase)

Dr Gihan Gawish 21
 Lead Toxicity

Vitamin
B6

lead

Dr Gihan Gawish 22
 PORPHYRIAS
• A group of rare disorders caused by deficiencies of enzymes of the heme
biosynthetic pathway

•The majority of the porphyrias are inherited in an autosomal dominant fashion -

thus, affected individuals have 50% normal levels of the enzymes, and can still
synthesize some heme

•Affected individuals have an accumulation of heme precursors (porphyrins),

which are toxic at high concentrations

•Attacks of the disease are triggered by certain drugs, chemicals, and foods, and
also by exposure to sun

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Mitochondria PORPHYRIAS
GLYCINE + SuccinylCoA Agent Orange
ALA synthase
d-aminolevulinic acid(ALA)
ALA-dehydratase
ALA dehydratase
Deficiency porphyria
Porphobilinogen(PBG)
Acute intermittent
PBG deaminase porphyria
hydroxymethylbilane
Uroporphyrinogen III Congenital erythropoietic
cosynthase porphyria
uroporphyrinogen III
Uroporphyrinogen Prophyria
decarboxylase cutanea tarda
coprophyrinogene III
Coproporphyrinogen Herediatary
oxidase coproporphyria
Protoporphyrinogene IX
Protoporphyrinogen Variegate
protoporphyrin IX oxidase porphyria

Ferrochelatase Erythropoietic
Heme protoporphyria
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 Acute Intermittent Porphyria
• Autosomal dominant

• Caused by a deficiency in porphobilinogen deaminase, which is

involved in the conversion of porphobilinogen (PBG) to
uroporphyrinogen III

•PBG, uroprophryin, and 5-ALA accumulate in the plasma

and the excreted in urine

•Patients have neuropyschiatric symptoms and abdominal pain.

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 Porphyria Cutanea Tarda
•Most common porphyria

•Autosomal dominant

•Disease is caused by a deficiency in uroporphyrinogen decarboxylase, which is

involved in the conversion of uroporphyrinogen III to coproporphyrinogen III

•Uroporphyrinogen accumulates inplasma and appear in urine

• Patients are photosensitive (cutaneous photosensitivity)

➢Accumulation of porphyrinogens results in their conversion to porphyrins by

light

➢Porphyrins react with molecular oxygen to form oxygen radicals

➢Oxygen radicals can cause severe damage to the skin

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Heme Catabolism

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 Destruction of Normal Red Cell

▪ Red cell destruction usually occurs after a mean life

span of 120 days.

▪ The cells are removed extravascularly by macrophages

of the reticuloendothelial system (RES), specially in the
bone marrow but also in the liver and spleen.

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 Normal Red Cell Destruction
▪ The breakdown of red cells liberates
1- Iron for recirculation via plasma transferrin to
marrow erythroblasts
2- Protoporphyrin which is broken down to
bilirubin.
3- Globins which are converted to amino acids.

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 Bilirubin
➢ The bilirubin circulates to the liver where it is conjugated
to glucuronides which are excreted into the gut via bile
and converted to stercobilinogen and stercobilin
(excreted in faeces).

➢ Stercobilinogen and stercobilin are partly reabsorbed and

excreted in urine as urobilinogen and urobilin.

➢ A small fraction of protoporphyrin is converted to carbon

monoxide (CO) and excreted via the lungs.
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 Normal red cell breakdown
haemoglobin

haem globin
iron protoporphyrin
Amino acids

CO Bilirubin
transferrin Expired air (free)
Liver
conjugation
erythroblast
Bilirubin glucuronides

Urobilin(ogen) Stercobilin(ogen)

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Urine faeces
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DEGRADATION OF HEME TO BILIRUBIN

• 75% is derived from RBCs

• In normal adults this

results in a daily load of
P450 cytochrome
250-300 mg of bilirubin

• Normal plasma
concentrations are less then
1 mg/dL

• Hydrophobic – transported
by albumin to the liver for
further metabolism prior to
its excretion
“unconjugated” bilirubin

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 BILIRUBIN PRODUCTION
Heme proteins Hemoglobin Erythroid cells
myoglobin, cytochromes (70 to 80%)
(20 to 25%)

Heme
ferritin apoferritin (250 to 400 mg/day)
3 [O]
Heme oxygenase
3+
Fe + CO

Biliverdin
NADPH + H+
Biliverdin reductase
NADP+ indirect
albumin
Bilirubin unconjugated
pre-hepatic

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BILIRUBIN PROCESSING

albumin-Bilirubin albumin

ligandin hepatocyte
ligandin-Bilirubin
2 UDP-glucuronate UDP-Glucuronyl
ER transferase

2 UDP
direct
Bilirubin diglucuronide conjugated
post-hepatic

bile (gall bladder)

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 BILIRUBIN EXCRETION

Bilirubin diglucuronide
liver
2 glucuronate Bacterial enzyme
Intrahepatic
Bilirubin urobilinogen cycle

8H Bacterial enzyme
kidneys
intestines
Urobilinogen Urobilin urine
kidneys
Bacterial enzymes

Stercobilinogen Stercobilin feces

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 HYPERBILIRUBINEMIA

•Increased plasma concentrations of

bilirubin (> 3 mg/dL) occurs when there is an
imbalance between its production and
excretion

•Recognized clinically as jaundice

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 Diseases of Haem catabolism
1. Jaundice
2. Haemoglobinopathies
3. Anaemia

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 Hyperbilirubinemia
A. Hemolytic B. Hepatitis C. Biliary duct
anemia stone

excess
hemolysis

 unconjugated bilirubin
 unconjugated bilirubin  unconjugated bilirubin
(in blood)
(in blood) (in blood)
 conjugated bilirubin
 conjugated bilirubin  conjugated bilirubin
(released to bile duct)
(in blood) (in blood) 40
 Causes of Jaundice
1- Increased production of bilirubin by hemolysis or blood disease:
•Increase in blood indirect bilirubin
•Called pre-hepatic jaundice
•Stool color remains normal.
2- Abnormal uptake or conjugation of bilirubin:
•Leads to non-hemolytic unconjugated hyperbilirubinemia
•Increased indirect bilirubin.
•Stool color turns gray.
•Caused by liver damage or disease.

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3- Cholestasis: Problems with bile flow.
1. Intrahepatic cholestasis: hyper conjugated bilirubinemia
•Increase in blood indirect and direct bilirubin
•Caused by liver damage or disease: eg cirrhosis, hepatitis
•Can also occur in pregnancy:
2. Extrahepatic cholestasis:
•Blockage of bilirubin transport in the bilary tract.
•Increased direct bilirubin.
•Stool color turns gray.
•Caused by: Tumors or gall stones.

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Diagnoses of Jaundice

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 Haemoglobinopathies
There are mainly two types normalities:

Quantitative abnormalities: where there is reduction in

the production of certain types of globins e.g.
A. thalassaemia & B thalassaemia

Qualitative abnormalities:
where there is production of abnormal haemoglobin e.g.
sickle cell anaemia.

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 Iron Deficiency Anemia
▪ The characteristic sequence of events ensues when the total body
iron level begins to fall:
1. decreases the iron stores in the macrophages of the liver,
spleen and bone marrow

2. increases the amount of free erythrocyte protoporphiryn (FEP)

3. begins the production of microcytic erythrocytes

4. decreases the blood haemoglobin concentration

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 Factors leading to Iron deficiency anemia
▪ The end result of a long period of negative iron balance
➢ Decreased iron intake
➢Inadequate diet, impaired absorption, gastric surgery, celiac
disease
➢ Increased iron loss
➢Gastrointestinal bleeding (haemorrhoids, salicylate
ingestion, peptic ulcer, neoplasm, ulcerative colitis)
➢Excessive menstrual flow, blood donation, disorders of
hemostasis
➢ Increased physiologic requirements for iron
➢Infancy, pregnancy, lactation

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