Types of Hb,Functions of Hb,Normal Value,Variations.First MBBS Lecture

1. HAEMOGLOBIN
 INTRODUCTION
 STRUCTURE
 TYPES
 SYNTHESIS
 FATE
 DERIVATIVES
 CLINICAL- ANAEMIAS
- HAEMOGLOBINOPATHIES
- PORPHYRIAS

2.  Pigment present inside RBCs
 Carries O2 and CO2
 Responsible for red color of blood
 Deficiency is called anaemia

3. Disadvantages of Free Hb
 increase in blood viscosity, causing a rise in blood
pressure.
 osmotic pressure of plasma to @ 100mm Hg
which interferes with fluid exchange between
capillaries & tissue spaces.
 Loss in urine (Haemoglobinuria) kidney
damage by forming acid haematin.
 Taken up & rapidly destroyed by the tissue
Macrophase system.

4. Normal values
 In foetus- just before birth the Hb concentration of
blood from umbilical cord is =16.5 to 18.5 gm/dl.
 After birth=23gm% this occurs due to –transfusion
of cells from placenta to infant &
haemoconcentration by reduction of plasma volume
 At the end of 3 months = 10.5 gm/dl
 At the end of 1 year = 12.5gm/dl
 Adult male =14-18 gm/dl
 Adult female =12-15.5 gm/dl
 Clinically 14.8gm% Hb is considered 100% Hb

5. Quantity of Haemoglobin in the Cells & amount
of O2 carried.
 Men: Hb: 15 grams per 100 ml of blood;
and
 Women: Hb: 14 grams per 100 ml.
 one gram of haemoglobin carries 1.34
ml of oxygen when fully saturated.
 Therefore, in man 100 ml of blood can
carry @ 20 ml of oxygen and in women@
19 ml of oxygen.

6. Haemoglobin
 Human hemoglobin A, present in adults, consists of four subunits:
two α-subunits and two ß-subunits.
 The α- and ß-subunits are homologous and have similar three-
dimensional structures.
 The capacity of hemoglobin to bind oxygen depends on the presence
of a bound prosthetic group (heme).
 The heme group is responsible for the distinctive red color of blood.

7.  The heme group consists of a protoporphyrin, and a
central iron atom.
 Protoporphyrin is made up of four pyrrole rings
linked by methene bridges to form a tetrapyrrole ring.
 Four methyl groups, two vinyl groups, and two
propionate side chains are attached.

10.  Iron-protoporhyrin-globin forms a subunit
 4 subunits join to form a molecule of Hb
 Iron is in ferrous ( Fe2+) form
 Fe2+ binds 4 pyrrole rings, polypeptide
chain and a molecule of O2
STRUCTURE OF HEMOGLOBIN

11.  4 pyrroles join to form a ring called
porphyrins
 4 polypeptide chains constitute
globin
 α,β,γ and δ are four important
polypeptide chains
 α chain has 141 a.a
 β and γ chain have 146 a.a

12. GLOBIN (protein part or apoprotein):
 It is composed of four polypeptide chains 2α and 2β chains.
 The α-chain contains 141 amino acids and β-chain contains
146 amino acids.
 Each β-polypeptide chain is folded into 8 right handed α-
helices ( starting from NH2-terminal)
 α-subunit is folded into 7α-helices.
 The ratio of haem to globin is 1:1. So each haem moiety is
linked to one peptide chain.

14. Attachment of haem to globin
 One Hb molecule contain 4 units of Haem
each attached to one of the 4 polypeptide
chains, constituting globin.
 Thus one Hb mol. Can carry 4 molecules
(8 atoms) of oxygen.
 Oxygenation of 1st haem mol. In Hb
increases the affinity of second mol. for O2

15. Attachment of haem to globin
 and affinity for O2 in 3rd haem mol.
 Therefore 4th haem-gr of Hb has highest
affinity for O2.
 This shifting affinity of Hb for O2 result in
sigmoid shape of O2-Hb curve.
 Oxygenation of Hb is very rapid <.01 seconds.
 Hb at it’s normal con. Increases the O2 carying
capacity of blood 70 folds

16. Hb-oxygen dissociation curve

17. Haemoglobin
 Reaction of Hb & oxygen
 Oxygenation not oxidation
 One Hb can bind to four O2 molecules
 Less than .01 sec required for oxygenation
 b chain move closer when oxygenated
 When oxygenated 2,3-DPG is pushed out
 b chains are pulled apart when O2 is unloaded,
permitting entry of 2,3-DPG resulting in lower affinity
of O2

18. Oxy & deoxyhaemoglobin

20. Types
4 types of poly peptide chains based on amino acid
composition and sequence.
alpha, beta, gamma, delta
Adult Hb
Hb A = 2 alpha (141 AA)+ 2 beta (146 AA) chains
(α2β2 )
Hb A2 = 2 alpha (141 AA)+ 2 delta (146 AA) chains
(2.5%) 1 (α2δ2) (10 AA differ)
Fetal Hb
Hb F = 2 alpha (141 AA)+ 2 gamma (146 AA) chains
( α2γ2) (37 AA differ)
99% replaced with adult Hb with in a year of birth.

21. TYPES OF HEMOGLOBIN
 HbA- Adult Type- α2β2
 HbA2- 2% of adult Hb- α2δ2
 HbF- Fetal Type- α2γ2
Hb gower1- ε2ζ2
Hb gower2- ζ2β2

22.  HbA- Adult Type- α2β2
 It is the main form of normal adult Hb.
 It is spheroidal molecule with a
molecular weight of 68000
 HbA2- 2% of adult Hb- α2δ2
 In Hb A2 10 individual Amino Acids
differ from those in beta chain of HbA

23. III. Fetal haemoglobin = HbF (α2 γ2):
 It is present normally in newborn and early fetal life
and at age of 5 months 90 % of fetal haemoglobin is
replaced by adult haemoglobin ( HbA1)
- It consists of 2 alpha chains and 2 gamma chains.
- In gamma chain there is 37 amino acid different from those in
HB A.
- HbF has a great affinity for O2 because γ-
chains do not bind 2,3 DPG well. DPG is
responsible for lowering the O2 affinity of Hb
and allowing Hb to release O2 at the low PO2
of tissues.

24. Fetal Hb-(HB-F)
 Due to this movement of o2 from maternal
to fetal circulation is facilitated.
 At PO2 20 mmHg ,Hb-F is 70% saturated
while Hb-A is only 30-35% saturated.
 Life span of Hb-F is much less (80 days) as
compared to that of Hb-A(120 days)

25. Hemoglobin Genes and Gene Products

26. Synthesis of globin
 Various types of globin combines with
haem to from different haemoglobin
 Eight functional globin chains, arranged in
two clusters the
 b- cluster (b, g, d and e globin genes) on the short
arm of chromosome 11
 a- cluster (a and z globin genes) on the short arm of
chromosome 16

27. Globin gene clusters

28. Globin synthesis, starts at 3rd week of
gestation
 Embryonic
Haemoglobin Gower I ( z2e2)
Haemoglobin Portland ( z2g2)
Haemoglobin Gower II (a2e2)
 Fetal : HbF (a2g2), HbA (a2b2)
 Adult : HbA, HbA2 ( a2d2), HbF.
Synthesis of globin

29. Globin chain switch

30. Hemoglobin (Hb)
 Red, oxygen carrying pigment present in RBCs.
Heme (4%)
Globin (96%)
 Quantity
700-900g in body
29-32 pico gram/RBC(MCH)
 RBCs (MCHC)
Male= 36g/100ml
Female = 34g/100ml
 Molecular Weight
68,000 30

31. deoxyHb
deoxyMb
Myoglobin and Hemoglobin Structure
b
b
a
a
oxyMb (MbO2)
O2

32. DERIVATIVES OF Hb
1) Oxyhaemoglobin (HbO2) – oxygenation reaction
– combination with Fe 2+
2) Carbaminohaemoglobin ( CO2Hb)  carbon
dioxide combines with globin part
3) Carboxyhaemoglobin (COHb)  carbon
monoxide binds with Fe 2+
{ 250 times more affinity than oxygen}

33. 4) Methhaemoglobin ( HHb) 
oxidized haemoglobin
5) Sulfhaemoglobin
6) Glycated haemoglobin (HbA1c)
Glucose attached to terminal
Valine in β chain. – integrated index
of Diabetic control over 4 to 6 weeks

34. Methemoglobin
 This is oxidized Hb,
 The Fe2+ normally present in heme being
replaced by Fe3+,the ability to react as an O2
carrier is lost.
 The normal erythrocyte contains small amount of
met Hb, formed by spontaneous oxidation of Hb.
 Met Hb is normally reconverted to Hb by reducing
systems in the RBC, the most important of which is
NADH-methemoglobin reductase.

35. Congenital methemoglobinemias
A. Hemoglobin M (Hb-M):
It is a congenital condition due to mutation in globin
biosynthesis in which distal or proximal histidine is
replaced by tyrosine.
B. Deficiency of NADH cytochrome b5 methemoglobin
reductase system
Acquired (toxic) methemoglobinemea
Usually arises following the ingestion of large amounts
of drugs e.g. phenacetin or the sulphonamides, excess
of nitrites or certain oxidizing agents present in the
diet.

36. II. Glycosylated haemoglobin (Hb A1c):
- It is modified form of haemoglobin .
- it contains glucose linked to amino group present on the NH2 -
terminal ends. The reaction is non enzymatic and its rate
depends on the concentration of glucose .It is present in
normal value -5% of the total haemoglobin.
- This glycohaemoglobin gives an idea about the blood glucose
level during the last three months and is useful in the
assessment of diabetic control
- This percentage is increased in diabetic patients up to 8-14%.

37. FUNCTIONS OF HEMOGLOBIN
 1. Transports O2 from lungs to tissues in the
form of oxy-hemoglobin
 2. Transports CO2 from tissues to lungs in the
form of carbamino-hemoglobin:- 30% of total
CO2 transport
 3. Acts as a buffer- important in acid-base
balance- 6 times more than plasma proteins

38. Hemoglobin (Hb)
 250 million Hb molecules / RBC
 So carry 1 billion oxygen molecules / RBC
 Synthesis of Hb
 Starts at proerythroblastic stage
 Synthesis steps:
 Heme is made from acetic acid and glycine in mitochondria
 Acetic Acid  α-ketoglutaric Acid  Succinyl Co A (Krebs
Cycle)
 Globin (polypeptide chain) is synthesized by Ribosomes
38

39. SYNTHESIS
SUCCINYL Co-A + GLYCINE
δ-AMINOLEVUNIC ACID
PORPHOBILINOGEN
PROTOPORPHYRIN IX
HAEM
HAEMOGLOBIN
ALA synthase
haem synthase
Fe2+
GLOBIN

40. Synthesis of Haemoglobin

41. FATE OF HAEMOGLOBIN
 RBCs are destroyed in RES ( mainly spleen
and bone marrow) after 120 days of life.
 Macrophages phagocytose – hemolyse -
degrade haemoglobin – form Bilirubin –
transport to liver with albumin – conjugated
and detoxified – secreted in bile.

43. Bilirubin
Biliverdin
Protoporphyrin
iron pool
Iron CO
Haem
Amino acid pool
Globin
Haemoglobin

44. HAEMOGLOBINOPATHIES
 Abnormal formation of Hb
 Due to disorders of Globin Synthesis
 Two main types
 Formation of abnormal polypeptide chain
e.g.-Sickle cell Anaemia
 Supression of synthesis of polypeptide
chain e.g- Thalassaemia

45. Sickle Cell anemia
 Sickle Cell Anemia is a
genetic disorder that is
characterized by the
formation of hard, sticky,
sickle-shaped red blood
cells, in contrast to the
biconcave-shaped red
blood cells (RBCs) found
in “normal” individuals.
 This disease is caused by a
mutation in hemoglobin.

46. Spectroscopic view of RBC in Sickle
cell anemia
46

47. Sickle cell anaemia:
Valine replaces glutamate in the 6th position of β chain.
 Is common in African blacks
 Hb crystallizes & takes sickle shape under hypoxic
conditions.
 Increased tendancy towards haemolysis

48. Heterozygous Half the circulating
hemoglobin is abnormal and half is
normal.
Have sickle cell trait
Homozygous  all of the
hemoglobin is abnormal.
Develop the full blown disease

49. Sickle Cell Disease
Hemoglobin S (HbS) ≥ 50% Hb present.
Homozygotic HbSS (sickle cell anemia) - HbS =
100% Hb present, Giving Sickle cell disease
HbSA disease - heterozygote for HbS and
HbA, with intermediate clinical severity. It is
called Sickle cell trait

50.  Basic abnormality - glutamic acid is replaced by
valine at the sixth position of the b-globin chain.
 2 normal a-globin and 2 abnormal b-globin chains
forms HbS.
 HbS carries O2 normally but begins to form
semisolid aggregate structures once O2 is unloaded
to the tissues.
 These HbS aggregates distort RBCs and cause
them to lose their normal elasticity.
Molecular and cellular changes of
hemoglobin S

52. Anemia - reduced O2 carrying
capacity of the blood
 Abnormal hemoglobin in RBCs:
Sickle Cell - one amino acid in the beta chains is wrong.
In low O2 conditions the beta chains form stiff rods which cause RBCs to sickle
blocking small vessels.

53. Hb-S polymerizes at low O2 tensions,
and this causes the red cells to become
sickle-shaped, hemolyze, and form
aggregates that block blood vessels.
The result is the severe hemolytic
anemia known as sickle cell anemia.

54. The sickle cell gene is an example of a gene
that has persisted and spread in the
population.
It originated in the black population in
Africa, and it confers resistance to one type
of malaria.
Africa = 40% of the black population have
the sickle cell trait.
In United States 10 %
Treatment:
Bone marrow Transplatation
Hb-F production by hydroxyurea.

55. Thalassemia
 Thalassemia is a group of inherited disorders of hemoglobin
synthesis characterized by a reduced or absent output of one or more
of the globin chains of adult hemoglobin .
 The name is derived from the Greek words Thalasso = Sea" and
"Hemia = Blood" in reference to anemia of the sea.
 Thalassemia result from over 150 mutations of the globin genes that
result in the absence or a reduction of the globin chains(α or β)
55

56. Thalassaemias:
 The name is derived from the Greek word
thalassa,which means sea.Greek identified this
disease present around Mediterranean sea.
 They are hereditary hemolytic diseases in which
the synthesis of either α- or β- globin chain is
reduced/absent.
 This decreased rate of synthesis of the globin
chains is due to mutation affecting the regulatory
gene rather than the structural gene.

57. Chromosomes
57

58. Alpha (a ) thalassemia
 It appears when a person does not produce enough
alpha chains for hemoglobin.
 It is mainly prevalent in the Africa, the Middle East ,
India, and occasionally in Mediterranean region
countries.
 There are four types categorized according to the
severity of their effects on persons with thalassemia :-
Silent Carrier State (1 affected gene)
 Alpha Thalassemia Trait (2 affected genes)
Hemoglobin H Disease (3 affected genes)
Alpha Thalassemia Major (also called hydrops fetalis, 4
affected genes)
58

59. A) α- thalassaemias: there are decreased or absent
synthesis of α-chains of haemoglobin with
compensatory increase in the synthesis of other
chains.
a.Homozygous α-chain thalassaemia (thalassaemia
α major):
Incompatible with life, and present as hydrops foetalis
usually die in utero , due to complete absence of α-
chains which are required for synthesis of HbF.
b.Heterozygous α-chain thalassaemia thalassaemia
α minor,(trait):

60. Alpha Thalassemia
 Alpha thalassemia is caused by mutations
in the alpha chain of the hemoglobin
molecule
 Major:all four alpha chain genes are
deleted, which is so severe that death can
occur in utero (prior to birth).
 Minor: two alpha chain genes are deleted
 Silent Carrier State ,Mild .

61. All 4 genes deleted
3 genes
defective & 1
normal
2 genes normal
2 genes
defective

62. Types of α thalassemia
 Silent Carrier State (1 affected gene)
The silent carrier will have normal hemoglobin levels and
red cell indices but can pass on the affected gene to their
offspring. Often, these individuals are identified only after
having a child with Hb H disease or alpha thalassemia trait.
 Alpha Thalassemia Trait (2 affected genes)
Patients who have alpha thalassemia trait have red blood
cells that are microcytic, hypochromic, have decreased
MCV, and have a mild chronic anemia, but they do not
generally experience any other symptoms. This is an
anemia that does not respond to iron supplements.
62

63. Types of α thalassemia
 Hemoglobin H Disease (3 affected genes)
With this condition, the decrease in the amount of alpha
globin chains produced causes an excess of beta chains,
which then aggregate into beta tetramers (groups of 4 beta
chains), known as Hemoglobin H.
Hb H disease can cause moderate to severe anemia and
splenomegaly (enlarged spleen).
Some individuals are asymptomatic while others have
severe anemia. Hemoglobin H disease is found most often
in individuals of Southeast Asian or Mediterranean
descent.
63

64. Types of α thalassemia
 Alpha Thalassemia Major (also called hydrops fetalis, 4
affected genes)
This is the most severe form of alpha thalassemia. In this
condition, no alpha globin is produced, therefore, no Hb A
or Hb F are produced.
64

65. Hydrops Fetalis
Foetuses affected by alpha thalassemia major become
anaemic early in pregnancy.
They become hydropic and frequently have enlarged hearts
and livers (swollen abdomen).
This diagnosis is frequently made in the last months of
pregnancy when a fetal ultrasound indicates a hydropic
foetus.
About 80% of the time, the mother will have toxaemia and
can develop severe postpartum bleeding (haemorrhage).
Foetuses with alpha thalassemia major are usually
miscarried, stillborn, or die shortly after birth.
65

66.  It appears when a person does not produce enough beta
chains for hemoglobin.
 It is mainly prevalent in the Mediterranean region
countries, such as Greece, Cyprus, Italy, Palestine and
Lebanon.
 There are 3 types categorized according to severity:
 Thalassemia minor
 Thalassemia intermedia
 Thalassemia major
Beta (ß) thalassemia
66

67. B) β- thalassaemias: Synthesis of of β-chains is decreased
or absent whereas synthesis of α-chains is normal and
will combine with δ-chains giving excess of HbA2 (α2δ2)
or it may combine with γ-chains producing excess of
HbF (α2γ2).
The abnormal haemoglobin do not function as normal
haemoglobin
- Homozygous (Thalassaemia β- major = Cooley's
anaemia = Mediterranian sea anaemia):
There is complete absence of β-globin chain and there is
marked increase of HbF.
- Heterozygous thalassaemia (Thalassaemia β- minor):
 There is slow rate of synthesis of β-globin chain.

70. THALASSEMIA DIAGNOSIS
 complete blood count (CBC) - a measurement of
size, number, and maturity of different blood cells
in a specific volume of blood.

hemoglobin electrophoresis with A2 and F
quantitation - a lab procedure that differentiates
the types of hemoglobin present.

FEP (free-erythrocyte protoporphyrin) and
ferritin - to exclude iron deficiency anemia

71. Management and treatment
Thalassemia minor (trait) :
No need for any treatment, since the carriers are usually
symptomless.
Thalassemia major:
The severe life-threatening anemia, requires regular life long
blood transfusion, to compensate for damaged red blood cells.
The continuous blood transfusion will eventually lead to iron
overload, which must be treated with chelation therapy to avoid
organ failure.
71

72. Management and treatment
Other novel treatments like bone-marrow transplantation are very
costly.
New treatments includes the use of oral chelators, to replace the
chelation treatment using Desferal delivered by infusion under
the skin through a battery-operated pump.
Gene therapy is also an option still researched
72

73. Other molecules containing Haem:-
i) Myoglobin – present in muscles
- combination with single
polypeptide chain.
ii) Neuroglobin – present in CNS
iii) Cytochrome enzymes – present in
mitochondria
iv) Peroxidases