Hematology 1

topic 5
hemoglobin
determination
 OVERVIEW OF Topic 5
1. Hemoglobin

2. Iron

3. Protoporphyrin

4. Globin

5. Porphyria

6. Functions of Hgb
 01
hemoglobin
 hemoglobin
• Conjugated globular protein consisting of 4 heme groups
and 2 heterogenous pairs of polypeptide chain

• Main cytoplasmic component of erythrocytes

• Serves as a vehicle for transportation of oxygen and carbon

dioxide

• 65% are synthesized in the nucleated RBC

• 35% are synthesized in the reticulocyte stage

 hemoglobin
3 Functions of Hemoglobin

1. Transport of oxygen form lungs to tissues

2. Transport of carbon dioxide from tissues to lungs

3. Buffering of the blood

 hemoglobin
4 significantly different constituent of Hemoglobin

1. A protein component called globin composed of sets of

two different polypeptide chain

2. Four molecules of nitrogenous substances

3. Four iron atoms in the Fe2+ state that combine with

protoporphyrin IX to form 4 heme molecules

4. One 2-3-DPG molecule as a sometime resident in the

center of the hgb
02
iron
 Iron
• Electron exchange

• Oxygen transport

• Control of toxic free radicals

• The iron from diet comes in either two forms

Iron MEtabolism
 Distribution of iron in the body
a. Hemoglobin

b. Myoglobin

c. Storage iron
 Iron absorption
• A normal individual absorb 4 – 10% of iron of the iron
ingested with an average of 6% hence about 1 – 2 mg of
iron is absorbed daily

• Mucosal Block Theory – a theory to which the ferritin-

apoferritin system in mucosal cells controls the amount of
iron absorb

• Influenced by :

o Amount & type of iron accessible from food

o Functional state of gastrointestinal mucosa and
pancreas
o Current iron stores
o EPO needs
 Iron excretion
• Normally excreted 0.9 – 1.3 mg or iron / day
 Iron overload occurs
1. Excessive mucosal absorption of dietary iron

2. Multiple or repeated blood transfusion

Methods of investigating iron metabolism
Serum (Plasma) Iron Concentration or Ferritin Level

1. Heme

2. Non-heme
Methods of investigating iron metabolism
Interacting forces involved in detection of plasma iron level:

a. Iron input comes from such sources as hemoglobin

destruction, iron absorption, and release of iron from iron
stores

b. Iron is utilized for hemoglobin synthesis, storage deposits,

myoglobin synthesis and cellular enzyme formation

c. Iron is lost through desquamation of cells in feces and

urine, etc.
Methods of investigating iron metabolism
Decreased in

• Iron deficiency anemia

• Chronic infection

• After gastrectomy

• Rheumatoid arthritis and malignant tumors

• During the period of recovery from hemolytic anemia

Methods of investigating iron metabolism
Increased in

• Anemias characterized by decreased hemoglobin

formation not due to iron deficiency such as aplastic and
sideroachrestic anemia

• Hemochromatosis and hemosiderosis

• After administration of iron

• Hemolytic anemia such as PA and thalassemia which are

characterized by ineffective erythropoiesis

• Acute hepatitis
Methods of investigating iron metabolism
Total Iron Binding Capacity

• Increases the maximal saturation of transferring with iron

and it then measures the total amount of transferring iron
available for iron binding

• Increased in: IDA and the use of oral contraceptives,

physiologically elevated in pregnancy
Methods of investigating iron metabolism
Unsaturated Iron – Binding Capacity (UIBC)

• Measures the concentration of transferring not bound to

iron and is arrived at by subtracting the serum iron level
from the maximal level of iron-saturated transferring

Patient Saturation of Transferrin

• Represent the proportion of iron-binding protein that is

saturated with iron
 03
protoporphyrin
protoporphyrin
 protoporphyrin
Porphyrinogen

• Are the intermediate of heme synthesis not porphyrins

Heme Protoporphyrin

• Name IX because it was the 9th of 15 possible isomers

synthesized by Hans Fischer who was the first to study and
describe porphyrin
04
globin
 globin
• Globin chains are synthesized in the ribosome

• Alpha chain are numbered beginning with 1 at the N-

terminal beginning of the chain at the C-terminal
 05
Porphyria
 Porphyria
• There is an excessive production of intermediate metabolite
or overproduction of porphyrin and or porphyria precursor
ALA and PBG

• Large amount of UPG I accumulating in the BM, RBC, and

urine
 Porphyria
Erythropoietic porphyria

• Accumulation of porphyrins in the BM associated with high

incidence of photosensitivity and dermatitis

• CEP (Congenital Erythropoietic Porphyria): rare inherited

disorder characterized to have red teeth, disfigured, hirsute,
photosensitivity, dermatitis (cutaneous blister and solar
sensitivity) and nocturnal (Werewolf legend)

• Excrete red pigmented urine due to excessive excretion of

Coproporphyrin I & Uroporphyrin I
 Porphyria
Hepatica porphyria

• Accumulation of porphyrin precursors in the liver associated

with neurological deficit causing excruciating pain

• Acute Intermittent Hepatic Porphyria

 Porphyria
Mixed Porphyria

• Accumulation in both BM and liver resulting in both

neurologic and dermatologic involvement

• Variegata porphyria

• Diagnosis may be made in a 24-hour urine collection

 06
Functions of hemoglobin
 Functions of hemoglobin
a. Transport of oxygen from the lungs to the tissues and of
carbon dioxide in the reverse direction

b. Assisting in the acid-base regulation by eliminating CO2 in

the lungs and by the buffering action of imidazole histidine
group of hemoglobin

Two Allosteric form of Hgb:

1. Tense or Taut or T form

2. Relaxed or Reactive or R Form

 Oxygen dissociation curve
• Represents the hemoglobin affinity of oxygen and used to
designate the partial pressure of oxygen in which the
hemoglobin molecule is 50% saturated with oxygen

• The sigmoidal shape of the ODC is due to the cooperative

binding of oxygen by hemoglobin

• The curve shows the relationship of the percentage of

oxygen saturation to the oxygen tension
 AbNormal derivatives of hemoglobin
Carboxyhemoglobin

• The combination is irreversible. The affinity of Hgb of Co is

210 greater than that of oxygen

• Cherry red or bright cherry pink on patient’s blood and skin

 AbNormal derivatives of hemoglobin
Sulfhemoglobin

• Irreversible reaction – damage is in the structural portion

within the protoporphyrin of the heme moiety

• Erythrocytes containing S-Hb have normal survival and

osmotic fragility

• Produces a mauve or lavender color in a sample screening

test
 AbNormal derivatives of hemoglobin
Methemoglobin

• Change from Ferrous to Ferric state due to exposure to an

oxidizing agent

• Absorption is at 630 – 633 nm

• Normally Met Hgb is continuously being formed within the

erythrocyte because of the oxidation of hgb, but is
prevented from accumulating within the red cells by specific
enzyme Met Hgb reductase or diaphorase
 07
Abnormal hemoglobin
 Abnormal hemoglobin
These congenital variants of normal hemoglobins from which
they differ in their globulin structure:

1. The exchange of a single amino acid for another amino

acid in an otherwise unchanged amino acid sequence

2. The deletion of one or more amino acid from a polypeptide

chains resulting on a shorter than normal chain

3. An abnormal arrangement of otherwise normal amino acid

chains

4. An abnormal linkage of portions of peptide chains

TYPES OF ABNORMAL HEMOGLOBINS
(HEMOGLOBINOPATHIES
Hemoglobin S

• Most common variant in about 8 – 11% in American

negroes

• Sickle cell disease

• Sickle cell anemia: caused by homozygous variant of sickle

cell disease

• Sickle cell trait: a heterozygous form of sickle cell disease

• Valine replaces glutamic acid

TYPES OF ABNORMAL HEMOGLOBINS
(HEMOGLOBINOPATHIES
Hemoglobin C

• Lysine replaces glutamic acid at 6th amino acid of beta chain

• Positive charge replaces negative

TYPES OF ABNORMAL HEMOGLOBINS
(HEMOGLOBINOPATHIES
Hemoglobin E

• Substitution of lysine for glutamine at 26th amino acid of

beta chain

• Second most common abnormal Hb in the world of SE

Asian
TYPES OF ABNORMAL HEMOGLOBINS
(HEMOGLOBINOPATHIES
Hemoglobin M

• Mutation altering alpha or beta chain in region of histidine

residue of heme pocket

• Leads to oxidation of heme iron to form methemoglobin