Hemoglobin & jaundice

1. Hemoglobin
Dr. Sai Sailesh Kumar G
Associate Professor
Department of Physiology
RDGMC

2. Learning objectives
 List the steps in the biosynthesis of
hemoglobin
 Describe the fate of hemoglobin
 List the normal and abnormal hemoglobins
 Jaundice

3. Introduction
 Synthesis of hemoglobin begins in the pro-
erythroblasts and continues even into the
reticulocyte stage of the RBC.
 When reticulocytes leave the bone marrow and
pass through the blood stream, they continue
to form minute quantities of hemoglobin for
another day or so until they become mature
RBC

5. Formation of hemoglobin
1. Succinyl-CoA, formed in the Krebs metabolic cycle,
binds with glycine to form a pyrrole molecule.
2. Four pyrroles combine to form protoporphyrin IX
3. Protoporphyrin IX then combines with iron to form
heme molecule
4. Each heme molecule combine with long poly peptide
chain, a globin synthesized by ribosomes, forming a
sub unit of hemoglobin called hemoglobin chain
5. Each chain has molecular weight of about 16000
6. Four of these in turn bind together loosely to form the
whole hemoglobin molecule

6. Formation of hemoglobin
 There are several slight variations in the different sub-
unit hemoglobin chain
 The variation depends on the amino acid combination
of the poly peptide portion
 The different types of chains are designated alpha
chains, beta chains, gamma chains and delta chains
 The most common form of hemoglobin in the adult
human being, hemoglobin A
 Hemoglobin A is the combination of two alpha chains
and two beta chains
 Hemoglobin A has molecular weight of 64,458

8. Hemoglobin binding with oxygen
 Each hemoglobin chain has a heme prosthetic
group containing an atom of iron
 There are four hemoglobin chains in each
hemoglobin molecule
 Each of these can bind loosely with one
molecule of oxygen
 Total four molecules of oxygen (8 oxygen
atoms) can be transported by each hemoglobin
molecule

9. Binding affinity of hemoglobin
 The types of hemoglobin chains in the
hemoglobin molecule determine the binding
affinity of hemoglobin for oxygen
 Abnormalities of chains can alter the physical
characteristics of the hemoglobin molecule as
well
 Sickle cell anemia, the amino acid valine is
substituted for glutamic acid at one point in
each of the two beta chains

10. Sickle cell anemia
 Sickle cell anemia, the amino acid valine is
substituted for glutamic acid at one point in each of
the two beta chains
 When this type of hemoglobin is exposed to low
oxygen, it forms elongated crystals inside the RBC
that are some times 15 micro meters in length
 This makes it almost impossible for the cells to
pass through many small capillaries
 Spiked ends of these crystals rupture the cell
membrane

11. Inherited disorders of hemoglobin
Condition Mutation Functional
abnormality
Hb S (Sickle cell anemia) Val - Glu Shortened survival
Hb C Lys - Glu Shortened survival
Hb E Glu - Lys Microcytosis
Hb M Tyr - His Methemoglobin
formation
Alfa- Thalassemia Reduced or no
production of alpha
globin chain
Dysfunctional Hb
Beta- Thalassemia Reduced or no
production of beta globin
chain
Unstable RBC
membrane

12. Combination of Hb with oxygen
 Hb has ability to combine loosely and reversibly
with oxygen
 Combines with oxygen in the lungs
 Releases oxygen in the peripheral tissue
capillaries
 Oxygen does not become ionic oxygen but is
carried as molecular oxygen (composed of two
oxygen atoms)

13. Iron metabolism
 Iron is important in the formation of Hb
 Also important in the formation of other
essential elements of the body (myoglobin,
cytochromes etc)
 Total quantity of iron in the body is 4-5 g
 65% of this iron is in the form of Hb
 4% is in the form of myoglobin
 1% in the form of various heme compounds that
promote intracellular oxidation

14. Iron metabolism
 0.1 % is combined with the protein transferrin in
the plasma
 15-30% stored for later use, mainly in the
reticulo-endothelial system and liver
 The storage form of iron is ferritin

15. Transport and storage of iron
 Iron is absorbed from the small intestine
 In blood it binds with beta globulin, apotransferrin
to form transferrin
 Transferrin is transported in plasma
 Iron is loosely bound in transferrin
 So iron can be released to any tissue cell at any
point in the body
 Excess iron in the blood is deposited especially in
the liver hepatocytes and less in reticulo
endothelial cells of the bone marrow

16. Transport and storage of iron
 In the cell cytoplasm, iron combines with
apoferritin to form ferritin
 The iron stored as ferritin is called storage iron
 If the body capacity to store ferritin is exhausted
 Then extra iron will be stored in the form of
hemosiderin
 Hemosiderin collects in the cells as large clusters
that can be visible microscopically
 In contrast, ferritin particles are so small and
dispersed and visible only with electron
microscope

17. Transport and storage of iron
 When plasma iron content decreases
 Iron in the ferritin pool is removed easily and
transported in the form of transferrin
 Transferrin binds with the receptors on the
erythroblasts membrane in the bone marrow
 Then it is ingested into the erythroblasts by
endocytosis
 There transferrin delivers iron directly to mitochondria
 Heme is synthesized in mitochondria
 People with low transferrin levels – hypochromic
anemia (less Hb in the RBC)

18. Transport and storage of iron
 After 120 days life span of RBC
 RBC’s are destroyed
 Hb is released from the cells
 Ingested by monocyte-macrophage cells
 Iron is liberated and stored in ferritin pool
 Used as needed for formation of new
hemoglobin

19. Transport and storage of iron

20. Daily loss of iron
 0.6 mg of iron is lost in the feces each day
 Additional loss occurs when bleeding occurs
 For a women, additional menstrual loss of the
blood brings long-term iron loss to an average
of about 1.3 mg/day

21. Absorption of iron from intestinal tract
 Iron is absorbed from all the parts of small intestine
 Liver secretes moderate amounts of apotransferrin into the
bile
 Bile flows through bile duct into duodenum
 In duodenum apotransferrin binds with free iron and other
iron compounds such as hemoglobin and myoglobin from
meat
 This combination is called transferrin
 It binds with the receptors of the intestinal epithelial cell
membranes
 Absorbed into epithelial cells by pinocytosis
 Released into the blood capillaries as plasma transferrin

22. Absorption of iron from intestinal tract
 Iron absorption from the intestines is extremely
slow
 Maximum rate of few milligrams per day
 Even when tremendous quantities of iron is
present in the food
 Only small proportions can be absorbed

23. Fate of hemoglobin
 Old RBC’s are destroyed in the reticulo endothelial
system especially in spleen
 Spleen – Graveyard of RBC’s
 Old RBC’s become fragile due to decrease in the
NADPH activity
 The fragile membrane of old RBC ruptures when
they pass through small capillaries
 Release of hemoglobin
 Hb is taken by reticulo endothelial cells (tisue
macrophages)

24. Fate of hemoglobin
 In tissue macrophages, Hb is breaks to heme and
globin
 Globin is degraded into amino acids, which enters
into amino acid pool of plasma
 Heme is acted upon by microsomal oxygenase
 Iron and carbon monoxide liberated (only step that
release CO) and product formed is biliverdin
 Iron is released into the circulation and carried to
bone marrow and other tissues.
 Storage form of iron - ferritin

25. Fate of hemoglobin
 Biliverdin is acted upon by NADPH-dependet
biliverdin reductase to form bilirubin
 When 1 gram of HB is destroyed, 35 mg of
bilirubin is formed
 Normal bilirubin is 0.2-0.8 mg/100mL of blood
 When it becomes more than 2 mg/100mL of
blood, the condition is called Jaundice

26. Fate of hemoglobin

27. Fate of hemoglobin
 Bilirubin enters circulation where it combines with
albumin and transported to liver
 In liver, uptake, conjugation and excretion steps
1. Uptake - Bilirubin splits from albumin and enters
liver cells
2. Conjugation – bilirubin is conjugated with 2
molecules of glucuronic acid to form birubin
diglucuronide (glucuronyl transferase)
Bilirubin glucuronide is water soluble conjugated bilirubin
3. Excretion – Bilirubin diglucuronide is extreted into
intestine through bile ducts

28. Fate of hemoglobin

29. Fate of hemoglobin
 In intestine, BDG is acted upon by bacterial enzymes in
the terminal ileum and large intestine to form
urobilinogen
 Some of the urobilinogen enters portal circulation and
reaches the liver. This is known as enterohepatic
circulation of bile pigments
 Some urobilinogen escapes into general circulation and
is excreted through urine as urobilinogen
 Rest of the urobilinogen present in the large intestine is
converted to stercobilinogen and excreted through
feces
 This gives golden yellow color to feces

30. Fate of hemoglobin
 Conjugated bilirubin is water soluble and is
excreted through urine
 Unconjugated bilirubin is insoluble and hence not
excreted through urine
 The reason is unconjugated bilirubin is bound to
albumin and so it is not filtered by kidneys.
 Severe obstructive jaundice, significant amounts of
conjugated bilirubin appears in urine
 This can be demonstrated by shaking the urine and
observing the foam, which turns an intense yellow.

31. Fate of hemoglobin

32. Jaundice
 Define jaundice
 Classify different types of jaundice
 Describe the biochemical tests done to
investigate
 Jaundice and how they can be interpreted

33. Jaundice
 Jaundice refers to yellowish tint to the body
tissues, including a yellowness of the skin and
deep tissues
 The usual cause of jaundice is large quantities of
bilirubin in the ECF (either conjugated or
unconjugated bilirubin)
 Normal plasma concentration of bilirubin (almost
entirely unconjugated form) averages 0.5 mg/dL
 In abnormal conditions, this can raise to 40 mg/dL
and much of it become conjugated form

34. Jaundice
 The skin usually begin to appear jaundiced
when the concentration rises to about three
times normal
 That is above 1.5 mg/dL
 The common cause of jaundice are ???

35. Common cause of Jaundice
 Increased destruction of RBC, with rapid
release of bilirubin into the blood – Hemolytic
jaundice
 Obstruction of the bile ducts (Obstructive
jaundice)
 Damage to the liver cells (Hepatic Jaundice)
 so that even usual amounts of bilirubin can not
be excreted into GIT

36. Hemolytic Jaundice
 Function of liver is not impaired
 No Obstruction of the bile ducts
 RBC’s are hemolyzed so rapidly
 Hepatic cells can not excrete the bilirubin as quickly as
it is formed
 Plasma concentration of free bilirubin raises above
normal levels (unconjugated bilirubin increases)
 Rate of formation of urobilinogen in the intestine
increases
 Much of this is absorbed into the blood and later
excreted in urine

37. Obstructive Jaundice
 Obstruction of bile ducts
 Most often occurs when a gall stone or cancer blocks the
common bile duct
 RBC’s are hemolyzed normally
 Rate of formation of bilirubin is normal
 Unconjugated bilirubin enters liver cells and becomes
conjugated bilirubin
 But conjugated bilirubin formed can not pass from the blood
into the intestines
 Conjugated bilirubin returned (regurgiteted) to the blood
 Plasma conjugated bilirubin increases
 Urine bilirubin increases

38. Obstructive Jaundice
 Conjugated bilirubin returned to the blood
 How??
 Probably by rupture of congested bile canaliculi
and direct emptying of bile into the lymph leaving
the liver

39. Obstructive Jaundice
 Obstruction of bile ducts
 No bilirubin can reach intestine
 No urobilinogen to be absorbed into blood
 No urine urbilinogen
 Urobilinogen in urine negative
 Stools become clay colored due to lack of
stercobilinogen and other bile pigments
 Steatorrhea – increased fat excretion in the
stools due to absence of bile pigments

40. Hepatic Jaundice
 Damage of hepatic cells which occurs in hepatitis
 RBC’s are hemolyzed normally
 Rate of formation of bilirubin is normal
 Liver function is abnormal
 All the three phases of liver are effected
 Decreased uptake – increase in plasma unconjugated
hemoglobin
 Decreased excretion – conjugated bilirubin regurgitates into
blood
 Both conjugated and unconjugated bilirubin increases in
blood.
 Steatorrhea is present

41. Diagnosis of Jaundice
 Clinical laboratory tests can be used to
differentiate unconjugated and conjugated
bilirubin in the plasma
 In hemolytic jaundice, almost all the bilirubin is
unconjugated form
 In obstructive jaundice, it is mainly in the
conjugated form
 In hepatic jaundice, both conjugated and
unconjugated forms

42. Diagnosis
 Conjugated bilirubin is water soluble and is
excreted through urine
 Unconjugated bilirubin is insoluble and hence not
excreted through urine
 The reason is unconjugated bilirubin is bound to
albumin and so it is not filtered by kidneys.
 Severe obstructive jaundice, significant amounts of
conjugated bilirubin appears in urine
 This can be demonstrated by shaking the urine and
observing the foam, which turns an intense yellow.

43. Tests for jaundice
 Serum unconjugated bilirubin (indirect Van den Bergh test)
 Serum conjugated bilirubin (direct Van den Bergh test)
 Urine urobilinogen
 Liver function tests
 Serum
1. Alanine Transaminases
2. Aspartate Transaminases
3. Alkaline phosphatase
4. Gamma- glutamyl transpeptidase
 Coagulation tests
1. Prothrombin time

45. THANK YOU