1. Lecturers of Medical Biochemistry and Molecular Biology
Heme Metabolism
(synthesis)
Dr. Dalia Abdelwahab
&
Dr. Marian Maher
Lecturers of Medical Biochemistry and Molecular Biology

2. Lecture 1

3. Intended Learning Outcomes ILOs
Describe the structure of Porphyrins
List important heme-proteins
Describe steps of heme biosynthesis
Identify the regulation of heme synthesis

4. Structure Of Porphyrins
They are cyclic compounds formed from:
4 pyrrole rings linked by methenyl bridges
4 methyl , 2 propionyl and 2 vinyl groups connected to the pyrrole rings

5. They constitute a system of conjugated double bonds which absorb light
Porphyrinogens are reduced forms of porphyrins .They contain methyl (- CH2-) rather than methenyl bridges (-HC=).
So,
they have no conjugated double bonds and colorless .

6. Iron porphyrins (Heme), Magnesium porphyrins (Clorophyll).
Porphyrins form complexes with metal ions , that bind to Nitrogen of pyrrole rings.:
Iron porphyrins (Heme), Magnesium porphyrins (Clorophyll).

7. Some important heme-proteins
Haemoglobin
Myoglobin
Cytochromes of electron transport chain (cyt aa3,cyt.c)
cytochrome P450
Catalse and Peroxidase (degradation of H2O2), ,
Tryptophan pyrrolase (Oxidation of tryptophan),
Cytoplasmic guanyl cyclase (activated by NO).

8. Biosynthesis of Heme
The principal tissues involved in heme biosynthesis are mainly the bone marrow and the liver.
Heme biosynthetic pathway is partly mitochondrial and partly cytosolic. The reactions are Irreversible.

9. Steps:
The initial
reaction and the last three steps in the formation of porphyrins occur
in mitochondria, whereas the intermediate steps of the biosynthetic
pathway occur in the cytosol
porphobilinogen synthase
(A pyrrol)
hydroxymethylbilane synthase
(tetrapyrrol)

10. 1- Formation of -aminolevulinic acid (ALA):
Glycine and succinyl coenzyme A that condense to form ALA by mitochondrial ALA synthase (ALAS) .
This reaction requires pyridoxal phosphate (PLP)
It is the committed and rate-limiting step in porphyrin biosynthesis.
There are two isoforms of ALAS 1 and 2.
Erythroid tissue produces only ALAS2.
TCA cycle
To produce one molecule of heme, 8 molecules each of glycine
and succinyl CoA are required.
COASH

11. 2-Formation of porphobilinogen:
By Zn-containing ALA dehydratase (porphobilinogen synthase)
It is extremely sensitive to inhibition by heavy metal ions e.g. lead that replace the zinc .
This inhibition cause the elevation in ALA and the anemia seen in lead poisoning.
porphobilinogen synthase
(cytosolic enzyme)

12. 3- Formation of hydroxymethylbilane:
The condensation of four
porphobilinogens produces
the linear tetrapyrrole,
hydroxymethylbilane,
porphobilinogen deaminase

13. 4- Formation of uroporphyrinogen:
Hydroxymethylbilane,
which is
isomerized and cyclized by uroporphyrinogen III
Synthase
to produce the
asymmetric uroporphyrinogen III.

14. 5- Formation of coproporphyrinogen:
This cyclic tetrapyrrole undergoes decarboxylation of its acetate groups, generating coproporphyrinogen III.
These reactions occur in the cytosol.

15. enters the mitochondrion
6- Formation of protoporphyrinogen, protoporphyrin, heme
enters the mitochondrion
Corpropophrynogen
2 CO2
2 P V
(IX)
CH CH
(IX)
inhibited by
lead

16. (porphobilinogen synthase)
zinc
4 NH4 Fe
4 CO2

18. MCQ Porphyrin ring present in all of the following except:
A. peroxidase
B. Xanthine oxidase
C. Tryptophan pyrrolase
D. catalase
E. guanyl cyclase

19. MCQ An amino acid required for porphyrin synthesis is: A. proline
B. serine
C. glycine
D. histidine
E. alanin

20. Regulation of Heme Synthesis
ALA synthase is the rate
limiting regulatory enzyme
Excess heme is converted to hemin by oxidation of Fe2+to Fe3+. Hemin decreases the amount of ALAS1 by repressing transcription of its gene (act as aporepressor), increasing degradation of its messenger RNA, and decreasing import of the enzyme into mitochondria.

21. In erythroid cells, ALAS2 is controlled by the availability of intracellular iron.

22. Substances metabolized by cytochrome P450 increases the activity of ALAS1 in liver e.g. barbiturates , alcohol and carcinogens.
Drugs metabolized by cytochrome P450 monooxygenase in the liver .
Synthesis of cytochrome P450
Consumption of heme
a component of cytochrome P450 proteins.
The concentration of heme in liver
Cells.
The synthesis of ALAS1(derepression)

23. ALA dehydratase and ferrochelatase inhibited by Lead poisoning

24. MCQ -Aminolevulinic acid synthase activity:
A. in liver is frequently decreased in individuals treated with drugs, such as the barbiturate phenobarbital.
B. catalyzes a rate-limiting reaction in porphyrin biosynthesis.
C. requires the coenzyme biotin.
D. is strongly inhibited by heavy metal ions such as lead.
E. occurs in the cytosol.

25. Lecture 2

26. Intended Learning Outcomes ILOs
Define Porphyrias
Identify clinical manifestations of porphyrias
List different types of porphyrias
Describe the treatment of porphyrias

27. Disorders of Heme Synthesis 1) sideroblastic anemia
Loss of function mutations in ALAS2 result in X-linked sideroblastic anemia. Low levels of ALA2 cause no porphyria (only anemia) no known defect of ALA1

28. Disorders of Heme Synthesis
( Porphyrias)
A group of diseases collectively called porphyrias are associated with abnormalities in biosynthesis of heme. They are characterized by accumulation and excretion of porphyrins or porphyrin precursors.
ALA dehydratase porphyria
Hydroxymethylbylane
(protoporphyria)

29. “Porphyria” refers to the purple color caused by pigment-like porphyrins in the urine of some patients
One common feature of the porphyrias is a decreased synthesis of heme. In the liver, heme normally functions as a repressor of the gene for ALAS1. Therefore, the absence of this end product results in an increase in the synthesis of ALAS1 (derepression). This causes an increased synthesis of intermediates that occur prior to the genetic block.
The accumulation of these toxic intermediates is
the major pathophysiology of the porphyrias.

30. The porphyrias are classified as 1-erythropoietic (Congenital erythropoietic porphyria & Protoporphyria (erythropoietic porphyria)). 2-hepatic (chronic or acute) depending on whether the enzyme deficiency occurs in the erythropoietic cells of the bone marrow or in the liver.

31. Most of porphyrias are
1-inherited as autosomal dominant manner except congenital erythropoietic porphyria (recessive).
2- acquired porphyrias can result from lead poisoning. The toxic effect of lead is due to inhibition of ferrochelatase and ALA dehydratase.

32. Clinical manifestations:
Individuals with an enzyme defect prior to the synthesis of the tetrapyrroles manifest abdominal and neuropsychiatric signs.
Whereas those with enzyme defects leading to the accumulation of tetrapyrrole intermediates show photosensitivity that is, their skin itches and burns (pruritus) when exposed to visible light.

33. Enzyme deficiency early in pathway before formation of tetrapyroles :
only abdominal pains and neuro-psychiatric symptoms are present as in acute intermittent porphyria which is characterized by deficiency of the enzyme porphobilinogen deaminase

34. to visible light, urine is red
Enzyme deficiency late in pathway after formation of tetrapyroles & porphyrinogen :
Porphyrinogens are oxidized to their corresponding porphyrins which react with molecular oxygen to form reactive oxygen species that cause oxidative damage membranes and cause the release of destructive enzymes from lysosomes.
photosensitivity (skin inflammation damage with ultimate disfigurement and scaring.) in response
to visible light, urine is red
Porphyria cutanea tarda which is the (most common type).
Due to low levels of  
Uroporphyrinogen decarboxylase

35. Symptoms of the acute hepatic porphyrias (as ALA dehydratase deficiency porphyria, acute intermittent porphyria, hereditary coproporphyria, and variegate porphyriaare) often precipitated by drugs that cause induction of cytochrome P450 e.g. steroids , alcohol, Phenobarbital ,are contraindicated for porphyria patients because they precipitate attacks.

36. Treatment
The severity of symptoms of porphyrias can be diminished by intravenous injection of hemin which decreases synthesis (represses) ALA synthase.
Avoidance of sunlight and ingestion of B-carotenes (anti-oxidant), are helpful in photosensitivity.

37. MCQ Biochemical basis of precipitation of porphyria by barbiturates is
A. Repression of ALA synthase
B. Derepression of ALA synthase
C. Rerepression of ALA synthase
D. MiRNA mediated

38. MCQ Most common porphyria is due to deficiency of A. PBG deaminase
B. Uroporphyrinogen decarboxylase
C. Ferrocheletase
D. Coproporphyrinogen oxidase
E.ALA synthase

39. Lecture 3

40. Catabolism of Heme
After RBCs reach the end of their life span ( average 120 days), they are phagocytosed by reticulo-endothelial cells of liver, spleen and bone marrow. Hemoglobin is degraded first into globin and heme

41. 1-Oxidation of Heme : a- Heme is degraded by microsomal enzyme; of the reticuloendothelial cells of liver, spleen and bone marrow which requires molecular oxygen and NADPH b- It catalyzes the cleavage of α methenyl bridge between the pyrrole rings I and II to from biliverdin with the release of carbon monoxide CO. c- Iron is liberated from heme d- In mammals, biliverdin is further reduced to bilirubin by NADPH – dependent biliverdin reductase.

42. 2- Transport of Bilirubin:
Bilirubin is slightly soluble in plasma. It transported to the liver by non covalent binding to albumin forming hemobilirubin (unconjugated or indirect bilirubin).
Liver uptake bilirubin by carrier mediated transport.
3- Uptake of bilirubin by liver: The solubility of bilirubin increase by conjugating with 2 molecules of glucuronic acid to form cholebilirubin (conjugated or direct bilirubin).
This reaction is catalyzed by glucuronosyl transferase enzyme

43. Extended Modular Program
But where do Gulcuronic acid come from???
Extended Modular Program

44. Obtain glucuronic acid
Uronic acid pathway
Occur in liver
Oxidize glucose (C6) to
Obtain glucuronic acid
Not ATP

45. G-1-P uridyl transferase
Steps:
G-1-P uridyl transferase

46. Importance Detoxification of some compounds
steroids, bilirubin and some drugs
Glucuronic acid is a highly polar molecule
so, it has the ability to be conjugated with less polar compounds make them more water soluble thus facilitating their renal excretion.
2) Glucuronic acid is also a component of the glycosoaminoglycans & proteoglycans

47. 3) Glucuronic acid is oxidized to L-Gulonic acid which has 2 fates:
In mammals, except human and guinea pigs, it is converted to L-ascorbic acid (vitamin C).
It is converted to L-Xylulose then to D-Xylulose which can enter HMP pathway to complete its metabolism.

48. 4- Excretion of conjugated bilirubin:
The conjugated bilirubin secreted with the bile into intestine (the unconjugated bilirubin not secreted) where it is hydrolyzed and reduced by the gut bacteria to urobilinogen (colorless compound).  
Most of urobilinogen is oxidized by intestinal bacteria to stercobilin, which give feces the characteristic brown color.
90%
By bacteria
10%
Some of urobilinogen is reabsorbed from the gut and enters the portal blood and resecreted to the kidney, where it is converted to urobilin that gives the urine the characteristic yellow color

49. urobilin
bacterial oxidation
stercobilin

50. Lecture 4

51. Hyperbilirubinemia The normal plasma bilirubin level range from 0
Hyperbilirubinemia The normal plasma bilirubin level range from 0.3 – 1 mg/dl . If the serum bilirubin exceeds 1 mg/dl, the condition is called hyperbilirubinemia. If the bilirubin level exceeds 2 mg/dl. Jaundice will occur with yellowish discoloration of sclera, conjunctiva and skin. The sclera is particularly affected because it is rich in elastin, which has a high affinity for bilirubin.

52. Note that:
unconjugated bilirubin can cross the blood-brain barrier into the central nervous system so encephalopathy due to hyperbilirubinemia (kernicterus) thus occurs only with unconjugated bilirubin.
Alternatively, because of its water-solubility, only conjugated bilirubin can appear in urine.
choluric jaundice (choluria is the presence of bile pigment in the urine) occurs only in regurgitation conjugated hyperbilirubinemia,
acholuric jaundice occurs if only the presence of an excess of unconjugated bilirubin.

53. MCQ The substance deposited in skin and sclera in jaundice is:
A. biliverdin.
B. only unconjugated bilirubin.
C. only direct bilirubin.
D. Both direct and indirect bilirubin
E. hematin.

54. Classification of Hyperbilirubinemia
Type of bil.
conjugated
unconjugated
Site of defect
Prehepatic
Hepatic
Posthepatic 55

55. Type of Bilirubin Unconjugated Conjugated Unconjugated Conjugated
Neonatal “physiological jaundice”
Hemolysis
Gilbert syndrome
Crigler-Najjar syndromes types I & II
Hepatic damage
Conjugated
Obstruction of the biliary tree
Dubin–Johnson syndrome
Rotor syndrome
Unconjugated
imbalance between the rates of production of bilirubin and its uptake or conjugation in the liver
Conjugated
Elevations of conjugated bilirubin levels in plasma are due to liver and/or biliary tract disease.

56. Site of defect Prehepatic Hepatic Posthepatic
Hemolytic
Neonatal jaundice”
Hepatic
Hepatitis
Crigler-Najjar syndromes I & II
Gilbert syndrome
Dubin–Johnson syndrome
Rotor syndrome
Posthepatic
Obstruction of the biliary tree
Prehepatic
Increase bilirubin production
Hepatic
Defect in conjugation and/or excretion of bil.
Posthepatic
Obstruction of bile duct

57. 1) Hemolytic Jaundice
Due to: In neonates Rh incompatibility between maternal and fetal blood.
In children and even in adult from enzyme deficiency of G-6-P dehydrogenase or pyruvate kinase or sickle cell anemia.
Extensive hemolysis produce bilirubin faster than it can be conjugated UCB levels in the blood become elevated
more CB is made and excreted into the bile, the
amount of urobilinogen entering the enterohepatic circulation is increased, and urinary urobilin and stercobilin is increased
causing jaundice, normal colour of urine and stool

58. 2) Neonatal “Physiological Jaundice”
This is transient hyperbilirubinemia
due to accelerated rate of destruction of RBCs and to the immature hepatic system of conjugation.
Elevated UCB, in excess of the binding capacity of albumin (20–25 mg/dl)
Causing diffuse into the basal ganglia, cause toxic encephalopathy (kernicterus)
ttt
Phototherapy (converts bilirubin to more polar water-soluble isomers) and barbiturates (promoter of bilirubin-metabolism)

59. 3) Hepatocellular Jaundice
due to cirrhosis or hepatitis
Results in: Damage to liver cells can cause UCB levels in the blood to increase as a result of decreased conjugation
Inflammatory oedema of hepatocytes will compress the intracellular canaliculi “mild obstruction” causing increased CB
Urobilinogen decreased if micro-obstruction is present
Causing the urine consequently darkens, whereas stools may be a pale, clay color

60. 4) Crigler-Najjar syndrome
Type I Crigler-Najjar: autosomal recessive disorder
due to mutations in the gene encoding bilirubin-UGT (UDP-glucuronyl-transferase)activity in hepatic tissues.
serum UCB usually exceeds 20 mg/dL
It is characterized by severe congenital jaundice
ttt Phototherapy reduces plasma bilirubin levels somewhat, but phenobarbital has no effect.
Type II
(UDP-glucuronyl-transferase), have some activity and the condition has a more benign course than type I. Serum UCB concentrations usually do not exceed 20 mg/dl,ttt: patients respond to treatment with large doses of phenobarbital

61. 5) Gilbert’s disease
due to congenital defect in conjugation by hepatocytes (70–80% reduction) and 30% of the bilirubin UDP-glucuronosyl
transferase activity is retained in Gibert syndrome the condition is harmless.

62. 6) Dubin Johnson syndrome It is a benign autosomal recessive.
caused by mutations in the gene encoding the protein involved in the secretion of conjugated bilirubin into bile.
So CB increases

63. Extended Modular Program
7) Rotor syndrome
Its cause has not been identified, but it is related to Dubin Johnson syndrome
This is a rare benign condition characterized by chronic conjugated hyperbilirubinemia and normal liver histology.
Extended Modular Program

64. 8) Obstructive Jaundice
Due to obstruction of the common bile duct as in Biliary cirrhosis – hepatoma - Gall stones- Cancer head of pancreas “The most severe form of Jaundice, bilirubin > 30 mg/dl”.
Preventing passage of CB into the intestine The liver “regurgitates” CB into the blood (hyperbilirubinemia).
The CB is eventually excreted in the urine (which darkens upon standing), and is referred to as “urinary bilirubin.” Urinary urobilinogen is absent.
Causing GI pain and nausea and produce stools that are a pale, clay color.

65. Obstruction of bile duct Hepatic jaundice
Post hepatic jaundice 
Obstruction of bile duct
Hepatic jaundice 
Defect in conjugation and/or excretion of bilirubin in bile
Prehepatic jaundice
Increase bilirubin production
biliary stones
cancer head of pancreas
1.Gilbert syndrome
2.Crigler-najjar syndrome
Dubin-johnson syndrome
Rotor syndrome
Hepatic damage
e.g;
Hepatitis
1.hemolysis of RBCs as in sickle cell anemia,
G6PD deficiency and RH incompatibility
2.Neonatal jaundice
Causes:
direct (conjugated)
Indirect
direct
Indirect + direct
Indirect (unconjugated)
Type of elevated bilirubin
Present
(choluric)   
Absent 
(choleric)
Decrease
Absent
(acholuric)
Normal color
 increase
Urine
1.Presence of conjugated bilirubin (dark colored urine)
2.urobilinogen
Pale clay colored and bulky(steatorrhea)
absent
Pale clay colored
decrease
Normal
increase
Stool
1.Color and consistency
2.stercobilin
Normal 
Increased 
………. 
……………. 
Present (itching)
Increased
…… 
…….. 
…….
Low 
Elevated
 ……..
Blood test:
Serum ALT and AST
Serum ALP
Blood hemoglobin 
Reticulocyte count
Serum bile salts
Serum cholestero