Degradation of Heme Mahmoud A. Alfaqih BDS PhD Jordan University of Science and Technology

ORGANIZATION OF THE GLOBIN GENES

Organization of the globin genes The genes for the α-globin-like and β-globin- like subunits of hemoglobins occur in two separate gene clusters (or families) Families are located on two different chromosomes

α-Gene family The α-gene cluster is located on chromosome 16 and contains the following genes: 1.Two genes for the α-globin chains 2.The ζ gene 3.A number of globin-like genes that are not expressed (psuedogenes)

β-Gene family Contains the following genes on chromosome 11: 1.A single gene for the β-globin chain 2.ε gene 3.Two γ genes 4.δ gene

Hemoglobinpathies

Hemoglobin electrophoresis

Distribution of sickle cell disease in Africa Distribution of malaria in Africa

Methemoglobinemias Oxidation of the heme component of hemoglobin to the ferric (Fe 3+ ) state forms methemoglobin Methemoglobin cannot bind oxygen Oxidation is caused 1. Drugs (such as nitrates) 2. Reactive oxygen intermediates 3. Deficiency of NADH-cytochrome b 5 reductase (the enzyme that converts methomoglobin back to hemoglobin)

Methemoglobinemias The methemoglobinemias are characterized by “chocolate cyanosis” Chocolate cyanosis is a brownish-blue coloration of the skin and membranes) and chocolate-colored blood Treatment is with methylene blue which promotes reduction of Fe +3 to Fe +2

Degradation of heme  After 120 days, RBCs are taken up and degraded by the reticuloendothelial system (Liver and spleen)  85% of heme destined for degradation come from mature RBCs.  15% is from turnover of immature RBCs and cytochromes from extraerythroid tissues

1. Formation of bilirubin  Catalyzed by the microsomal heme oxygenase system of the reticuloendothelial cells  In the presence of NADPH and O2, the enzyme adds a hydroxyl group to the methenyl bridge between two pyrrole rings, with a concomitant oxidation of ferrous iron to Fe3 +  A second oxidation by the same enzyme system results in cleavage of the porphyrin ring.  The green pigment biliverdin is produced as ferric iron and CO are released  Biliverdin is reduced, forming the red-orange bilirubin

2. Uptake of bilirubin by the liver  Bilirubin is slightly soluble in plasma and is transported to the liver by binding non-covalently to albumin  Salicylates and sulfonamides can displace bilirubin from albumin, causing bilirubin to enter CNS  This causes the potential for neural damage in infants  Bilirubin dissociates from albumin molecule and enters a hepatocyte where it binds to ligandin

3. Formation of bilirubin diglucuronide  In liver cells, the solubility of bilirubin is increased by the addition of two molecules of glucuronic acid (conjugation).  The is catalyzed by microsomal bilirubin glucuronyltransferase using (UDP-glucoronic acid) as the glucuronate donor.  Varying deficiency of this enzyme result in Crigler-Najjar I and II and Gilbert syndrome

4. Secretion of bilirubin into the bile  Conjugated bilirubin is actively transported into the bile.  This energy-dependent, rate-limiting step is susceptible to impairment in liver disease  A deficiency in the protein required for transport of conjugated bilirubin out of the liver results in Dubin-Johnson syndrome  Unconjugated bilirubin is normally not secreted

5. Formation of urobilins in the intestine  Bilirubin diglucuronide is hydrolyzed and reduced by bacteria in the gut to yield urobilinogen (colorless)  Urobilinogen is then oxidized by bacteria to stercobilin (brown color)  Some of the urobilinogen is reabsorbed and enters the portal blood, is taken up by the liver and re-secreted in the bile  The remainder of the urobilinogen is transported by the blood to the kidney, where it is converted to yellow urobilin and excreted

Jaundice (Icterus)  Jaundice is yellow color of skin, nail beds, and sclerae (whites of the eyes) caused by deposition of bilirubin  Secondary to increased bilirubin levels in the blood (hyper- bilirubinemia)  Jaundice is not a disease but a symptom of an underlying disorder

Types of Jaundice 1. Hemolytic jaundice  Caused by lysis of RBCs (sickle cell anemia, pyruvate kinase, glucose 6-phosphate dehydrogenase deficiency)  Bilirubin is pruduced faster than it can be conjugated  Biochemical changes: More bilirubin is excreted into the bile Urobilinogen entering the enterohepatic circulation is increased Urinary urobilinogen is increased Unconjugated bilirubin levels become elevated in the blood

Haptoglobins A family of circulating plasma proteins that have a high affinity for oxyhemoglobin dimers Deoxyhemoglobin does not dissociate into dimers in physiological settings and does not bind to haptoglobins Haptoglobins are synthesized in the liver Haptoglobin-Hemoglobin complex is too large to be filtered through the renal glomerulus Haptoglobin delivers hemoglobin to the reticulo- endothelial cells

Haptoglobins Heme in hemoglobin is resistant to the action of heme oxygenase. Binding to hapatoglobin makes it succeptible Measurement of haptoglobin is used clinically as an indication of the degree on intravascular hemolysis Patients with significant hemolysis have little haptoglobin because of removal of haptoglobin-hemoglobin complex by reticuloendothelial system

B. Hepatocellular jaundice  Damage to liver cells (for example, in patients with cirrhosis or hepatitis) can cause jaundice  Biochemical features, signs and symptoms: unconjugated bilirubin levels increase in the serum Conjugated bilirubin is not efficiently secreted into the bile, and diffuses (“leaks”) into the serum Urobilinogen levels increase in urine(reduced enterohepatic circulation) Dark urine Stools are a pale, clay-like color Serum levels of ALT and AST are elevated

C. Obstructive jaundice  Jaundice is caused by obstruction of the bile duct (hepatic tumor or bile stones)  Biochemical features, signs and symptoms: Gastrointestinal pain and nausea Pale, clay-like color stools Urine that darkens upon standing The liver “regurgitates” conjugated bilirubin into the blood causing its levels to increase Conjugated bilirubin is eventually excreted in the urine Prolonged obstruction of the bile duct leads to liver damage and a rise in unconjugated bilirubin

D. Neonatal Jaundice  The activity of hepatic bilirubin glucuronyltransferase is low at birth  Activity reaches adult levels in about four weeks  Newborn infants, particularly if premature, often accumulate bilirubin  Bilirubin, in excess of the binding capacity of albumin, can diffuse into the basal ganglia and cause toxic encephalopathy (kernicterus)  Newborns with neonatal jaundice are treated with blue fluorescent light  Blue florescent light converts bilirubinto more polar (water-soluble) isomers

Gilbert syndrome Benign autosomal recessive hereditary disorder that affects 5% of the population One of the hepatic causes of increased levels of un- conjugated bilirubin in the blood Results from a genetic mutation in the gene (UGT1A1) that produces UDPGT It carries no morbidity, mortality or clinical consequences. Characterized by intermittent unconjugated hyperbilirubinemia

Crigler-Najjar syndrome Crigler-Najjar syndrome is an inherited disorder resulting from a defect in the gene involved in bilirubin conjugation Crigler-Najjar syndrome is rare and may result in death May be divided into two types: Type 1: Complete absence of enzymatic bilirubin conjugation Type 2: Severe deficiency of the enzyme responsible for bilirubin conjugation

Dubin-Johnson syndrome Rare autosomal recessive inherited disorder Caused by a deficiency of the canalicular multidrug resistance transporter Liver’s ability to uptake and conjugate bilirubin is functional Removal of conjugated bilirubin from the liver cell and the excretion into the bile are defective Accumulation of conjugated bilirubin in the blood, leading to hyperbilirubinemia and bilirubinuria

Dubin-Johnson Syndrome A distinguishing feature is the appearance of dark- stained granules on a liver biopsy sample People with Dubin-Johnson syndrome have a normal life expectancy, so no treatment is necessary

Rotor Syndrome Rotor syndrome is clinically similar to Dubin- Johnson syndrome The defect causing Rotor syndrome is not known Liver biopsy does not show pigmented granules More rare than Dubin-Johnson syndrome It has an excellent prognosis

Determination of Bilirubin Concentration  Bilirubin is most commonly determined by the van den Bergh reaction (Colorimetric method)  In aqueous solution, the water-soluble, conjugated bilirubin reacts rapidly with the reagent (within one minute), and is said to be “direct-reacting.  The unconjugated bilirubin is less soluble in aqueous solution and reacts more slowly  When the reaction is carried out in methanol, both conjugated and unconjugated bilirubin are soluble and react with the reagent (total bilirubin)  The “indirect-reacting” bilirubin (unconjugated bilirubin), is obtained by subtracting the direct-reacting from the total bilirubin  In normal individuals, only 4% of the bilirubin is conjugated