Sickle β-Thalassaemia


Sickle cell anaemia and β-thalassaemia are two common haemoglobinopathies. Co-inheritance of the two is called sickle β-thalassaemia. Sickle β-thalassaemia seen in Africa, throughout the  Mediterranean, Arabian Peninsula and sporadically in india. It has heterogeneous clinical presentation. The severity depends on the severity of the thalassaemia allele and the extent to which the impaired haemoglobin synthesis is compensated by foetal haemoglobin synthesis.

Pathophysiology

With a very few exceptions (Blood 1989; 74: 1817-22) the sickle cell and the thalassaemia gene are arranged in trans i.e on different chromosomes (βsthal). One allele is inherited from the mother and one from the father. One parent carries the a β-thalassaemia trait the other parent has a sickle cell disease that may be sickle cell anaemia, sickle β-thalassaemia or a trait. Sickle β-thalassaemia in Africa and India/Arabia is mild whereas the patients from the Mediterranean region have severe disease. As mentioned above the differences in severity have to do with severity of the β-thalassaemia and the degree to which the impaired haemoglobin A synthesis is compensated by HbF. Weatherall suggested that patients with HbA <15% follow a course similar to severe HbA and those with HbA 20-30% follow a mild course.

  1. African sickle β-thalassaemia: African patients have a mild β-thalassaemia resulting in a relatively higher HbA level and a lower risk of sickling. These patients run a mild clinical course.
  2. Arab/Indian sickle β-thalassaemia: Patients from India and the Arabian peninsula have a sickle cell haplotype that is associated with a high HbF production. The HbF retards sickling. High levels of HbF attenuate symptoms. Patients carrying this haplotype have mild symptoms even when the inherit a severe β- chain defect. Another reason of a mild phenotype in India is the interaction with α thalassaemia.
  3. Mediterranean sickle β-thalassaemia: Mediterranean patients usually inherit a severe form of  β-thalassaemia. These patients have severe sickling because there is very little HbA or HbF to offset inhibit the crystallisation of HbS. Despite only one chromosome carrying HbS the phenotype of these patients resembles sickle cell anaemia.

Clinical Picture of Sickle-β Thalassaemia

The features of sickle-β thalassaemia resemble those of other sickling disease. It is a chronic haemolytic anaemia the course of which is interrupted by acute exacerbations known as crisis. The manifestations include haemolytic anaemia, painful and other crisis, leg ulcers, priapism and complications of pregnancy. The severity of symptoms is variable. One end of the spectrum are patients, usually of origin Mediterranean descent, whose presentation is indistinguishable from sickle cell anaemia. These patients have inherit severe forms of β (β0) chain defects. Those with sickle cell-β+ thalassaemia have milder symptoms. These patients are typically of African ancestory. Unlike patients with sickle cell anaemia patients with sickle-β thalassaemia may have splenomegaly that is more prominent patients with sickle cell-β+ thalassaemia. The spleen is usually moderately enlarged but massive splenomegaly that may be associated hypersplenism neccesisating splenectomy has been reported. The effect of co-inheritance of α-thalassaemia is small. A decrease in the frequency of acute chest syndrome and leg ulcers and a higher persistence of splenomegaly is seen. Co-inheritance of α thalassemia is one of the reasons that sickle-β thalassaemia runs a milder course in India (the other being the high HbF due to the Arab-Indian haplotype of HbS).

Diagnosis

The haematological findings vary with severity. More severe phenotypes shows greater anaemia, lower MCHC, higher reticulocytes, HbF and HbA2. A variable number of sickle cells may be found. Unlike sickle cell anaemia both forms of sickle cell-β thalassaemia have an elevated HbA2. The distribution of HbA2 is very similar to heterozygous β thalassaemia. The levels of HbF are variable. High levels are found in patients with the Arab-Indian and Senegal haplotype of HbS.

Sickle cell-β0 thalassaemia needs to be differentiated from sickle cell anaemia. The presentation of both may be identical. However an offspring of a sickle cell-β0 thalassaemia patients and a carrier of β-thalassamia trait has a 25% risk of suffering from β-thalassaemia major. The offspring of a patients with sickle cell anaemia and a carrier of β thalassaemia trait does not carry the risk of β thalassaemia major. Though sickle cell-β0 thalassaemia is characterised by an elevated HbA2 and splenomegaly this can not be relied upon to differentiate between the two conditions. Family and DNA studies are needed. If the studies show one parent to be heterozygous for HbS and the other a carrier of β thalassaemia trait no further studies are needed. If any of the parent has a phenotype of sickle cell anaemia DNA studies may be the only way to make the diagnosis.

Sickle Cell β thalassaemia in cis

Almost all patients with sickle-β thalassaemia have the disorder in trans i.e. the one β globin gene is thalassaemic and the other has a the sickle mutation. Patients with HbS and thalassaemia gene in cis have been described. These patients have a mild hemolysis, HbA2 levels were 6%–7%, HbF approximately 3% and HbS of 10%–11%.

Treatment

The symptoms of sickle-β thalassaemia are due to sickling need to be treated accordingly.

Evolution and Spread of HbS


The gene for β globin (OMIM  is present on chromosome 11 (11p15.4) along with other globin genes (ε, γ, γ and δ). This is known as the β-globin cluster . Individuals carrying identical genes on the β-globin gene cluster may not have identical DNA sequences in non-codeing regions of the DNA of the cluster. The non-coding regions include segments of DNA between genes and introns within genes. . Differences in DNA exist between individuals every 1000-2000 bases in the form of single nucleotide polymorphisms (SNPs). Single nucleotide polymorphisms are variations in a single nucleotide that occurs at a specific position in the genome. Many of these differences have no consequences on gene expression because either they do not result in change in amino acid sequence or they occur in regions of DNA that neither code for the gene nor regulate the gene. SNPs evolve by spontaneous mutations over time. The lesser the number of such differences between two individuals closer the individuals are the each other genetically (and in terms of evolution). Fewer differences in SNPs between individuals mean a more recent common ancestor.

One of the meanings of the word haplotype is a pattern of SNPs. A haplotype may be considered as a DNA “environment” in which the gene(s) occurs. This “environment” is created by the sequence of single nucleotide polymorphisms in which the gene(s) exists. As mentioned above differences in SNPs (and hence the “environment” the gene(s) exist in) evolve by spontaneous mutations over period of time. Fewer the differences between the “environments” the genes occurs in the more the likelihood that they come from related individuals.

HbS results from a single base substitution in the codon 6 of the β-globin gene. GAG becomes GTA resulting in substitution of valine for glutamate. This change results in a haemoglobin that crystallizes in hypoxic conditions resulting in a haemolytic anaemia. HbS occurs in diverse population groups including African, Mediterranean, Middle-Eastern and Indian. Is the haplotype of the HbS gene in these regions similar?

The HbS mutation occurs on five different haplotypes four African and one Arab-Indian. The mutation is the same (GAG to GTA on codon 6) but the SNPs are different. The haplotypes are

  1. Senegal: The Senegal HbS haplotype is found in Atlantic West Africa and Portugal
  2. Benin: The Benin HbS haplotype is found Central West Africa, Northern Africa and Mediterranean Europe (Greece, Sicily)
  3. Central African Republic or Bantu: The Central African Republic or Bantu is found in South Central and Eastern Africa
  4. Cameroon: The Cameroon haplotype is found in the Eton ethnic group of eastern Cameroon
  5. Arab-Indian: The Arab-Indian haplotype is the only non-African phenotype of HbS found in the eastern oasis of Saudi Arabia and India.

Origin of Haplotypes

There are two theories about the origin of haplotypes. The first, and the more accepted one, states that the five haplotypes arose from five independent mutations. An alternative hypothesis states that HbS mutation occurred only once and spread to other haplotypes by gene conversion.

 

Haplotypes and Severity of Symptoms

Symptoms of sickle cell anaemia are a consequence of crystallisation of haemoglobin under hypoxic conditions. HbF inhibits sickling. Patients with high HbF have fewer symptoms. The Arab-Indian and the Senegal haplotype are associated with higher HbF levels (17% and 12.4% respectively). In general patients carrying these haplotypes have milder symptoms than the Bantu or Benin haplotypes (Blood 2014; 123: 481)

 

Haplotypes and Human Migrations

Trade, conquests and human migrations (voluntary and slave trade) have disseminated the African haplotypes beyond Africa.

  1. The Mediterranean: Most of the Mediterranean (Greece and Scilly) has the Benin haplotype. This reflects pre-historic migrations from Central West Africa along the then fertile Sahara to North Africa. From here it spread to the Mediterranean via the interactions (Trade and wars) between the two regions. The only exception is Portugal. Portugal has the Senegal haplotype which reflects the trading contacts between Portugal and Atlantic West Africa (Angola and Mozambique).
  2. Americas: Neither the native americans nor the original European settlers to the Americas carried the HbS gene. HbS was imported to the Americas with the slaves from Africa. Jamaica was an important slave import hub and records for where tthe slaves arrived from are available. Jamaica has 73% Benin haplotype, 17% Bantu and 10% Senegal haplotypes. These numbers are close to the actual number of slaves who arrived in Jamaica from regions of Africa where these haplotypes are prevalent. Similarly the distribution of haplotype correspond to the origins of slaves in Baltimore and South Carolina (Mariam Bloom. Understanding Sickle Cell Disease, Page 34).
  3. Arab or Indian: It is not clear if the Arab-Indian haplotype originated in India or Saudi Arabia. But considering that all of tribal India has only one haplotype but the East and West Arabian Peninsula have different haplotypes it is possible that the haplotype originated in India.
  4. Spread to Other Parts: As opposed to the era of slave trade modern migration of people in the recent past have been voluntary. These populations have spread across the world as have those form mediterranean but to a lesser extent. These migrations have introduced the HbS gene in areas where it was not indigenous.

 

Sickle Cells


Sickle Cell - 100X - IMG_0542

Sickle Cells 40X

Sickle Cell 100X - IMG_0540

Sickle Cells – 100X

Sickle Cell 100X - IMG_0538

Sickle Cells – 100X


The three photomicrographs above show sickle cells from a patients with sickle cell anemia. Sickle cell anaemia occurs because an A→T substitution in codon 6 of the β globin chain of haemoglobin. The single nucleotide polymorphism results in valine substituting for glutamic acid resulting in the formation of haemoglobin S (HbS). HbS crystallizes in hypoxic conditions resulting in sickling of erythrocytes.

Related articles
Sickle Haemoglobin and Variants

Sickle Haemoglobin and Varients


Substitution of the amino acid glutamic acid by valine at position 6 of the beta globin gene (HBB glu6val) results in a mutant haemoglobin that polymerizes at low oxygen pressure. This mutation results in sickle shaped cells under hypoxic conditions. The haemoglobin gets its name, sickle haemoglobin, from the phenomena. Sickling is responsible for symptoms of sickle cell anaemia. Shown above are sickle cells from the smear of a patient with sickle cell anaemia.

HbS is an autosomal co-dominant trait. Homozygous individuals suffer from sickle cell anaemia (SS). The clinical profile of compound heterozygous depends on the non-HbS allele.

  1. HbA: HbA does not participate in sickling. Patients with AS have about 35% HbS and 65% HbA there is little sickliness and symptoms. These patients said to have sickle cell trait. A patient with as with an apparent AS pattern can have see sickling (see below).
  2. β-Thalassaemia: The severity of interaction between HbS and β-thalassaemia depends on the severity of the co-thalassaemia. β0-thalassaemia is identical to SS and β+-thalassaemia is a milder disease with severity depending on the degree of impairment of the β-chain synthesis.
  3. Other Sickling haemoglobinopathies: Co-inheritnce of Hb-O Arab and Hb D Punjab produces a sickle cell anaemia like disease. Co-inheritance of Hb E produces a sickling disease milder than SS.
  4. Haemoglobinopathies than mimic AS on electrophoresis but produce sickling: There are two categories in this class. First, is a disease that resulting from co-inheritance of an abnormal haemoglobin, Hb Quebec Chori, migrates like HbA but promotes sickling. Patients compound heterozygous for Hb S and and Hb Quebec Chori show and electrophoretic pattern of AS but show sickling. Second are variants of HbS where a second mutation has resulted in an increased tendency to precipitate making a individual heterozygous for these variants symptomatic. These include Hb South End, Hb Jamican Plain Hb S Antilles and Hb S-Oman. The first three have mutations reducing the affinity of the haemoglobin increasing polymerization and sickling.

Diagnosis of Haemolytic Anaemia


Haemolysis is decreased erythrocytes lifespan (normal about 100-120 days). The bone marrow responds to haemolysis by increasing erythrocyte production. Anaemia occurs only when the erythrocyte life span is reduced to about 15-20 days. A compensated haemolytic state is when a patient has a shorted erythrocyte lifespan that is adequately compensated by increased erythrocyte production. These patients may have features of haemolysis except anaemia.


Figure 1. Manifestations of Haemolysis

Clinical Manifestations of Haemolysis

The site of haemolysis, intravascular or extravascular, determines the clinical manifestations of haemolysis (figure 1). Extravascular haemolysis is an exacerbation of a physiological catabolic process. The end product of extravascular haemolysis, like that of normal erythrocyte breakdown, is unconjugated bilirubin. Unconjugated bilirubin can cause brain damage in the forms of kernicterus. The conjugation of bilirubin is so efficient that the serum bilirubin rarely increase to more than 5mg/dL only due to bilirubin overproduction. The blood brain barrier prevents entry of bilirubin in the brain. Both the mechanisms are compromised in a neonate making them prone to kernicterus in case of haemolysis as is seen in haemolytic disease of the newborn. The only consequences of extravascular haemolysis occurring in patients beyond the neonatal period are anaemia, unconjugated hyperbilirubinaemia, mild to moderate splenomegaly and pigment gallstones.

Intravascular haemolysis is lysis of cells within the blood vessels. Cell free haemoglobin is oxidizing and pro-inflammatory. Haptoglobin binds and detoxifies cell free haemoglobin but it has a limited capacity. Haemoglobinaemia resulting from intravascular haemolysis causes serious clinical manifestations including renal failure, hypertension, smooth muscle spasm and a prothrombotic state. Many of these result from the nitric oxide scavenging capacity of haemoglobin (Rother et al JAMA 293:1653;2005). Persistent haemoglobinuria is a feature of chronic intravascular haemolysis, e.g. those with haemolysis due to prosthetic valves and paroxysmal nocturnal haemoglobinuria. Iron deficiency may accompany chronic intravascular haemolysis because of haemoglobinuria. Extravascular haemolysis is not associated with haemoglobin loss and does not cause iron deficiency.

Intravascular haemolysis often presents with acute severe haemolysis as may be seen with drug induced immune haemolysis, haemolysis in G6PD deficient patients, severe malaria and mismatched transfusion reactions. The diseases causing extravascular haemolysis have a less acute presentation.

The Diagnosis of Haemolytic Anaemia

Estimation of erythrocyte lifespan is too cumbersome to be used routinely in clinical practice. The diagnosis of haemolytic anaemia instead relies on indirect evidence of increased erythrocyte destruction. This includes:

  1. Increased production of erythrocytes in an anaemic patient with no evidence of blood loss
  2. Increase in products of erythrocyte destruction – Lactate dehydrogenase (LDH) and haemoglobin
  3. Consequences of haemoglobinaemia – low haptoglobin, haemoglobinuria, renal failure and hemosiderinuria

Increased erythrocyte production manifests as reticulocytosis (see The Reticulocyte Count for performing and interpreting reticulocyte count). If polycythaemia is not seen despite reticulocytosis, an equivalent erythrocyte loss is presumed to be occurring. Erythrocytes loss may internal, e.g., haemolysis, or external, e.g., bleeding. Reticulocytosis is seen when a large amount of blood is lost over a short period. Such losses are rarely occult. If there is no obvious blood loss, reticulocytosis in a patient whose haemoglobin is not increasing indicates haemolysis. Large occult haematomas, typically occurring retroperitoneally, may mimic haemolysis.

Lactate dehydrogenase (LDH) is abundant in brain, erythrocyte, liver and lung. Injury to any of these organs increases LDH. LDH is also increased in diseases characterized by increased cell proliferation e.g. acute leukaemia, lymphomas (particularly high grade non-Hodgkin lymphoma), myeloproliferative diseases and myelodysplastic syndrome. The clinical picture, radiology and laboratory investigations can exclude non-erythrocyte sources of LDH.

Haemolytic anaemias are characterized by an increase in LDH. The increase in LDH is more pronounced in patients with intravascular haemolysis and is matched by that seen in patients with nutritional megaloblastic anaemia. Unlike megaloblastic anaemia, intravascular haemolysis is characterized by reticulocytosis, haemoglobinaemia and haemoglobinuria.

The alterations caused by catabolism of haemoglobin released during haemolysis depend on the site of haemolysis (see haemoglobin catabolism). Haemoglobin released during extravascular haemolysis is metabolized to unconjugated bilirubin. The liver responds by increasing conjugation and excretion of bilirubin which increases urinary urobilinogen. Unconjugated hyperbilirubinaemia (unconjugated bilirubin >85% of total bilirubin) with increased urobilinogen is a characteristic of haemolytic anaemia. It differentiates haemolytic anaemia from Gilbert’s Syndrome, a common disorder of bilirubin conjugation that is seen in 3-7% of population. The urobilinogen levels in Gilbert’s syndrome are low. Crigler-Najjar syndrome type I and II are present with a more pronounced increase in bilirubin (>5mg/dL and >20mg/dL respectively) and are rarely considered as a differential diagnosis of an uncomplicated haemolytic anaemia. Bilirubin congugation defects do not show reticulocytosis. The bilirubin levels in patients with haemolytic anaemia rarely exceed 5mg/dL. Higher levels indicate a co-existing illness. If the bilirubin is unconjugated then an incidental co-inheritance of Gilbert’s syndrome should be suspected. Haemolytic anaemia increases the risk of pigment stones. These may cause obstructive jaundice. These patients, unlike those with haemolysis have a higher proportion of conjugated bilirubin with an elevated alkaline phosphatase. Donor screening for hepatitis B and C have made transfusion induced hepatitis a thing of the past.

Haemolysis releases haemoglobin. Cell free haemoglobin has oxidizing and pro-inflammatory properties. Haptoglobin is a haemoglobin scavenger that protects the body from the toxic effects of cell free haemoglobin. Haemolysis decreases serum haptoglobin (see haemoglobin catabolism). Extravascular haemolysis does not cause haemoglobinaemia, but contrary to what may be expected, it does cause low haptoglobin levels.  Low haptoglobin is of no value in differentiating intravascular and extravascular haemolysis. It appears that there is some haemoglobinaemia during extravascular haemolysis. It is possible that there is some intravascular component to extravascular haemolysis or there is regurgitation of haemoglobin from the macrophage during phagocytosis. Haptoglobin is synthesized by the liver. Decrease synthesis limits the usefulness of haptoglobin in the diagnosis of haemolysis in the presence eof chronic liver disease. Haptoglobin is an acute phase reactant that in increased by corticosteroid administration. Patients with acute inflammation and those on corticosteroid therapy may have a normal haptoglobin despite haemolysis.

Haemoglobin filtered into the glomerular fluid results in haemoglobinuria when the absorptive capacity of renal tubules (5g/day, Turgeon, ML. Clinical Hematology: Theory and procedures 4th ed. Lippincott Williams and Wilkins, 2005:89) is exceeded. Haemoglobin can precipitate in the tubules and causes renal failure. The haemoglobin absorbed by the renal tubules in metabolized by haemoglobin oxygenase to iron, bilirubin and amino acids. The iron is stored in the renal tubular cells as hemosiderin. These cells desquamate and cause hemosiderinuria. Hemosiderinuria appears a few days after hemolysis and may persist for a week or more after hemolysis has abated. Iron deficiency anaemia caused by hemosiderinuria may complicate chronic low grade intravascular haemolysis as is seen in prosthetic valve haemolysis, paroxysmal nocturnal haemoglobinuria or march haemoglobinuria.

Diagnosis of haemolysis by transfusion requirements

Even with complete cessation of erythropoiesis only about one unit of blood is needed every week in an adult to maintain haemoglobin of about 10g/dL. A greater requirement in the absence of evidence of blood loss suggests haemolysis. Transfused blood will only be lysed if there is an extrinsic defect e.g. autoimmune haemolytic anaemia. Cells transfused to patients with an intrinsic defect e.g. hereditary spherocytosis will have a normal life span. A patient who needs more than one unit of blood per week to maintain haemoglobin of about 10g/dL is likely to have a haemolytic anaemia if blood loss can be excluded. This haemolysis is likely to be due to an extrinsic cause.

Differential Diagnosis of Haemolysis

Haemolysis may be confused with conditions associated with unconjugated hyperbilirubinaemia, anaemia and reticulocytosis. These include bilirubin conjugation defects, acute blood loss and megaloblastic anaemia.

Bilirubin Conjugation Defects Bilirubin conjugation defects include Gilbert’s syndrome and Crigler-Najjar syndrome types I and II. The bilirubin levels in Crigler-Najjar syndrome type I and II are more than 5mg/dL so these entities are rarely confused with haemolysis. Gilbert’s syndrome is characterized by bilirubin levels which can be seen in haemolysis but there is no associated reticulocytosis. Bilirubin conjugation defects are characterized by low urobilinogen levels.

Acute blood loss: Acute blood loss, like haemolysis, causes anaemia and reticulocytosis but these are not the presenting complains. Blood loss needed to cause reticulocytosis is rarely ignored by the patient and is in fact the reason for seeing the doctor. When severe, it may be accompanied by signs of hypovolemia. The presenting complains of patients with haemolytic anaemia are related to anaemia and hyperbilirubinaemia. Chronic occult blood loss has a different presentation that acute blood loss or haemolysis. The gradual fall in haemoglobin associated with occult blood loss makes the patients tolerant to anaemia unless a cardiovascular or respiratory co-morbidity exist. These patients present with a clinical picture of iron deficiency namely, fatigue, malaise and hypochromic microcytic anaemia with anisocytosis. There is no jaundice and there is reticulocytopenia. Large occult haematomas cause anaemia and reticulocytosis. As the haematoma resolves jaundice may be seen.

Megaloblastic anaemia Megaloblastic anaemia, like haemolytic anaemia, presents with macrocytic anaemia, indirect hyperbilirubinaemia, and a low serum haptoglobin. These patients have reticulocytopenia. The macrocytosis is more pronounced. An MCV of >110 fl usually indicates a megaloblastic anaemia. Patients with nutritional megaloblastic anaemia respond to treatment with pronounced reticulocytosis, usually seen by 5-7 days after initiating therapy and lasting for up to 2 weeks.  Reticulocyte counts may be as high as 20-25%. A history of treatment with vitamin B12 and/or folic acid must be sought in every patient of haemolytic anaemia to avoid confusion with megaloblastic anaemia.

 

Related Pages

  1. Reticulocyte Count
  2. Evaluating Anaemia
  3. Haemoglobin Catabolism
  4. Sickle Haemoglobins and Variants
  5. Red Cell Indices

Haematology Atlas Pages

  1. Sickle Cells
  2. Pappenheimer Bodies

Pappenheimer Bodies


Described by Pappenheimer in 1945, the Pappenheimer bodies are basophilic erythrocytic inclusions that are usually located at the periphery of the cell. They contain iron and stain with Prussian blue. They also stain with Romanowsky stains because of co-precipitation of ribosomes. Pappenheimer bodies seen following splenectomy in patients without haematological disease are composed of ferritin. Whereas Pappenheimer bodies seen in pathological conditions like sideroblastic anaemia are also composed of iron laden mitochondria and phagosomes.

Pappenheimer bodies are seen in sideroblastic anaemia and haemolytic anaemia. The spleen clears Pappenheimer bodies. Splenectomy is associated with an increase in Pappenheimer bodies. The increase is more pronounced in patients with haemolytic anaemia and sideroblastic anaemia. Cells containing Pappenheimer bodies can be confused with late reticulocytes. Prussian blue stain, which is not taken up by reticulocytes, is helpful in differentiating the two. Pappenheimer bodies can also cause a false elevation of platelet counts when performed with electronic counters.

There is an intererting article about the history of Pappenheimer bodies published in The American Journal of Haematology (Am. J. Hematol 75:249;2004)

Haemoglobin Catabolism


Figure 1. Synthesis and breakdown of haemoglobin

Haemoglobin is made up of heme, an iron containing porphyrin and globin, a protein. Normally the erythrocyte lives about 120 days. Ageing or damaged erythrocytes are destroyed by the macrophages of the reticuloendothelial system of the spleen. Other sites notably the, liver and the bone marrow, are also capable of destroying erythrocytes. As the life span of erythrocytes is not increased in splenectomized patients, these sites can completely take over the function in the absence of the spleen. The spleen, unlike other reticuloendothelial sites, is sensitive to subtle damage to the erythrocytes.

Heme splits into globin and hemin and globin. The amino acids released from the catabolism of globin chain are reused for protein synthesis. Hemin is acted upon by heme oxygenase to give biliverdin and iron. The iron is reused for haemoglobin synthesis. Biliverdin, released from the catabolism of protoporphyrin, is finally excreted as conjugated bilirubin in the bile (Figure 1).

Biliverdin is converted to bilirubin by biliverdin reductase. Bilirubin is water insoluble and needs to be conjugated with glycuronic acid in the liver to make it water soluble and make excretion in bile possible. Unconjugated bilirubin binds to albumin and is carried to the liver. The hepatocyte takes up the unconjugated bilirubin by both simple and facilitated diffusion and converts it to bilirubin diglucuronide in two steps. Some bilirubin monoglucuronide is also formed. The enzyme uridine diphophate glucuronyl transferase (UDPGT) facilitated bilirubin conjugation.

Bilirubin is secreted into bile against a concentration gradient. MRP-2 (multidrug resistance like protein 2) is one of the proteins involved in bilirubin secretion.

In the intestine bilirubin is converted to urobilinogen, a colourless compound, by the intestinal flora. Urobilinogen is converted to a pigment responsible for the colour of faeces, urobilin. Some urobilinogen is absorbed and excreted in urine.

Bilirubin metabolism is affected in conjugation defects, secretion defects and in states of increased haemoglobin catabolism.

  1. Bilirubin conjugation defects: Mutations in the UDGPT result in three syndromes. These are, in decreasing severity, Crigler-Najjar Syndrome I (CN-I), Crigler-Najjar Syndrome II (CN-II) and Gilbert’s syndromes. While CN-I is fatal except in those who undergo liver transplantation, Gilbert’s syndrome causes no symptoms otherthan jaundice. Patients with all three diseases have unconjugated hyperbilirubinemia which may range from usually >20mg/dL in CN-I, usually <20mg/dL in CN-II and <4mg/dL in Gilbert’s syndrome. Gilbert’s syndrome can mimic haemolysis. The absence of other evidence of red cell destruction, viz. increase in the LDH and decrease in the haptoglobin, and the absence reticulocytosis differentiates it from haemolysis.
  2. Bilirubin secretion defects: Defects in secretion of conjugated bilirubin include Dubin-Johnson syndrome, Rotor syndrome, benign recurrent intrahepatic cholestasis (types 1 and 2) and progressive familial intrahepatic cholestasis (types 1, 2 and 3). (links take you to the OMIM page for the disease)

Increased haemoglobin catabolism: Increased haemoglobin catabolism, seen in patients with haemolytic anaemia and states associated with ineffective erythropoiesis, increases bilirubin production and overwhelms the hepatic uptake/conjugation capacity. This increases the unconjugated bilirubin. Increased bilirubin production in patients with haemolytic anaemia is a result of increased erythrocyte destruction by the spleen. Typically, hyperbilirubinemia is associated with extravascular haemolysis. Splenomegaly may accompany unconjugated hyperbilirubinemia due to haemolysis because of the increased workload. Patients of haemolytic anaemia show elevated LDH levels and reticulocytosis. Patients with ineffective erythropoiesis, e.g. megaloblastic anaemia due to folate/B12 deficiency, have increased haemoglobin catabolism due to destruction of haemoglobinized precursors in the bone marrow. There is reticulocytopenia, the increase in LDH is more pronounced than haemolytic anaemia, there is no splenomegaly. Haemolysis and ineffective erythropoiesis may co-exist in megaloblastic crises of haemolytic anaemia. Unconjugated hyperbilirubinemia due to increased bilirubin production is associated with increased urinary urobilinogen, a features not seen in inherited syndromes of bilirubin conjugation (CN-I, CN-II and Gilbert’s syndrome). It is unusual for the bilirubin to increase beyond 4mg/dL only from increased haemoglobin breakdown. When higher values are encountered other reasons for an increased bilirubin must be sought. All patients with increased haemoglobin breakdown do not show hyperbilirubinemia.