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Bilirubin is the end product of heme degradation. From 70–90% of bilirubin is derived from degradation of the hemoglobin of senescent red blood cells. Bilirubin produced in the periphery is transported to the liver within the plasma, where, due to its insolubility in aqueous solutions, it is tightly bound to albumin.

Under normal circumstances, bilirubin is removed from the circulation rapidly and efficiently by hepatocytes.

Transfer of bilirubin from blood to bile involves four distinct but interrelated steps:

1) Hepatocellular uptake: Uptake of bilirubin by the hepatocyte has carrier-mediated kinetics.

2) Intracellular binding: Within the hepatocyte, bilirubin is kept in solution by binding as a non substrate ligand to several of the glutathione-S-transferases, formerly called ligandins.

3) Conjugation: Bilirubin is conjugated with one or two glucuronic acid moieties by a specific UDP-glucuronosyl transferase to form bilirubin mono- and diglucuronide, respectively. Conjugation disrupts the internal hydrogen bonding that limits aqueous solubility of bilirubin, and the resulting glucuronide conjugates are highly soluble in water. Conjugation is obligatory for excretion of bilirubin across the bile canalicular membrane into bile. The UDP-glucuronosyl transferases have been classified into gene families based on the degree of homology among the mRNAs for the various isoforms. Those that conjugate bilirubin and certain other substrates have been designated the UGT1 family. These are expressed from a single gene complex by alternative promoter usage.

4) Biliary excretion: Bilirubin mono- and diglucuronides are excreted across the canalicular plasma membrane into the bile canaliculi by an ATP-dependent transport process mediated by a canalicular membrane protein called multidrug resistance–associated protein 2 (MRP2). Mutations of MRP2 result in the Dubin-Johnson syndrome.

Extra hepatic Aspects of Bilirubin Disposition

Bilirubin in the Gut

Following secretion into bile, conjugated bilirubin reaches the duodenum and passes down the gastrointestinal tract without reabsorption by the intestinal mucosa. An appreciable fraction is converted by bacterial metabolism in the gut to the water-soluble colorless compound, urobilinogen. About 80–90% of these products are excreted in feces, either unchanged or oxidized to orange derivatives called urobilins. The remaining 10–20% of the urobilinogens are passively absorbed, enter the portal venous blood, and are reexcreted by the liver. A small fraction (usually <3 mg/dL) escapes hepatic uptake, filters across the renal glomerulus, and is excreted in urine.(See figure 1)


Figure-1 – showing formation and fate of bilirubin


Unconjugated bilirubin ordinarily does not reach the gut except in neonates or, by ill-defined alternative pathways, in the presence of severe Unconjugated hyperbilirubinemia [e.g., Crigler-Najjar syndrome, type I (CN-I)]. Unconjugated bilirubin that reaches the gut is partly reabsorbed, amplifying any underlying hyperbilirubinemia.

Renal Excretion of Bilirubin Conjugates

Unconjugated bilirubin is not excreted in urine as it is too tightly bound to albumin for effective glomerular filtration and there is no tubular mechanism for its renal secretion. In contrast, the bilirubin conjugates are readily filtered at the glomerulus and can appear in urine in disorders characterized by increased bilirubin conjugates in the circulation.


Jaundice is a yellow discoloration of the skin, mucous membranes, and sclera caused by increased amounts of bilirubin in the blood. Jaundice is a sign of an underlying disease process.

Jaundice results from the accumulation of bilirubin. Hyperbilirubinemia may be due to abnormalities in the formation, transport, metabolism, and excretion of bilirubin. Total serum bilirubin is normally 0.2–1.2 mg/dL (mean levels are higher in men than women and higher in whites and Hispanics than blacks), and jaundice may not be recognizable until levels are about 3 mg/dL.

In the normal adult the rate of systemic bilirubin production is equal to the rates of hepatic uptake, conjugation, and biliary excretion. Jaundice occurs (bilirubin levels may reach 30-40 mg/dL in severe disease) when the equilibrium between bilirubin production and clearance is disturbed by one or more of the following mechanisms (1) excessive production of bilirubin, (2) reduced hepatic uptake, (3) impaired conjugation, (4) decreased hepatocellular excretion, and (5) impaired bile flow (both intra hepatic and extra hepatic). The first three mechanisms produce unconjugated hyperbilirubinemia, and the latter two produce predominantly conjugated hyperbilirubinemia. More than one mechanism may operate to produce jaundice, especially in hepatitis, which may produce Unconjugated and conjugated hyperbilirubinemia. In general, however, one mechanism predominates, so that knowledge of the predominant form of plasma bilirubin is of value in evaluating possible causes of hyperbilirubinemia

Classification of jaundice

 1) Pre-hepatic (before bile is made in the liver)

Jaundice in these cases is caused by rapid increase in the breakdown and destruction of the red blood cells (hemolysis), overwhelming the liver’s ability to adequately remove the increased levels of bilirubin from the blood.

Examples of conditions with increased breakdown of red blood cells include:


-Sickle cell crisis,



-Glucose-6-phosphate dehydrogenase deficiency (G6PD),

-Drugs or other toxins, and

Autoimmune disorders.

2) Hepatic (the problem arises within the liver)

Jaundice in these cases is caused by the liver’s inability to properly metabolize and excrete bilirubin.


 Viral – hepatitis A, B, or C, yellow feverBacterial sepsis, tuberculosis,

AlcoholDrugs e.g. estrogens, contraceptive pills and Pregnancy

Carcinoma: metastases. Lymphoma. Adenocarcinoma of kidney (non-metastatic)

Preoperative hypo perfusion/shock

-Chronic active hepatitis

3) Post-hepatic (after bile has been made in the liver)

Jaundice in these cases, also termed obstructive jaundice, is caused by conditions which interrupt the normal drainage of conjugated bilirubin in the form of bile from the liver into the intestines. The obstruction may be intrahepatic or extra hepatic

Causes of obstructive jaundice include:

a) Intra hepatic Obstruction

-Biliary atresia

-Primary Biliary Cirrhosis

-Malignant infiltration of ducts

 b) Extra hepatic obstruction

-Gallstones in the bile ducts,

-cancer (pancreatic and gallbladder/bile duct carcinoma),

-strictures of the bile ducts,

-Pressure on the common bile duct from enlarged lymph nodes,


-congenital malformations, 



-pregnancy, and

-Newborn jaundice.

 Congenital conditions that may cause jaundice

1) Crigler-Najjar syndrome: an inherited condition that may lead to severe Unconjugated hyperbilirubinemia (high bilirubin concentrations); a gene mutation leads to a deficiency in an enzyme necessary for bilirubin conjugation.

2) Dubin-Johnson syndrome: an inherited disorder that causes the retention of conjugated bilirubin (and other compounds that turn the liver black) in liver cells; patients may have intermittent jaundice.

3) Rotor’s syndrome: an inherited conjugated hyperbilirubinemia that causes intermittent jaundice; similar to Dubin-Johnson without the retention of other compounds or a black liver.

4) Gilbert syndrome-There is compensatory hemolysis, impaired uptake and conjugation of bilirubin.


Figure-2- showing the causes of jaundice


Physiologic Neonatal Jaundice

Bilirubin produced by the fetus is cleared by the placenta and eliminated by the maternal liver. Immediately after birth, the neonatal liver must assume responsibility for bilirubin clearance and excretion. However, many hepatic physiologic processes are incompletely developed at birth. Levels of UDP –Glucuronyl Transferase levels are low, and alternative excretory pathways allow passage of unconjugated bilirubin into the gut. Since the intestinal flora that convert bilirubin to urobilinogen are also undeveloped, an enterohepatic circulation of Unconjugated bilirubin ensues. As a consequence, most neonates develop mild unconjugated hyperbilirubinemia between days 2 and 5 after birth. Peak levels are typically <85–170 mol/L (5–10 mg/dL) and decline to normal adult concentrations within 2 weeks, as mechanisms required for bilirubin disposition mature. Prematurity, with more profound immaturity of hepatic function, or hemolysis, results in higher levels of unconjugated hyperbilirubinemia. 


A rapidly rising Unconjugated bilirubin concentration, or absolute levels >340 mol/L (20 mg/dL), puts the infant at risk for bilirubin encephalopathy, or kernicterus. Under these circumstances, bilirubin crosses an immature blood-brain barrier and precipitates in the basal ganglia and other areas of the brain. The consequences range from appreciable neurologic deficits to death. Treatment options include phototherapy, which converts bilirubin into water-soluble photo isomers that are excreted directly into bile, Administration of inducing agents(Phenobarbitone), Albumin infusion and exchange transfusion. The canalicular mechanisms responsible for bilirubin excretion are also immature at birth, and their maturation may lag behind that of UGT, this can lead to transient conjugated neonatal hyperbilirubinemia, especially in infants with hemolysis.

Maternal-fetal blood group incompatibility (Rh, ABO)

This form of jaundice occurs when there is incompatibility between the blood types of the mother and the fetus. This leads to increased bilirubin levels from the breakdown of the fetus’ red blood cells (hemolysis).

Breast milk jaundice

This form of jaundice occurs in breastfed newborns and usually appears at the end of the first week of life. Certain chemicals in breast milk are thought to be responsible for inhibition of UDP Glucuronyl transferase. It is usually a harmless condition that resolves spontaneously.

Clinical Features

1) Unconjugated Hyperbilirubinemia (Pre hepatic jaundice)Stool and urine color are darker than normal, and there is mild jaundice and indirect (Unconjugated) hyperbilirubinemia with no bilirubin in the urine. Splenomegaly occurs in hemolytic disorders except in sickle cell anemia.

2) Hepatocellular disease (Hepatic jaundice)

Malaise, anorexia, low-grade fever, and right upper quadrant discomfort are frequent. Dark urine, jaundice, and, in women, amenorrhea occur. An enlarged tender liver, depending on the cause, severity, and chronicity of liver dysfunction is observed

3) Conjugated Hyperbilirubinemia (Post hepatic jaundice)

There may be right upper quadrant pain, weight loss (suggesting carcinoma), jaundice, dark urine, and light-colored stools. Symptoms and signs may be intermittent if caused by stone, carcinoma of the ampulla, or cholangiocarcinoma. Pain may be absent early in pancreatic cancer. Occult blood in the stools suggests cancer of the ampulla. Hepatomegaly and a palpable gallbladder (Courvoisier’s sign) are characteristic, but neither specific nor sensitive, of a pancreatic head tumor. Fever and chills are more common in benign obstruction with associated cholangitis.


The goal of testing is to determine the cause of the jaundice and to evaluate the severity of the underlying condition. The initial step is to obtain appropriate blood tests to determine if the patient has an isolated elevation of serum bilirubin. If so, is the bilirubin elevation due to an increased Unconjugated or conjugated fraction? If the hyperbilirubinemia is accompanied by other liver test abnormalities, is the disorder hepatocellular or cholestatic? If cholestatic, is it intra- or extra hepatic? All of these questions can be answered with a thoughtful history, physical examination, and interpretation of laboratory and radiologic tests and procedures.

Laboratory Studies

Measurement of Serum Bilirubin

The terms direct- and indirect-reacting bilirubin are based on the original van den Bergh reaction. This assay, or a variation of it, is still used in most clinical chemistry laboratories to determine the serum bilirubin level. In this assay, bilirubin is exposed to diazotized sulfanilic acid, splitting into two relatively stable dipyrrylmethene azopigments that absorb maximally at 540 nm, allowing for photometric analysis.

The direct fraction is that which reacts with diazotized sulfanilic acid in the absence of an accelerator substance such as alcohol. The direct fraction provides an approximate determination of the conjugated bilirubin in serum. The total serum bilirubin is the amount that reacts after the addition of alcohol. The indirect fraction is the difference between the total and the direct bilirubin and provides an estimate of the Unconjugated bilirubin in serum.

With the van den Bergh method, the normal serum bilirubin concentration usually is 17 mol/L (<1 mg/dL). Up to 30%, or 5.1 mol/L (0.3 mg/dL), of the total may be direct-reacting (conjugated) bilirubin. Total serum bilirubin concentrations are between 3.4 and 15.4 mol/L (0.2 and 0.9 mg/dL) in 95% of a normal population.

Measurement of Urine Bilirubin

Unconjugated bilirubin is always bound to albumin in the serum, is not filtered by the kidney, and is not found in the urine. Conjugated bilirubin is filtered at the glomerulus and the majority is reabsorbed by the proximal tubules; a small fraction is excreted in the urine. Any bilirubin found in the urine is conjugated bilirubin. The presence of bilirubinuria implies the presence of liver disease. A urine dipstick test (Ictotest) gives the same information as fractionation of the serum bilirubin. This test is very accurate. A false-negative test is possible in patients with prolonged cholestasis due to the predominance of conjugated bilirubin covalently bound to albumin.

Liver biochemical tests: changes in three types of jaundice.

Tests Prehepatic jaundice Hepatocellular Jaundice Uncomplicated Obstructive Jaundice
  Direct Increased Increased
  Indirect Increased Increased Increased
Urine bilirubinUrine Urobilinogen NoneIncreased IncreasedIncreased IncreasedAbsent
Serum albumin/total protein Normal Albumin decreasedTotal protein, 6.5–8.4 g/dL Unchanged
Alkaline phosphatase Normal Increased (+) Increased (++++)
Prothrombin time Normal Prolonged if damage severe and does not respond to parenteral vitamin K Prolonged if obstruction marked, but responds to parenteral vitamin K
ALT, AST Normal Increased in hepatocellular damage, viral hepatitis Minimally increased

ALT, alanine aminotransferase; AST, aspartate aminotransferase.

Serum alanine and aspartate aminotransferase (ALT and AST) levels vary with age and correlate with body mass index. Elevated alkaline phosphatase levels are seen in cholestasis or infiltrative liver disease (such as tumor or granuloma). Alkaline phosphatase elevations of hepatic rather than bone, intestinal, or placental origin are confirmed by concomitant elevation of γ-glutamyl transpeptidase or 5′-nucleotidase levels. The differential diagnosis of any liver test elevation includes toxicity caused by drugs, herbal remedies, and toxins.

Liver Biopsy

Percutaneous liver biopsy is the definitive study for determining the cause and histological severity of hepatocellular dysfunction or infiltrative liver disease.


Demonstration of dilated bile ducts by ultrasonography or CT scan indicates biliary obstruction (90–95% sensitivity). Ultrasonography, CT scan, and MRI may also demonstrate hepatomegaly,intrahepatic  tumors, and portal hypertension. Magnetic resonance cholangiopancreatography (MRCP) is a sensitive, noninvasive method of detecting bile duct stones, strictures, and dilation; however, it is less reliable than endoscopic retrograde cholangiopancreatography (ERCP) for distinguishing malignant from benign strictures.


The treatment is entirely dependent on the cause of the jaundice. In some cases it will be curative e.g. surgical removal of a gallstone blocking the common bile duct. Viral infections e.g. hepatitis A, may recover spontaneously. In other cases treatment will modify the disease process and the jaundice will improve or disappear. Treatment may be purely symptomatic e.g., drugs to relieve itching or palliative as in malignant disease. Some types of advanced liver disease-causing jaundice may be treated by liver transplantation.

Phototherapy for Neonatal Jaundice

The goal of therapy is to lower the concentration of circulating bilirubin or keep it from increasing. Photo therapy achieves this by using light energy to change the shape and structure of bilirubin, converting it to molecules that can be excreted even when normal conjugation is deficient

Phototherapy converts bilirubin to yellow photo isomers and colorless oxidation products that are less lipophilic than bilirubin and do not require hepatic conjugation for excretion. Photo isomers are excreted mainly in bile, and oxidation products predominantly in urine.

Prognosis is fairly good in the treated cases, except in cases of malignant obstruction of common bile duct.

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Case study-1

A 24 –year –old male suffering from Malaria was put on Primaquine. He developed malaise, fatigue and yellow discolouration of sclera and skin.

On examination – There was pallor- ++, icterus ++, Pulse – 100/min., Temperature 102°F. Liver and spleen were palpable.

The investigation report was as follows-


TLC-13000 cmm esp. polymorphs

Serum bilirubin- 6 mg%

Van den Bergh- Indirect positive.

Urine- Hemoglobin + and  Urobilinogen +

The color of the urine was brownish black

What is the probable diagnosis?

What is the relationship of primaquine intake and the present manifestations?

Case Details- This is a case of primaquine induced Hemolytic anemia, progressing to jaundice. Glucose-6-P dehydrogenase deficiency seems to be the underlying defect. High fever is due to malaria, while pallor and icterus are due to hemolytic anemia and underlying jaundice as apparent from low Hb and high bilirubin levels. Indirect positive Van Den Bergh indicates Uncinjugated Bilirubinemia. Urine is positive for hemoglobin and urobilinogen indicating the underlying hemoglobinuria and hemolytic jaundice

Primaquine being an oxidant drug precipitates the underlying defect to induce hemolysis. (See the details below).The liver has the capacity to conjugate and excrete over 3000 mg of bilirubin per day, whereas the normal production of bilirubin is only 300mg/day. The excess capacity allows the liver to respond to increased haem degradation with a corresponding increase in conjugation and secretion of bilirubin diglucuronide. 

However massive lysis of red blood cells, as in Glucose-6 –phosphate dehydrogenase deficiency, may produce bilirubin faster than it can be conjugated. More bilirubin is excreted in to the bile, the amount of urobilinogen entering the enterohepatic circulation is increased and urinary urobilinogen is  also increased. Unconjugated bilirubin levels become elevated in the blood causing jaundice.


 Case study-2

A 10 –year- old boy received a sulfonamide antibiotic as prophylaxis for recurrent urinary tract infections. Although he was previously healthy and well nourished, he became progressively ill and presented with pallor and irritability. A blood count revealed that he was severely anaemic with jaundice due to hemolysis of the red blood cells.

What is the problem with the boy?

What is the cause of anemia and jaundice in this boy?

What is the simplest way for the diagnosis of this problem?

Case details The child is suffering from Glucose-6-phosphate dehydrogenase deficiency. The individuals with G-6-P-D deficiency present with excessive hemolysis on exposure to certain drugs like antibiotics, analgesics and Antimalarials. Acute HA can develop as a result of three types of triggers: (1) fava beans, (2) infections, and (3) drugs. Glucose 6-phosphate dehydrogenase (G6PD) is an enzyme critical in the redox metabolism of all aerobic cells .In red cells, its role is even more critical because it is the only source of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which, directly and via reduced glutathione (GSH), defends these cells against oxidative stress.

figure showing the role of G-6-P-Dehydrogenase in Glucose metabolism.

NADPH is a required cofactor in many biosynthetic reactions which also maintains glutathione in its reduced form. Reduced glutathione acts as a scavenger for dangerous oxidative metabolites in the cell. With the help of the enzyme glutathione peroxidase, reduced glutathione converts harmful hydrogen peroxide to water. The inability to decompose hydrogen peroxide results in free radical induced membrane disruption and reduced life span as a result of haemoglobin formation.G6PD deficiency is a prime example of a haemolytic anemia due to interaction between an intracorpuscular and an extracorpuscular cause, because in the majority of cases hemolysis is triggered by an exogenous agent. People deficient in glucose-6-phosphate dehydrogenase (G6PD) are not prescribed oxidative drugs, because their red blood cells undergo rapid hemolysis under this stress. Although in G6PD-deficient subjects there is a decrease in G6PD activity in most tissues, this is less marked than in red cells, and it does not seem to produce symptoms.

Clinical manifestations The vast majority of people with G6PD deficiency remain clinically asymptomatic throughout their lifetime.

However, all of them have an increased risk of developing neonatal jaundice (NNJ) and a risk of developing acute HA when challenged by a number of oxidative agents. Typically, a haemolytic attack starts with malaise, weakness, and abdominal or lumbar pain. After an interval of several hours to 2–3 days, the patient develops jaundice and often dark urine, due to hemoglobinuria. The onset can be extremely abrupt, especially with favism in children. The anemia is moderate to extremely severe, usually normocytic and normochromic, and due partly to intravascular hemolysis; hence, it is associated with haemogobinemia, hemoglobinuria, and low or absent plasma Haptoglobin. Jaundice is prehepatic.

The laboratory workup for glucose-6-phosphate dehydrogenase (G6PD) deficiency includes the following:

  • Measure the actual enzyme activity of G6PD rather than the amount of glucose-6-phosphatase dehydrogenase (G6PD) protein.
  • Obtain a complete blood cell (CBC) count with the reticulocyte count to determine the level of anemia and bone marrow function.
  • Indirect bilirubinemia occurs with excessive hemoglobin degradation and can produce clinical jaundice.
  • Urinary urobilinogen is high

Treatment- Identification and discontinuation of the precipitating agent is critical in cases of glucose-6-phosphate dehydrogenase (G6PD) deficiency. Affected individuals are treated with oxygen and bed rest, which may afford symptomatic relief. Prevention of drug-induced hemolysis is possible in most cases by choosing alternative drugs. When acute HA develops and once its cause is recognized, no specific treatment is needed in most cases. However, if the anemia is severe, it may be a medical emergency, especially in children, requiring immediate action, including blood transfusion. 

 Case study-3

A 50 –year-old woman had 8 day history of loss of appetite, nausea and flu-like symptoms. She had noticed that her urine had been dark in color over the past two days. On examination she had tenderness in the right upper quadrant. 

Laboratory investigations showed;

Serum Total bilirubin 4.5 mg%

Direct bilirubin 2.5 mg%

Indirect bilirubin 2.0 mg%

Serum AST- 40 IU/L

Serum ALT-115 IU/L

Serum ALP- 20 Units (KA)

What is the probable diagnosis?

What will be the observation regarding bile pigments in urine?

Case details Flu like symptoms are indicative of viral hepatitis. Damage to liver cells can cause unconjugated bilirubin to increase in the blood as a result of decreased conjugation. The bilirubin that is conjugated is not efficiently secreted in to the bile, but instead diffuses in to the blood. Urobilinogen is increased in urine because hepatic damage decreases the enterohepatic circulation of this compound allowing more to enter blood, from which it is filtered in to the urine. The urine thus becomes dark in color, whereas stools are pale colored. Plasma levels of AST and ALT are elevated. This is a case of hepatic jaundice.


Based on the following clinical laboratory data, give the most probable diagnosis

Serum bilirubin 4 mg%

Direct bilirubin 0.2 mg%

Serum Alkaline phosphatase 6 units( KA)



Urine Bilirubin- Negative

Urine urobilinogen-Positive

Urine Bile Salts- Negative

Case details- Normal enzyme profile, Hyperbilirubinemia, absence of urinary bilirubin and positive urobilinogen are indicative of Hemolytic jaundice.

 Case study-5

 A 40 –year- old, fat female, presents with intolerance to fatty foods, pain in the right side of abdomen, yellowing of eyes and passage of clay colored stools.

Laboratory Investigations revealed


Total Bilirubin – 20 mg%

Direct Bilirubin- 16 mg%

ALP- 800 U(KA)



Color- deep yellow

Bilirubin- ++

Urobilinogen- absent


Clay colored

Stercobilnogen- absent

What is the likely diagnosis?

Which other enzymes are likely to increase?

 Case details This is a case of obstructive jaundice due to gall stones. This patient fits the “classic” criteria of gallbladder disease: female, middle-aged, overweight. Gallstones are collections of solid material (predominantly crystals of cholesterol) in the gallbladder. Gallstones may cause pain. Pain develops when the stones pass from the gallbladder into the cystic duct, common bile duct, or ampulla of Vater and block the duct. Then the gallbladder dilates, causing pain called biliary colic. The pain is felt in the upper abdomen, usually on the right side. Eating a heavy meal can trigger biliary colic, but simply eating fatty foods does not.

In this instance jaundice is not due caused due to overproduction of bilirubin, but instead results from obstruction of  the bile duct  from the gall stones. The liver regurgitates conjugated bilirubin in to the blood (Hyperbilirubinemia)

High direct bilirubin (Conjugated hyperbilirubinemia), high alkaline phosphatase (marker of cholestasis),slightly increased SGPT level are suggestive of post hepatic or obstructive jaundice. Furthermore the diagnosis is supported by the presence of bilirubin (since it is conjugated) and absence of urobilinogen (Since there is obstruction to the out flow of bile) in urine. Due to the same reason of obstruction stool is clay colored as stercobilnogen is absent.

Treatment is based on the relieving the obstruction surgically. Prolonged obstruction of the bile duct can lead to liver damage and a subsequent rise in unconjugated hyperbilirubinemia and a rise in SGPT levels.

 Case Study- 6

 An Rh negative mother delivers a baby who develops jaundice immediately after birth.

 General Examination reveals

Heart Rate    80/min

Icterus          +

Irritability     +

Liver            Palpable

 Laboratory Investigations



  • Total            10 mg%
  • Indirect        7 mg%
  • Direct           3 mg%

Alkaline phosphatase    50 U/L


Urobilinogen           +++


Stercobilnogen        +++

What is your likely diagnosis?     

 Case details  This is a case of haemolytic jaundice due to Rh incompatibility. Indirect hyper bilirubinemia (Unconjugated hyperbilirubinemia),  high urinary urobilinogen and fecal stercobilnogen are indicative of haemolytic jaundice. Typically bilirubin is absent in urine since unconjugated bilirubin being water insoluble and albumin bound (macromolecule), can not pass through glomeruli to appear in urine.






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A 30-year-old woman had severe abdominal pain, nausea, vomiting and diarrhea. Evaluations including upper and lower endoscopies did not establish any intestinal infection. She gradually improved and was discharged after 2 weeks. 2 years later she was admitted to a psychiatric unit with acute mental changes and hallucinations, she had to be transferred to the emergency department due to abdominal pain, a grand mal seizure and hyponatremia.

Her pulse was 120 and BP 174/114 mm Hg. She was disoriented but had no focal neurological signs. MRI showed sub cortical abnormalities, and the spinal fluid was normal. After cholecystectomy for a distended gallbladder, she was discharged but she stayed with a family member in another city because her symptoms were worse and muscle weakness had developed. She was hospitalized and progressed to quadriparesis, respiratory failure and aspiration pneumonia. Urinary porphobilinogen (PBG) was reported as 44 mg/24 hours (reference range 0-4).

What is the diagnosis and defect in this disease?

How is the diagnosis done and what is its prognosis?

What is the genetic basis of this disease?  


Case Discussion

The patient is suffering from acute intermittent Porphyria as evident from the typical combination of abdominal pain, motor neuropathy, psychiatric symptoms and increased amounts of urinary Porphyrins and their precursors.  Acute intermittent Porphyria (AIP) is a rare autosomal dominant metabolic disorder affecting the production of heme. It is characterized by deficiency of enzyme porphobilinogen deaminase.


Basic concept

Haem synthesis

Heme is required for a variety of haemoproteins such as hemoglobin, myoglobin, respiratory Cytochromes, and the cytochrome P450 enzymes (CYPs). Hemoglobin synthesis in erythroid precursor cells accounts for approximately 85% of daily heme synthesis in humans. Hepatocytes account for most of the rest, primarily for synthesis of CYPs, which are especially abundant in the liver endoplasmic reticulum, and turn over more rapidly than many other haemoproteins, such as the mitochondrial respiratory cytochromes.

Heme biosynthesis involves eight enzymatic steps in the conversion of glycine and succinyl-CoA to heme (Fig.1). These eight enzymes are encoded by nine genes, as the first enzyme in the pathway, 5′-aminolevulinate synthase (ALA-synthase), has two genes that encode unique housekeeping (ALAS1) and erythroid-specific (ALAS2) isozymes. The first and last three enzymes in the pathway are located in the mitochondrion, whereas the other four are in the cytosol. As shown in Fig.1, pathway intermediates are the porphyrin precursors, ALA and PBG, and porphyrins (mostly in their reduced forms, known as porphyrinogens). At least in humans, these intermediates do not accumulate in significant amounts under normal conditions or have important physiologic functions.


Figure-1- showing the steps of Haem synthesis


Steps of Haem synthesis


1) The first and rate-controlling step is the condensation of glycine and succinyl–coenzyme A (CoA) to form δ-aminolevulinic acid (ALA). The enzyme, ALA-synthase is activated by Pyridoxal phosphate. In the liver, this rate-limiting enzyme can be induced by a variety of drugs, steroids, and other chemicals. Defects in the erythroid gene, cause X-linked Sideroblastic anemia (XLSA).

2) The ALA formed is transported into the cytoplasm, where the second enzyme, ALA dehydratase (also known as porphobilinogen synthase), condenses two molecules of ALA to form the monopyrrole porphobilinogen.

3) The third enzyme, porphobilinogen deaminase (also known as hydroxymethylbilane synthase), forms a linear tetrapyrrole, hydroxymethylbilane, which is normally rapidly converted, mainly to the cyclic intermediate Uroporphyrinogen III, by the enzyme Uroporphyrinogen III synthase (also known as Uroporphyrinogen cosynthase). When Uroporphyrinogen III synthase is deficient, as in congenital erythropoietin Porphyria (Guenther’s disease), hydroxymethylbilane rapidly undergoes nonenzymatic ring closure to form Uroporphyrinogen I.

4) The enzyme Uroporphyrinogen decarboxylase carries out the stepwise decarboxylation of Uroporphyrinogen I or III to form intermediates with 7-, 6-, 5-, and 4-carboxyl groups. Coproporphyrinogen is the common name for the 4-carboxyl–containing intermediate.

5) Coproporphyrinogen III is transported back into mitochondria, where the enzyme Coproporphyrinogen III oxidase carries out the stepwise oxidative decarboxylation of two of the remaining propionate beta side chains, at positions 2 and 4 (on rings A and B, respectively), to vinyl groups, forming protoporphyrinogen IX.

6) Next, the enzyme protoporphyrinogen oxidase carries out the oxidation of protoporphyrinogen IX to form protoporphyrin IX, after which the enzyme Ferrochelatase (also called heme synthase) inserts ferrous iron into the protoporphyrin IX macrocycle to form the end product heme. (See figure-1)

Regulation of Heme Biosynthesis

Regulation of heme synthesis differs in the two major heme-forming tissues, the liver and erythron. In the liver, “free” heme regulates the synthesis and mitochondrial translocation of the housekeeping form of ALA-synthase. Heme represses the synthesis of the ALA-synthase mRNA and interferes with the transport of the enzyme from the cytosol into mitochondria. Hepatic ALA-synthase is increased by many of the same chemicals that induce the cytochrome P450 enzymes in the endoplasmic reticulum of the liver. Because most of the heme in the liver is used for the synthesis of cytochrome P450 enzymes, hepatic ALA-synthase and the cytochrome P450s are regulated in a coordinated fashion, and many drugs that induce hepatic ALA-synthase also induce CYPs. The other hepatic heme biosynthetic enzymes are presumably expressed at constant levels, although their relative activities and kinetic properties differ. For example, normal individuals have high activities of ALA-dehydratase but low activities of HMB-synthase, the latter being the second rate-limiting step in the pathway.

In the erythron, novel regulatory mechanisms allow for the production of the very large amounts of heme needed for hemoglobin synthesis. The response to stimuli for hemoglobin synthesis occurs during cell differentiation, leading to an increase in cell number. The erythroid-specific ALA-synthase is expressed at higher levels than the housekeeping enzyme, and erythroid-specific control mechanisms regulate other pathway enzymes as well as iron transport into erythroid cells.



The Porphyrias are a group of rare metabolic disorders arising from reduced activity of any of the enzymes in the heme biosynthetic pathway. The disorders may be either acquired or inherited through a genetic defect in a gene encoding these enzymes. These deficiencies disrupt normal heme production, and produce symptoms when increased heme is required. Porphyrin precursors, overproduced in response to synthetic pathway blockages, accumulate in the body and cause diverse pathologic changes thereby becoming the basis for diagnostic tests.

The diagnosis of acute porphyrias can be confirmed by repeating the quantitation of urinary porphyrin during an acute episode and finding elevated levels (2–5 times of normal) of porphobilinogen.


The prevalence of this condition is unknown, but probably ranges from 1 in 500 to 50,000 worldwide. Certain types of porphyrias are more common in specific populations, such as whites in South Africa and Scandinavians.


The Porphyrias can be classified as either hepatic or erythropoietic, depending on whether the heme biosynthetic intermediates that accumulate arise initially from the liver or developing erythrocytes, or as acute or cutaneous, based on their clinical severity.


Most of the Porphyrias are inherited. Inheritance patterns depend on the type of porphyria. Some forms of the condition are inherited in an autosomal dominant pattern, which means one copy of the altered gene is sufficient to cause the disorder. Other Porphyrias are inherited in an autosomal recessive pattern, which means two copies of the gene must be altered for a person to be affected by the condition.

Typically, patients with the autosomal dominant varieties present initially in adulthood, and those with homozygous variants present in early childhood. Symptomatic porphyria is thought to be more common in female than male patients with a female-male ratio of 5 to 1.

A) The Hepatic Porphyrias

1) ALA-Dehydratase Deficient Porphyria (ADP)

ADP is a rare autosomal recessive acute hepatic porphyria caused by a severe deficiency of ALA-dehydratase activity. The clinical presentation depends on the amount of residual ALA-dehydratase activity. Symptoms resemble those of AIP (Acute intermittent Porphyria) including abdominal pain and neuropathy. Infant with more severe disease manifest failure to thrive beginning at birth. Diagnosis is confirmed by significantly elevated levels of plasma and urinary ALA, urinary coproporphyrin III and ALAD activity in erythrocytes &lt;10% of normal.

The treatment of ADP acute attacks is similar to that of AIP.

2) Acute Intermittent Porphyria (AIP)

Acute intermittent porphyria is inherited as an autosomal dominant, though it remains clinically silent in most patients who carry the trait. Clinical illness usually develops in women. Symptoms begin in the teens or 20s, but onset can begin after menopause in rare cases. The disorder is caused by partial deficiency of porphobilinogen deaminase activity, leading to increased excretion of aminolevulinic acid and porphobilinogen in the urine. The diagnosis may be elusive if not specifically considered. The characteristic abdominal pain may be due to abnormalities in autonomic innervations in the gut. In contrast to other forms of porphyria, cutaneous photosensitivity is absent in acute intermittent porphyria. Attacks are precipitated by numerous factors, including drugs and intercurrent infections. Hyponatremia may be seen, due in part to inappropriate release of antidiuretic hormone, though gastrointestinal loss of sodium in some patients may be a contributing factor.

Clinical Findings

Symptoms and Signs

Patients show intermittent abdominal pain of varying severity, and in some instances it may so simulate acute abdomen as to lead to exploratory laparotomy. Complete recovery between attacks is usual. Any part of the nervous system may be involved, with evidence for autonomic and peripheral neuropathy. Peripheral neuropathy may be symmetric or asymmetric and mild or profound; in the latter instance, it can even lead to quadriplegia with respiratory paralysis. Other central nervous system manifestations include seizures, psychosis, and abnormalities of the basal ganglia. Hyponatremia may further cause or exacerbate central nervous system manifestations.

Laboratory Findings

Often there is profound hyponatremia. The diagnosis can be confirmed by demonstrating an increased amount of porphobilinogen in the urine during an acute attack. Freshly voided urine is of normal color but may turn dark upon standing in light and air.


Avoidance of factors known to precipitate attacks of acute intermittent porphyria—especially drugs can reduce morbidity. Starvation diets also cause attacks and so must be avoided.


Treatment with a high-carbohydrate diet diminishes the number of attacks in some patients and is a reasonable empiric gesture considering its benignity. Acute attacks may be life-threatening and require prompt diagnosis, withdrawal of the inciting agent (if possible), and treatment with analgesics and intravenous glucose and hematin. Electrolyte balance requires close attention. Liver transplantation may provide an option for patients with disease poorly controlled by medical therapy.

3) Porphyria Cutanea Tarda

PCT, the most common of the Porphyrias, can be either sporadic (type 1) or familial (types 2 and 3) and can also develop after exposure to halogenated aromatic hydrocarbons. Hepatic URO-decarboxylase is deficient in all types of PCT, and for clinical symptoms to manifest, this enzyme deficiency must be substantial (~20% of normal activity or less).

Clinical Features

Blistering skin lesions that appear most commonly on the backs of the hands are the major clinical feature .These rupture and crust over, leaving areas of atrophy and scarring. Lesions may also occur on the forearms, face, legs, and feet. Occasionally, the skin over sun-exposed areas becomes severely thickened, with scarring and calcification that resembles systemic sclerosis. Neurologic features are absent.

A number of susceptibility factors, can be recognized clinically and can affect management. These include hepatitis C, HIV, excess alcohol, elevated iron levels, and estrogens. Excess alcohol is a long-recognized contributor, as is estrogen use in women. Multiple susceptibility factors that appear to act synergistically can be identified in the individual patient with PCT. Patients with PCT characteristically have chronic liver disease and sometimes cirrhosis and are at risk for hepatocellular carcinoma.


Porphyrins are increased in the liver, plasma, urine, and stool. The urinary ALA level may be slightly increased, but the PBG level is normal. Urinary porphyrins consist mostly of uroporphyrin with lesser amounts of coproporphyrin. Plasma porphyrins are also increased.

Porphyria Cutanea Tarda: Treatment

Alcohol, estrogens, iron supplements, and, if possible, any drugs that may exacerbate the disease should be discontinued, but this step does not always lead to improvement. A complete response can almost always be achieved by the standard therapy, repeated phlebotomy, to reduce hepatic iron. A unit (450 mL) of blood can be removed every 1–2 weeks. The aim is to gradually reduce excess hepatic iron until the serum ferritin reaches the lower limits of normal. Because iron overload is not marked in most cases, remission may occur after only five or six phlebotomies; however, PCT patients with hemochromatosis may require more treatments to bring their iron levels down to the normal range.

An alternative when phlebotomy is contraindicated or poorly tolerated is a low-dose regimen of chloroquine or hydroxychloroquine, both of which complex with the excess porphyrins and promote their excretion. Hepatic imaging can diagnose or exclude complicating hepatocellular carcinoma. Treatment of PCT in patients with end-stage renal disease is facilitated by administration of erythropoietin.

Sun screen lotions and beta carotene are recommended to prevent  skin damage caused by sun light.

 4) Hereditary Coproporphyria

HCP is an autosomal dominant hepatic porphyria that results from the half-normal activity of COPRO-oxidase. The disease presents with acute attacks, as in AIP. Cutaneous photosensitivity also may occur, but much less commonly than in VP. HCP patients may have acute attacks and cutaneous photosensitivity together or separately. HCP is less common than AIP and VP.

Clinical Features

HCP is influenced by the same factors that cause attacks in AIP. The disease is latent before puberty, and symptoms, which are virtually identical to those of AIP, are more common in women. HCP is generally less severe than AIP. Blistering skin lesions are identical to PCT and VP.


COPRO III is markedly increased in the urine and feces in symptomatic disease and often persists, especially in feces, when there are no symptoms. Urinary ALA and PBG levels are increased (but less than in AIP) during acute attacks but may revert to normal more quickly than in AIP when symptoms resolve. Plasma porphyrins are usually normal or only slightly increased, but they may be higher in cases with skin lesions. The diagnosis of HCP is readily confirmed by increased fecal porphyrins consisting almost entirely of COPRO III, which distinguishes it from other porphyrias. An increase in the fecal COPRO III/COPRO I ratio is useful for detecting latent cases.

Although the diagnosis can be confirmed by measuring COPRO-oxidase activity, the assays for this mitochondrial enzyme are not widely available and require cells other than erythrocytes.

Hereditary Coproporphyria: Treatment

Neurologic symptoms are treated as in AIP .Phlebotomy and chloroquines are ineffective when cutaneous lesions are present.

5) Variegate Porphyria

VP is an autosomal dominant hepatic porphyria that results from the deficient activity of PROTO-oxidase, the seventh enzyme in the heme pathway, and can present with neurologic symptoms, photosensitivity, or both. VP is particularly common in South Africa, where 3 of every 1000 whites have the disorder.

Clinical Features

VP can present with skin photosensitivity, acute neurovisceral crises, or both. Acute attacks are identical to those in AIP and are precipitated by the same factors as AIP. Blistering skin manifestations are similar to those in PCT but are more difficult to treat and usually are of longer duration. Homozygous VP is associated with photosensitivity, neurologic symptoms, and developmental disturbances, including growth retardation, in infancy or childhood.


Urinary ALA and PBG levels are increased during acute attacks but may return to normal more quickly than in AIP. Increases in fecal protoporphyrin and COPRO III and in urinary COPRO III are more persistent. Plasma porphyrin levels also are increased, particularly when there are cutaneous lesions. VP can be distinguished rapidly from all other porphyrias by examining the fluorescence emission spectrum of porphyrins in plasma at neutral pH since VP has a unique fluorescence peak at neutral pH.

Assays of PROTO-oxidase activity in cultured fibroblasts or lymphocytes are not widely available.

Variegate Porphyria: Treatment

Acute attacks are treated as in AIP, and hemin should be started early in most cases. Other than avoiding sun exposure, there are few effective measures for treating the skin lesions. Beta-Carotene, phlebotomy, and chloroquine are not helpful.

B) The Erythropoietic Porphyrias

In the erythropoietic porphyrias, excess porphyrins from bone marrow erythrocyte precursors are transported via the plasma to the skin and lead to cutaneous photosensitivity.

1) X-Linked Sideroblastic Anemia

XLSA results from the deficient activity of the erythroid form of ALA-synthase and is associated with ineffective erythropoiesis, weakness, and pallor.

Clinical Features

Typically, males with XLSA develop refractory hemolytic anemia, pallor, and weakness during infancy. They have secondary hypersplenism, become iron overloaded, and can develop hemosiderosis. The severity depends on the level of residual erythroid ALA-synthase activity and on the responsiveness of the specific mutation to Pyridoxal 5´-phosphate supplementation.


Peripheral blood smear reveals a hypochromic, microcytic anemia with striking anisocytosis, poikilocytosis, and polychromasia; the leukocytes and platelets appear normal. Hemoglobin content is reduced, and the mean corpuscular volume and mean corpuscular hemoglobin concentration are decreased.

Bone marrow examination reveals hypercellularity with a left shift ,megaloblastic erythropoiesis with an abnormal maturation. A variety of Prussian blue–staining sideroblasts are observed.

Levels of urinary porphyrin precursors and of both urinary and fecal porphyrins are normal. The level of erythroid ALA-synthase is decreased in bone marrow, but this enzyme is difficult to measure in the presence of the normal ALA-synthase housekeeping enzyme. Definitive diagnosis requires the demonstration of mutations in the erythroid ALAS gene.

X-Linked Sideroblastic Anemia: Treatment

The severe anemia may respond to pyridoxine supplementation. This cofactor is essential for ALA-synthase activity, and mutations in the pyridoxine binding site of the enzyme have been found in several responsive patients. Cofactor supplementation may make it possible to eliminate or reduce the frequency of transfusion. Unresponsive patients may be transfusion-dependent and require chelation therapy.

2) Congenital Erythropoietic Porphyria

CEP, also known as Günther disease, is an autosomal recessive disorder. It is due to the markedly deficient, but not absent, activity of URO-synthase and the resultant accumulation of uroporphyrin I and coproporphyrin I isomers. CEP is associated with hemolytic anemia and cutaneous lesions.

Clinical Features

Severe cutaneous photosensitivity begins in early infancy. The skin over light-exposed areas is friable, and bullae and vesicles are prone to rupture and infection. Skin thickening, hypo- and hyperpigmentation, and hypertrichosis of the face and extremities are characteristic. Secondary infection of the cutaneous lesions can lead to disfigurement of the face and hands. Porphyrins are deposited in teeth and in bones. As a result, the teeth are reddish-brown and fluoresce on exposure to long-wave ultraviolet light. (Erythrodontia)-See figure  Hemolysis is probably due to the marked increase in erythrocyte porphyrins and leads to splenomegaly. Adults with a milder form of the disease also have been described.


Uroporphyrin and coproporphyrin (mostly type I isomers) accumulate in the bone marrow, erythrocytes, plasma, urine, and feces. The predominant porphyrin in feces is coproporphyrin I. The diagnosis of CEP can be confirmed by demonstration of markedly deficient URO-synthase activity and/or the identification of specific mutations in the UROS gene. The disease can be detected in utero by measuring porphyrins in amniotic fluid and URO-synthase activity in cultured amniotic cells or chorionic villi, or by the detection of the family’s specific gene mutations.

 Figure-2 showing Erythtrodontia in Congenital Erythropoietic porphyria

Congenital Erythropoietic Porphyria: Treatment

Severe cases often require transfusions for anemia. Chronic transfusions of sufficient blood to suppress erythropoiesis are effective in reducing porphyrin production but results in iron overload. Splenectomy may reduce hemolysis and decrease transfusion requirements. Protection from sunlight and from minor skin trauma is important. Beta Carotene may be of some value. Complicating bacterial infections should be treated promptly. Recently, bone marrow and cord blood transplantation has proven effective in several transfusion-dependent children, providing the rationale for stem-cell gene therapy.

3) Erythropoietic Protoporphyria

EPP is an inherited disorder resulting from the partial deficiency of ferrochelatase activity, the last enzyme in the heme biosynthetic pathway. EPP is the most common erythropoietic porphyria in children and, after PCT, the second most common porphyria in adults. EPP patients have ferrochelatase activities as low as 15–25% in lymphocytes and cultured fibroblasts. Protoporphyrin accumulates in bone marrow reticulocytes and then appears in plasma, taken up in the liver, and excreted in bile and feces.

Clinical Features

Skin photosensitivity, which differs from that in other porphyrias, usually begins in childhood and consists of pain, redness, and itching occurring within minutes of sunlight exposure .Photosensitivity is associated with substantial elevations in erythrocyte protoporphyrin and occurs only in patients with genotypes that result in Ferrochelatase activities below ~35% of normal. Redness, swelling, burning, and itching can develop shortly after sun exposure Symptoms may seem out of proportion to the visible skin lesions.

Although EPP is an erythropoietic porphyria, up to 20% of EPP patients may have minor abnormalities of liver function, and in about 5% of these patients the accumulation of protoporphyrins causes chronic liver disease that can progress to liver failure and death.


A substantial increase in erythrocyte protoporphyrin, which is predominantly free and not complexed with zinc, is the hallmark of this disease. Protoporphyrin levels are also variably increased in bone marrow, plasma, bile, and feces.

Erythropoietic Protoporphyria: Treatment

Avoiding sunlight exposure and wearing clothing designed to provide protection for conditions with chronic photosensitivity are essential. Oral Beta-carotene improves tolerance to sunlight in many patients. The beneficial effects of -carotene may involve quenching of singlet oxygen or free radicals.

Treatment of hepatic complications, which may be accompanied by motor neuropathy, is  difficult. Cholestyramine and other porphyrin absorbents such as activated charcoal may interrupt the enterohepatic circulation of protoporphyrin and promote its fecal excretion, leading to some improvement. Splenectomy may be helpful when the disease is accompanied by hemolysis and significant splenomegaly. Plasmapheresis and intravenous hemin are sometimes beneficial.

Liver transplantation has been carried out in some EPP patients with severe liver complications and is often successful in the short term. Bone marrow transplantation is considered after the liver





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