2. In this reaction (which needs, heme oxygenase enzyme , O 2 and NADPH), iron (Fe++) is removed for re-use. The remaining of heme ring is cleaved between pyrrole rings number I and II to form biliverdin (green pigment) and carbon monoxide (CO)
3. Biliverdin is then reduced into bilirubin (golden yellow) in a reaction requires biliverdin reductase enzyme .
1-This is a reaction between bilirubin and Ehrlich diazo reagent giving a reddish purple compound.
2- Conjugated bilirubin reacts directly with the reagent. Thus it is called: direct bilirubin
3 - Unconjugated bilirubin does not react with the reagent directly except after addition of methyl alcohol . Thus it may be called: indirect bilirubin
Differences between unconjugated bilirubin and conjugated bilirubin
Heme catabolism is classified into several stages
Formation of bilirubin in reticuloendothelial system. In reticuloendothelial system (RES), hemoglobin is degraded into heme and globin . Globin is either degraded to amino acids or reused for synthesis of hemoglohin . Heme is converted into biliverdin (green) then to bilirubin (yellow).
One gram of hemoglobin yields 35 mg of bilirubin . The daily bilirubin formation in adult human is about 250 mg . Bilirubin is transported to the liver in conjunction with plasma albumin (termed indirect or unconjugated bilirubin ). Each molecule of albumin has one high affinity site and one low affinity site for bilirubin.
This stage occurs in the liver and can be divided into three processes :
1-Uptake of bilirubin by liver parenchymal cells.
This is followed by removal of albumin and release of free bilirubin.
2-Synthesis of bilirubin-diglucuronide.
This is catalyzed by bilirubin-glucuronyl-transterase to form bilirubin diglucuronide . UDP-glucuronate (UDP-GlcUA) acts as donor or the glucuronate group to form bilirubin-monoglucuronide then bilirubin-diglucuronide (termed chole bilirubin. direct or conjugated bilirubin ) and it is water soluble .
3-Bilirubin-diglucuronide is actively secreted into bile.
Stage III : This is produced by the action of intestinal bacteria.
Bilirubin is deconjugated from glucuronate , and then reduced to urobilinogens (colorless) . Most of urobilinogens are excreted in feces and oxidized to the colored pigments urobilins by oxygen. This explains darkening of feces upon standing in air. Normally small amounts of urobilinogens are re absorbed by portal blood and excreted by liver cells into bile (enterohepatic circulation) and traces escape and excreted in urine.
The nonnal level of bilirubin in plasma (or serum) is less than 1.2 mg/dL and it is present in two forms , indirect and direct .
1-Indirect reacting (bilirubin-albumin complex or unconjugated bilirubin).
It requires the addition of methanol to react with the diazo reagent of Van den Bergh (used for its estimation). It is the main fonn present nonnally in plasma and it ranges from 0.3 to 0.8 mg/dL . It increases in all cases of hemolytic anemia and when liver fails to take up or conjugate bilirubin .
2-Direct reacting (bilirubin-diglucuronide or conjugated bilirubin).
It is water-soluble and reacts directly with the diazo reagent of van den Bergh. It represents the bilirubin-diglucuronide which escapes from liver to systemic blood. It increases when liver fails to excrete bilirubin after conjugation or when there is obstruction in the biliary system that prevents its passage to the intestine. Normally its level in plasma is less than 0.3 mg/ldL.
It is due to overproduction of bilirubin by reticuloendothelial system over the capacity of the liver to remove and clear from blood. It is characterized by high level of indirect or unconjugated bilirubin. This type of bilirubin can cross the blood-brain barrier into the central nervous system and cause kernicterus (encephalopathy). Unconjugated bilirubin is water insoluble and not excreted in urine ( acholuric jaundice ).
Unconjugated hyperbilirubinemia occurs in the following conditions
I-Neonatal or Physiologic Jaundice : This is the most common cause of jaundice in neonatal age. It results from accelerated hemolysis and immature hepatic system for uptake, conjugation and secretion of bilirubin. It is characterized by high plasma level of unconjugated bilirubin, if it exceeds 20-25 mg/dL
Conjugated hyperbilirubinemia is due to reflux of direct or conjugated bilirubin into blood due to biliary obstruction , conjugated bilirubin is water soluble, so it is excreted in urine and darken its colour (Choluric jaundice) .
It occurs in the following conditions:
1-Obstructive Jaundice (Cholestatic Jaundice)
Conjugated hyperbilirubinemia results from blockage of hepatic or common bile duct (stones and tumors), bilirubin diglucuronide is not excreted and returns to hepatic veins and lymphatics and appears in blood and urine.
It may be produced by micro-obstruction of intrahepatic biliary ductules by swollen damaged hepatocytes e.g. viral hepatitis and liver cirrhosis . Both cases are associated with marked increase of conjugated bilirubin and slight to moderate increase of unconjugated bilirubin (mixed hyperbilirubinemia).