CHEMISTRY OF BILLIRUBIN FORMATION;
What is billirubin;
Bilirubin may be a yellow compound that happens within the normal catabolic pathway that breaks down heme in vertebrates. This catabolism may be a necessary process within the body's clearance of waste products that arise from the destruction of aged or abnormal red blood cells.
FORMATION OF BILLIRUBIN;
Roughly, 80% of bilirubin is formed from the breakdown of hemoglobin in senescent red blood cells, and prematurely destroyed erythroid cells within the bone marrow. the rest originates from the turnover of varied heme-containing proteins found in other tissues, primarily the liver and muscles.
FATE OF BILLIRUBIN;
1. Uptake ;
Bilirubin formed by the destruction of RBCs is free or unconjugated bilirubin. It is lipid soluble and bound to albumin in plasma (protein conjugation), which prevents its excretion by the kidneys in urine.
2. Conjugation;
On reaching the liver, free (unconjugated) bilirubin is split from albumin and enters the hepatic cells. In the hepatic cells it undergoes conjugation with 'uridine diphosphate glucuronic acid' (UDPGA) in a reaction catalyzed by enzyme glucurony1 transferase' to form bilirubin mono and di-glucuronides. these are water soluble i.e. conjugated bilirubin, also called cholebilirubin.
3. Excretion;
Excretion of conjugated bilirubin by the hepatic cells into the bile canaliculi is an active process i.e. it is transported against a concentration gradient into bile canaliculi. This is the rate limiting step in the liver for handling of bilirubin and needs energy. Some of it escapes into the general circulation and is excreted via kidneys in urine as urine bilirubin; while most of it passes via the bile ducts to the small intestine.
4. Degradation;
When this 'conjugated' water soluble bilirubin reaches the large intestine it is degraded by colonic bacteria Glucuronic acid is split from bilirubin, and bilirubin undergoes reduction forming a colourless Compound stercobilinogen, also called urobilinogen.
5. Re-Excretion
Some 20% of stercobilinogen (urobilinogen) is reabsorbed into the portal circulation to reach the liver.
A. A part of which is re-excreted by the liver into the bile and returns to the small intestine by enterohepatic circulation.
B. The remaining absorbed stercobilinogen (urobilinogen) enters the general circulation. It gets filtered by the kidneys and is excreted in urine as urine urobilinogen. If urine is allowed to stand, urobilinogen is oxidised to urobilin.
The remaining 80% of stercobilinogen which is not reabsorbed from the intestine is excreted in amounts of 20-250 mg/day in the faeces. Some of this stercobilinogen is oxidised to a brownish compound stercobilin which is responsible for the brown colour of the faeces.
The total serum bilirubin includes conjugated bilirubin (mainly) plus free (unconjugated) bilirubin.
One of the tests which helps in determining the type of bilirubin present in the serum is The van den Bergh Test.
Principle: "Diazo' reagent (mixture of sulphanilic acid, hydrochloric acid and sodium nitrite) on reacting with serum containing excess of 'conjugated bilirubin' (water soluble) within 30 seconds i.e. almost immediately gives a reddish violet colour. This is said to be Direct positive reaction.
However, when Diazo reagent reacts with serum containing an excess of 'unconjugated' bilirubin (lipid soluble), no color develops until some solvent like alcohol is added. This is called Indirect positive reaction.