In this article we will discuss about:- 1. Origin and Formation of Bile Pigments 2. Chemistry and Varieties of Bile Pigments 3. Circulation and Fate.
Origin and Formation of Bile Pigments:
The old and worn-out red blood cells disintegrate and are removed from the circulation by the cells of the reticuloendothelial system; the bone-marrow appears to be the most active site. Haemoglobin is released and by degradation, opening of the porphyrin ring system occurs. The degraded compound is known as verdohaemoglobin or choleglobin. In the next stage it is broken down into protein and haem. Protein is broken down into amino acids which enter the general amino acid pool of the body.
The iron present in the haem remains stored in the body as apoferritin, ferritin and haemosiderin which help in the formation of new haemoglobin. The rest of the haem is converted into yellow pigment bilirubin which is oxidised into green pigment biliverdin or the green pigment biliverdin is formed first which by reduction forms the yellow pigment bilirubin. Biliverdin reductase is the enzyme which catalyses the reduction of biliverdin to bilirubin. These are also derived to some extent from myohaemoglobin.
A schematic representation of bile pigment formation is given below in the Fig. 9.26. It does not represent proved steps of the reactions, but only attempts to summarise the facts and supplies possible pathways.
The oxidation and reduction take place by transference of hydrogen from the substrate and NAD/NADH or NADP/NADPH system. The bilirubin then probably combines with albumin of the plasma. When it enters the liver cells, plasma albumin is separated from bilirubin. In the liver cells and to lesser extent in kidney cells it is conjugated with glucuronic acid (UDP glucuronic acid) and forms monoglucuronide and diglucuronide.
In hepatic bile these are bound in addition with protein and in gall-bladder bile with lipoprotein, cholesterol and bile acids. The reaction is catalised by glucuronyl transferase. Some bilirubin is also esterified by sulphuric acid as bilirubin sulphate.
Chemistry and Varieties of Bile Pigments:
A number of pigments are present in bile. The two chief pigments are bilirubin (golden-yellow) and biliverdin (green). Bilirubin (C33H36N4O6) is the chief pigment of human and carnivorous bile. Biliverdin (C33H36N4O8) is the oxidation product of bilirubin. It is present chiefly in the bile of birds and of herbivorous animals.
Biliprasin is supposed to be an intermediate product formed during oxidation of bilirubin into biliverdin. Bilicyanin (blue), bilifuscin (red) and choletelin (yellow) are three other pigments formed by the successive oxidation of biliverdin. They are found in the gall-stones. The bile pigments are porphyrin compounds and constitute about 15 – 20% of the total solids of the liver bile. [They can be detected by Gmelin’s test.]
Circulation and Fate of Bile Pigments:
Liver (Kupffer cells), spleen and bone-marrow, being the chief seats of the reticulo-endothelial system, take the main part in bilirubin formation. Blood leaving the spleen and bone-marrow has a much higher bilirubin content than the arterial blood. Normal blood serum contains traces of bilirubin which on the average amounts to about 0.5 – 0.8 unit. It is to be noted that bilirubin, as it is present in blood (haemobilirubin), is not the same as that present in bile (cholebilirubin). Haemobilirubin remains combined with serum albumin and cholebilirubin remains in combination with glucuronic acid.
The main differences between the two bilirubins are summarised below in table 9.3:
Van Den Bergh Reaction:
This test helps in detection of bile pigment in blood serum.
There are three types of reactions:
Two types of solutions are used:
i. Solution I:
Sulphanilic acid (0.1 gm), concentrated HCI (1.5 ml), distilled water—(100 ml).
ii. Solution II:
Sodium nitrate (0.5 gm) and water (100 ml).
25ml of No. I solution is mixed with 0.75 ml of No. II solution—Diazo reagent. 1 ml of serum is taken in a small test-tube.
To it equal amount of Diazo reagent is added and any one of the following reactions may occur:
1. Direct Reactions:
i. Immediate or Prompt:
A bluish-violet colour immediately appears (within 10 – 30 sec).
Reddish colour appears which gradually becomes violet and this takes from 5-15 minutes or even half an hour.
2. Biphasic Reaction:
A reddish colour appears promptly and after much longer time becomes violet.
3. Indirect Reaction:
At first 1 ml of serum is treated with 2 ml of 95% alcohol. It is shaken and centrifuged. 1 ml of supernatant fluid is taken and to it 0.25 ml of Diazo reagent is added. A reddish-violet colour appears immediately.
In Jaundice there is excessive accumulation of bile pigments in blood which causes yellowish discolouration of skin, mucous membrane and conjunctiva.
There are three types of jaundice:
1. Obstructive Jaundice:
Van den Bergh test is direct and prompt.
2. Haemolytic Jaundice:
Van den Bergh test is indirect.
3. Toxic or Infective Jaundice: (Where there is Parenchymatous Liver Damage):
Van den Bergh test is delayed direct or biphasic.
After passing through the hepatic cells, conjugated bilirubin and biliverdin enter bile channels and then into the intestine along with bile.
In the intestine following changes take place:
Bilirubin → mesobilirubin → mesobilirubinogen → stercobilinogen. On being exposed to air stercobilinogen is further oxidised into yellowish-brown stercobilin and is responsible for the normal colour of the faeces. About half the amount of total bile pigments is excreted in the faeces, which varies from 40 – 280 mgm per day.
The remaining part of stercobilinogen is reabsorbed from the intestine and is carried back to liver. Under normal conditions, this reabsorbed stercobilinogen is almost fully re-excreted in the bile. A trace of stercobilinogen may fail to pass through the liver, and is excreted in the urine. This excretory product is named as urobilinogen which is quickly oxidised into urobilin by the air after the urine is voided (Fig. 9.27).
It is believed that some urobilinogen passes directly to the kidney escaping the liver for excretion (not shown in Fig. 9.27). Urobilinogen is identical with stercobilinogen and urobilin is identical with stercobilin. Normally faecal excretion of bile pigments varies from 50 – 250 mgm per day, only 1-2 mgm being excreted through urine.
When liver is damaged, urobilinogen reabsorbed from the intestine, fails to pass through the liver cells and appears in the urine in a larger amount. Under such conditions, urine contains considerable amounts of urobilinogen and urobilin. Presence of urobilinogen in urine in excess, therefore, indicates functional deficiency of liver.