Bilirubin and Bilirubin Fractions

Bilirubin and Bilirubin Fractions

Synonyms - Specimen - Reference Value - Method - Description - Indications - Interpretation 

Synonyms : Conjugated / direct bilirubine , unconjugated / indirect bilirubin , delta bilirubin , TBil.

Specimen : Serum , plasma is also acceptable

Reference Value : 0.1 - 1.2mg/dL ,Direct bilirubin 0.1 - 0.4 mg/dL , Indirect ( unconjugated )bilirubin :0.2 - 0.7 mg/dL , Newborn TBil:1 - 12 mg/dL .

Total bilirubin levels in infants should decrease to adult levels by day 10 as the development of hepatic circulatory system matures . Values in breastfed infants may take longer to reach normal adult levels . Values in premature infants may initially be higher than in full term infants and also take longer to decrease to normal levels . 

Method : Spectrophotometry

Technique for measuring serum bilirubin 

( Van den Bergh's test ) 

-  The determination of total bilirubin and conjugated bilirubin in the serum is based on Van Den Bergh's reaction .This reaction is based on the ability of bilirubin to couple with the diazo benzo sulfo chlorid (diazo reagent ) to form a characteristic red violet color . 

- Science unconjugated bilirubin is insoluble in water and the diazo reagents is aqueous solution , the detection of unconjugated bilirubin require the use of substance in which both bilirubin and diazo reagent are mutually soulble as 95% ethyl alcohol . So that this reaction is called indirect reaction and the bilirubin is called indirect bilirubin .

- The addition of alcohol is not required for coupling of the conjugated  bilirubin to the diazo reagent science they are both soluble in an aqueous solution . this reaction is called direct reaction and the bilirubin is called direct reacting bilirubin

The accepted method for the Van Den Bergh's test is to measure the direct bilirubin and the total bilirubin as follow : Mix serum withdiazo reagent and color developed after one minutes is measured as direct bilirubin by using spectrophotometer . Then add 95% ethyl alcohol to the mixture of serum and diazo reagent and the color developed after 30 minutes represent total bilirubin . 

- The measurement is done using spectrophotometer with standard solution for comparing . Indirect bilirubin is obtained by subtracting the direct bilirubin from total bilirubin

(i.e indirect bilirubin = total bilirubin - direct bilirubin ) .  

Description 




- The majority of bilirubin ( 80 % ) is produced from the degradation of hemoglobin from erythrocytes undergoing normal or abnormal destruction . 

- A small percentage (20 % ) is derived from the catabolism of various hepatic hemoproteins ( myoglobin , cytochrome P450 ) as well as from the overproduction of hem  by bone marrow  .

- Hemoglobin released from red cells is phagocytized by mononuclear phagocytes . The iron portion of hem is transferred to transferrin or stored in cells as ferritin . The globin portion is broken down to amino acids , which are then used for protein synthesis .

- The remaining porphyrin ring is transformed to unconjugated bilirubin by hem oxygenase . This enzyme is also located in renal and hepatic parenchyma,enabling this tissues to take up heme and convert it to bilirubin unconjugated or free bilirubin is then transferred to plasma where it binds to albumin . Uptake of unconjugated bilirubin occurs in the liver , and is carrier-mediated . The carrier mediated uptake is shared with unconjugated bile acids and dyes such as BSP .

- Once within the hepatocyte, unconjugated bilirubin is transported with ligand ( or Y protein ) and the majority is conjugated to glucuronic acid  by glucuronyl transferase . The remainder is conjugated to a variety of neutral glycosides ( glucose , xylose ) . Bilirubin must be conjugated, a process that makes it water soluble ,before excretion into bile .

- Excretion into biliary canaliculi is rate-limiting and energy dependent . 

- Within the intestine , bile forms micelles facilitating absorption of fat . Under the action of bacteria in the intestine , bilirubin is converted to urobilinogen .

- urobilinogen is reabsorbed (20% ) or broken down into urobilin and sterocobilin and excrated with stool . Of the reabsorbed urobilinogen , 10% is excreted in the urine and the remaining 90% undergoes enterohepatic circulation . 

- Remember , only conjugated bilirubin  can be found in urine as it is water soluble . The renal epithelium is capable of converting heme to bilirubin , however this only occurs when there is intravascular hemolysis with hemoglobinuria . The renal epithelium absorbs hemoglobin from the urine , converting it to bilirubin . This may be responsible for the bilirubinuria in intravascular hemolysis .

-Circulating bilirubin exists in two main forms as determind by the Van Den Bergh's reaction , which which differentiates bilirubin into conjugated ( direct ) and unconjugated ( indrect )forms .

 

There is a third form of bilirubin called delta bilirubin or ( biliprotein ) which is conjugated bilirubin bound to proteins . Delta bilirubin increases in serum when hepatic excretion of conjugated bilirubin is impaired ( cholestasis ) and the liver retains intact conjugation mechanisms . It has a long half-life and is not excreted in the urine ( as it is protein bound ) . Delta bilirubin may be responsible for a persistent bilirubinemia without bilirubinuria seen in cholestasis . It does react with the diazo dyes , similar to direct or conjugated bilirubin .

Indications

- Assist in the differential diagnosis of obstructive jaundice .

- Assist in the evaluation of liver and biliary disease .
- Monitor the effects of drug reactions on liver function .
- Monitor jaundice in newborn patients

- Monitor the effects of photo therapy on jaundiced newborns .

Interpretation

When bilirubin concentration increases the yellowish pigment deposits in skin and sclera . This increase in yellow pigmentation is termed jaundice or icterus .