1. Bilirubin

2. Bilirubin
One of the most important liver functions, and one that is disturbed in a large number of diseases is the excretion of bile
Bile comprises bile acids or salts, bile pigments & others
Bilirubin is the principal pigment in bile
Derived from breakdown of hemoglobin

3. Bilirubin There are two types of bilirubin:
conjugated (direct),
and unconjugated (indirect).
Total bilirubin is composed of the conjugated bilirubin plus the unconjugated bilirubin.
The total bilirubin level increases with any type of jaundice.

5. Conjugated Bilirubin
Normally, direct or conjugated, bilirubin is excreted by the gastrointestinal (GI) tract, with only minimal amounts entering the bloodstream.
Its level rises in the blood when obstructive jaundice (as from gallstones) or hepatic jaundice occurs,
In this case the bilirubin is unable to reach the intestines for excretion and instead, enter the bloodstream for excretion by the kidneys.
Conjugated bilirubin is the only type of bilirubin able to cross the glomerular filter; thus it is the only type of bilirubin that can be found in the urine.

6. Unconjugated Bilirubin
Indirect bilirubin, or unconjugated bilirubin, is normally found in the bloodstream.
Unconjugated bilirubin rises in cases of hemolytic jaundice,
the breakdown of hemoglobin results in a higher than normal level of unconjugated bilirubin being present in the bloodstream.
This is the type of bilirubin elevated in cases of hepatocellular dysfunction, such as hepatitis.
Alcohol must be added for the reaction to occur.

7. Unconjugated Bilirubin
Direct
Indirect
Water soluble
Water insoluble
Found in urine
Not found in urine
Does not need accelerator
Needs accelerator
Total bilirubin = Conjugated + Unconjugated bilirubin
Typically, only the total bilirubin is reported.
If the total bilirubin is abnormal, further testing is done to differentiate the level of direct and indirect bilirubin.
Total bilirubin – Conjugated = Unconjugated bilirubin

8. Clinical Significance
Increased Conjugated Bilirubin
Biliary obstruction
Cancer of the head of the pancreas
Dubin-Johnson syndrome
Increased Unconjugated Bilirubin
Autoimmune hemolysis
Crigler-Najjer syndrome
Gilbert’s syndrome
Hemolytic transfusion reaction
Hepatitis

9. Specimen & Storage
Total bilirubin determinations using a diazo method require either serum or plasma.
Bilirubin is very sensitive to and is destroyed by light and heat; therefore specimens should be protected from ambient light prior to and during analysis.
Concentrations may decrease by 30% to 50% per hour if exposed to direct sunlight.
If separated and stored in the dark, serum or plasma is stable for:
1 day at °C
7 days at °C
3 months at - 20 °C (if frozen immediately)

10. Specimen & Storage
Conjugated bilirubin may be determined in either serum or plasma,
Urine samples can be analyzed by direct diazo methods, since the polar conjugated bilirubin is not protein bound and is filtered at the glomerulus and excreted into urine.

11. Assay Principle
Bilirubin in normal serum reacted with diazo reagent (diazotized sulfanilic acid) only when alcohol was added
The pigment that reacted in the absence of alcohol was termed “direct” (conjugated).
The pigment that required the presence of alcohol was termed the “indirect” (unconjugated) bilirubin fraction.
The unconjugated bilirubin is a nonpolar molecule and is not soluble in water.
Consequently it will react with diazo reagent only in the presence of an agent (historically called an accelerator), such as alcohol, in which both bilirubin and the diazo reagent are soluble.

12. Procedure for total Bilirubin
standard
Sample
Sample blank
Reagent blank -
100 u DW
Standard
1 ml
Reagent (AT)
1ml
Working reagent
2. Mix thoroughly and let stand the tubes for 2 min at room temperature.
3. Read the absorbance of the sample blanks at 540 nm against DW
4. Read the absorbance of the sample and standard at 540 nm reagent blank.

13. Procedure for Direct Bilirubin
Sample
Sample blank
Reagent blank -
100 u DW
1 ml
Reagent (AD)
Working Reagent
2. Mix thoroughly and let stand the tubes for 5 min at water bath.
3. Read the absorbance of the sample blanks at 540 nm against DW
4. Read the absorbance of the sample at 540 nm reagent blank.

14. Calculation:
The bilirubin concentration in the sample is calculated using the following formula:
C sample =
(Abs sample – Abs sample blank) * C standard / Abs standard
Note:
In calculation of direct bilirubin, use the absorbance value obtained for the standard in the total bilirubin procedure

15. Serum bilirubin levels
Normal
0.2 – 0.8 mg/dL
Unconjugated (indirect):
0.2 – 0.7 mg/dL
Conjugated (direct):
0.1 – 0.4 mg/dL
Latent jaundice:
Above 1 mg/dL
Jaundice:
Above 2 mg/dL

16. Bilirubin levels and jaundice
Class of Jaundice
Causes
Pre-hepatic or hemolytic
Abnormal red cells; antibodies; drugs and toxins; thalessemia
Hemoglobinopathies, Gilbert’s, Crigler-Najjar syndrome
Hepatic or Hepatocellular
Viral hepatitis, toxic hepatitis, intrahepatic cholestasis
Post-hepatic
Extrahepatic cholestasis; gallstones; tumors of the bile duct, carcinoma of pancreas