Bilirubin is a yellow compound produced during the breakdown of heme in vertebrates, processed by the liver for elimination. It exists in two forms: unconjugated (indirect) bilirubin, which is not water-soluble, and conjugated (direct) bilirubin, which is water-soluble and can be excreted in urine. Testing for bilirubin levels is essential for diagnosing liver health, jaundice, and hemolytic conditions, with the DXC800 system measuring total bilirubin concentration using a diazo method.

1. Jaber Al Ahmed Armed forces Hospital
Medical Laboratory Department.
Biochemistry unit
Bilirubin
Presented by :
Cpl md Shohel Rana

2. What is Bilirubin?
Bilirubin is a Yellow Compound that occurs in the normal catabolic pathway that
breaks down heme in vertebrates. When heme, a part of the haemoglobin, Is
broken down ,Bilirubin is produced .Bilirubin processed by the liver for
elimination from the body. There are a number of medical conditions that can
cause an increased Bilirubin presence in the blood.

3. Function:
 Bilirubin is created by the activity of biliverdin reductase on biliverdin.
Bilirubin, when oxidized, reverts to become biliverdin once again. This cycle
in addition to the demonstration of the potent antioxidant activity of
bilirubin, has led to the hypothesis that bilirubin’s main physiologic role is as
a cellular antioxidant.

4. Source: The majority of bilirubin (80%) is produced from
 a. Degradation of haemoglobin from erythrocytes undergoing normal (removal
of aged of effete cells)
 b. Abnormal destruction (I.E intravascular or extravascular haemolysis) within
mononuclear phagocytes (principally splenic hepatic and bone marrow
macrophages).
Small percentage 20% is derived from
 a. Catabolism of various hepatic haemoproteins(myoglobin cytochrome
P450).
 b. Overproduction of heme from ineffective erythropoiesis in the bone
marrow.

5. Metabolism:
 Within macrophages, a free heme group (iron + porphyrin ring ) is oxidized by microsomal
heme oxygenase into biliverdin and the iron is released (the iron is then stored as ferritin or
released into plasma, where it is bound to the transport protein, trasnsferrin ).
 Biliverdin reductase then reduces the green water soluble biliverdin in to unconjugated
bilirubin.
 Heme oxygenase is also located in renal and hepatic parenchyma, enabling these tissues to
take up heme and convert it to bilirubin.
 Unconjugated or free bilirubin is then released into 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 (Y protein) or
other proteins (e.g. Z protein) and the majority is conjugated to glucuronic acid by
glucuronyl transferase. The remainder is conjugated to a variety of neutral glycosides
(xylose).

6.  Bilirubin must be conjugated before it can excreted
into bile (conjugation makes bilirubin water soluble).
 Bilirubin is secreted into the intestine.
 In the intestine, bacterial degrade it to urobilinogen.
 Urobilinogen is reabsorbed (about 10%) or broken
down (90%) into urobilin and stercobilin (both of which
are excreted in the feces).
 Of the resorbed urobilinogen, most is taken up by the
liver (enterohepatic circulations, i. e. the urobilinogen
is absorbed into the portal vein, taken up by the liver
and re-excreted into bile, whilst the rest bypasses the
liver and is excreted into the urine.
 In all species , bilirubinuria may precede an increase in
serum bilirubin in cholestatic disorders. Remember,
only conjugated bilirubin can be excreted in urine as it
is water soluble. circulating bilirubin exists in two main
forms as determined by the van den Bergh reaction,
which differentiates bilirubin into conjugated (direct)
and unconjugated (indirect) forms.

7. Types of bilirubin: Bilirubin circulates in the bloodstream in two
forms.
 Indirect (or unconjugated) Bilirubin: This form does not dissolved in water indirect bilirubin travels through the bloodstream to the liver,
where it is changes in to a soluble form(Direct or conjugated).
 Increases in unconjugated bilirubin occur with
 Haemolysis
 Liver disease
 Cholestasis
 Direct (or conjugated) Bilirubin: Direct Bilirubin dissolves in water, it’s made by the liver form indirect bilirubin.
 Cause of increase conjugated bilirubin
 Alcohol
 Infectious hepatitis
 Drug reaction
 Autoimmune disorder
 Total bilirubin and direct bilirubin levels are measured directly in the blood.
 Indirect bilirubin levels are derived from the total and direct bilirubin measurements.
 Only conjugated bilirubin is excreated by the kidney. That is why unconjugated bilirubin is not elevated in the bloodstream.

8. Why it is Done:
 Bilirubin testing is usually done as part of a group of tests to check the health of
our liver. bilirubin testing may be done.
 Investigate jaundice a Yellow of the skin and eyes caused by elevated levels of
Bilirubin. A common use of this test is to measure Bilirubin levels in New-borns to
check for infants jaundice
 Determine whether there might be blockage in our bile ducts, In either the liver or
the gallbladder.
 Helps detect liver disease, Particularly hepatitis or monitor its progression.
 Help evaluate anaemia caused by the destruction of red blood cells.
 Help to follow how a treatment is working.
 Help evaluate a suspected drug toxicity.

9. Summary of test principle and clinical relevance:
 The DxC800 uses a timed-endpoint Diazo method(jandrassik-Grof)to measure
the concentration of total bilirubin in serum or plasma.in the reaction
bilirubin reacts with diazo reagent in the presence of caffeine,banzoat and
acetate as accelerators to form azobilirubin. The system monitors the change
in absorbance at 520nm at a fixed time interval. The change in absorbance is
directly proportional to the concentration of total bilirubin in the sample.
 Total bilirubin measurements are used in the diagnosis and treatment of liver
and haematological disorder.

10. Does haemolysis affect bilirubin result:
Measurement of bilirubin in samples is interfered by haemolysis. over a method
depending cut off value of measured haemolysis, bilirubin value is not accepted
and a new sample is required for evaluation although this is not always possible,
especially with new-borns and cachectic oncological patients.

11. Normal values:
 Serum or plasma
Age group
0-1 day-------------2.0-6.0 mg/dl
1-2 day-------------6.0-10.0mg/dl
3-5 day-------------4.0-8.0mg/dl
>5 day--------------0.2-1.3mg/dl

12. Hope you liked the presentation
Thank you