The document discusses bilirubin, including its formation from the breakdown of heme, transport via albumin in plasma, uptake and conjugation in the liver, excretion in bile, and role as a diagnostic marker for liver and blood disorders. Methods for measuring total and direct bilirubin are also presented, based on the reaction of bilirubin with diazotized sulphanilic acid to form colored azobilirubin derivatives.

1. DR. GURJINDER SINGH
BIOCHEMISTRY

2. INTRODUCTION
Bilirubin (formerly referred to as haematoidin) is the yellow breakdown product
of normal haem catabolism.
Bilirubin is a linear tetrapyrrole M.W. 585 Da.
Having molecular formula C33H36N4O6.
Bilirubin is excreted in bile and urine, and elevated levelsmay indicate certain
diseases.
It is responsible for the yellow color of bruises, the background straw-yellow color
of urine.
the brown color of faeces (via its conversion to stercobilin), and the yellow
discoloration in jaundice.
Bilirubin is the end product of haemoglobin and serve as a
diagnostic marker of liver and blood disorders.

3. HISTORY
Discovered by virchow in 1849, in blood extravasates , named ‘hematoidin’
Term ‘bilirubin’ coined by Stadeler in1864
In 1874 Tarachanoff demonstrated direct assoc to Hb
In 1942 Fisher and Plieninger synthesized bilirubin IX alpha
and proposed its structure
Bilirubin is nonpolar, and is insoluble in plasma. Therefore it binds by noncovalent
bonds to plasma albumin. This form is called: unconjugated or indirect bilirubin.

4. HAEM SYNTHESIS
CATALYSED BY
δ- AMINOLAVULINIC ACID
LIVER / BONE MARROW
GLYCINE + SUCCINYL CO A
δ- AMINOLAVULINATE
SYNTHASE
- LEAD
PORPHOBILINOGEN
HYDROXYMETHYLBILANE
UROPORPHYRINOGEN III
COPROPORPHYRINOGEN III
PROTOPORPHYRINOGEN IX
PROTOPORPHYRIN IX
- LEAD
INHERITED
ENZYME
DEFICIENCYIN
LIVER
LEADS TO
PORPHYRIA
Haem
RATE
LIMITING
STEP
HAEM DEGRADATION
LIVER / SPLEEN
ERYTHROCYTES
ERYTHROID CELLS
HAEMOGLOBIN,CYTOCHROME
Haem PROTEINS,
MYOGLOBIN
AMINO
ACIDS
Haem
CO, FE3+
Haem OXIGENASE
BILIVERDIN
NADPH + H+
BILIVERDIN REDUCTASE
CONJUGATED BILIRUBIN
UDP GLUCURONYL
TRNSFERASE
3 O
BILIRUBIN
ALBUMIN
NADP+
UNCONJUGATED BILIRUBIN
2 UDP GLUCURONATE
2 UDP

5. FORMATION OF BILIRUBIN
The breakdown of Haem produces bilirubin (an insoluble waste product) and other
bile pigments. Bilirubin must be made water soluble to be excreted. This
transformation occurs in 5 steps;-
1. Formation
2. Plasma transport
3. Liver uptake
4. Conjugation
5. Biliary excretion
One gram of hemoglobin yields 35 mg of bilirubin. The daily bilirubin formation in
adult human is about 250 mg.

6. 1.1FORMATION
Bilirubin is formed in the monocytic macrophages of the spleen and bone marrow and
in hepatic Kupffer cells, from Haem by opening of the ring at the α carbon bridge. This
cleavage is catalyzed by the microsomal enzyme haem-oxygenase of RE cells forming
biliverdin IXα.
NADPH is used as the reducing agent, molecular oxygen enters the reaction, carbon
monoxide(CO) is produced and the iron is released from the molecule as the ferric
ion (Fe3+). CO acts as a cellular messenger and functions in vasodilatation.
Biliverdin IXα is then reduced to bilirubin IXα by cytosolic enzyme biliverdin
reductase.
This is unconjugated bilirubin, which is not soluble in water, due to intramolecular
hydrogen bonding and they also shield the central –CH 2 –, which thus becomes
inaccessible for the diazo- reagent.
7

7. Cleavage at non-α sites is possible; it is probably non-enzymic and occurs only to a
minor extent. This results in the formation of other isomers.
The IXβ isomer is present in neonatal urine and in meconium.
Because intramolecular hydrogen bonds cannot be formed in these isomers, they are
more hydrophilic, and appear in urine or bile as an unconjugated pigment.
Phototherapy, leads to the formation of another group of more hydrophilic
derivatives of the natural UCB IXα, such as the 4E,15Z ; 4Z,15E and 4E,15E
photoisomers, which can be excreted in bile without conjugation.
1.2 FORMATION

8. 2. PLASMA TRANSPORT
From R.E. cells bilirubin enters the circulation binds to albumin.It cannot pass
through the glomerular membrane into the urine. Albumin binding weakens under
certain conditions (eg, acidosis), and some substances (eg, salicylates, certain
antibiotics) compete for the binding sites.
Depending on the pH of the plasma, bile or urine UCB can be present as uncharged
diacid, as a monoanion or as a dianion.
The uncharged diacid is by far the dominant species at low and
physiological pH (>80%).
The ionized fractions become more important in an alkaline pH, because the pK’a
values have been determined to be 8.12 and 8.44, respectively, for the first and for
the second anion.

9. 3. LIVER UPTAKE
Entry into the hepatocytes appears to be partly passive and partly mediated by
organic anion transporter proteins (OATP 1B1 has the highest binding affinity).
In the hepatocytic cytosol, UCB is mostly bound to glutathione-S-transferase
A (ligandin), and a small partis bound to the fatty acid-binding protein. As in
serum, this binding keeps the free fraction low. (which is potentially toxic).

10. 4. CONJUGATION
Bilirubin is conjugated in hepatocytic microsomes in an ester linkage with sugar
moieties donated by uridine diphosphate
The conjugation is catalyzed by UDP- glucuronyltransferase.
Sugar moieties are coupled to the –COOH of the propionic acid
side chain of UCB, resulting in mono or diconjugated bilirubin.
The esterification disrupts the intramolecular hydrogen bonds, thereby opening the
molecule and rendering the (CB) more
water-soluble. Conjugation also decreases the binding to albumin
or to intracellular proteins, and prevents intestinal re-absorption.
The bilirubin conjugates formed in the hepatocytes are excreted in bile against a
concentration gradient and mediated by the canalicular membrane transporter
multidrug resistance-related protein 2 (MRP2) also termed ABC-C2, belonging to the
adenosine triphosphate (ATP)-binding cassette family.

11. 5.1 BILIARY EXCRETION
The conjugates are incorporated into mixed micelles (with bile acids, phospholipids
and cholesterol) and pass with the bile into the intestine, where reductive
breakdown into urobilinogen occurs by intestinal or bacterial enzymes.
A minor part undergoes deconjugation mainly the intestinal and bacterial enzyme
β- Glucuronidase, forming UCB and reabsorbed through enterohepatic
circulation.
In neonates, the bacterial flora is not yet developed, deconjugation will thus
prevail and this adds to the enhanced serum bilirubin levels observed in neonatal
jaundice.
Bile salts incorporate bilirubins in micelles and protectthem from deconjugation.
Normally, 95% of bile salts are re- absorbed in the terminal ileum.

12. 1. Intestinal bacteria acts on bilirubin diglucuronide leading to:
a) Removal of glucuronides (by -glucuronidases enzymes).
b) Reduction of bilirubin to colorless compounds called:
Urobilinogens
2. A small fraction of urobilinogens are reabsorbed from intestine to the liver again and
re-excreted in the bile (95%), forming the enterohepatic urobilinogens cycle.
some part of urobilinogen is excreted in urine (5%)in the form of urobilin (is
responsible for the yellow colour of urine)which is the product of oxidation of
urobilinogen.
3. The remainder travels down the digestive tract and is
converted to stercobilinogen. This is oxidized to stercobilin. which is excreted
and is responsible for the color of faeces
17
5.2 BILIARY EXCRETION

13. BILIRUBIN PRODUCTION
Heme
Heme oxygenase
Hemoglobin
(80 to 85%) Erythroid cells
Heme proteins myoglobin,
cytochromes (15 to 20%)
Biliverdin
NADPH + H+
NADP+ apoferritin
ferritin
Biliverdin reductase
indirect
unconjugated
pre-hepatic
Bilirubin albumin
Globin
Amino acid
H2O
met
CO
NADPH + H+
NADP+
fp
O2
fp
Fe3+

14. BILIRUBIN PROCESSING
Bilirubin-albumin
ligandin hepatocyte
UDP-GLUCURONYL
TRANSFERASE
ligandin-Bilirubin
bile (gall bladder) Intestine
albumin
2 Udp-glucuronate
2 UDP
Bilirubin diglucuronide

15. Bilirubin diglucuronide
Stercobilinogen
Bacterial enzymes
2 glucuronate -glucuronidases enzyme
Bilirubin
Bacterial enzyme
Urobilinogen
8H
bile
Intrahepatic urobilinogen
cycle 15%
Urobilin (5%)
kidneys urine
Stercobilin faeces
INTESTINE

16. Background
Bilirubin (formerly referred to as haematoidin) is the yellow breakdown product of
normal haeme catabolism.
Having molecular formula C33H36N4O6.
Bilirubin is excreted in bile and urine, and elevated levels may
indicate certain diseases.
Itis responsible for the yellow color of bruises, the background
straw-yellow color of urine.
the brown color of faeces (viaits conversion to stercobilin), and
the yellow discoloration in jaundice.
Bilirubin is the end product of hemoglobin and serve as a
diagnostic marker of liver and blood disorders.

17. Formation of Bilirubin
The breakdown of haeme produces bilirubin (an insoluble waste product)
and other bile pigments. Bilirubin must be made water soluble to be
excreted. This transformation occurs in 5 steps;-
1. Formation… Macrophages converts heme to Biliverdin
2. Plasma transport… via albumin and
3. Liver uptake… bilirubin is taken by hepatocytes
4. Conjugation… to glucoronic acid via UDP-glucoranyle transferase
5. Biliary excretion… in bile intestine stercobilin/urobilin formed and
excreted
One gram of hemoglobin yields 35 mg of bilirubin. The dailybilirubin
formation in adult human is about 250mg.

18. Difference Between Conjugated and
Unconjugated Bilirubin
Conjugated Bilirubin
Present Normally in Bile
Conjugated to glucoronic
acid
Can be filtered by kidney
Cannot cross BBB
Unconjugated Bilirubin
Present Normally in Plasma
Conjugated to Albumin
Cannot be filtered by kidney
Can cross BBB

19. Functions of Bilirubin
Levels of serum bilirubin are inversely related to risk of certain heart
diseases
Acts as uncoupler in Neonates and thus maintain body heat
Bile pigments such as Biliverdin naturally possess significant anti-
mutagenic and antioxidant properties
Biliverdin and bilirubin have been shown to be potent scavengers of
peroxyl radicals
inhibit the effects of polycyclic aromatic hydrocarbons, heterocyclic
amines, and oxidants—all of which are mutagens.

20. Diagnostic Importance of Bilirubin
Clinically hyperbilirubinemia appears as jaundice or icterus
Jaundice can usually be detected when the serum bilirubin level exceeds 2.0 to
2.5 mg/dl
When the level of bilirubin is between 1to 2 mg/dl ,it is known as latent jaundice
Inneonates, unconjugated bilirubin can cross BBB and thus lead to accumulation
in Brain of neonates. This interfere with nervous system development and
permanent Nervous impairment.

21. Normal Range
Bilirubin Form Normal Value
Total (elderly/Adult) 0.2 — 1mg/dl
Newborn 0.8 — 12mg/dl
Critical Value(adult) >12mg/dl
Critical Value (Newborn) >15mg/dl
Fecal urobilinogen 40 – 280 mg/dl
Urine 0.0 – 0.02 mg/dl

22. Clinical Correlates

23. Hypo Bilirubinemia
No clinical significance of low bilirubin level.
Some researches suggest that low bilirubin may contribute to Cerebrovascular or
cardiovascular events.

24. Hyper Bilirubinemia
Two Types
Unconjugated Hyperbilirubinemia
Conjugated Hyperbilirubinemia

25. Unconjugated Hyperbilirubinemia
Overproduction
Hemolysis (intra and
extravascular)
Decreased hepatic uptake
Decreased bilirubin conjugation
(Transferase deficiency)
Gilbert’s Syndrome
Crigler-Najjar Syndrome
Neonatal Jaundice
Acquired Transferase Deficiency
Drug inhibition (chloramphenicol
and pregnanediol)
Breast milk jaundice (T
ransferase
inhibition by pregnanediol and
fatty acids in breast milk)
Hepatocellular disease (hepatitis,
cirrhosis)
Sepsis

26. Conjugated Hyper Bilirubinemia
Impaired hepatic excretion
Dubin-Johnson syndrome
Recurrent (benign) intrahepatic
cholestasis
Cholestatic jaundice of pregnancy
Acquired disorders
Hepatocellular disease (e.g. Viral
or drug induced hepatitis,
cirrhosis)
Drug induced cholestasis
(e.g. Oral contraceptives,
androgens, chlorpromazine)
Alcoholic liver disease.
Sepsis
Biliary cirrhosis
Extra-hepatic Biliary
obstruction
Gallstones
Biliary malformation .
Infection
Malignancy
Hemophilia (trauma, tumor)
Sclerosing cholangitis
Malignancy
Inflammation (pancreatitis)

28. Requirements
Working Reagent
R1 Sulphanilic Acid + HCl
R2 Sodium Nitrite
R3 Caffeine + Sodium Benzoate
R4 Tartarate + Sodium Hydroxide
Reaction/ Method is called “Jandrassik-groff Method”

29. Principle
Total bilirubin concentration is determined in presence of caffeine by the
reaction with diazotized sulphanilic acid to produce colored diazo dye.
Intensity of the color of the dye, measured at 540-550 nm, is proportionate
to the concentration of the totalbilirubin.
Direct Bilirubin is determined in absence of caffeine by direct reaction with
diazotized sulphanilic acid to form Red Colored azobilirubin.
Intensity of the color of the dye, measured at 546 nm, is proportionate to
the concentration of the direct bilirubin.
Sulphanilic Acid + NaNO2------------ Diazotized sulphanilicacid
Bilirubin + Diazotized Sulphanilic Acid---------- Azobilirubin

30. Procedure for Total Bilirubin
Take Two test tubes marked as Sample Blank(B) and Unknown(U)
Add R1reagent about 200 µl to both test tubes.
Add R2reagent about one drop to the Test tube marked asunknown
Add R3reagent about 1.0 ml to both TestTubes
Now add Serum about 200 µl to both testtubes.
Mix and incubate for 10 minutes at 20—25 degree Celsius
Now add R41.0 ml to both Testtubes
Mix and incubate for 5 minutes at 20—25 degree Celsius
Check color intensity at 578 nm
Calculation: Unknown Intensity x 10.8

31. Procedure for Direct Bilirubin
Take Two test tubes marked as Sample Blank(B) and Unknown(U)
Add R1reagent about 200 µl to both test tubes.
Add R2reagent about one drop to the Test tube marked asunknown
Add Normal Saline about 2.0 ml to both TestTubes
Now add Serum about 200 µl to both testtubes.
Mix and incubate for 10 minutes at 20—25 degree Celsius
Check color intensity at 546 nm
Calculations: Unknown intensity x 14.4

32. METHOD IN OUR DEPARTMENT
Diazo method End-Point analysis
•Modified method of pearlman & lee in which surfactant is used as solublizer
•Bilirubin glucuronate reacts directly with sulphodiazodium salt and forms colored derivative azobilirubin.
•Thje color intensity of formed azobilirubin measured at 540-550 nm
•It is proportional to direct bilirubin concentration in sample.
Reagent composition
Reagent 1: bilirubin total reagent
Surfactant 1.0%
HCL 100mmol/L
Sulphanilic Acid 5mMol/L
Reagent 2: Sodium nitrite reagent
Sodium nitrite 144mmol/L

33. Reagent Preparation
TEST Vol. of working
regent
ADD
REAGENT 1 (R1)
Reagent 2)
Total Bilirubin 1ml 1ml 0.02ml
Assay Procedure
Pipette into tube
Tubes marked
Blank test
Working reagent 500ul 500ul
Distilled water 25ul -
Test - 25ul
Mix well incubate for 5 mins at 37 C
Read absorbance at 546 nm
Calculation
T. Bilirubin=ABS.X TestX Factor

34. Precautions
The only acceptable anticoagulants are heparin and oxalate.
Fresh Serum to be obtained for Experiment.
Ideally, after Centrifugation, serum should be immediately separated from blood
cells.
Do not try to ingest or inhale the reagent Solution. In case ofcontact, wash
thoroughly and seek medical help.

35. Limitations
Analytical Range of this reaction:
Total Bilirubin: 0.1 – 30 mg/dl
Direct Bilirubin: 0.1 – 10 mg/dl
Avoid hemolysis, asit interferes with reaction
Lipemic specimen may interfere with the reaction
Drugs, theophylline and propranolol, may cause artificially lower level of
bilirubin in sample taken.

36. Resources Used:
Resources used:
Textbook of Medical Biochemistry by M.N.CHATTERJEA
Textbook of biochemistry for medical students by Sreekumari and DM Vasudevan
Wikipedia and Internet