Billirubin estimation

DR. RAJENDER KUMAR
PG 1ST YEAR
BIOCHEMISTRY

INTRODUCTION
 Bilirubin (formerly referred to as haematoidin) is the yellow
breakdown product of normal haeme catabolism.
 Bilirubin is a linear tetrapyrrole M.W. 585 Da.
 Having molecular formula C33H36N4O6.
 Bilirubin is excreted in bile and urine, and elevated levels may
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.

HISTORY
 Discovered by virchow in 1849, in blood extravasates , named
‘hematoidin’
 Term ‘bilirubin’ coined by Stadeler in 1864
 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.

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

STRUCTURE OF HAEME
HAEME
1. Haeme consists of protoporphyrin IX , Containing 4 pyrrole
rings linked togethor by methenyl bridge
2. Iron incorporated is in ferrous form Fe2+
3. Haeme synthesis occurs in all cells

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
 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.

1.1FORMATION
 Bilirubin is formed in the monocytic macrophages of the spleen
and bone marrow and in hepatic Kupffer cells, from haeme 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

 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

STRUCTURE OF BILIRUBIN
Structure of naturally occuring
unconjugated bilirubin IXα 4Z,15Z
Formation of bilirubin.

STRUCTURE OF BILIRUBIN
•Overall structure of bilirubin was
established by Xray
crystallography.
•Bilirubin assumes a ridge tiled
configuration stabilised by six
intramolecular hydrogen bonds.
•Two additional festures have
been noted:- 1. Z-Z (trans)
conformation for double bonds b/t
carbon 4 & 5 and 15&16.
2. An involuted hydrogen bonded structure in which propoinic acid-
carboxylic acid groups are hydrogen bonded to nitrogen atoms of the
pyrrole rings.these bonds stabilizes Z-Z configuration of bilirubin and
prevents its interaction with polar groups. when exposed to light Z-Z
configuration is converted to E-E & other configuration like E-Z, Z-E.

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.

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 part is
bound to the fatty acid-binding protein. As in serum, this
binding keeps the free fraction low. (which is potentially
toxic).

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.

15
Structure of Conjugated Bilirubin

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 protect them
from deconjugation. Normally, 95% of bile salts are re-
absorbed in the terminal ileum.

1. Intestinal bacteria acts on bilirubin diglucuronide leading to:
a) Removal of glucuronides (by b-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

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

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

Bilirubin diglucuronide
Intrahepatic
urobilinogen cycle
15%
Stercobilinogen
Bacterial enzymes
Bilirubin
b-glucuronidases enzyme2 glucuronate
Bacterial enzyme
Urobilinogen
8H
bile
Urobilin (5%)
kidneys urine
Stercobilin faeces
INTESTINE

TYPES OF BILIRUBIN
 The decomposition of hemoglobin in the body results in the
formation of the Z,Z-isomer of bilirubin.
 Phototherapy results in the conversion of the Z,Z-isomer of
bilirubin into the E,E-isomer. Two terminal rings of bilirubin
favour lactam form.
 A cyclic system containing the grouping —CONH— is
called a lactam, and the isomeric form, —COH & bond;NH—, a
lactim. However both the forms are available and are
interchangeable.
 Four bilirubin fractions have been isolated from serum:-
 1. Unconjugated bilirubin (α-bilirubin)
 2. Monoconjugated bilirubin (β-bilirubin)
 3. Diconjugated bilirubin (γ-bilirubin)
 4. A fraction irreversibly bound to protein (δ-bilirubin)

22
Unconjugated Bilirubin and Conjugated
Bilirubin

DELTA-BILIRUBIN (BD)
 Delta-bilirubin arises through a non-enzymatic covalent
coupling reaction between glucuronated bilirubin and albumin
 this fraction is found in patients with hepatic and posthepatic
icterus or with Dubin-Johnson syndrome
 Delta-bilirubin is not excreted by liver or kidneys, but is slowly
metabolized with a half-life of 20 days.
 It can constitute up to 90% of the total bilirubin in the
convalescence phase of cholestatic disorders.
 Free bilirubin (Bf):- Free bilirubin, i.e., unconjugated and not
bound to albumin, represents a significant component of the
neurotoxicity of bilirubin, which is made responsible for the
bilirubin encephalopathy of the neonate (resulting in kernicterus)
 The toxic effect is thought to occur even at a concentration of
0.005 mg/dL .

Bilirubin Fraction in Different Methods
HPLC
PEAK
δ
IRREVERSIBLY
BOUND TO PROTEIN
γ
DICONJUGATED
β
MONOCONJUGATED
VITROS
METHOD
DIAZO
METHOD
α
UNCONJUGATED
TOTAL BILIRUBIN
INDIRECT
BILIRUBIN
T-D
? DIRECT BILIRUBIN
TOTAL BILIRUBIN
UNCONJUGAT
ED BILIRUBIN
Bu
CONJUGATED BILIRUBIN
Bc
DELTA
BILIRUBIN
= T-Bn
NEONAL BILIRUBIN=Bu+Bc
INDRECT DIRECT BILIRUBIN =T-Bu
? In Diazo method reactivity with direct reaction varies

Physiological Role Of Bilirubin In Our Body
 Studies have found higher levels of bilirubin in elderly
individuals are associated with higher functional independence.
 Studies have also revealed that levels of serum bilirubin are
inversely related to risk of certain heart diseases.
 An uncoupler is a compound that impedes ATP generation but
has no effect on electron transport chain.The energy released by
electron transport is mostly dissipated Some portion of it
generates heat.
 Bilirubin levels well above physiological range can act as an
physiological uncoupler and thus helps maintain body
temperature in infants.
 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.

Physiological Role Of Bilirubin In Our Body
 They have also been shown to inhibit the effects of polycyclic
aromatic hydrocarbons, heterocyclic amines, and oxidants—all of
which are mutagens.
 In vitro experiments showed that biliverdin and bilirubin
competitively inhibited HIV-1 proteases at low micromolar
concentrations, reducing viral infectivity.
 In1950s, bilirubin was reported to protect against the oxidation of
lipids such as linoleic acid and vitamin A.
 In the late 1980s, Ames and colleagues demonstrated that the
antioxidant effect of bilirubin exceeds that of vitamin E toward
lipid peroxidation.
 Higher bilirubin levels, 0.9 mg/dL were associated with lowered
risk of myocardial infarction and other cardiovascular disease
events.

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 1 to 2 mg/dl ,it is known
as latent jaundice.
 unconjugated plasma bilirubin that is not bound to albumin can
cross the blood brain barrier.In conditions such as neonatal
jaundice or type-I or type-II Crigler-Najjar syndrome extremely
high concentrations (>20mg/dl) of unconjugated bilirubin can
accumulate, and the resulting diffusion of bilirubin into the
central nervous system can cause encephalopathy and permanent
impairment of nervous function.

Clinico-pathological classes of Jaundice
 1. Pre-hepatic:- Excessive destruction of red blood cells
increases bilirubin formation. However, with normal hepatic
function, the liver is able to remove the excess bilirubin fairly
rapidly so this jaundice is usually mild. As the amounts of bile
pigments entering the gut are greater than normal, the amount
of urobilinogen reabsorbed from the gut is also increased, this
raises it’s levels in the urine. There is often also a small
increase in the serum conjugated bilirubin.
 2. Hepatic:-This type of jaundice results either because of
defective uptake or conjugation.
 In neonate because of immature hepatic enzyme glucuronyl
transferase and lack of intestinal bacteria preventing
conversion of bilirubin into urobilinogen, unconjugated
hyperbilirubinemia results.

 Glucuronyl transferase inhibition by variety of agents
such as chloramphenicol, novobiocin, vitamin K,
breast milk , pregnanediol, free fatty acids,
hypothyroidism.
 Flavaspidic acid, used in the treatment of tape worm
infestation, competes with bilirubin for binding to
ligandin, may cause unconjugated
Hyperbilirubinemia.
 Damaged liver cells are less able to transfer bilirubin
from the blood into the bile.
 Hepatic jaundice may also be caused by conditions
which lead to intrahepatic obstruction.

3.Post hepatic:-
A. Familial defects in hepatic excretory function-
I. Dubin-Johnson syndrome-The serum contains more
diconjugated than monoconjugated bilirubin.
II. Rotor syndrome- Serum conjugated bilirubin has more
monoconjugates than diglucuronide conjugates.
B. Acquired defects of hepatic excretory function-
I. Drug induced and post operative. Obstruction of the bile
ducts after they have left the liver, sometimes referred to as
extrahepatic Cholestatic jaundice.. As there is no bilirubin in
the gut none is reabsorbed into the blood to be excreted by the
kidneys as urobilinogen.This means the urine contains little or
none of this pigment.However as the levels of bile pigment in
the blood continue to rise it is excreted in the urine, once a
renal threshold is reached, causing dark colored urine.
II. Hepatitis and Cirrhosis.

Role of bilirubin in Neonate
 1.Physiologic Jaundice:- Newborns produce bilirubin at a rate of
approximately 6 to 8 mg per kg per day.The average total serum bilirubin
level usually peaks at 5 to 6 mg per dL (86 to 103 μ mol per L) on the third
to fourth day of life and then declines over the first week after birth.
 Bilirubin elevations of up to 12 mg per dL, with less than 2 mg per dL of the
conjugated form, Infants with multiple risk factors may develop an
exaggerated form of physiologic jaundice in which the total serum bilirubin
level may rise as high as 17 mg per dL.
 2.Jaundice and breast feeding:- Total serum bilirubin levels vary from 12
to 20 mg per dL (340 μ mol per L) and are non pathologic. Substances in
maternal milk, such as β-glucuronidases , and nonesteriied fatty acids, may
inhibit normal bilirubin metabolism.
 3.Pathologic Jaundice:- Jaundice is considered pathologic if it presents
within the first 24 hours after birth. a rapidly rising total serum bilirubin
concentration (increase of more than 5 mg per dL per day), and a total serum
bilirubin level higher than 17 mg per dL in a full-term newborn and elevation
of the serum conjugated bilirubin level to greater than 2 mg per dL.

Predominantly Unconjugated Hyperbilirubinemia
 I. Overproduction
 A. Hemolysis (intra and
extravascular)
 B. Ineffective erythropoesis
 II. Decreased hepatic uptake
 III. Decreased bilirubin conjugation
(decreased hepatic glucuronyl
transferase activity)
 A.Hereditary transferase deficiency
 1. Gilbert’s syndrome
 2. Crigler-Najjar type II (moderate
transferase deficiency)
 3. Crigler Najjar type I (absence of
transferase)
 B. Neonatal jaundice ( transient
transferase deficiency)
 C. Acquired transferase deficiency
 1. Drug inhibition (chloramphenicol
pregnanediol)
 2. Breast milk jaundice (transferase
inhibition by pregnanediol and fatty
acids in breast milk)
 3. Hepatocellular disease (hepatitis,
cirrhosis)
 D. sepsis

Predominantly Conjugated Hyperbilirubinemia
 I. Impaired hepatic excretion
 A. Hereditary disorders
 1. Dubin-Johnson syndrome
 2. Rotor syndrome
 3. Recurrent (benign)
intrahepatic cholestasis
 4. Cholestatic jaundice of
pregnancy
 B. Acquired disorders
 1. Hepatocellular disease
(e.g.,viral or drug
 induced hepatitis,cirrhosis)
 2. Drug induced cholestasis
(e.g.,oral
 contraceptives,androgens,chlorp
romazine)
 3. Alcoholic liver disease.
 4. Sepsis
 5. Postoperative state
 6. Parenteral nutrition
 7. Biliary cirrhosis
 II. Extrahepatic Biliary
obstruction
 1. Gallstones
 2. Biliary malformation .
 3. Infection
 4. Malignancy
 5. Hemobilia (trauma,tumor)
 6. Sclerosing cholangitis
 7. Malignancy
 8. Inflammation (pancreatitis)

BILIRUBIN LAB VALUES
Bilirubin form Normal value
Total (elderly, adult, child) 0.2 to .8 mg/dL
(newborn) .8 to 12.0 mg/dL
Critical value (adult) >12 mg/dL
Critical value (newborn) >15 mg/dL
Pre-hepatic,unconjugated, indirect 0.2 to 0.7 mg/dL
Post-hepatic, conjugated, direct 0.1 to 0.4 mg/dL
Fecal urobilinogen 40 to 280 mg/day
Urine 0.0 to 0.02 mg/dL

Specimen Collection and Storage
 Serum or plasma A fasting morning sample collected
 Excessive hemolysis (serum hemoglobin >300mg/ dL) should
be avoided because it may falsely lower the bilirubin value
 Specimens should be protected from direct exposure to either
artificial light or sunlight as soon as they are drawn
 assay should be carried out within 2 hours of sample collection.
If a longer delay is unavoidable, refrigerate the sample
 The sensitivity to light is temperature-dependent; for optimal
stability, storage of specimens in the dark and at low
temperatures is essential.
 point of care direct spectrophotometric measurement of bilirubin
in neonates heel prick samples may be taken directly in
hematocrit capillary tubes.

Methods Of bilirubin estimation
A. Non invasive :- I. Clinical method – using cephalocaudal progression
II. Icterometer –simple colored Perspex which has grades of
yellow color compaired with body skin
III. Transcutaneous billirubinometer :- using the principle of skin
reflectance .
B.Invasive :-I. Capillary billirubin estimation–based on the principle of spectrophotometry.
II. Filter paper with billirubinometer.
III. Laboratory estimation–1. Diazo reaction
2. Bilirubinometer–a. Direct spectrophotometry
b. The Jendrassik-Grof method.
c. The Malloy-Evelyn method
3. Reflectance spectrophotometry
4. high pressure liquid chromatography
5. Peroxidase method method
6. Peroxidase Diazo method
7. Simple colorimetric method
8. New enzymatic assay

CLINICAL METHODS
I.using cephalocaudal progression II. Icterometer
Gossett Icterometer
CRAMMER’S METHOD

Transcutaneous billirubinometer
 These meters work by directing light into the skin and measuring
the intensity of specific wavelength that is returned.
 The meter analyzes the spectrum of optical signal reflected from
subcutaneous tissues.These optical signals are converted to
electrical signal by a photocell. Signals are analyzed by a
microprocessor to generate a serum bilirubin value.
 The major skin components, which impart the spectral reflectance
in neonate, are (i) melanin, (ii) dermal maturity, (iii) hemoglobin,
and (iv) bilirubin.Hyperemia at the test site may affect the results.
 The available meters can be divided into 2 categories:
1.Multi wavelength Spectral Reflectance meters (Bilicheck) TM.
2.Two wavelength (460,540 nm) Spectral Reflectance meters
(Minolta, Bili-test).
Transcutaneous bilirubinometers can serve as a screening tool,
However, this cannot serve as a substitute for TSB estimations.

Transcutaneous billirubinometer
Transcutaneous billirubinometer the
dual optical path system
Multi wavelength Spectral
Reflectance meters

Diazo Reaction
 Ehrlich in 1883 treated bilirubin in urine with diazo reagent and found
that a red –blue coloured pigment was formed.
 Introduction of the diazo reaction for serum bilirubin by van den
Bergh in 1918 led to its widespread adoption for quantitating the
pigment in serum.In an aqueous solution, Ehrlich's diazo reagent reacts
with the direct bilirubin in the serum to form a pink to reddish-purple
colored compound (azobilirubin). read at one minute.
 In a 50% methyl alcohol solution, Ehrlich's diazo reagent reacts with
the total bilirubin in the serum to form a pink to reddish-purple colored
compound. (Read at 30 minutes.)
 Malloy & Evelyn used diazo reagent for the colorimetric estimation of
bilirubin which was read at 540 nm.
 These methods require preliminary step of protein precipitation in
alcoholic solution at about pH 4.This leads to low & poorly
reproducible results because of co precipitation of bilirubin esters
along with protein.

Jendrassik and Grof –doumas et al – Reference Method
In 1938 Jendrassik & Grof modified the diazo method by using
caffeine benzoate as an accelerator.
PRINCIPLE:- Bilirubin reacts with diazotized sulfanilic acid in the
presence of caffeine benzoate-acetate & converted to azopigments
The reaction occurs at the methylene carbon atom between rings B
and C , with the formation of one molecule each of azopigment &
hydroxypyromethenecarbinol.A second molecule of azopigment is
formed from the reaction between this carbinol & diazo reagent.
The tartrate buffer makes the mixture alkaline and converts the red
acid bilirubin to a green coloured compound which shows peak
absorbance at 598 nm. At this wavelength the absorbance due
to haemoglobin or carotene is minimal.
Ascorbic acid is used to stop the coupling reaction, and to eliminate
interference by haemoglobin.

Salient features
 Ease of standardization, high precision, excellent linearity,
minimum interference from haemoglobin, rapid colour
development
 constancy of the molar absorptivity of the azopigment in
various protein matrices, and adaptability to various instruments
for automated chemical analysis.
 Suitable for neonatal sera.
 Measures all 4 bilurubin fractions ( T.bil values with HPLC
corelate well with Jendrasic and Grof method)
 Colour Stability - with BIL in HSA or BSA, the color of the
azopigment is stable for at least 30 min. With biirubin in serum,
there is a small increase in absorbance with time in both tests
and sample blanks, caused by a gelatinous precipitate, slowly
formed in both tests and sample blanks.

For direct bilirubin estimation
Dilution of serum with dilute HCl is done allowing it to stand for 5
minutes before adding the diazo reagent , this keeps the unconjugated
bilirubin from reacting as direct.
Ascorbic acid is added to destroy excess diazo reagent, which would react
with unconjugated bilirubin when alkaline tartrate is added.
The caffeine reagent suppresses the absorptivity of the azopigment at
598nm by about 12%. Omission of the caffeine reagent in this assay
has been responsible for the paradox of sometimes having direct
bilirubin values exceeding those of total bilirubin.
Interference by hemoglobin is far stronger than in the method for total
bilirubin.The negative bias is due to oxidation of conjugated bilirubin
to the diazo negative biliverdin and to the destruction of the
azopigment by H2O2 produced by the conversion of hemoglobin to
methemoglobin at the low pH of the reaction medium; the negative
bias is substantially reduced by addition of potassium iodide to the
serum diluent (HCl).

Other Diazo Reaction
 Powell(1944) added conventional diazo reagent to the plasma
followed by sodium benzoate urea solution. In this method a plasma
blank test is carried out in which HCL replaces diazo reagent. Colour
densities were read in EEL colorimeter at Ilford filter 625.This
method was used for plasma containing bilirubin conc. upto 5mg/dl.
 Method of King & Coxon(1950) modified in a manner as suggested
by Perryman et al(1957)-Ammonium sulphamate(0.15%) was
incorporated in diazo reagent and few crystals of sodium azide were
added after the addition of ethanol (85%v/v).Readings were made at
530nm and 425nm,and the azo pigment extinction was then corrected
for interference by haeme pigments by means of a simple formula.
 Method of Lathe & Ruthven Synthetically prepared taurobilirubin
was used as a model for direct bilirubin. It was found that extinction
and absorption maximum values depend on pH, alcohol & albumin.

Method used in our deptt.
Principle:- Van Den Berg devised colorimetric estimation
Reagents required:
Diazo reagent;10 ml of Diazo A + 0.3 ml of Diazo B
DiazoA;1g sulphanillic acid & 15ml of Conc. HCl in water
Diazo B; 0.5 g of sodium nitrite/100 ml in water. This solution
must be prepared fresh as is unstable.
Diazo blank; 0.1 N HCl
Methanol.
Standard stock: An artificial standard is used i.e. methyl red
290mg/100ml glacial acetic acid that gives an optical density
(0.32) equal to 8 mg/dl of bilirubin. Methyl red is an azo dye
with an absorbance curve very similar to azobilirubin and it can
therefore be used as a standard. Original standard is not used
because it is photosensitive and thus unstable, so cannot be
obtained easily and is costly.

Procedure:-
take 2 test tubes and label them test and control.
TEST CONTROL
Distilled Water 1.8 ml 1.8 ml
Serum 0.2 ml 0.2 ml
Diazo reagent 0.5 ml --------
Diazo blank -------- 0.5 ml
Methanol 2.5 ml 2.5 ml
Keep the test tubes in dark. Readings taken at 1 minute is
direct/conjugated bilirubin, at 30 minutes is total bilirubin.
Unconjugated bilirubin= Total bilirubin – conjugated bilirubin.
Calculations:- S. Bilirubin (mg%)=[ODt – ODc/ODs ]X 8

Enzymatic methods
 Bilirubin oxidase from the fungus species Myrothecium
verrucaria.
 BOX is a 52 kDa enzyme with one copper ion attached and
with a maximum activity at pH 8.0
 Near pH 8 in presence of sodium cholate and sodium
dodecylsulphate , all 4 fractions are oxidized to biliverdin
which is oxidized to purple and finally colorless products
 Decrease in absorbance at 425 / 460 nm is proportional to conc
of total bilirubin.
 Direct bil is measured at pH 3.7 – 4.5 , when enzyme oxidizes
conjugated and delta bil but not unconjugated bil
 At pH 10 enzyme activity is selective for the two glucuronides ,
delta fraction is not oxidized , and about 5% of unconjugated bil
is oxidized.

Peroxidase method-
 The peroxidase method obtains the unbound conjugated bilirubin
(bc ) and unbound unconjugated bilirubin ( bu) using horse
radish peroxidase (HRP)-catalyzes oxidation of bc and bu by a
peroxide (usually hydrogen or ethyl hydrogen peroxide) to
colorless products at sample dilutions of about 1:40.
 Bilirubin bound to albumin (A:bc and A:bu) is protected from
oxidation.
 The unbound bilirubin concentration is calculated by dividing the
reaction velocity (rate of change in bilirubin light absorption at
460 nm) by Kp z [HRP], where Kp (min21) is the first-order rate
constant for the oxidation of bilirubin by peroxide in an albumin-
free solution containing 1 mg/ml HRP.

Peroxidase Diazo method
 Add a small volume of HRP and peroxide to an aliquot of
sample (usually 25 ml) and allowing the reaction to proceed 0
or t min before initiating the diazo test. The sulfanilic acid
denatures the HRP, stopping the bilirubin oxidation reaction.
Since the bilirubin oxidation products are diazo negative , only
nonoxidized bilirubin will form diazo derivatives. The
volumes of HRP and peroxide added determine the sample
dilution at which the unbound bilirubin is measured. For
example, if 25 ml of HRP and 10 ml of peroxide are added to
25 ml of sample, the unbound bilirubin is measured at a
dilution of 1:2.4. The reaction would be stopped at the selected
times with 0.5 ml of sulfanilic acid solution and the diazo test
completed by adding 25 ml of nitrite followed by 0.5 ml of
diluted methanol.

High Pressure Liquid Chromatography
 HPLC methods allow for relatively rapid separation and
quantification of the four bilirubin fractions. The bilirubin
mono and diglucuronide conjugates are converted to mono and
dimethyl esters by treatment with alkaline methanol.
Unconjugated bilirubin is not affected by the reaction and is
extracted into chloroform with the methyl ester derivatives.
The pigments can then be separated and quantified by high
performance liquid chromatography(HPLC) or thin layer
chromatography and detected spectrophotometrically in the
effluent. Use of an internal standard and calibration of the
method with crystalline reference bilirubin and bilirubin
methyl esters permit direct measurement of the individual
pigment fractions in the sample

HPLC
 In earlier methodological studies the mono-
and diglucuronide were first converted into
the stable methyl ester by methanol, and then
extracted together with the Bu with
chloroform.
 The separation then proceeded either by
normal-phase HPLC on a silica gel column
or with reversed-phase HPLC on a C18
column. This procedure also permits
separation of the C8 and C12 isomers of the
monomethyl ester. Since serum proteins are
denatured and completely removed,
measurement of Bd is not possible with this
method

 Lauff et al. developed a HPLC method in which the
serum is pretreated with a saturated sodium sulfate
solution. This method precipitates mainly proteins that
 are larger than albumin, while albumin itself, together
with Bd, remains in solution. The serum is then
transferred to a reversed-phase column, which is
eluted, according to decreasing polarity, with a linear
gradient from a diminishing, acid, aqueous phase
based on phosphoric acid and an increasing
isopropanol-based phase. The Bd is eluted most
rapidly through the column. Then the other bilirubin
fractions follow in the order bilirubin-diglucuronide,
bilirubin-monoglucuronide and Bu .Since the
precipitation with sodium sulfate is not completely
selective, there is a risk that variable proportions of
albumin will also precipitate leading to a loss of Bd.

Direct Spectrophotometery
Direct photometric measurements
are based on direct measurements
of serum at the wavelength of
455nm, which is the absorption
maximum of bilirubin.
oxyhaemoglobin absorbs light at
the wavelength of 455nm.

Simple colorimetric method for the estimation of
plasma biliverdin
 A new colorimetric method for the assay of
biliverdin in biological fluids is described. The
method, based upon the reaction of biliverdin
with barbituric acid, offers improved sensitivity
and selectivity when compared to direct
spectrophotometric measurements. Using this
method biliverdinaemia was observed in two
patients with obstructive jaundice of malignant
origin

New enzymatic assay
 New enzymatic assay- for total (TBil) and direct bilirubin (DBil),
the principle of which involves measuring the decrease in
absorbance at 450 nm produced by bilirubin oxidase from
Myrothecium verrucaria. Since TBil and DBil are oxidized at pH 7.2
and 3.7, respectively, the degree of bilirubin oxidation is measurable
in each case. An analysis of bilirubin by high-performance liquid
chromatography, before and after the enzymatic reaction with
bilirubin oxidase, verified the specificity of the enzyme. The results
obtained using this method varied linearly with TBil and DBil
concentrations up to at least 250 mg/L and 150 mg/L, respectively.
Reducing substances, commonly used anticoagulants and
hemoglobin showed no apparent interference. The degree of day-to-
day precision (CV) for TBil and DBil ranged from 1.2% (206.2
mg/L) to 10.6% (3.5 mg/L) and from 1.8%(84.3 mg/L) to 12.4%
(2.1 mg/L), respectively. Values measured using this new method
correlated well with those obtained by Malloy-Evelyn's method and
the slide method employing the Kodak Ektachem analyzer

CSF BILIRUBIN
 CSF Bilirubin is an important test for excluding
subarachnoid haemorrhage.
 The “Gold Standard” test is scanning
spectrophotometer
 “New” Diazonium ion
 Sample volume 3 uL
 Limit of detection 1.7 umol/L
 Haemolysis – no interference up to 1000 mg/dL
 The bilirubin assay is resistant to haemolysis
improving clinical sensitivity compared to
spectrophotometry.

Linearity, Precision, and Accuracy
 Linearity –the relation is linear and that
there is adherence to Beer’s law up to an
absorbance of at least 1.92 A.
 Precision -The within-run precision,
calculated from duplicate determinations is
excellent (CV 0.1%).
 Accuracy-

Interference is negligible by-
1.oxyhemoglobin(up to 2 gIL), mechanism – oxyhb in acidc
conditions produces acid hematin and H2O2. H2O2
oxidizes bilirubin to biliverdin due to pseudoperoxidase
activity of acid hematin
2.ascorbic acid (up to 20 mg/L), (with ingestion of 1 to 3 g,
ascorbic acid concentration in serum does not exceed 35
mg/L)
3.zinc (at physiological concentrations)
4. L-dopa and a-methyldopa can interfere
5. A propranolol metabolite, a conjugate of 4-
hydroxypropranolol that is normally excreted in the urine,
accumulates in the plasma of undialysed patients with
chronic renal failure and interferes with the widely used
diazo reaction to give falsely raised bilirubin
concentrations, which may cause confusion clinically and
lead to unnecessary investigations. The Bilirubinometer,
available in most neonatal units, provides a simple way of
avoiding this source of error.

WHO – STANDARD OPERATING
PROTOCOL
Principle is same as reference method
Modifications-
1. half reaction volumes are used
2. waveleanght used is 607 nm
3. simplified procedure is used for total and
direct bil estimation for ease in routine use
4. Commercially available bilirubin is used to
make standard by dissolving in pooled non
icteric human sera(tested –ve for HIV) with
the aid of of a small amount
of dimethyl sulphoxide (DMSO) and NaOH.
The exact value of bil in it has to be
determined against a primary standard

Precautions
Storage of standard
Aliquot small volumes of standard into screw-capped
vials and store in the freezer on the same day it is
prepared. Stable for 2 months at -200C. Do not
refreeze leftover standard after use
Hazardous materials
This method uses sulphanilic acid and sodium
hydroxide. Avoid contact with eyes, skin and mucous
membranes
Samples with bilirubin concentrations higher than
20mg/dl should be diluted with an equal volume of
distilled water and the result obtained should be
multiplied by 2

QUALITY CONTROL
 Include one internal QC in every batch of
samples analysed every day irrespective of the
number of samples in a batch.
Since bilirubin is analysed in a single batch in a day
in an intermediate laboratory, it will not be possible
to analyse several QC samples and calculate within-
day precision. However, even if a single QC sample
is analysed in a day, this value can be pooled with
the preceding 10 or 20 values obtained in the
previous days and between-day precision can be
calculated and expressed as %CV. Ensure that this is
well within the acceptable limit, i.e, 10%.
 Once a week it is good to analyse another QC serum
from either a low QC or high QC pool.

Method used in our deptt.
Reagents required:
Diazo reagent: 10 ml of Diazo A + 0.3 ml of Diazo B
Diazo A: 1g sulphanillic acid and 15ml of Conc. HCl/L in water.
Diazo B: 0.5 g of sodium nitrite/100 ml in water. This solution
must be prepared fresh as is unstable
Diazo blank: 0.1 N HCl, Methanol
Standard stock: An artificial standard is used i.e. methyl red
290mg/100ml glacial acetic acid. working standard – 1ml of
stock , 5ml glacial acetic acid,14.4 gm hydrated Na acetate in
1L water gives an optical density (0.32) equal to 8 mg/dl of
bilirubin. Methyl red is an azo dye with an absorbance curve very
similar to azobilirubin and it can therefore be used as a standard.
Original standard is not used because it is photosensitive and
thus unstable, so cannot be obtained easily and is costly.

DPD method
 in an acid medium with 2,5-dichlorophenyl-
diazonium-tetrafluoroborate (DPD), bilirubin forms
an azopigment that can be measured
photometrically at 540 to 560 nm.
 The release of the Bu from the albumin is achieved
with the detergent Triton X which also helps to
avoid protein precipitations
 There is good agreement between the measurement
results of the Jendrassik-Grof principle and the
DPD method however, the latter is much less
laborious

Enzymatic methods
 bilirubin oxidase (BOX, EC 1.3.3.5) from the fungus species
Myrothecium verrucaria MT-1 has permitted the development
of an enzymatic method for measuring total bilirubin.
 BOX is a 52 kDa enzyme with one copper ion attached to every
enzyme molecule, and with a maximum activity at pH 8.0
 Near pH 8 in presence of sodium cholate and sodium
dodecylsulphate , all 4 fractions are oxidized to biliverdin
which is oxidized to purple and finally colorless products
 Decrease in absorbance at 425 / 460 nm is proportional to conc
of total bil
 Results of total bil are slightly lower than diazo methods
 Direct bil is measured at pH 3.7 – 4.5 , when enzyme oxidizes
conjugated and delta bil but not unconjugated bil
 At pH 10 enzyme activity is selective for the two glucuronides ,
delta fraqction is not oxidized , and about 5% of unconjugated
bil is oxidized

 Lauff et al. developed a HPLC method in which
the serum is pretreated with a saturated sodium
sulfate solution. This method precipitates mainly
proteins that
 are larger than albumin, while albumin itself,
together with Bd, remains in solution. The serum
is then transferred to a reversed-phase column,
which is eluted, according to decreasing polarity,
with a linear gradient from a diminishing, acid,
aqueous phase based on phosphoric acid and an
increasing isopropanol-based phase. The Bd is
eluted most rapidly through the column. Then the
other bilirubin fractions follow in the order
bilirubin-diglucuronide, bilirubin-
monoglucuronide and Bu .Since the precipitation
with sodium sulfate is not completely selective,
there is a risk that variable proportions of albumin
will also precipitate leading to a loss of Bd.

 In another method for differentiation of bilirubin
with HPLC, serum diluted with acetic acid is
filtrated through a 0.45 mm filter, transferred to a
polyacrylic-ester column and finally eluted with a
linear pentasulfonic acid– acetonitrile gradient.
 Five bilirubin fractions can be detected in the
eluate; the additional fifth fraction in comparison
to the both methods described above is the (Z,E)
or (E,Z) photoisomer of bilirubin.
 Disadvantage is the high cost of the procedure and
the use of microfilters through which possible
losses of Bd may arise.
 Additionally, the solvent acetonitrile is a
carcinogenic substance

However,the accuracy and precisionof HPLC in the
measurement of serum TBIL are at present
inadequate because-
(a) Calibration is performed with Bu with the
ssumption that the molar absorptivity of the other
three bilirubin species are identical to that of Bu
although in fact they are not known
(b) errors in the measurements of the fractions are
cumulative
(c) Some BIL may be lost during the complex pre
treatment of samples.

DETERMINATION OF BILIRUBIN IN
INFANTS BY DIRECT
SPECTROPHOTOMETRYBilirubin in human serum exhibits an absorption
maximum near 460nm..
Because carotenes and other pigments are virtually
absent in the newborn’s blood, hemoglobin is the
only pigment in serum that interferes with the
bilirubin measurement
This interference is eliminated by analysis of a
two-component system
Turbidity (lipemia) will cause a negative bias if the
secondary wavelength is shorter than the
primary, and a positive bias if it is longer. This is
because light scattering (and hence absorbance)
varies inversely with the wavelength

Spectrophotometric methods following
dilution serum sample is diluted with caffeine benzoate solution
 With the use of a photometer with monochromatic light
(spectral band width of 2 nm) the method does not need
to be calibrated.
 The bilirubin concentration is calculated from the
following formula:
c (micromol/l) =f X 21.26 X(E 465 nm–E 528
nm)
where f is volume of the test/volume of the sample.
 Interferences through oxyhaemaglobin and turbidity are
well corrected
 However, since the method cannot be automated, its use
remains restricted to situations where sample numbers
are low.

Spectrophotometric measurement without
dilution (‘‘bilirubinometer’’)
 Since the molar extinction coefficient of the
haemoglobin is identical at 454 nm and 540 nm, the
bilirubin concentration can be calculated from the
difference
Bilirubin, mg/dL = (A454 - A540) X 1.19 X 51
 To obtain sufficiently accurate absorbance readings
required by this method, a spectrophotometer with a
bandpass of less than 8 nm should be used
 A haematocrit capillary tube serves as cuvette; it is
filled with (capillary) blood and centrifuged in a
special centrifuge.

Direct spectrophotometry in whole blood
 The direct, spectrophotometric measurement of total bilirubin in
whole blood is possible with the kits ABL 730, 735, 830 Flex,
835 Flex and 837 Flex (Radiometer), Rapidlab 1200 (Siemens
Medical Solutions) or cobas b 221 (formerly OMNI S) (Roche
Diagnostics). The measurement principle is identical for all kits
 in the Co-oximetry module, bilirubin is determined in the
haemolysed (ABL, cobas) or not-haemolysed (Rapid lab)
sample together with the haemoglobin fractions by means of a
multi-wavelength measurement (ABL, 128 wavelengths in the
range 478–672 nm; cobas, 512 wavelengths in the range 460–
660 nm, Rapidlab 256 wavelengths in the range 500–680 nm)..
The bilirubin concentration is calculated from the results of the
measurements of absorption with the help of a multi-component
analysis.

Measurements with carrier-bound reagents
(‘‘dry chemistry’’)  Multilayer film technology (Vitros,
Ortho-Clinical Diagnostics)
The required reagents are applied to a
thin film, that together with a
carrier layer forms a ‘‘slide’’ with a
reaction zone of about 1 cm2. A
serum or plasma sample is applied
and low-molecluar components
and water diffuse into the
underlying layer, the latter thereby
dissolving the reagents enclosed in
the slide. After completion of the
indicator reaction, the pigment
formed is measured
reflectometrically through the
transparent carrier layer.

Determination of total bilirubin by the diazo-
procedure
(TBIL slide)
The slide contains as reagents -
a stabilized diazonium salt (4-(N-carboxy-
methylaminosulfonyl)-
benzoldiazoniumhexafluorphosphate) and
diphyllin and Triton X-100 as accelerators.
All bilirubin fractions react quantitatively to a
pigment, which is bound to a mordant and
measured at 540/460 nm.
The range of measurement reaches from 0.1
mg/dL to 27 mg/dL

Measurement of unconjugated and conjugated bilirubin
with the help of direct spectrophotometry
(‘‘BuBc slide’’)
 The reagent-carrying layer of the slide contains caffeine, sodium benzoate
and surfactants, which cause a dissociation of bilirubin and albumin.
 The dissociated, unconjugated bilirubin migrates together with the bilirubin
glucuronides through a barrier layer that retains proteins – and with these
delta-bilirubin too.
 After binding to a mordant the glucuronides show an absorption maximum
at 420 nm, whereas that of unconjugated bilirubin is at 460 nm. At 400 nm
the molar extinction effects are virtually equal. Using a two-wavelength
measurement and an elaborate calculation it is possible to determine the
concentrations of unconjugated and conjugated bilirubin separately.
 Since haemoglobin is held back by the barrier layer, the test is practically
not influenced by haemolysis

Transcutaneous
measurement
 Uses reflectance photometry
 Measurement is required at mulltiple (8)
sites
 ‘Bili check’ provides results within +/- 2 mg
/dl of diazo procedure
 Underestimates bil when conc is greater than
10 mg/dl
 Provides instaneous information and spares
heel sticks
 Aids in predicting those babies who require
folow up by “hour- specific” normogram

Reference Ranges
In adults and infants older than 1 month, the normal
reference interval for total serum bilirubin is
total- 0.2 to 1.2mg/dL (3.4 to 20.5mmol/L)
conjugated bilirubin - 0 to 0.2mg/dL(0 to 3.4mmol/L)

URINE BILIRUBIN
 Presence indicates conjugated hyperbilurubinemia
 Dipstick methods can detect conc as low as 0.5
mg/dl
 Fresh sample is required
 In case of delay store at 2-8 C for max 24 hrs,
protected from light
 Strip is immersed in specimen for 1 sec and read at
60 sec
 Use diazo reaction
 Ictotest tablet is semi quantitative method
 Medications that color urine red or give red color in
acidic media , e.g. phenazopyridine give false +ve
 Large quantities of vit C and nitrite interfere

Urine – Urobilinogen
 Ehrlich’s test
 Principle: Urobilinogen reacts with p-dimethylamino-benzaldehyde
in chloroform to form a pink coloured aldehyde complex.
 Reagents:
- Ehrlich’s reagent
- Saturate sodium acetate
- Chloroform
 Procedure:
 5 ml Urine + 5 ml Ehrlich’s reagent  Mix and allow to stand for
10 min  + 5 ml saturated sodium acetate and mix  + 5 ml
chloroform  shake vigourously and allow layers to separate
 Appearance of pink colour in the chloroform layer indicates
presence of urobilinogen
 Colour is easily detected when viewed from top of the test tube
81

and faeces
 Laboratory determination of fecal or urine urobilinogen is based on Ehrlich’s
reaction, which uses para-dimethylaminobenzaldehyde to form a red color.
 Ascorbic acid may be added to the sample to maintain urobilinogen in its
reduced state.
The Specimen
 The test requires fresh sample; urobilinogen may be oxidized to urobilin on
standing.
Interferences
 Urobilin in the sample is reduced by alkaline ferrous hydroxide to urobilinogen.
Sodium acetate further reduces other chromogens, which may interfere with
Ehrlich’s reagent.
 Bilirubin may interfere with the reaction; significant amounts of bilirubin must
be precipitated with barium chloride and removed by filtration.
Reference Ranges
 Urine 0.5–4.0 Ehrlich units/day
 Feces 75–400 Ehrlich units/day
Increased in prehepatic / hepatic jaundice . Absent in post hepatic jaundice

Free bilirubin (Bf)
 Free bilirubin, i.e., unconjugated and not bound to albumin,
represents a significant component of the neurotoxicity of
bilirubin, which is made responsible for the bilirubin
encephalopathy of the neonate (resulting in kernicterus)
 The toxic effect is thought to occur even at a concentration of
0.005 mg/dL .
 Up to now, there has been no really reliable method for
measuring free bilirubin in plasma .

Mechanisms and Causes Unconjugated hyperbilirubinemia
Mechanism Examples Suggestive Findings
Increased
bilirubin
production
Common: Hemolysis
Less common: Resorption of
large hematomas, ineffective
erythropoiesis
Few or no clinical
manifestations of hepatobiliary
disease; sometimes anemia,
ecchymoses Serum bilirubin
level usually < 3.5 mg/dL (<
59 μmol/L), no bilirubin in
urine, normal aminotransferase
levels.
Decreased
hepatic
bilirubin
uptake
Common: Heart failure
Less common: Drugs, fasting,
portosystemic shunts
Decreased
hepatic
conjugation
Common: Gilbert syndrome
Less common: Ethinyl estradiol ,
Crigler-Najjar syndrome,
hyperthyroidism

Conjugated hyperbilirubinemia
Hepatocellular
dysfunction
Common: Drugs, toxins, viral hepatitis
Less common: Alcoholic liver disease,
hemochromatosis, primary biliary
cirrhosis, primary sclerosing
cholangitis,steatohepatitis, Wilson disease
Aminotransferase levels usually > 500 U/L
Intrahepatic
cholestasis
Common: Alcoholic liver disease, drugs,
toxins, viral hepatitis
Less common: Infiltrative disorders (eg,
amyloidosis, lymphoma, sarcoidosis, TB),
pregnancy, primary biliary cirrhosis,
steatohepatitis
Gradual onset of jaundice, sometimes
pruritus If severe, clay-colored stools,
steatorrhea If long-standing, weight loss
Alkaline phosphatase and GGT usually > 3
times normal Aminotransferase levels <
200 U/L
Extrahepatic
cholestasis
Common: Common bile duct stone,
pancreatic cancer
Less common: Acute cholangitis,
pancreatic pseudocyst, primary sclerosing
cholangitis, common duct strictures caused
by previous surgery, other tumors
manifestations possibly similar to those of
intrahepatic cholestasis or a more acute
disorder (eg,common bile duct stone or
acute pancreatitis) Alkaline phosphatase
and GGT usually > 3 times normal
Aminotransferase levels < 200 U/L
Other, less
common
Hereditary disorders (mainly Dubin-
Johnson syndrome and Rotor syndrome)
Normal liver enzymes

Some Drugs and Toxins That Can Cause Jaundice
Mechanism Drugs or Toxins
Increased bilirubin production Drugs that cause hemolysis (In G6PD
deficiency), such as sulfa drugs and
nitrofurantoin
Decreased hepatic uptake Chloramphenicol , probenecid , rifampin
Decreased conjugation Ethinyl estradiol
Hepatocellular dysfunction Acetaminophen (high dose), amiodarone
, isoniazid , NSAIDs, statins, many others, many
drug combinations. Amanita phalloides
mushrooms, carbon tetrachloride, phosphorus
Intrahepatic cholestasis Amoxicillin/clavulanate, anabolic steroids,
chlorpromazine , pyrrolizidine alkaloids (eg, in
herbal preparations), oral contraceptives,
phenothiazines

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