Chapter 2
Liver Function tests
By Zerihun Ataro (HU, Assistant Professor)
Objectives
Upon completion of this chapter the student will be able to:
• Describe the anatomy and physiological role of the liver.
• Discuss the metabolic processes and organs involved in the
formation & excretion of forms of bilirubin
• Explain the clinical significance of bilirubin
• Describe the types of jaundice
• Describe methods of analysis of serum bilirubin, sources of errors
and interpretation of bilirubin results
• Describe the Evelyn-Malloy, Jendrassik-Grof, Walters-Gerarde and
the Icterus Index methods for measuring bilirubin in serum
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Outline
• Anatomy of the liver
• Physiological role of the liver
• Tests for liver function
• Bilirubin
– Formation & excretion of bilirubin
– Clinical significance of bilirubin
– Determination of serum Bilirubin (Direct & total)
– Interpretation of bilirubin results
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Gross anatomy of Liver
• The largest organ and weigh 1.2-1.5 kg
• Has abundant blood supply (receives about 15 ml/min) from:
– Hepatic artery: provide oxygen enriched blood
– Portal vein: about 70% of blood supply
• Provide nutrient enriched blood from GIT
• Significance of portal flow
– All nutrients(except fats) pass through liver first before
entering general circulation
– Enables the liver to carry out many metabolic
function(metabolic factory)
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Microscopic anatomy
Lobule
• Forms the structural and functional unit of the liver.
• Composed of cords of hepatocytes radiating from central vein.
• Its boundary is formed by portal tract containing a branch of
hepatic artery, portal vein and bile duct
• Contains Sinusoids:
• vascular space between the cords of liver cells.
• Lined by endothelial cells and kupffer’s cells(phagocytic
macrophages which ingests bacteria or other foreign material
from blood that flow in sinusoids)
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Physiologic role of liver
• Metabolic function
– Carbohydrate metabolism and Lipid metabolism
• Excretory function
– Liver secrete bile into small intestine
– bile contains bilirubin esters, bile acid conjugates, cholesterol,
phospholipid, organic anions, electrolytes, soluble derivatives of drugs
and steroid hormones, most of them are waste products
– Serve as excretory mechanism for certain pigments and waste products
• Synthesis of all plasma proteins(except gamma-globulin) and
coagulation factors
• Storage Function: for glycogen, and vitamin A,D and B12, Iron
• Function in blood coagulation
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Physiologic role of liver cont’d
• Protective function: from foreign or dangerous materials by:
– Phagocytic action: lining the sinusoids there are kupffer
cells(phagocytic cells) which act as scavengers and remove
foreign materials from blood.
– Detoxification: by converting toxic subs into less toxic form
or by converting insoluble compounds into water soluble
form(excreted by kidney)
• Mechanism:
– Reaction with glucuronic acid: eg bilirubin, drugs,
steroid hormones
– Esterification, acetylation, methylation, oxidation or
reduction. Eg: NH3(toxic) into urea by hepatocytes
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Disorder of liver
• Jaundice/icterus
– The yellowish dicoloration of the skin and sclera resulting
from hyperbilirubinemia
• Cirrhosis
– The irreversible scaring process by which normal liver
architecture is transformed into abnormal nodular
architecture
• Tumors/hepatocellular carcinoma
– Most cases can be related to previous infection with a
hepatitis virus
– The liver is frequently involved secondarily by tumors
arising in other organ
• Drug and alcohol related disorder 9
Lab Tests for liver function
• Tests of hepatic excretory function
– By serum conc of endogenously produced compounds: bilirubin, bile acids
– By rate of clearance of administered exogenous compounds:
bromsulfophthalein(BSP), Hippuric acid test
• Tests measuring hepatic synthetic ability
– Not very sensitive to minimal liver damage
– However, used to quantitate severity of hepatic dysfunction
– The most commonly used plasma proteins to assess liver disease: albumin,
immunoglobulins, clotting factors
• Enzymes tests in liver disease
– Used to assess the extent of liver damage and differentiate
hepatocellular(functional) from obstructive disease
– Include: AST, ALT, ALP, GGT, LDH
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Bilirubin
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Bilirubin
• Used to assess hepatic excretory function
• The principal orange-yellow pigment in the bile
• Source:
– 85% from the breakdown of hemoglobin released when
aged RBC phagocytized by the reticuloendothelial system
– 15% from the destruction of other heme-containing
proteins [myoglobin, cytochromes, and catalase] and from
the catabolism of heme released from red cell precursors
destroyed in the bone marrow
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13
Metabolism of bilirubin
• Formation of bilirubin from Hgb and other source
– Hgb destroyed→ Globin and Heme (Fe and Porphyrin)
• Porphyrine-further break down as waste product and excreted
– After Fe and globine is removed→biliverdin(green)
– In RES, biliverdin is reduced to bilirubin(lipid soluble)
• Bilirubin leaves RE cell and bound to albumin and transported to
liver as bilirubin-albumin complex
• In liver, bilirubin separated from albumin and taken-up by
hepatocytes
– Conjugation of bilirubin in ER of liver cells by uridyldiphosphate
glucuronyl transferase(UDPG-T)
– Secretion of conjugated bilirubin into bile canaliculi→bile duct→ intestine
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Metabolism of bilirubin cont’d
• In small intestine
– Then bacteria convert it to three colorless compounds
collectively known as urobilinogen (sterco-, meso-, and
urobilinogen).
• 20% of the urobilinogen reabsorbed and enter
enterohepatic circulation
– Majority of them taken by liver cells (for re-excretion
into the bile)
– Small portion(2-5%) that escape excretion by
hepatocyte, reach peripheral circulation(excreted by
kidney in urine)
• About 80% of urobilinogen (not absorbed in the colon) is
oxidized spontaneously to urobilin (orange
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brown)→excreted in stool.
From circulation & RES – Globin to protein
hemolysis of senescent reserves &
RBCs releases Hgb Iron to Iron stores
Blood
Unconjugated Bilirubin + Albumin Kidney
2%-5% reabsorbed
•Albumin removed in hepatic sinusoids
Liver urobilinogen re-enters
•Unconj. Bili conjugated to glucoronic acid circulation and
by hepatocytes → Conjugated Bilirubin excreted in urine.
(soluble) enters biliary tree
Portal Vein
Intestine
Bilirubin+ bacterial flora + alk. pH 15% reabsorbed
→ Urobilinogen
Urobilin (85%
in feces)
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Bilirubin cont’d
• 200-300mg bilirubin is produced daily in health adults
– Must be removed by normal functioning liver, which
requires the bilirubin be in conjugated form
– Excretion
• >99% bilirubin: in feces(in the form of urobilin)
• <1% bilirubin: in urine(in the form of urobilinogen)
• Low concentration of bilirubin(0.2-1 mg/dl) found in serum
– The majority is unconjugated form
– Small portion(0-0.2mg/dl): conjugated form
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Types of bilirubin
• Unconjugated bilirubin (indirect bilirubin):
– nonpolar and water-insoluble substance that is found in
plasma bound to albumin.
– only react with the diazotized sulfanilic acid (diazo reagent) in
the presence of an accelerator (solubilizer).
• Conjugated bilirubin (direct bilirubin):
– polar and water-soluble compound that is found in plasma in the free
state (not bound to any protein
– reacts in the absence of an accelerator
• Delta Bilirubin: Conjugated bilirubin covalently bound to albumin,
usually a very small fraction of total
• Total Bilirubin: made up of three fractions: conjugated, unconjugated, and
delta bilirubin.
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Disorder of bilirubin metabolism
Jaundice
• Yellowish pigmentation of skins and sclera due to accumulation
of bilirubin or retention of bilirubin in general circulation
• Manifested when serum bilirubin >2 mg/dl(though the upper
limit is 1 mg/dl)
• The serum appears darker in color→icteric
• Cause:
– Increased bilirubin load to liver cell
– Disturbance in uptake and transport within the liver
– Defect in conjugation or excretion
– Obstruction of large bile duct before bilirubin reach small
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intestine
Jaundice
• The yellow staining of
connective tissue from excess
bilirubin.
• Prominently - sclera of the
eyes and skin.
• Icterus describes the dark
yellow-brown color of serum
with increased bilirubin.
• Normal serum
• Icteric serum
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Jaundice classification
• Prehepatic jaundice
• Hepatic jaundice
• Post hepatic jaundice
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Pre-Hepatic Jaundice(hemolytic jaundice)
• Due to over production of bilirubin
• Caused by hemolysis of RBC in hemolytic anemia, fatal
erythroblastosis, hemolytic disease of the new born.
• Increased destruction of RBC→↑ load of Bilirubin-albimin
complex to liver
• Characterized by unconjugated hyperbilirubinemia
• Laboratory Results:
– ↑ serum bilirubin (mostly unconjugated), ↑ urine urobilinogen, ↓ Hgb ↓
Hct MCV ↓ MCHC
– Bilirubin not detected in urine
– Urobilinogen is greatlly increased in urine and faeces(urobilin)
• Bilirubin rarely exceeds 5 mg/dl
• In HDN unconjugated bilirubin>20 mg/dl 22
Hepatic Jaundice
• Associated with liver disease (it can’t metabolize bilirubin
correctly)
• The defect associated with liver disease include:
– Conjugation failure: absence or deficiency of UDPG-T,
characterized by inconjugated hyperbilirubinemia
– Preconjugation transport failure: defect in hepatocellular
uptake of bilirubin, ↑unconjugated bilirubin,
↓urobilinogen
– Post conjugation transport failure: defect in hepatocellular
excretion, ↑conjugated bilirubin, slight ↓urobilinogen
– Diffuse hepatocellular damage or necrosis: by viral
hepatitis, toxic drugs, cirrhosis
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Hepatic Jaundice cont’d
• Intrahepatic obstruction(cholestasis)
– Obstruction of bile flow due to
• Mechanical obstruction to the flow of bile (stones in
bile duct, gall bladder, tumors around common bile
duct)
• Hepatocellular inflammation/damage as in viral
hepatitis, drug toxicity
– Characterized by elevation of serum conjugated
bilirubin and bilirubin in urine
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Post-Hepatic Jaundice
• Result from impaired excretion of bilirubin
• Caused by mechanical obstruction of the flow of bile into
intestine
– Gallstone, tumor in or near to the bile ducts impede bile flow
• During complete obstruction
– No bilirubin glucuronides reach the intestine→no urobilinogen
is produced
– Urobilin is lacking, faeces appear light brown to chalky white,
no urobilinogen in urine, but urine contain bilirubin
• In sever cases
– Conjugated bilirubin: 12 mg/dl
– Total bilirubin: 18 mg/dl
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Bilirubin determination
Specimens for Bilirubin
• Non-hemolyzed serum or heparinized plasma
• Fresh urine
• Protect from light (e.g., wrap collection tube in aluminum
foil)
– Direct sunlight and artificial light cause 50% decrease in
bilirubin/hour
• Stability: for a week if refrigerated in the dark; for 30 months
if frozen.
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Assay methods
• Spectral method: direct measurement of its natural color
– The icterus index test
– Direct spectrophotometry
– Direct-reading bilirubinometry
• Chemical methods: based on diazo colorimetric reaction
– Malloy-evelyn method
– Jendrassic-Groff method
• Other: Paper and thin layer chromatography, high
performance liquid chromatograph, capillary electrophoresis
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Icterus Index Test
• Measures the degree of icterus in plasma or serum and
correlates with a rough estimation for bilirubin
concentration.
• Serum/plasma diluted with saline/sodium citrate and
absorbance read at 420nm,
– Result is expressed in icterus index units obtained in
comparison with standard potassium dichromate
solution of assigned icterus index value
• Low specificity because of interference due to presence
of hemoglobin, carotene, and different yellow pigments
found in sample.
• Has limited clinical significance
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Direct Spectrophotometry
• Based on linear relation ship between concentration of
bilirubin and its Absorbance
• Absobance of diluted serum is measured at two
wavelenth(454 & 540 nm) and correction is made for
oxyhemoglobin and turbidity
– A 454= absorbance of bilirubin, hemoglobin, and turbidity
– A 540= absorbance of hemoglobin and/or turbidity only
Absorbance of bilirubin= A454-A540
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Direct Spectrophotometry cont’d
• Source of error
– Turbidity, hemolysis: can be blanked out by measuring a
second wavelength
– Yellow lipochrome pigments(carotene, xantopyll): not
corrected
• It is restricted to new born infants(since serum
doesn’t contain carotene and other lipochrome)
• Not used for older children and adults
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Chemical method (diazo colorimetric tests)
• Bilirubin is commonly measured by photometric methods based
upon the diazo reaction
– Bilirubin + diazotized sulfanilic acid → azobilirubin
• Direct/conjugated/cholebilirubin: react with diazo reagent in
acqueous solution, in absence of accelerator
• Indirect/unconjugated/hemobilirubin: react with diazo reagent
only in presence of accelerating agent
• Accelerators/coupling agents/solubilizers
– Facilitates the reaction of unconjugated bilirubin with diazo
reagent
– Ethanol, methanol, caffeine with/without benzoates,
acetamide, NaOH, bile salts, sodium salicylate, citric acid 31
Diazo colorimetric tests cont’d
• Two methods
– Malloy-Evelyn method
– Jendrassic-Groff method
• Malloy-Evelyn method
• Principle
– Bilirubin + diazotized sulfanilic acid → azobilirubin(purple)
– intensity of the color measured at 540 nm ~ concentration of
bilirubin
– Conjugated bilirubin + diazo reagent in aqueous solution form
color within 1 minutes
– Subsequent addition of 50% methanol accelerates the
reaction of unconjugated fraction in the specimen and a value
for total bilirubin is obtained after 30 min 32
Jendrassic-Groff method
Principle:
• For determination of total bilirubin, the specimen is added to a
solution of
– Sodium acetate: buffers the PH of the diazotization reaction
– Caffeine-sodium benzoate: accelerates the coupling of
bilirubin with diazotized sulfanilc acid
– Diazotized sulfanilic acid
– The diazotization reaction is terminated by the addition of
ascorbic acid, which destroys the excess diazo reagents.
– A strong alkaline tartarate solution is then added to convert
the purple azobilirubin to blue azobilirubin (intensity of the
color is read at 600 nm). 33
Jendrassic-Groff method cont’d
• For the determination of conjugated bilirubin,
– The specimen is first added to a dilute acid solution (HCl) followed by
the diazo reagent.
– The absence of accelerator allow only for the reaction of
conjugated bilirubin with diazotized sulfanilic acid
• The azobilirubin color produced by conjugated bilirubin is
developed for 1 minute, where as that from total bilirubin is
essentially completely developed in 10 minutes.
• After addition of ascorbic acid and the tartarate solution, the
color is stable for 30 minutes.
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Remarks on diazo techniques
• Highly colored serum not necessarily indicate
bilirubin(since hemoglobin, carotene has pigment),
no accurate conversion of bilirubin conc to icterus
index
• Biliverdin
– Green, oxidation product of bilirubin
– Not interfere, as it does not react within the diazo
reagent
– Indicator of bilirubin is lost from specimen
through oxidation
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• Urine bilirubin
– Bilirubin is not detected in the urine of normal
healthy person
– When it present in urine
• Suggests that the presence of conjugated bilirubin
• Indicates damage of hepatocytes, obstructive jaundice
• Increased urine urobilinogen
– Found in hemolytic disease, defective liver cell function
• Absence of urobilinogen in urine and stool
– Complete obstruction of biliary (obstructive jaundice)
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Reference Ranges for Bilirubin for
Interpretation of Results
Patient conjugated total bilirubin Urine Urine
(direct) unconj. + bilirubin, urobilinogen
bilirubin conjugated conjugated
Newborn Not < 12.0 mg/dL
(1-2 d old) applicable
Adult 0-0.2 mg/dL 0.3-1.2 negative <0.8 EU*
mg/dL
• Compare patient result with reference range
• * EU = “Ehrlich unit”, is equivalent to 1 mg/dL
• Newborn range for full term,1-2 days old
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Clinical Correlations
**Urobilinogen = UBG; *Total Bili = (conjugated + unconjugated)
Jaundice Clinical Serum Serum Urine Urine
Type Condition Conjugated Total Bilirubin (UBG)**
Bilirubin Bilirubin *
None Normal Normal level Normal Neg Normal
level <1 mg/dL
Pre-hepatic Hemolytic Normal or Increase Neg Increase
anemia Sl Increase
Hepatic Hepatitis Increase Increase Positive Normal or
increase
Post-hepatic Obstruct-ion Increase Increase Positive Normal or
of bile duct none.
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Summary
• This chapter reviewed anatomy and
physiology of the liver to relate to liver
function testing, the biochemistry, clinical
significance and methods of bilirubin and
correlation with liver function
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Review Questions
• Differentiate unconjugated bilirubin, conjugated
bilirubin and urobilinogen in terms of:
– A. Where they are produced
– B. General chemical characteristics
– C. How they react in the Jendrassik-Groff method
– D. Normal amounts in serum and urine
• Mention the lab finding during
– Pre-hepatic jaundice
– Hepatic jaundice
– Post hepatic jaunduce 40
• The tube containing patient’s serum was left
in the test rack for 7 hours on the laboratory
bench before it was analyzed for total and
direct bilirubin. Evaluate this situation for any
possible source of errors.
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