Al Asmarya Islamic
University
Faculty of Pharmacy
Clinical biochemistry
Lectured by : ESMAEIL B. MUSA
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Liver function tests
• Introduction
• The liver plays a major role in protein, carbohydrate and lipid
homeostasis
• The metabolic pathways of glycolysis, the Krebs cycle, amino acid
synthesis and degradation, and the processes of oxidative
phosphorylation are all carried out in the hepatocytes, which are well
endowed with mitochondria
• It is responsible for the synthesis of albumin and many other plasma
proteins, as well as most of the coagulation factors
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• It synthesizes bile acids, major
constituents of bile, and is the
key organ for detoxification,
metabolism and inactivation of
drugs and the metabolism and
excretion of many endogenous
compounds,
including
cholesterol, amino acids,
steroid and other hormones
• It also protects the body
against potential carcinogens
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• Liver disease is relatively common, and the measurements of serum levels
of bilirubin, hepatic enzymes and albumin, as well as the prothrombin time
(PT), provide simple tests to determine whether disease is present and give
some guidance as to its nature
• Structure of the liver
• Only about 80% of the cells in the liver are hepatocytes
• The functional unit of the liver lobules, each lobule is primarily composed
of a central vein (CV), the portal triad consists of a portal vein, biliary duct
and hepatic artery
• Hepatocyte cords are separated by sinusoids that carry blood from the portal
triads to the central vein
• Hepatocytes secrete bile salts into the bile canaliculi that lead to the bile
duct
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• Stellate cells are in the space of Disse between the hepatocyte cords and
sinusoids
• Kupffer cells, which are the specialized macrophages of the liver, that
form part of the reticulo‐endothelial system, the epithelial cells that line
the bile ducts are called Cholangiocytes
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• Liver function tests
• Most laboratories perform a standard tests which are useful
for:
1. Obstruction to the biliary tract
2. Acute hepatocellular damage
3. Chronic liver disease
CLASSIFICATION OF LIVER FUNCTION
TESTS
A. Tests of the liver’s capacity to transport organic anions and
to metabolize drugs: (Serum bilirubin, urine bilirubin,
urobilinogen)
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B. Tests that detect injury to hepatocytes [serum enzyme tests]:
(Aminotransferases, alkaline phosphatase, γ-glutamyl
transpeptidase, 5-nucleotidase, leucine aminopeptidase)
C. Tests of the Liver’s biosynthetic capacity: (Serum proteins,
albumin, prealbumin, serum ceruloplasmin, procollagen III
peptide, a 1 antitrypsin, α-feto protein, prothrombin time)
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A. Tests of the liver’s capacity to transport
organic anions and to metabolize drugs
• Bilirubin production and metabolism
• Bilirubin is derived from haem, an iron-containing protoporphyrin
mainly found in haemoglobin, from other haem proteins such as
myoglobin and the cytochromes
• Iron is removed from the haem molecule, and the porphyrin ring is
opened to form bilirubin
• An adult normally produces about 450 μmol of bilirubin daily
• It is insoluble in water and is transported in plasma almost totally
bound to albumin
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• It is taken up by liver cells and conjugated to
form mono- and diglucuronides, which are much
more soluble in water than unconjugated bilirubin
• The conjugated bilirubin is excreted into the bile
• Normal bile contains bilirubin monoglucuronide
as 25% and the diglucuronide as 75% of the total,
accompanied by traces of unconjugated bilirubin
• The main functional constituents of the bile are
the bile salts, which are involved in fat digestion
and absorption from the small intestine
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• Serum bile acid concentrations are more sensitive indices of hepatic
transport function than are total bilirubin measurements
• In the terminal ileum and colon the bilirubin conjugates are attacked
by bacteria to form a group of compounds that are known collectively
as stercobilinogen, most of which are excreted in faeces
• Some are absorbed and eventually re-excreted from the body by way
of bile (the enterohepatic circulation)
• Small amounts of these tetrapyrroles are found in urine in which they
are known as urobilinogen
• When the biliary tract becomes blocked, bilirubin is not excreted and
serum concentrations rise and the patient becomes jaundiced
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1. SERUM BILIRUBIN
• Bilirubin is altered by exposure to light so serum and plasma samples
must be kept in dark before measurements are made
• When the liver function tests are abnormal and the serum bilirubin
levels more than17μmol/L suggest underlying liver disease
Types of bilirubin
1. Total bilirubin: This is measured as the amount, which reacts in 30
minutes after addition of alcohol
• Normal range is 0.2-0.9 mg/dl (2-15μmol/L)
• It is slightly higher by 3-4 μmol/L in males as compared to females,
which helps to diagnose Gilbert syndrome in males easily
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2. Direct Bilirubin : This is the water-soluble fraction
• Measured by the reaction with diazotized sulfanilic acid in 1 minute
and this gives estimation of conjugated bilirubin
• Normal range 0.3mg/dl( 5.1μmol/L)
3. Indirect bilirubin: it is calculated by the difference of the total and
direct bilirubin and is a measure of unconjugated fraction of
bilirubin
A newer highly accurate method of estimation involves alkaline
methanolysis of bilirubin followed by chloroform extraction of
bilirubin methyl esters and later separation of these esters by
chromatography and spectrophotometric determination at 430 nm
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i. Diagnostic value of bilirubin levels :
• Bilirubin in body is a careful balance between production and removal
of the pigment in body
Hyperbilirubinemia: It results from overproduction / impaired uptake,
conjugation or excretion / regurgitation of unconjugated or conjugated
bilirubin from hepatocytes to bile ducts
• Increased
unconjugated
bilirubin:
This
results
from
overproduction/impaired uptake, no conjugation
• Increased conjugated bilirubin: Impaired intrahepatic excretion /
regurgitation of conjugated bilirubin from hepatocytes of bile ducts
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ii. Prognostic value of bilirubin levels
• Bilirubin may be of prognostic value in conditions like hepatic failure
where deep jaundice is associated with increased mortality
Hyperbilirubinemia and Hemolysis
• Bilirubin itself is not soluble in water and is bound to albumin and
thus does not appear in urine
• Hemolysis with overproduction of bilirubin and concomitant reduced
GFR cause decreased excretion and can lead to high bilirubin levels
• Bilirubin levels in excess of 25 mg/dl may be seen in hemolysis in
association with liver disease
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2. URINE BILIRUBIN
• Normal blood test range from 0 to 0.3 mg/dL in adults
• Unconjugated bilirubin is tightly bound to albumin and not filtered by
the glomerulus and thus not present in urine
• Measurable amounts of conjugated bilirubin in serum are found only
in hepatobiliary disease
• The presence of urine bilirubin indicates hepatobiliary disease
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3. UROBILINOGEN
• Normal range: 0.5-4mg is excreted in urine of 24 hours
• It is markedly increased in hemolysis
• In cholestatic jaundice urobilinogen disappears from urine
• Urobilinogen gives a purple reaction to Ehrlich’s aldehyde reagent
• Freshly voided urine should be used
Cause of increased urobilinogen
• It can be increased in case of hemolysis and hemorrhage in tissue
Cause of decreased urobilinogen
• Obstructive jaundice and reduction of intestinal bacterial flora
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B. Tests that detect injury to hepatocytes( serum
enzyme tests
1. Enzymes that detect hepatocellular damage – aminotransferases
• The aminotransferases (formerly transaminases)are the most
frequently utilized and specific indicators of hepatocellular damage
• These enzymes are AST and ALT
• ALT is primarily localized to the liver but the AST is present in a wide
variety of tissues like the heart, skeletal muscle, kidney, brain and liver
• Whereas the AST is present in both the mitochondria and cytosol of
hepatocytes, ALT is localized to the cytosol
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Mild , moderate and severe elevations of aminotransferases
1. Severe ( > 20 times, 1000 U/L)
• The highest elevations occur in severe viral hepatitis, drug or toxin
induced hepatic necrosis and circulatory shock
2. Moderate (3-20 times)
• The AST and ALT are moderately elevated in (acute, neonatal,
chronic, autoimmune, drug induced, alcoholic) hepatitis and acute
biliary tract obstructions
• The ALT is usually more frequently increased as compared to AST
except in chronic liver disease
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3. Mild (1-3 times)
• These elevations are usually seen in sepsis induced neonatal hepatitis,
extrahepatic biliary atresia (EHBA), fatty liver, cirrhosis, non
alcoholic steato hepatitis(NASH), drug toxicity, myositis, Duchenne
muscular dystrophy and even after vigorous exercise
2. Enzymes that detect cholestasis
a) ALKALINE PHOSPHATASE (ALP)
• Highest levels of alkaline phosphatase occur in cholestatic disorders
• Elevations occur as a result of both intrahepatic and extrahepatic
obstruction to bile flow
• Alkaline phosphatase levels are likely to be very high in EHBA
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• Mildly elevated levels of alkaline phosphatase may be seen in cirrhosis
and hepatitis of congestive cardiac failure
• Low levels of alkaline phosphatase occur in hypothyroidism,
pernicious anemia, zinc deficiency and congenital hypophosphatasia
• Drugs like cimetidine, frusemide, phenobarbitone and phenytoin may
increase levels of alkaline phosphatase
b) γ-GLUTAMYL TRANSPEPTIDASE (GGT)
• GGT is a membrane bound glycoprotein which catalyzes the transfer
of γ-glutamyl group to other peptides, amino acids and water
• Widely distributed in tissues including liver and renal tubules, and
prostate
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• The activity of γ-GT in plasma is raised whenever there is cholestasis,
and it is a very sensitive index of liver pathology
• It is also affected by ingestion of alcohol, even in the absence of
recognizable liver disease
• Drugs such as phenytoin induce enzyme activity
• In acute hepatic damage, changes in γ-GT activity parallel those of the
aminotransferases
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C. Tests of the Liver’s biosynthetic capacity
a) SERUM PROTEINS (albumin)
• The liver is the major source of most the serum proteins
• The parenchymal cells are responsible for synthesis of albumin,
fibrinogen and other coagulation factors and most of the α and ß
globulins
• It is the most useful indicator of hepatic function
• Because the half life of albumin in serum is as long as 20 days, the
serum albumin level is not a reliable indicator of hepatic protein
synthesis in acute liver disease
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• Albumin synthesis is affected not only in liver disease but also by
nutritional status, hormonal balance and osmotic pressure. Liver is the
only site of synthesis of albumin
• The serum levels are typically depressed in patients with cirrhosis and
ascites
• Normal serum values range from 3.5g/dl - 4.5 g/dl
• Corticosteroids and thyroid hormone stimulate albumin synthesis by
increasing the concentration of albumin mRNA and tRNA in
hepatocytes
• Albumin levels below 3g/dl in hepatitis should raise the suspicion of
chronic liver disease like cirrhosis which usually reflects decreased
albumin synthesis
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• Hypoalbuminemia is not specific for liver disease and may occur in
protein malnutrition, nephrotic syndrome and chronic protein losing
enteropathies
b) SERUM CERULOPLASMIN
• Ceruloplasmin is the major copper-carrying protein in the blood,
synthesized in the liver
• Normal plasma levels are 0.2-0.4g/L
• This is an important diagnostic marker in Wilson disease, in which the
plasma level is usually low
c) PROTHROMBIN TIME (PT)
• Clotting is the end result of a complex series of enzymatic reactions
that involve at least 13 factors
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• The liver is the major site of synthesis of 11 blood coagulation
proteins
• Most of these are present in excess and abnormalities of coagulation
only result when there is substantial impairment in the ability of the
liver to synthesize these factors
• Normal control usually is in the range of 9-11 seconds
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