CLINICAL MEDTECH LEC

CHEMISTRY II TERM 02

LIVER FUNCTION TESTS

LABORATORY
LIVER ANATOMY potentially toxic substances from the
blood.
Largest organ of the body located at the
upper right quadrant of the abdominal From the sinusoid, blood flows to the
cavity, extending from 5th rib to the lower central canal (central vein) of each lobule.
rib cage. It is through the central canal that blood
leaves the liver.
Weight: 1.2 to 1.5 kg
1500 ml of blood passes through the liver
Consisting of 2 major lobes and is per minute.
suspended from the diaphragm and
abdominal wall by a delicate mesentery
cord, the falciform ligament.
Functional unit of the liver is known as the
lobules, mainly consists a major central
vein and clusters of hepatocytes, portal
vein, hepatic artery, bile duct and hepatic
macrophage known as the Kupffer cells.

The excretory system of the liver begins at

the bile canaliculi. The bile canaliculi are
small spaces between hepatocytes that
form intrahepatic ducts, where excretory
products of the cell can drain.
The intrahepatic ducts join to form the
right and left hepatic ducts, which drains
the secretions from the liver.
The right and left hepatic ducts merge to
form a common hepatic duct, which is
Liver is an extremely vascular organ that eventually joined with cystic duct of the
receives blood supply from two supply: gall bladder to form the common bile duct.
hepatic artery and the portal vein.
Combined digestive secretions are then
The hepatic artery, a branch of the aorta, expelled into the duodenum.
supplies oxygen rich blood from the heart
to the liver and responsible for providing
approximately 25% of the total blood
supply.
The portal vein, supplies nutrient-rich
blood (collected as food is digested) from
the digestive tract, and responsible for
providing approximately 75% of the total
blood supply
The two blood supplies eventually merge
into the hepatic sinusoid, which is lined
with hepatocytes capable of removing

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 CLINICAL MEDTECH LEC
CHEMISTRY II TERM 02

LIVER PHYSIOLOGY The liver is responsible for metabolizing

both lipids and the lipoproteins and is
SITE OF MAJOR BIOMOLECULE SYNTHESIS responsible for gathering free fatty acids
from the diet and those produced by the
• Biomolecules such as plasma proteins,
liver itself, and breaking them down to
lipids, enzymes, hormones and many other
produce acetyl-COA.
substances are synthesized here.
acetyl-COA can then enter several
FILTRATION, DETOXIFICATION and
pathways to form triglycerides,
EXCRETION
phospholipids, or cholesterol.
• Converts dangerous substances into non-
Almost all proteins are synthesized by the
toxic metabolites (urea, ammonia,
liver except for the immunoglobulins and
cholesterol, bilirubin, drugs)
adult hemoglobin.
STORAGE
The liver plays an essential role in the
• Stores excess plasma constituents such as development of hemoglobin in infants.
glycogen, protein, lipids, vitamins,
One of the most important protein
minerals, etc.
synthesized by the liver is albumin.
DIGESTION
The liver is also responsible for
• Produces bile which aids in breakdown of synthesizing the positive and negative
fat. acute phase reactants and coagulation
proteins, and also serves to store a pool of
amino acids through protein degradation.
METABOLISM DETOXIFICATION AND DRUG METABOLISM
Metabolism of carbohydrates is one of the The liver serves as a gatekeeper between
most important functions of the liver. subtances absorbed by the
When carbohydrates are ingested and gastrointestinal tract and those released
absorbed, the liver can do three things: into systemic circulation.

1. use the glucose for its own cellular Every substances that is absorbed in the
energy requirements. gastrointestinal tract must first pass
through the liver that is referred to as first
2. circulate the glucose for use at the pass.
peripheral tissues
The body has two mechanism for
3. store glucose as glycogen (principal detoxification of foreign materials (drugs
storage form of glucose) within the liver or and poisons) and metabolic products
other tissues. (bilirubin and ammonia)
Detoxification of drugs: oxidation,
reduction, hydrolysis, hydroxylation,
carboxylation, and demythylation.

Lipids are metabolized in the liver under

normal circumstances when nutrition is
adequate and the demand for glucose is
being met.

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CHEMISTRY II TERM 02

LIVER FUNCTION TEST Tests for Hepatocellular injury or

damage
Liver Function tests is performed for
determination of liver integrity by • Aspartate aminotransferase
measuring the levels of several analytes • Alanine aminotransferase
such as protein, enzymes, and bilirubin in • Lactate dehydrogenase
blood.
Tests for cholestasis
Detect general types of diseases.
• Alkaline phosphatase
Evaluate diagnosis and prognosis of
• Gamma-glutamyl transferase
disease.
Assessment of treatment BILIRUBIN
It is helpful in the following conditions: Bilirubin is an orange-yellow pigment
derived from senescent RBCs.
▪ Monitor liver damage caused by
infections Extracted and biotransformed by the liver
into several analytes and is excreted in bile
▪ Determine the detrimental effects to the and urine.
liver by certain medications
BILIRUBIN BIOSYNTHESIS
▪ Patients experiencing symptoms
associated with liver and gallbladder Bilirubin is produced by
disorder reticuloendothelial system (splenic
macrophage) from the breakdown of old RBC
▪ Alcoholic patients
(heme, globin)
▪ High levels of lipid profile, diabetics,
Heme is converted into biliverdin by the
hypertensives and anemics.
enzyme heme oxygenase producing products
CLASSIFIED INTO SEVERAL TESTS: such iron and carbon monoxide.

Protein Determination Free iron is converted to intracellular

ferritin or stored in iron pools.
• Albumin
• Globulin Carbon monoxide can be used as
• A/G Ratio vasodilative, anti-thrombotic and anti-
• Coagulation Factor levels inflammatory.
Liver Enzymology
• Biliverdin is converted into
• Alkaline Phosphatase (ALP) unconjugated bilirubin (B1) by the
enzyme biliverdin reductase.
• Alanine Aminotransferase (ALT)
• Unconjugated bilirubin forms a complex
• Aspartate Aminotransferase (AST)
with albumin for transport to the liver.
• Gamma-glutamyl Transferase This form of bilirubin is water insoluble.
(GGT)
• Bilirubin binds to ligandin and is
Bilirubin Tests conjugated in the hepatocytes
• Unconjugated Bilirubin endoplasmic reticulum with glucuronic
• Conjugated Bilirubin acid to form bilirubin diglucuronide or
• Total Bilirubin conjugated bilirubin (B2). It is catalyzed
• Urobilinogen by the reaction with UDPGT or uridine
diphosphate glucuronyl transferase
BROAD CLASSIFICATION OF LIVER TESTS
Tests for Liver dysfunction

• Total and Conjugated bilirubin

• Urine bile salts and urobilinogen
• Total protein test and A/G ratio
• Prothrombin time

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CHEMISTRY II TERM 02

B2 is excreted in the bile for storage in the MALLOY-EVELYN METHOD

gallbladder, and reduced by intestinal
flora to urobilinogen. Based on van den Berg method and is a
colorimetric technique.
Some intestinal urobilinogen is
reabsorbed, a portion returns to the liver Used to detect indirect bilirubin (B1).
and some enters the kidney for excretion Uses methanol as an accelerator to
in the urine in the form of urobilinogen accelerate precipitation of proteins,
and urobilin. permitting diazotization of unconjugated
The remaining portion in the intestines in bilirubin to azobilirubin.
oxidized by intestinal anaerobic flora for Red to violet color o is produced and is
excretion into stool as urobilin and further measure spectrophotometrically.
converted to stercobilinogen and into
stercobilin which gives the stool its Has a substantial effects, especially
orange-brown to black color. negative interference by hemoglobin.

JENDRASSIK-GROF METHOD
Detects both conjugated and unconjugated
bilirubin.
Based on the same principle but uses
caffeine and sodium benzoate.

Bilirubin + sodium acetate + caffein-

sodium benzoate + diazotized sulfanilic
acid → purple azobilirubin + alkaline
tartrate → green-blue azobilirubin.
The tartrate buffer makes the solution
alkaline and converts the initial acid red
bilirubin to a green azobilirubin and is
measured at 600nm.
ENZYMATIC METHOD
Based on the principle of bilirubin oxidase
BILIRUBIN DETERMINATION by oxidation of bilirubin to biliverdin.
Bilirubin can be detected in serum, urine
At pH near 8, and in the presence of
and feces.
sodium cholate and sodium
Diazo reaction by Ehrlich which uses dodecylsulfate, all four bilirubin fractions
sulfanilic acid for diazotization is are oxidized into biliverdin, which is
modified by Van den Berg and Muller is the further oxidized to purple and finally
basis of bilirubin determination. colorless products.

Other modifications of van den Berg Direct bilirubin is measured at pH 3.7-4.5

method is also used for reproducibility when enzyme oxidizes conjugated and
and reliability. delta bilirubin but not unconjugated
bilirubin.
 Malloy and Evelyn
Delta bilirubin – bilirubin that is
 Jendrassik and Grof irreversibly bound to albumin. It is due to
More sensitive techniques includes HPLC prolonged cholestasis.
and enzymatic methods.
INTERFERENCES
Transcutaneous bilirubin is available with
• Hemolysis
the use of bilirubinometer based on
reflectance photometry. • Lipemia

• Exposure to light (direct sunlight or

fluorescent)

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CHEMISTRY II TERM 02

REFERENCE VALUES
Bilirubin Reference value

Total bilirubin (elderly) 0.2-0.8 mg/dL

Newborn 0.8-12 mg/dL

Critical values in adult >12 mg/dL

JAUNDICE

Critical values in >15 mg/dL French word jaune- yellow

newborn Upper limit of normal Total Bilirubin is 1.0
to 1.5 mg/dl, jaundice not noticeable to the
Unconjugated bilirubin 0.2-0.7 mg/dL naked eye (overt jaundice) until bilirubin
reach 3.0 to 5.0 mg/dl

Conjugated bilirubin 0.1-0.4 mg/dL Classification of Jaundice: Pre-hepatic,

hepatic jaundice and post-hepatic jaundice

Fecal urobilinogen 40-280 mg/dL

Urine bilirubin 0-0.02 mg/dL

PRE-HEPATIC JAUNDICE
Problem causing jaundice occurs prior to
liver metabolism.
Increased amount of bilirubin being
presented to the liver.
Acute and chronic hemolytic anemia

Not seen in the urine

CLINICAL SIGNIFICANCE
Unconjugated hyperbilirubinemia
Jaundice – is characterized by
hyperbilirubinemia and deposition of bile HEPATIC JAUNDICE
pigments in the skin, mucous membranes,
and sclera, resulting into a yellow Primary problem causing jaundice resides
appearance of the patient, termed as in the liver (intrinsic liver defect or
icterus. disease)

Characterized by elevation of levels of It can be due to disorders of bilirubin

unconjugated and conjugated bilirubin in metabolism and transport defects
the absence of other abnormal liver tests. (Crigler-Najjar Syndrome, Dubin-Johnson
Clinical fractionation of bilirubin is useful Syndrome, Gilbert Syndrome and
only in detection of inherited disorders. Neonatal Physiologic Jaundice of the
Newborn)

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 CLINICAL MEDTECH LEC
CHEMISTRY II TERM 02

Or due to diseases resulting to

hepatocellular injury or destruction
(Gilbert disease, CN syndrome )
Elevated unconjugated bilirubin
Dubin-Johnson and Rotors Syndrome
(elevated conjugated bilirubin)

POSTHEPATIC JAUNDICE
Results from biliary obstructive disease
usually from physical obstruction
(gallstones or tumors) that prevent the
flow of conjugated bilirubin into the bile
canaliculi

JAUNDICE

CAUSES OF JAUNDICE

REFERENCE HEMOLYTIC INTRAHEPATIC EXTRAHEPATIC

RANGE JAUNDICE (EARLY (OBSTRUCTIVE)
HEPATITIS)

SERUM 0-0.2 mg/dL Normal or Increased Markedly

CONJUGATED slightly increased
BILIRUBIN increased

SERUM 0.2-0.8 mg/dL Increased Markedly Increased

UNCONJUGATED increased
BILIRUBIN

FECAL 75-400 EU (+2) Markedly Decreased (+1) Decreased or

UROBILINOGEN increased (+4) negative

URINE 0.5-4.0 EU (+1) Markedly Increased Decreased or

UROBILINOGEN increased (+4) negative

URINE Negative Negative Increased Increased

BILIRUBIN

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CHEMISTRY II TERM 02

CIRRHOSIS GILBERT SYNDROME

Clinical conditions in which scar tissue Gilbert's syndrome, first described in the
replaces normal, healthy liver tissue early twentieth century, is a benign
autosomal recessive hereditary disorder
Cirrhosis rarely causes signs and
that affects approximately 5% of the US
symptoms in its early stages, but as its
population.
deteriorates signs and symptoms appears:
fatigue, nausea, unintended weight loss, Gilbert's syndrome results from a genetic
jaundice, bleeding from GIT, intense mutation in the UGT1A1 gene that
itching and swelling in the legs and produces the enzyme uridine diphosphate
abdomen glucuronosyltransferase, one of the
enzymes important for bilirubin
Chronic alcoholism-most common cause
metabolism. The UGT1A1 gene is located
Other causes: chronic Hep-B, Hep-C, Hep- on chromosome 2, and other mutations of
D, autoimmune hepatitis, inherited this same gene produce Crigler-Najjar
disorders, non alcoholic steatohepatitis, syndrome, a more severe and dangerous
blocked bile ducts, drugs, toxin, infections form of hyperbilirubinemia.

TUMORS
Cancer of the liver are classified as
primary or metastatic
Common benign tumors of the liver:
hepatocellular adenoma and hemangioma

REYE’S SYNDROME
Reye’s syndrome is a term used to
described a group of disorders caused by
infectious, metabolic, toxic, or drug-
induced disease found almost exclusively
in children.
The molecular basis of Gilbert's syndrome
Often preceded by a viral syndrome such
(in whites) is related to the UDPGT
as varicella, gastroenteritis, URTI such as
superfamily, which is responsible for
influenza
encoding enzymes that catalyze the
Aspirin ingestion during viral syndrome conjugation of bilirubin. The UGT1A1 (the
developed to Reye’s syndrome hepatic 1A1 isoform of UDPGT)
contributes substantially to the process of
Characterized by noninflammatory conjugating bilirubin.
encephalopathy and fatty degeneration of
the liver The UGT1A1 promoter contains the
sequence (TA)6TAA. The insertion of an
Fatty degeneration of liver: Inc-ammonia, extra TA in the sequence, as seen in
AST, ALT Gilbert's syndrome, reduces the
expression of the UGT1A1 gene to 20% to
INHERITED BILIRUBIN DISORDERS
30% of normal values. That is, the liver's
conjugation system in Gilbert's syndrome
is working at approximately 30% of
normal.
Mild unconjugated hyperbilirubinemia
(1.5-4 mg/dL)
Misdiagnosed as chronic hepatitis but can
be differentiated by the absence of anemia
and bilirubin in urine and by normal liver
function tests.
Low levels of biliary bilirubin.

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Acetaminophen toxicity may lead to this Partial deficiency of UDPGT 1A1 with an
disorder due to metabolism of enzymatic activity usually less than 10%
acetaminophen by glucuronidation. of normal activity.

CRIGLER-NAJJAR SYNDROME TYPE I Unconjugated bilirubin is usually 5-

20mg/dL.
Crigler-Najjar syndrome was first
described by Crigler and Najjar in 1952 as Can be treated by phenobarbital and
a syndrome of chronic nonhemolytic phototherapy, especially to pregnant
unconjugated hyperbilirubinemia.Crigler- patients without affecting the mother of
Najjar syndrome, like Gilbert's syndrome, fetus.
is an inherited disorder of bilirubin
metabolism resulting from a molecular
LUCEY-DRISCOLL SYNDROME
defect within the gene involved with Also known as maternal serum jaundice or
bilirubin conjugation. transient familial hyperbilirubinemia.
Crigler-Najjar syndrome may be divided Caused by circulating inhibitor of
into two types: type 1, where there is a bilirubin conjugation (inhibits bilirubin
complete absence of enzymatic bilirubin glucuronide)
conjugation, and type II, where there is a
mutation causing a severe deficiency of the Transplacentally transferred from
enzyme responsible for bilirubin plasma or present in milk of mothers
conjugation. Unlike Gilbert's syndrome, (breast milk hyperbilirubinemia)
Crigler-Najjar syndrome is rare and is a Usually mild and lasts for the first 2-3
more serious disorder that may result in weeks of life
death.
DUBIN-JOHNSON SYNDROME
Transport deficit
Characterized by elevated conjugated
bilirubin and a minor elevation of
unconjugated bilirubin.
Due to mutation of the ABCC2 gene, the
transport of the bile salts to bind with
bilirubin is impaired.

It causes conjugation of various organic

anions and bilirubin, impairing the
excretion to bile to the bile canaliculi.
Liver has a greenish black appearance
and dark brown pigmentation of
hepatocytes and Kupffer cell upon liver
Conjugation deficit biopsy.
CN type I is the complete absence of
UDPGT 1A1, and is often characterized
by high concentrations of unconjugated
bilirubin (>20mg/dL).
No conjugation occurs therefore no
detectable levels of conjugated bilirubin.
Most patients die of severe brain damage
caused by kernicterus.

Can be controlled by phlebotomy and

plasmapheresis.
The only effective therapy is early liver
transplantation.

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ROTOR SYNDROME • Sclerosing cholangitis

• Intrahepatic obstruction (drugs,
Another form of conjugated granulomas, primary biliary cirrhosis,
hyperbilirubinemia similar to Dubin- etc.)
Johnson syndrome, but without pigment in
liver.

PHYSIOLOGIC CLASSIFICATION OF JAUNDICE (B1)

Increased Production of Unconjugated
Bilirubin from Heme
▪ Hemolysis (hereditary, acquired)
▪ Ineffective erythropoiesis
▪ Rapid turnover of increased red blood
cell mass (due to replacement of older
proteins)
Decreased delivery of Unconjugated
Bilirubin in Plasma
▪ Right sided congestive heart failure
UROBILINOGEN
▪ Portocaval shunt
Is a colorless end product of bilirubin
Decreased Uptake of Unconjugated metabolism that is oxidized by intestinal
bilirubin across hepatocyte membrane bacteria brown pigment urobilin.
▪ Competitive inhibition (drugs, etc.) It is either excreted in urine and feces,
▪ Gilbert syndrome or reabsorbed into the portal blood and
returned liver.
▪ Sepsis
Absence of this substance in urine or
▪ Fasting stool denotes complete biliary obstruction.
Decreased Storage of Unconjugated In the collection of the sample, avoid
Bilirubin in Cytosol exposure to direct light.
▪ Competitive inhibition Specimen: 2 hour freshly collected
▪ Fever urine or freshly collected stool.

Decreased Biotransformation Method: Ehrlich’s method (p-dimethyl

(conjugation) aminobenzaldehyde reagent)

▪ Neonatal jaundice Reference value:

▪ Inhibition (drugs) Urine= 0.1 – 1.0 Ehrlich units/2hour or 0.54

Ehrlichs units/day-urine
▪ Hereditary (Crigler-Najjar syndrome)
Stool= 75-275 Ehrlich units/100g of feces.
▪ Hepatocellular destruction
III. TEST FOR DETOXIFICATION FUNCTION
▪ Gilbert syndrome
It involves enzyme and ammonia tests
Decreased Secretion of Conjugated
Bilirubin into Canaliculi A. Enzyme Tests

• Hepatocellular disease (Hepatitis, is used to assess the extent of liver

cholestasis) damage and to differentiate hepatocellular
• Dubin-Johnson and Rotor syndromes (functional) from obstructive
• Drugs (estradiol) (mechanical)disease

Decreased Drainage Any injury to the liver that results in

cytolysis and necrosis causes the liberation of
• Extrahepatic obstruction (stones, various enzyme.
carcinoma, atresia, stricture)

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Enzyme tests are often the only Plasma levels of ammonia are not
indication of cell injury in early or localized dependent on renal function, though an
liver disease. Enzymes secreted by the liver: NPN, but on liver function thus are not
ALP, aminotransferases, 5’nucleotidase, GGT, useful in the study of kidney diseases.
OCT, LAP and LD
Preferred specimen: arterial blood
B. Ammonia (venous blood is not recommended, if
used, tourniquets should be used
It arises from deamination of amino acids
minimally, and first clenching and relaxing
which occurs mainly through the action of
avoided during collection.
digestive and bacterial enzymes (bacterial
proteases, ureases and amine oxidases) on Specimen requirements: Heparin or EDTA
proteins in the intestinal tract. plasma/serum kept in ice water
immediately; hemolysis should be
It is also released from metabolic reactions
avoided.
that occur in skeletal muscles during
exercise.

The liver normally removes most of this • Common Methods: Berthelot and
NPN via the portal vein circulation and Glutamate Dehydrogenase.
converts it to urea, then eliminated by the 1. Digestion (Kjeldahl) Method
kidneys (urine) • Nitrogen ion in a potential-free filtrate
Reference value: 19-60 ug/dl (11-35 (PFF) of the specimen is converted to
mmol/l) ammonia using hot concentrated
sulphuric acid in the presence of
Diagnostic Significance catalyst.
For the diagnosis of hepatic failure
(hepatic coma) and Reye’s syndrome. H2SO4
Nitrogen NH3
In severe liver disorder, it accumulates CuSO4
and reaches the systematic circulation Hg
which is then converted to glutamine in Selenium
the brain, thus compromising the Kreb’s
cycle leading to coma due to lack of ATP for 2. Measurement of Ammonia
the brain-ammonia increases CNS pH. a. Nesslerization Reaction
Elevated plasma levels of ammonia are Gum Ghatti
neurotoxic and are often associated with NH3 + K2Hg2I2 NH2HgI2
encephalopathy- an important mechanism • yellow end color – N2 low to moderate
by which ammonia can cause toxicity to • Orange brown end color- N2 high
the CNS is its ability to lower the 3. Berthelot Reaction
concentration of y-aminobutyric acid Na Nitroprusside
(GABA), a critically important NH3 + Phenol + Hypochlorite Indophenol
neurotransmitter in the CNS, by reacting Blue
with glutamic acid to form glutamine via
reversal of the glutamine-catalyzed 4. Glutamate Dehydrogenase
reaction.
Increased levels: cirrhosis, hepatitis,
Reye’s syndrome, chronic renal disease
and acetaminophen poisoning.
METHODS:

Smoking is a source of contamination

(patient and the phlebotomy) which leads
to elevated concentrations.
Prolonged standing of the specimen rises
ammonia level due to enzymatic
deamination of labile amides like
glutamine.

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