Liver Function
Reference: Bishop, M.L., Fody, E.P., Schoeff, L.E (2010). Clinical Chemistry: Techniques, Principles,
Correlation (6th Edition). Lippincott Williams & Wilkins, a Walter Kluwer business
Other references: Sir Mario’s Notes and Clinical Chemistry (2019) by Rodriquez
FUNCTION
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Biochemical role in the metabolism
Digestion
Detoxification
Elimination of substances from the body
Excretory, synthetic, and metabolic
functions
Regenerate cells
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ANATOMY
A. Gross Anatomy
▪
▪
Weight: ~1.2-1.5 kg
Located beneath and is attached to the
diaphragm, is protected by the lower rib
cage, and is held in place by ligamentous
attachments.
▪ Has two lobes where the right lobe is ~6
times larger than left lobe
▪ Vascular organ that receives its blood
supply from two sources:
- Hepatic artery: branch of the aorta,
supplies oxygen-rich blood from the
heart to the liver and is responsible
for providing ~25% of the total blood
supply to the live.
- Portal vein: supplies nutrient-rich
blood (collected as food is digested)
from the digestive tract, and it is
responsible for providing ~75% of the
total blood supply to the liver.
- Sinusoids: where the two blood
supply merge.
▪ ~1,500 mL of blood passes through the
liver per minute
- Drained by a collecting system of
veins that empties into the hepatic
veins and ultimately into the inferior
vena cava
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Bile canaliculi
- where the excretory system of the
liver begins
- small
spaces
between
the
hepatocytes that form intrahepatic
ducts, where excretory products of
the cell can drain.
Intrahepatic ducts join to form the right
and left hepatic ducts, which drain the
secretion from the liver.
Right and left hepatic ducts merge to form
the common hepatic duct, which is
eventually joined with the cystic duct of
the gallbladder to form the common bile
duct.
Combined digestive secretions are then
expelled into the duodenum
B. Microscopic Anatomy
▪
Lobules: functional unit of the liver; they
are responsible for all metabolic and
excretory functions performed by the
liver.
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A six-sided structure with central
vein that has portal triads at each of
the corners.
- Portal triads contains a hepatic
artery, portal vein, and a bile duct
surrounded by connective tissue.
Two major cell types:
- Hepatocytes
- ~80% of the volume of the organ
- Large cells that radiate outward
from the central vein in plates to
the periphery of the lobule.
- Major functions associated with
the liver and are responsible for the
regenerative properties of the liver
- Kupffer cells
- Macrophages that line the
sinusoids of the liver and act as an
active phagocyte capable of
engulfing bacteria, debris, toxins,
and other substances flowing
through the sinusoids.
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BIOCHEMICAL FUNCTIONS
A. Excretory and Secretory
▪ One of the most important functions of
the liver is the processing and excretion of
endogenous and exogenous substances
into the bile or urine such as the major
heme waste product, bilirubin.
▪ Bile: made up of bile acids or salts, bile
pigment,
cholesterol,
and
other
substances extracted from the blood.
- The body produces approximately 3
L of bile per day and excretes 1 L of
what is produced.
▪ Bilirubin: principal pigment in bile, and it
is derived from the breakdown of RBCs.
- ~126 days after the emergence
from the reticuloendothelial tissue,
red blood cells are phagocytized
and hemoglobin is released.
▪ Hemoglobin: broken down into heme,
globin, and iron.
- Iron is bound to transferrin and is
returned to iron stores in the liver
or bone marrow for reuse.
- Globin is degraded to its
constituent amino acids, which are
reused by the body.
- Heme portion of hbg is converted
to bilirubin in 2-3 hours.
▪
Unconjugated or Indirect Bilirubin:
- Bilirubin is bound by albumin and
transported to the liver.
- Insoluble in water and cannot be
removed from the body until it has
been conjugated by the liver.
- Once
at
the
liver
cell,
unconjugated bilirubin flows into
the sinusoidal spaces and is
released from albumin so it can be
picked up by a carrier protein
called ligandin.
- Ligandin: which is located in the
hepatocyte, is responsible for
transporting
unconjugated
bilirubin to the endoplasmic
reticulum, where it may be rapidly
conjugated.
- The conjugation (esterification) of
bilirubin occurs in the presence of
the enzyme uridyldiphosphate
glucuronyl transferase (UDPGT),
which transfers a glucuronic acid
molecule to each of the two
propionic acid side chains of
bilirubin
to
form
bilirubin
diglucuronide, also known as
conjugated bilirubin.
Conjugated Bilirubin
- Water soluble and is able to be
secreted from the hepatocytes into
the bile canaliculi
- Once in the hepatic duct, it
combines with secretions from the
gallbladder through the cystic duct
and is expelled through the
common bile duct in to the
intestines.
- Intestinal bacteria (especially the
bacteria in the lower portion of the
intestinal
tract)
work
on
conjugated bilirubin to produce
mesobilirubin, which is reduced to
form mesobilirubinogen and then
urobilinogen (a colorless product).
- Most of the urobilinogen formed
(roughly 80%) is oxidized to an
orange-colored product called
urobilin (stercobilin) and is
excreted in the feces. The urobilin
or stercobilin is what gives stool its
brown color.
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There are two things that can
happen to the remaining 20% of
urobilinogen formed.
~200–300 mg of bilirubin is produced per
day.
Almost all the bilirubin formed is
eliminated in the feces, and a small
amount of the colorless product,
urobilinogen, is excreted in the urine.
The healthy adult has very low levels of
total bilirubin (0.2–1.0 mg/dL) in the
serum, and of this amount, the majority is
in the unconjugated form.
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concentrations due to its ability to store
glucose as glycogen through glycogenesis
and
degrade
glycogen
through
glycogenolysis depending on the body’s
needs.
Under conditions of stress or in a fasting
state when there is an increased
requirement for glucose, the liver will
break down stored glycogen through
glycogenolysis to liberate glucose.
Gluconeogenesis: supply of glycogen
becomes depleted, the liver will create
glucose from nonsugar carbon substrates
like pyruvate, lactate, and amino acids.
Lipids
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B. Synthetic
▪ Liver is responsible for synthesizing many
biological
compounds
including
carbohydrates, lipids, and proteins.
Proteins
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Carbohydrates
The metabolism of carbohydrates is one
of the most important functions of the
liver.
▪ When carbohydrates are ingested and
absorbed, the liver can do three things:
a. use the glucose for its own cellular
energy requirements
b. circulate the glucose for use at the
peripheral tissues
c. store
glucose
as
glycogen
(principal storage form of glucose)
within the liver itself or within other
tissues
▪ The liver is the major player in
maintaining
stable
glucose
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Synthesized in the liver under normal
circumstances when nutrition is adequate
and the demand for glucose is being met.
The liver is responsible for gathering free
fatty acids from the diet, and those
produced by the liver itself, and breaking
them down to produce acetyl-CoA.
Acetyl-CoA can then enter several
pathways
to
form
triglycerides,
phospholipids, or cholesterol.
~70% of the daily production of cholesterol
(roughly 1.5–2.0 g) is produced by the
liver.
The liver is also involved in the
metabolism of lipids and their removal
from the body through the use of
lipoproteins and apoproteins.
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Almost all proteins are synthesized by the
liver except for the immunoglobulins and
adult hemoglobin.
The liver plays an essential role in the
development of hemoglobin in infants.
One of the most important proteins
synthesized by the liver is albumin, which
carries with it a wide range of important
functions.
The liver is also responsible for
synthesizing the positive and negative
acute-phase reactants and coagulation
proteins, and it also serves to store a pool
of amino acids through protein
degradation.
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C. Detoxification and Drug Metabolism
▪ First pass
- Substance that is absorbed in the
GIT through the liver.
- It can allow important substances to
reach the systemic circulation and
can serve as a barrier to prevent
toxic or harmful substances from
reaching the systemic circulation.
▪ The body has two mechanisms for
detoxification of foreign materials (drugs
and poisons) and metabolic products
(bilirubin and ammonia).
▪ The most important mechanism is the
drug-metabolizing system of the liver.
▪ This system is responsible for the
detoxification of many drugs through
oxidation,
reduction,
hydrolysis,
hydroxylation,
carboxylation,
and
demethylation.
▪ Many of these take place in the liver
microsomes via the cytochrome P-450
isoenzymes.
LIVER FUNCTION ALTERATIONS DURING
DISEASE
A. Jaundice
▪ Comes from the French word jaune,
which means “yellow,” and it is one of
the oldest known pathologic conditions
reported.
▪ Jaundice, or icterus, is used to describe
the yellow discoloration of the skin, eyes,
and mucous membranes most often
resulting from the retention of bilirubin;
however, it may also occur due to the
retention of other substances.
▪ Although the upper limit of normal for
total bilirubin is 1.0–1.5 mg/dL, jaundice
is usually not noticeable to the naked
eye (known as overt jaundice) until
bilirubin levels reach 3.0 mg/dL.
▪ Icterus: most commonly used in the
clinical laboratory to refer to a serum or
plasma sample with a yellow
discoloration due to an elevated bilirubin
level.
▪ Jaundice is most commonly classified
based on the site of the disorder:
a. Prehepatic Jaundice
- Referred to as unconjugated
hyperbilirubinemia
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Occurs when the problem causing
the jaundice occurs prior to liver
metabolism
Caused by increased amount of
bilirubin being presented to the
liver such that seen in acute and
chronic hemolytic anemia.
Hemolytic anemia causes an
increased amount of red blood cell
destruction and the subsequent
release of increased amounts of
bilirubin presented to the liver for
processing.
People with from prehepatic
jaundice rarely have bilirubin levels
that exceed 5 mg/dL because the
liver is capable of handling the
overload.
This
fraction
of
bilirubin
(unconjugated bilirubin) is not
water soluble, is bound to albumin,
and is not filtered by the kidneys
and therefore will not be seen in the
urine.
b. Hepatic Jaundice
- Occurs when the primary problem
causing the jaundice resides in the
liver (intrinsic liver defect or
disease).
- This intrinsic liver defect or disease
can be due to disorders of bilirubin
metabolism and transport defects
(Crigler-Najjar syndrome, DubinJohnson
syndrome,
Gilbert
disease, and neonatal physiologic
jaundice of the newborn) or due to
diseases resulting in hepatocellular
injury or destruction.
- Hepatic causes of jaundice that
results in the elevation in
unconjugated bilirubin.
a. Gilbert disease
b. Crigler-Najjar Syndrome
c. Physiologic jaundice of the
newborn
- Hepatic causes of jaundice that
results in elevation in conjugated
bilirubin.
a. Dubin-Johnson Syndrome
b. Rotor Syndrome
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Gilbert Syndrome
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A benign hereditary disorder
Most common cause, and interestingly, it
carries no morbidity or mortality of those
affected and carries generally no clinical
consequences
It is characterized by intermittent
unconjugated hyperbilirubinemia in the
absence of hemolysis and underlying liver
disease due to a defective conjugation
system.
Usually
manifests
during
adolescence or early adulthood.
Total serum bilirubin: 20–50 mol/L, and it
rarely exceeds 85 mol/L.
Molecular basis: related to UGT (uridine
diphosphoglucose
glucuronyltransferase)
superfamily, which is responsible for
encoding enzymes that catalyze the
conjugation of bilirubin.
UGT1A1 (the hepatic 1A1 isoform of UGT):
contributes substantially to the process of
conjugating bilirubin. The UGT1A1 promoter
contains the sequence (TA)6TAA.
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Rotor Syndrome
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Rare and is more serious disorder
Syndrome of chronic non-hemolytic
unconjugated hyperbilirubinemia.
An inherited disorder of bilirubin metabolism
resulting from a molecular defect within the
gene involved with bilirubin conjugation.
Type 1: where there is a complex absence of
enzymatic bilirubin conjugation.
Type 2: where there is a mutation causing
severe deficiency of the enzyme responsible
for bilirubin conjugation.
Dubin-Johnson Syndrome
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Is a rare inherited disorder caused by a
deficiency of the canalicular multidrug
resistance/multispecific organic anionic
transporter protein (MDR2/cMOAT). The
liver’s ability to uptake and conjugate
bilirubin is functional; however, the removal
of conjugated bilirubin from the liver cell and
the excretion into the bile are defective.
A condition that is obstructive in nature, so
much of the conjugated bilirubin circulates
bound to albumin.
This type of bilirubin (conjugated bilirubin
bound to albumin) is referred to as delta
bilirubin.
A reduction in the concentration or activity
of intracellular binding proteins such as
ligandin.
Relatively benign condition and carries an
excellent prognosis, and therefore treatment
is not warranted.
However, an accurate diagnosis is required
to aid in distinguishing it from more serious
liver diseases that require treatment
Physiologic Jaundice of the Newborn
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Crigler-Najjar Syndrome
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Distinguishing feature: appearance of darkstained granules (thought to be pigmented
lysosomes) on a liver biopsy sample.
Total bilirubin concentration: 2–5 mg/dL with
more than 50% due to the conjugated
fraction.
Relatively mild in nature with an excellent
prognosis.
No treatment is necessary
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Result of a deficiency in the enzyme
glucuronyl transferase, one of the last liver
functions to be activated in prenatal life
since bilirubin processing is handled by the
mother of the fetus.
In premature births, infants may be born
without glucuronyl transferase, the enzyme
responsible for bilirubin conjugation.
This deficiency results in the rapid buildup of
unconjugated bilirubin, which can be life
threatening.
Kernicterus often results in cell damage and
death in the newborn, and this condition will
continue until glucuronyl transferase is
produced.
Treatment: UV radiation to destroy the
bilirubin as it passes through the capillaries
of the skin.
c. Post hepatic Jaundice
- Results from biliary obstructive
disease, usually from physical
obstructions
(gallstones
or
tumors), that prevent the flow of
conjugated bilirubin into the bile
canaliculi.
- Since the liver cell itself is
functioning, bilirubin is effectively
conjugated; however, it is unable
to be properly excreted from the
liver.
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Since bile is not being brought to
the intestines, stool loses its source
of normal pigmentation and
becomes clay-colored.
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B. Cirrhosis
▪ A clinical condition in which scar tissue
replaces normal, healthy liver tissue.
▪ As the scar tissue replaces the normal
liver tissue, it blocks the flow of blood
through the organ and prevents the liver
from functioning properly.
▪ Signs and Symptoms: fatigue, nausea,
unintended weight loss, jaundice,
bleeding from the gastrointestinal tract,
intense itching, and swelling in the legs
and abdomen.
▪ Generally, have a poor prognosis.
▪ Most common cause:
a. Chronic alcoholism
b. Hepatitis C virus infection
▪ Other causes:
a. Chronic Hepatitis B and D virus
infection
b. Autoimmune hepatitis
c. a1-antitrypsin deficiency
d. Wilson disease
e. Hemochromatosis
f. Galactosemia
g. Non-alcoholic
h. Steatohepatitis
i. Blocked bile ducts
j. Drugs
k. Toxins
Cirrhosis caused by alcohol abuse is
treated by abstaining from alcohol.
Treatment for hepatitis-related cirrhosis
involves medications used to treat the
different types of hepatitis, such as
interferon for viral hepatitis and
corticosteroids for autoimmune hepatitis.
C. Tumors
▪ Primary Liver Cancer: is cancer that
begins in the liver cells.
▪ Metastatic Liver Cancer
- occurs when tumors from other
parts of the body spread
(metastasize)
- much more common
- 90%–95%
of
all
hepatic
malignancies are classified as
metastatic.
▪ Cancers that commonly spread to the
liver:
- Colon cancer
- Lung cancer
- Breast cancer
- Liver cancer: benign or malignant
▪ Common benign cancers of the liver:
- Hepatocellular adenoma (rare
condition
occurring
almost
exclusively in females of childbearing age)
- Hemangiomas (masses of atypical
blood
vessels
usually
mesenchymal in origin with no
known etiology)
▪ Malignant tumors of the liver:
- Hepatocellular carcinoma
▪ most common malignant
tumor
▪ develops in those with liver cell
damage
that
eventually
progresses to cirrhosis, which
is predisposing condition
▪ Treatment: liver
transplantation
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Hepatocarcinoma
Hepatoma
D. Reye Syndrome
▪ A term used to describe a group of
disorders caused by infectious, metabolic,
toxic, or drug induced disease found
almost exclusively in children, although
adult cases of Reye syndrome have been
reported.
▪ Often preceded by a viral syndrome such
as varicella, gastroenteritis, or an upper
respiratory tract infection such as
influenza.
▪ Cause: ingestion of aspirin during a viral
syndrome
and
the
subsequent
development of Reye syndrome (not
precise).
▪ Centers for Disease Control and
Prevention cautioned physicians and
parents to avoid salicylate use in children
with a viral syndrome
▪ Reyes syndrome is an acute illness
characterized
by
noninflammatory
encephalopathy and fatty degeneration
of the liver with a clinical presentation of
profuse vomiting accompanied with
varying degrees of neurologic impairment
such as fluctuating personality changes
and deterioration in consciousness.
▪ The encephalopathy is characterized by a
progression from mild confusion (stage 1)
through progressive loss of neurologic
function to loss of brainstem reflexes
(stage 5).
▪ The degeneration of the liver is
characterized
by
a
mild
hyperbilirubinemia
and
threefold
increases in ammonia and the
aminotransferases (AST and ALT).
E. Drug- and Alcohol-Related Disorders
▪ Liver is a primary target organ for
adverse drug reactions because it plays a
central role in drug metabolism.
▪ Immune-mediated
injury
to
the
hepatocytes
- In this type of mechanism, the drug
induces an adverse immune
response directed against the liver
itself and results in hepatic and/or
cholestatic disease.
▪
Ethanol
- Most important drug associated
with hepatic toxicity
- Small amount of ethanol causes
very mild, transient, and unnoticed
injury to the liver; however, with
heavier
and
prolonged
consumption, it can lead to
alcoholic cirrhosis.
- Long-term excessive consumption
of alcohol:
a. Alcoholic fatty liver
- Represents the mildest category
where very few changes in liver
function are measurable.
- This stage is characterized by
slight elevations in AST, ALT, and
GGT, and on biopsy, fatty
infiltrates are noted in the
vacuoles of the liver.
- This stage tends to affect young
to middle-aged people with a
history of moderate alcohol
consumption.
- A complete recovery within 1
month is seen when the drug is
removed.
b. Alcoholic hepatitis
- Presents with far more evidence
of liver damage such as
moderately elevated AST, ALT,
GGT, and ALP and elevations in
total bilirubin up to 30 mg/dL.
- Serum
proteins,
especially
albumin, are decreased and the
prothrombin time is prolonged.
- Prognosis is dependent on the
type and severity of damage to
the liver
c. Alcoholic cirrhosis
- Most severe stage
- The prognosis associated with
alcoholic cirrhosis is dependent
on the nature and severity of
associated conditions such as a
gastrointestinal bleeding or
ascites; however, the 5-year
survival rate is 60% in those who
abstain from alcohol and 30% in
those who continue to drink.
- This condition appears to be
more common in males than in
females, and the symptoms
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tend to be nonspecific and
include weight loss, weakness,
hepatomegaly, splenomegaly,
jaundice,
ascites,
fever,
malnutrition, and edema.
- Laboratory abnormalities:
a. increased liver function
tests (AST, ALT, GGT, ALP,
total bilirubin)
b. decreased albumin
c. a prolonged prothrombin
time
- Diagnosis: liver biopsy
Acetaminophen
- Most common drugs associated
with serious hepatic injury.
- When taken in massive doses, it
produces fatal hepatic necrosis
unless rapid treatment is initiated.
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ASSESSMENT OF LIVER FUNCTION/LIVER
FUNCTION TESTS
A. Bilirubin
A.1. Analysis of Bilirubin: A Brief
Overview
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Principle: rxn of bilirubin with a diazotized
sulfuric acid sol’n (diazo rxn) to form a
colored product.
Malloy and Evelyn: developed the first
clinically useful methodology for the
quantitation of bilirubin in serum samples
using the classic diazo reaction with a
50% methanol solution as an accelerator.
Commonly used method.
Jendrassik and Grof: described a method
using the diazo reaction with caffeinebenzoate-acetate as an accelerator.
Total bilirubin and conjugated bilirubin
are measured and unconjugated bilirubin
is determined by subtracting conjugated
bilirubin from total bilirubin.
Bilirubinometry
- Useful in the neonatal population
because of the presence of
carotinoid compounds in adult
serum that causes strong positive
interference in the adult population.
- Involves the measurement of
reflected light from the skin using
two wavelengths that provide a
numerical index based on spectral
reflectance.
Microspectrophotometer: determine the
optical densities of bilirubin, hemoglobin,
and melanin in the subcutaneous layers
of the infant’s skin.
Three fractions of bilirubin/Total Bilirubin
a. Unconjugated bilirubin (indirect)
- A nonpolar and water-insoluble
substance that is found in plasma
bound to albumin.
- Because of these characteristics,
unconjugated bilirubin will only react
with the diazotized sulfanilic acid
solution (diazo reagent) in the
presence
of
an
accelerator
(solubilizer).
b. Conjugated bilirubin (direct)
- A polar and water-soluble compound
that is found in plasma in the free
state (not bound to any protein).
- This type of bilirubin will react with the
diazotized sulfanilic acid solution
directly (without an accelerator).
c. Delta Bilirubin
- Conjugated bilirubin that is covalently
bound to albumin.
- This fraction of bilirubin is seen only
when there is significant hepatic
obstruction.
- Because the molecule is attached to
albumin, it is too large to be filtered by
the glomerulus and excreted in the
urine.
- This fraction of bilirubin, when
present, will react in most laboratory
methods as conjugated bilirubin
A.2. Specimen Collection and Storage
▪
Spx
a. Serum: preferred for Malloy-Evelyn
procedure because the addition of the
alcohol in the analysis can precipitate
proteins and cause interference with
the method
b. Plasma
c. Fasting sample: preferred as the
presence of lipemia will increase
measured bilirubin concentrations.
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NOTE
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Hemolyzed samples should be avoided as
they may decrease the reaction of
bilirubin with the diazo reagent.
Bilirubin is very sensitive to and is
destroyed by light; therefore, specimens
should be protected from light.
If left unprotected from light, bilirubin
values may reduce by 30%–50% per hour.
If serum or plasma is separated from the
cells and stored in the dark, it is stable for
2 days at room temperature, 1 week at
4°C, and indefinitely at -20°C.
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A.3. Methods
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Modified
Jendrassik-Grof
Method:
reference method for total bilirubin using
caffeine-benzoate as a solubilizer.
Jendrassik-Grof or Malloy-Evelyn: most
frequently used method to measure
bilirubin.
Advantage of Jendrassik-Grof method:
a. Not affected by pH changes
b. Insensitive to a 50-fold variation in
protein concentration of the sample
c. Maintains optical sensitivity even at
low bilirubin concentrations
d. Has minimal turbidity and a
relatively constant serum blank
e. Is not affected by hemoglobin up to
750 mg/dL
A.3.1. Malloy-Evelyn Procedure
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Bilirubin pigments in serum or plasma are
reacted with a diazo reagent.
The diazotized sulfanilic acid reacts at the
central methylene carbon of bilirubin to
split the molecule forming two molecules
of azobilirubin.
pH: 1.2
Azobilirubin produced: red-purple in color
Absorption: 560 nm.
Accelerator: methanol, to solubilize
unconjugated bilirubin.
A.3.2. Jendrassik-Grof Method for
Total and Conjugated Bilirubin
Determination
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acid in hydrochloric acid and sodium
nitrite), resulting in the production of the
purple product azobilirubin.
Measured spectrophotometrically.
Determined by taking two aliquots of
sample and reacting one aliquot with the
diazo reagent only and the other aliquot
with the diazo reagent and an accelerator
(caffeine-benzoate).
Caffeine-benzoate will solubilize the
water-insoluble fraction of bilirubin and
will yield a total bilirubin value (all
fractions).
The reaction of the aliquots with the diazo
reagent is terminated by the addition of
ascorbic acid.
- The ascorbic acid destroys the
excess diazo reagent.
It is then alkalinized using an alkaline
tartrate solution, which shifts the
absorbance spectrum of the azobilirubin
to a more intense blue color that is less
subject to interfering substances in the
sample.
The final blue product is measured at 600
nm with the intensity of color produced
directly
proportional
to
bilirubin
concentration.
Indirect (unconjugated) = conjugated
bilirubin concentration - total bilirubin
concentration.
Comments and Sources of Error
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Instruments should
be frequently
standardized to maintain reliable
bilirubin results, and careful preparation
of bilirubin standards is critical as these
are subject to deterioration from
exposure to light.
Hemolysis and lipemia should be avoided
as they will alter bilirubin concentrations.
Serious loss of bilirubin occurs after
exposure to fluorescent and indirect and
direct sunlight; therefore, it is imperative
that exposure of samples and standards
to light be kept to a minimum.
Specimens and standards should be
refrigerated in the dark until testing can
be performed.
Principle
▪
Bilirubin pigments in serum or plasma are
reacted with a diazo reagent (sulfanilic
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B. Urobilinogen in Urine and Feces
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Urobilinogen: is a colorless end product of
bilirubin metabolism that is oxidized by
intestinal bacteria to the brown pigment
urobilin.
Increased levels of urinary urobilinogen
are found in hemolytic disease and in
defective liver-cell function, such as that
seen in hepatitis.
Absence of urobilinogen from the urine
and stool is most often seen with
complete biliary obstruction.
Fecal urobilinogen is also decreased in
biliary obstruction, as well as in HCC.
B.1.
Determination
of
Urine
Urobilinogen (Semiquantitative)
Principle
- Reacts
with
p-dimethyl
aminobenzaldehyde (Ehrlich’s reagent)
to form a red color, which is then
measured spectrophotometrically.
- Ascorbic acid is added as a reducing
agent to maintain urobilinogen in the
reduced state.
- The use of saturated sodium acetate
stops the reaction and minimizes the
combination of other chromogens with
the Ehrlich’s reagent.
▪ Specimen: A fresh 2-hour urine specimen
is collected. This specimen should be kept
cool and protected from light.
▪ Reference range: Urine urobilinogen, 0.1–
1.0 Ehrlich units every 2 hours or 0.5–4.0
Ehrlich units per day (0.86.8 mmol/day); 1
Ehrlich unit is equivalent to approximately
1 mg of urobilinogen
b. Compounds, other than urobilinogen,
that may be present in the urine and react
with
Ehrlich’s
reagent
include
porphobilinogen, sulfonamides, procaine,
and 5-hydroxyindoleacetic acid. Bilirubin
will form a green color and, therefore,
must be removed, as previously
described.
c. Fresh urine is necessary, and the test
must be performed without delay to
prevent oxidation of urobilinogen to
urobilin.
Similarly,
the
spectrophotometric readings should be
made within 5 minutes after color
production because the urobilinogenaldehyde color slowly decreases in
intensity
B.2. Fecal Urobilinogen
▪
▪
▪
COMMENTS AND SOURCES OF ERROR
It is carried out in an aqueous extract of
fresh feces, and any urobilin present is
reduced to urobilinogen by treatment
with alkaline ferrous hydroxide before
Ehrlich’s reagent is added.
Reference range: A range of 75–275
Ehrlich units per 100 g of fresh feces or 75–
400 Ehrlich units per 24-hour specimen is
considered a normal reference range
C. Serum Bile Acids
▪
Rarely performed because the
methods required are very
complex.
D. Enzymes
▪
▪
Liver enzymes play an important role in
the assessment of liver function because
injury to the liver resulting cytolysis or
necrosis will cause the release of enzymes
into circulation.
Enzymes also play an important role in
differentiating hepatocellular (functional)
from obstructive (mechanical) liver
disease, which is an important clinical
distinction because failure to identify an
obstruction will result in liver failure if the
obstruction is not rapidly treated.
a. The results of this test are reported in
Ehrlich units rather than in milligrams of
urobilinogen because substances other
than urobilinogen account for some of the
final color development.
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where it may remain elevated up to
several weeks post delivery.
D.1. Aminotransferases
▪
▪
▪
▪
▪
Two most common aminotransferases:
a. AST/SGOT
- widely distributed in equal amounts
in the heart, skeletal muscle, and liver
b. ALT/SGPT
- found mainly in the liver (lesser
amounts in skeletal muscle and
kidney)
- a more “liver-specific” marker
Responsible for catalyzing the conversion
of aspartate and alanine to oxaloacetate
and pyruvate, respectively.
In the absence of acute necrosis or
ischemia of other organs, these enzymes
are most useful in the detection of
hepatocellular (functional) damage to the
liver.
Remain elevated for up to 2–6 weeks.
The highest levels of AST and ALT are
found in acute conditions such as viral
hepatitis, drug- and toxin-induced liver
necrosis, and hepatic ischemia.
D.2.2. 5’-Nucleotidase
▪
▪
▪
▪
D.2.3. y-Glutamyltransferases
▪
D.2. Phosphatases
D.2.1. Alkaline Phosphatase
▪
▪
▪
▪
▪
▪
Zinc metalloenzymes that are widely
distributed in all tissues; however, highest
activity is seen in the liver, bone, intestine,
kidney, and placenta.
Ability to differentiate hepatobiliary
disease from osteogenic bone disease.
In the liver, the enzyme is localized to the
microvilli of the bile canaliculi, and
therefore it serves as a great marker of
extrahepatic biliary obstruction, such as a
stone in the common bile duct, or in
intrahepatic cholestasis, such as drug
cholestasis or primary biliary cirrhosis.
Found in very high concentrations in
cases of extrahepatic obstruction with
only slight to moderate increases seen in
those with hepatocellular disorders such
as hepatitis and cirrhosis.
ALP, may also elevated in bone-related
disorders such as Paget’s disease, bony
metastases, diseases associated with an
increase in osteoblastic activity, and
rapid bone growth during puberty.
ALP is also found elevated in pregnancy
due to its release from the placenta,
A phosphatase that is responsible for
catalyzing the hydrolysis of neucleoside5-phosphate esters.
Found in a wide variety of cells, serum
levels become significantly elevated in
hepatobiliary disease.
Levels of both 5NT and ALP are elevated
in liver disease, whereas in primary bone
disease, ALP level is elevated, but the
5NT level is usually normal or only
slightly elevated.
This enzyme is much more sensitive to
metastatic liver disease than is ALP
because, unlike ALP, its level is not
significantly elevated in other conditions,
such as pregnancy or childhood.
▪
▪
▪
▪
Is a membrane-localized enzyme found
in high concentrations in the kidney,
liver, pancreas, intestine, and prostate
but not in bone.
Plays a role in differentiating the cause
of elevated levels of ALP as the highest
levels of GGT are seen in biliary
obstruction.
GGT is a hepatic microsomal enzyme;
therefore, ingestion of alcohol or certain
drugs
(barbiturates,
tricyclic
antidepressants, and anticonvulsants)
elevates GGT.
It is a sensitive test for cholestasis
caused by chronic alcohol or drug
ingestion.
Measurement of this enzyme is also
useful if jaundice is absent for the
confirmation of hepatic neoplasms
D.2.4. LDH
▪
▪
is an enzyme with a very wide
distribution throughout the body. It is
released into circulation when cells of
the body are damaged or destroyed,
serving as a general, nonspecific marker
of cellular injury.
Moderate elevations of total serum LDH
levels are common in acute viral
hepatitis and in cirrhosis, whereas
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biliary tract disease may produce only
slight elevations.
High serum levels may be found in
metastatic carcinoma of the liver
Fractionation of LDH into its five tissuespecific isoenzymes may give useful
information about the site of origin of the
LDH elevation.
▪
Tests Measuring Hepatic Synthetic Ability
▪
▪
▪
▪
▪
▪
▪
▪
▪
▪
▪
▪
▪
▪
▪
▪
The measurement of serum proteins can be
used to assess the synthetic ability of the
liver.
Useful in quantitating the severity of hepatic
dysfunction.
A decreased serum albumin may be a result
of decreased liver protein synthesis, and the
albumin level correlates well with the
severity of functional impairment and is
found more often in chronic rather than in
acute liver disease.
Serum a-globulins: decrease with chronic
liver disease.
Low or absent a-globulin: a-antitrypsin
deficiency as the cause of the chronic liver
disease.
Serum y-globulin levels
- increased in acute liver disease
- remain elevated in chronic liver disease
- found in chronic active hepatitis and
post necrotic cirrhosis.
IgG and IgM levels: elevated in chronic active
hepatitis
IgM: in primary biliary cirrhosis
IgA: in alcoholic cirrhosis.
Prothrombin time: increased in liver disease
because the liver is unable to manufacture
adequate amounts of clotting factor or
because the disruption of bile flow results in
inadequate absorption of vitamin K from the
intestine.
Serial measurements of prothrombin: useful
in following the progression of disease and
the assessment of the risk of bleeding.
A marked prolongation of the prothrombin
time indicates severe diffuse liver disease
and a poor prognosis.
▪
▪
▪
▪
Plasma ammonia level: is a reflection of the
liver’s ability to perform this conversion.
In liver failure, ammonia and other toxins
increase in the bloodstream and may
ultimately cause hepatic coma.
Adsorption of NADP+: 340 nm
Spx: plasma collected in EDTA, heparin, or
potassium oxalate
Samples is placed on ice to prevent
metabolism
of
other
nitrogenous
compounds to ammonia.
Frozen samples are stable for several days.
Hemolyzed sample should be rejected for
analysis as RBCs have a concentration of
ammonia 2-3 times higher than that of
plasma.
Hepatitis
▪
▪
▪
▪
▪
Characterized by presence of inflammation
in the liver tissue.
Viral infections account for the majority of
hepatitis cases observed in the clinical
setting.
Symptoms: jaundice, dark urine, fatigue,
nausea, vomiting, and abdominal pain
Hepatitis B and C: can lead to the prolonged
elevation of serum transaminase level
(longer than 6 months), a condition termed
chronic hepatitis
Transmission
- Hepatitis A and E:
ingestion of
contaminated food or water.
- Hepatitis B, C, and D: parenteral contact
with infected body fluids (e.g., from
blood transfusions or invasive medical
procedures
using
contaminated
equipment) and sexual contact.
Tests Measuring Nitrogen Metabolism
▪
The liver plays a major role in removing
ammonia from the bloodstream and
converting it to urea so that it can be
removed by the kidneys.
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recommended that all children receive the
HAV vaccine as early as age 12–23 months.
Hepatitis A
▪
▪
▪
▪
▪
▪
▪
▪
▪
▪
Known as infectious hepatitis or shortincubation hepatitis.
Most common form of viral hepatitis
worldwide. It is caused by a nonenveloped
RNA virus of the Picornavirus family.
Source of infection:
in the household
occurring via contaminated or improperly
handled food.
HAV is excreted in bile and shed in the feces,
which can contain up to 109 infectious
virions per gram.
Transmission: fecal oral route
Symptoms: fever, malaise, anorexia,
nausea, abdominal discomfort, dark urine,
and jaundice.
Symptoms are generally self-limited and
resolve within 3 weeks. However, in rare
instances, patients develop fulminant liver
failure.
Chronic infection with HAV is not found and
there is no evidence of a carrier state or
long-term sequelae in humans
Diagnosis:
A. Serologic Abs
- IgM anti-HAV:
▪ Detectable at or prior to the onset of
clinical illness and decline in 3–6
months,
when
it
becomes
undetectable
by
commercially
available diagnostic tests.
▪ Primary marker of acute infection
- IgG anti-HAV:
▪ Persist for years after infection,
and confer lifelong immunity.
▪ The presence of elevated titers of
IgG anti-HAV in the absence of IgM
indicates a past infection.
B. Viral antigen: another reliable method to
detect acute infection in px is assaying for
the presence of viral Ag.
C. Amplification of Viral RNA by RT-PCR
D. Nucleic Acid Detection Techniques
- more sensitive than immunoassays for
viral antigen to detect HAV in samples
of different origins (e.g., clinical
specimens, environmental samples, or
food).
- useful to determine the extent to which
unidentified infection occurs.
Food and Drug Administration/Centers for
Disease Control and Prevention (FDA/CDC)
Hepatitis B
▪
▪
▪
▪
▪
▪
▪
Known as serum hepatitis or longincubation hepatitis
Can cause both acute and chronic hepatitis
and is the most ubiquitous of the hepatitis
viruses.
Highest incidence of acute hepatitis B was
among adults aged 25–45 years.
Stable in the environment and remains
viable for longer than 7 days on
environmental
surfaces
at
room
temperature.
Detected in virtually all body fluids, including
blood, feces, urine, saliva, semen, tears, and
breast milk
Three major routes of transmission:
a. Parenteral
b. Perinatal
c. Sexual
Who is at risk:
a. Persons who engage in the sharing of
body fluids, such as high-risk sexual
behaviors (e.g., prostitution, male
homosexuality) and the sharing of druginjection needles.
b. Children born to mothers who are
hepatitis B surface antigen–positive at
the time of delivery
c. Immigrants from endemic areas
d. Sexual partners and household contacts
of patients who have hepatitis B are
high-risk groups for HBV infection.
e. Health care workers: laboratory
personnel
Serologic Markers of HBV Infection
▪
▪
▪
▪
▪
HBV is a 42-nm DNA virus classified in the
Hepadnaviridae family.
The liver is the primary site of HBV
replication.
The core of the antigen is synthesized in the
nuclei of hepatocytes and then passed into
the cytoplasm of the liver cell, where it is
surrounded by the protein coat.
An antigen presents in the core of the virus
(HBcAg) and a surface antigen present on
the surface protein (HBsAg) have been
identified by serologic studies.
Another antigen, called the e antigen
(HBeAg), also has been identified
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Hepatitis B Surface Antigen
Previously known as the Australia antigen
and hepatitis associated antigen (HAA)
▪ HBsAg
- Is the antigen for which routine testing is
performed on all donated units of blood.
- Useful serologic marker in patients before
the onset of clinical symptoms because it
is present during the prodrome of acute
hepatitis B.
- Only serologic marker detected during
the first 3–5 weeks after infection in newly
infected patients.
- The average time from exposure to
detection of HBsAg is 30 days (range, 6–
60 days).
- HBsAg positivity has been reported for up
to 18 days after hepatitis B vaccination
and is clinically insignificant.
▪ Highly sensitive single sample nucleic acid
tests can detect HBV DNA in the serum of an
infected person 10–20 days before detection
of HBsAg.
▪
In patients who have chronic HBV infection,
the IgM anti-HBc antibody titer can persist
during chronic viral replication at low levels.
▪ DN-dependent DNA polymerase
- Another marker for acute infection
- Closely associated with the presence of
the core antigen.
- This viral enzyme is required for viral
replication and is detectable in serum
early in the course of viral hepatitis,
during the phase of active viral
replication.
▪
Hepatitis B e Antigen
▪
▪
▪
e Antigen
- Closely associated with the core of the
viral particle, is detected in the serum of
persons with acute or chronic HBV
infection.
- Appears to correlate well with both the
number of infectious virus particles and
the degree of infectivity of HBsAgpositive sera.
The presence of HBeAg in HBsAg carriers is
an unfavorable prognostic sign and predicts
a severe course and chronic liver disease.
Conversely, the presence of anti-HBe
antibody in carriers indicates a low
infectivity of the serum
Hepatitis B Core Antigen
▪
▪
▪
▪
This antigen is present only in the nuclei of
hepatocytes during an acute infection with
HBV.
Anti-HBc usually develops earlier in the
course of infection than the antibody to the
surface antigen.
A test for the IgM antibody to HBcAg was
recently developed as a serologic marker for
clinical use.
The presence of this IgM antibody is specific
for acute hepatitis B infection.
▪
The e antigen is detected in serum only when
surface antigen is present
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Persons who recover from natural infection
typically will be positive for both anti-HBs
and anti-HBc, whereas persons who respond
to hepatitis B vaccine have only antiHBs.
▪ Persons who become chronically infected fail
to develop antibody to the HBsAg, resulting
in the persistent presence of HBsAg as well
as the presence of anti-HBc in patient serum,
typically for life.
▪ HBeAg and anti-HBe screenings: are used for
the management of patients with chronic
infection.
▪ Diagnosis: Nucleic acid hybridization or PCR
technique
- used to detect HBV DNA in the blood and
is another method used to measure
disease progression
-
▪
-
Provides a more sensitive measurement
of infectivity and disease progression than
serology.
It may be used to monitor the
effectiveness of antiviral therapy in
patients with chronic HBV infection, but it
supplements rather than replaces current
HBV serologic assays.
Chronic Infection With HBV
▪
▪
Approximately 25% of persons who were
chronically infected since childhood and 15%
of those who were chronically infected since
adulthood die prematurely from cirrhosis or
liver cancer.
The presence of HBsAg in chronically infected
patients is an indication that they are
infectious and at risk for developing
complications, including cirrhosis and HCC.
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HBV Treatment and Prevention
Chronic Hepatitis C Infection
Hepatitis B vaccination: is the most effective
measure to prevent HBV infection and
therefore
obviate
its
consequences,
including cirrhosis of the liver, liver cancer,
liver failure, and death.
▪ HBsAg: is the antigen used for hepatitis B
vaccination.
▪ The hepatitis B immune globulin (HBIG)
- Provides passively acquired anti-HBs and
temporary protection (i.e., 3–6 months)
when administered in standard doses.
- used as an adjunct to hepatitis B vaccine
for postexposure immunoprophylaxis to
prevent HBV infection.
- For nonresponders to hepatitis B
vaccination, HBIG administered alone is
the primary means of protection after an
HBV exposure.
▪
▪
▪
▪
▪
▪
▪
Hepatitis C
▪
▪
▪
▪
▪
originally “non-A non-B hepatitis”
caused by a virus with an RNA genome that
is a member of the Flaviviridae family.
Transmission:
parentally;
transmitted
primarily
by
blood
transfusion
of
inappropriately screened blood product.
HCV infection has a high rate of progression
to chronic hepatitis, cirrhosis, and liver
carcinoma, making HCV a major cause of
chronic hepatitis in the United States.
HCV infection is a leading cause of liver
transplantation in this country.
Laboratory Tests for Hepatitis C
▪
▪
Almost always be present in the later stages
Two laboratory tests are commonly used to
diagnose HCV infection in clinical practice:
a. Anti-HCV
detection
by
enzyme
immunoassay (EIA)
▪ most common
▪ Detect antibodies generated in
response to HCV infection.
▪ These patients (anti-HCV positive but
HCV RNA negative) are recommended
to retake the test for HCV RNA on a
second occasion, 3–6 months after the
first HCV RNA test
b. Quantitative nucleic acid PCR assays for
serum HCV RNA
▪ If anti-HCV gives a positive result, it is
the next step to perform
Most chronically infected patients are
asymptomatic and manifest only mild
elevations of liver function tests, especially
transaminases.
Alcohol consumption concomitant with
chronic HCV infection significantly increases
the risk of cirrhosis.
Liver biopsies are performed periodically in
these patients, with the degree of
inflammation and fibrosis correlating with
the risk of cirrhosis.
Patients with chronic HCV infection are
usually treated with pegylated interferon
and ribavirin.
Therapeutic efficacy is monitored by using
PCR to determine the number of viral copies
in serum.
A prototypic envelope peptide-based
vaccine has been reported to induce
antibodies in human subjects, but there is no
evidence for the presence of a neutralizing
antibody against HCV.
Hepatitis D
▪
▪
▪
▪
▪
▪
▪
▪
▪
Is a unique subvirus satellite virus infection.
It is a small, defective RNA-containing virus
that cannot replicate independently but
rather requires the HBsAg of HBV for
replication.
Incapable of causing any illness in patients
who do not have HBV infection.
Mode of Transmission: similar to HBV
~5% of the global HBV carriers are coinfected
with HDV, leading to a total of 10–15 million
HDV carriers worldwide.
HDV virions possess an outer envelope
composed of HBsAg proteins and host
membrane lipids and an inner nucleocapsid
consisting of viral RNA and hepatitis delta
antigen (HDAg).
HDV infection can occur concurrently with
HBV infection (coinfection) or in a patient
with
established
HBV
infection
(superinfection)
HDV superinfection is likely to become
chronic simply because HBV infection is
already chronic.
In general, in the acute phase, HDV
superinfected carriers may develop severe
hepatitis, and around 70%–90% will progress
to chronicity.
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▪
▪
▪
▪
▪
Diagnosis: detection of antibodies against
HDAg and serum HDV RNA, as well as HBV
markers.
Accurate diagnosis is made by a negative
test for IgM anti HBc and confirmed by the
detection of HDV markers.
HBV vaccine has resulted in a decline in the
incidence of hepatitis D.
Interferon-a: therapy used for treating
chronic HDV infection.
Treatment: requires a higher dosage and a
longer duration of treatment, and
posttreatment relapses are common.
Hepatitis E
▪
▪
▪
▪
▪
▪
▪
▪
RNA-containing HEV
Nonenveloped RNA virus
Size: 27–34 nm in diameter
Genus: Hepevirus
Family: Hepeviridae.
After infection, the incubation period is short,
generally between 21 and 42 days prior to
the onset of symptoms.
Detected in: feces and bile by about 7 days
after infection.
Transmission:
- primarily by the fecal-oral route, and
waterborne
epidemics
are
characteristic of hepatitis E in many
developing countries.
- In industrialized countries, several
nonhuman primates such as pigs, cows,
and sheep are susceptible to infection
with HEV, leading to the potential spread
of the virus through zoonosis
▪ Diagnosis
- Is made by biochemical assessment of
liver function.
- Acute hepatitis E is diagnosed when the
presence of IgM anti-HEV is detected.
- The presence of a high or increasing antiHEV IgG titer may support the diagnosis
of acute HEV infection, and in such cases
acute hepatitis E can be presumed even
in the absence of IgM anti- HEV.
- EIA and immunochromatography: are
most convenient for the detection of IgM
and/or IgG antiHEV.
- RT-PCR has a limited confirmatory role.
- Acute-phase HEV RNA can be detected in
feces by PCR in approximately 50% of
cases.
- Experimental
immune
prophylaxis
against HEV based on recombinant
antigens appears to confer short-term
protection and may be useful for
pregnant women in endemic areas and
travelers coming into these region
Other Forms of Hepatitis
▪
▪
▪
▪
Hepatitis G: currently unclear
Hepatitis F: is an enteric agent that may be
transmitted to primates
The GB group of flavo-like viruses, GBV-A,
GBVB, and GBV-C, are also associated with
acute and chronic hepatitis
Cytomegalovirus, Epstein-Barr virus, and
probably several other agents can also
cause hepatitis.
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