Liver Function

Reference: Bishop, M.L., Fody, E.P., Schoeff, L.E (2010). Clinical Ch

Chemistry:
emistry: Te
Techniques,
chniques, Principles,
Correlation (6th Edition). Lippincott Williams & Wilkins, a Walter Kluwer busines
businesss

Other references: Sir Mario’s Notes and Clinic

Clinical
al Chemistry (2019) by Rodriquez

FUNCTION
▪ Biochemical role in the metabolism
▪ Digestion
▪ Detoxification
▪ Elimination of substances from the body ▪ Bile canaliculi
▪ Excretory, synthetic, and metabolic - where the excretory system of the
functions liver begins
▪ Regenerate cells - small spaces between the
hepatocytes that form intrahepatic
ANATOMY ducts, where excretory products of
A. Gross Anatom
Anatomy
y 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 he
hepatic
patic ducts merge to form
the common hepatic duct, which is
eventually joined with the cystic duct of
the gallbladder to form the common bile
▪ Weight: ~1.2-1.5 kg duct.
▪ Located beneath and is attached to the ▪ Combined digestive secretions are then
diaphragm, is protected by the lower rib expelled into the duodenum
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 B. Microscopic Anatom
Anatomy
y
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 ▪ Lobules: functional unit of the liver; they
veins and ultimately into the inferior are responsible for all metabolic and
vena cava 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.
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.
- Li
Ligandin:
gandin: 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 bilir
bilirubin.
ubin.
▪ 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.
B. Synthetic
▪ Liver is responsible for synthesizing many
biological compounds including
carbohydrates, lipids, and proteins.
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
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
▪ 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.
Proteins
▪ 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. Detoxifi
Detoxification
cation 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 FUNCTI
FUNCTION
ON 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
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 Jau Jaundice
ndice
- Referred to as unc unconjugated
onjugated
hyperbi
hyperbilirubinemia
lirubinemia
- 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, Dubin-
Johnson 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 ▪ Distinguishing feature: appearance of dark-
stained granules (thought to be pigmented
▪ A benign hereditary disorder
lysosomes) on a liver biopsy sample.
▪ Most common cause, and interestingly, it
▪ Total bi
bilirubin
lirubin concentration: 2–5 mg/dL with
carries no morbidity or mortality of those
more than 50% due to the conjugated
affected and carries generally no clinical
fraction.
consequences
▪ Relatively mild in nature with an excellent
▪ It is characterized by intermittent
prognosis.
unconjugated hyperbilirubinemia in the
▪ No treatment is necessary
absence of hemolysis and underlying liver
disease due to a defective conjugation Rotor Syndrome
system. Usually manifests during
▪ A reduction in the concentration or activity
adolescence or early adulthood.
of intracellular binding proteins such as
▪ Total serum bilirubin: 20–50 mol/L, and it
ligandin.
rarely exceeds 85 mol/L.
▪ Relatively benign condition and carries an
▪ Molecular basis
basis:: related to UGT (uridine
excellent prognosis, and therefore treatment
diphosphoglucose glucuronyltransferase)
is not warranted.
superfamily, which is responsible for
▪ However, an accurate diagnosis is required
encoding enzymes that catalyze the
to aid in distinguishing it from more serious
conjugation of bilirubin.
liver diseases that require treatment
▪ UGT1A1 (the hepatic 1A 1A11 isoform of UG
UGT):
T):
contributes substantially to the process of Physiologic Jaundice of the Newborn
conjugating bilirubin. The UGT1A1 promoter
▪ Result of a deficiency in the enzyme
contains the sequence (TA)6TAA.
glucuronyl transferase, one of the last liver
Crigler
Crigler-Najjar
-Najjar Syndrome functions to be activated in prenatal life
since bilirubin processing is handled by the
▪ Rare and is more serious disorder
mother of the fetus.
▪ Syndrome of chronic non-hemolytic
▪ In premature births, infants may be born
unconjugated hyperbilirubinemia.
without glucuronyl transferase, the enzyme
▪ An inherited disorder of bilirubin metabolism
responsible for bilirubin conjugation.
resulting from a molecular defect within the
▪ This deficiency results in the rapid buildup of
gene involved with bilirubin conjugation.
unconjugated bilirubin, which can be life
▪ Type 1: where there is a complex absence of
threatening.
enzymatic bilirubin conjugation.
▪ Kernicterus often results in cell damage and
▪ Type 2: where there is a mutation causing
death in the newborn, and this condition will
severe deficiency of the enzyme responsible
continue until glucuronyl transferase is
for bilirubin conjugation.
produced.
Dubin-Johnson Syndrome ▪ Treatment: UV radiation to destroy the
bilirubin as it passes through the capillaries
▪ Is a rare inherited disorder caused by a
of the skin.
deficiency of the canalicular multidrug
resistance/multispecific organic anionic
c. Post hepatic Ja
Jaundice
undice
transporter protein (MDR2/cMOAT). The
- Results from biliary obstructive
liver’s ability to uptake and conjugate
disease, usually from physical
bilirubin is functional; however, the removal
obstructions (gallstones or
of conjugated bilirubin from the liver cell and
tumors), that prevent the flow of
the excretion into the bile are defective.
conjugated bilirubin into the bile
▪ A condition that is obstructive in nature, so
canaliculi.
much of the conjugated bilirubin circulates
- Since the liver cell itself is
bound to albumin.
functioning, bilirubin is effectively
▪ This type of bilirubin (conjugated bilirubin
conjugated; however, it is unable
bound to albumin) is referred to as delta
to be properly excreted from the
bilirubin.
liver.
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 - Since bile is not being brought to c. a1-antitrypsin deficiency
the intestines, stool loses its source d. Wilson disease
of normal pigmentation and e. Hemochromatosis
becomes clay-colored. 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 com commonly
monly spread to the
liver:
- Colon cancer
- Lung cancer
- Breast cancer
- Liver cancer: benign or malignant
B. Cirrhosis ▪ Common benign cancers of the liver:
▪ A clinical condition in which scar tissue - Hepatocellular adenoma (rare
replaces normal, healthy liver tissue. condition occurring almost
▪ As the scar tissue replaces the normal exclusively in females of child-
liver tissue, it blocks the flow of blood bearing age)
through the organ and prevents the liver - Hemangiomas (masses of atypical
from functioning properly. blood vessels usually
▪ Signs and Symptoms: fatigue, nausea, mesenchymal in origin with no
unintended weight loss, jaundice, known etiology)
bleeding from the gastrointestinal tract, ▪ Malignant tumors of the liver:
intense itching, and swelling in the legs - Hepatocellular carcinoma
and abdomen. ▪ most common malignant
▪ Generally, have a poor prognosis. tumor
▪ Most common cause: ▪ develops in those with liver cell
a. Chronic alcoholism damage that eventually
b. Hepatitis C virus infection progresses to cirrhosis, which
▪ Other causes: is predisposing condition
a. Chronic Hepatitis B and D virus ▪ Treatment: liver
infection transplantation
b. Autoimmune hepatitis
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 - Hepatocarcinoma
- Hepatoma
D. Reye Synd
Syndrome
rome
▪ 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 cconsumption
onsumption
of alcohol:
a. Alco
Alcoholic
holic 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.
ASSESSMENT OF LIVER FUNCTION/LIVER
FUNCTION TESTS
A. Bilirubin
A.1. Analysis of Bilirubin: A Brief
Overview
▪ 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 caffeine-
benzoate-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/
bilirubin/Total
Total Bilirubin
a. Unc
Unconjugated
onjugated 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. Conj
Conjugated
ugated bilir
bilirubin
ubin (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 Bilir
Bilirubin
ubin
- 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. Fa
Fasting
sting sample: preferred as the
presence of lipemia will increase
measured bilirubin concentrations.
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 NOTE
- 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.
A.3. Methods
▪ Modified Jendrassik-Grof Method:
reference method for total bilirubin using
caffeine-benzoate as a solubilizer.
▪ Jendrassik-Gr
Jendrassik-Gro of 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-E
Malloy-Evelyn
velyn Procedure
▪ 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
pH:: 1.2
▪ Azobilirubin produced
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
Principle
▪ Bilirubin pigments in serum or plasma are
reacted with a diazo reagent (sulfanilic
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
▪ 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.
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