Download Liver-and-Pancreatic

Bilirubin &
Amylase
Lab. 10
Bilirubin
• One of the most important liver
functions, and one that is disturbed in
a large number of diseases is the
excretion of bile
• Bile comprises bile acids or salts, bile
pigments & others
• Bilirubin is the principal pigment in bile
o Derived from breakdown of hemoglobin
Bilirubin
• There are two types of bilirubin:
o conjugated (direct),
o and unconjugated (indirect).
• Total bilirubin is composed of the
conjugated bilirubin plus the
unconjugated bilirubin.
• The total bilirubin level increases with
any type of jaundice.
Conjugated Bilirubin
• Normally, direct or conjugated, bilirubin is
excreted by the gastrointestinal (GI) tract, with
only minimal amounts entering the bloodstream.
• Its level rises in the blood when obstructive
jaundice (as from gallstones) or hepatic
jaundice occurs,
• In this case the bilirubin is unable to reach the
intestines for excretion and instead, enter the
bloodstream for excretion by the kidneys.
• Conjugated bilirubin is the only type of bilirubin
able to cross the glomerular filter; thus it is the
only type of bilirubin that can be found in the
urine.
Unconjugated Bilirubin
• Indirect bilirubin, or unconjugated bilirubin,
is normally found in the bloodstream.
• Unconjugated bilirubin rises in cases of
hemolytic jaundice,
• the breakdown of hemoglobin results in a higher than
normal level of unconjugated bilirubin being present in the
bloodstream.
• This is the type of bilirubin elevated in cases
of hepatocellular dysfunction, such as
hepatitis.
Conjugated Bilirubin
Direct
Water soluble
Unconjugated Bilirubin
Indirect
Water insoluble
Found in urine
Does not need accelerator
Not found in urine
Needs accelerator
Total bilirubin = Conjugated + Unconjugated bilirubin
• Typically, only the total bilirubin is reported.
• If the total bilirubin is abnormal, further testing is
done to differentiate the level of direct and indirect
bilirubin.
Total bilirubin – Conjugated = Unconjugated bilirubin
Clinical Significance
• Increased Conjugated Bilirubin
• Biliary obstruction
• Cancer of the head of the pancreas
• Dubin-Johnson syndrome
• Increased Unconjugated Bilirubin
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Autoimmune hemolysis
Crigler-Najjer syndrome
Gilbert’s syndrome
Hemolytic transfusion reaction
Hepatitis
Specimen & Storage
• Total bilirubin determinations using a diazo method
require either serum or plasma.
• Bilirubin is very sensitive to and is destroyed by light
and heat; therefore specimens should be protected
from ambient light prior to and during analysis.
• Concentrations may decrease by 30% to 50% per
hour if exposed to direct sunlight.
• If separated and stored in the dark, serum or plasma
is stable for:
o 1 day at 15 - 25 °C
o 7 days at 2 - 8 °C
o 3 months at - 20 °C (if frozen immediately)
Specimen & Storage
• Conjugated bilirubin may be determined in
either serum or plasma,
• Urine samples can be analyzed by direct
diazo methods, since the polar conjugated
bilirubin is not protein bound and is filtered
at the glomerulus and excreted into urine.
Assay Principle
• Bilirubin in normal serum reacted with diazo reagent
(diazotized sulfanilic acid) only when alcohol was
added
• The pigment that reacted in the absence of alcohol
was termed “direct” (conjugated).
• The pigment that required the presence of alcohol was
termed the “indirect” (unconjugated) bilirubin fraction.
• The unconjugated bilirubin is a nonpolar molecule and
is not soluble in water.
• Consequently it will react with diazo reagent only in the
presence of an agent (historically called an
accelerator), such as alcohol, in which both bilirubin
and the diazo reagent are soluble.
Amylase
• Amylase is an enzyme found primarily
in the pancreas and salivary glands.
• Its function is to assist in the digestion of
complex carbohydrates into simple
sugars.
• Measurement of serum amylase is
often performed to differentiate:
o abdominal pain due to acute pancreatitis
o from other causes of abdominal pain that may require
surgical treatment.
• The serum amylase begins to rise 3 to 6 hours after
the onset of acute pancreatitis and peaks in
approximately 24 hours.
• The values return to normal within 5 days after
onset.
• For confirmation of an acute pancreatitis
measurement of lipase should be additionally
performed.
• The amylase present in the blood is eliminated
through the kidney and excreted into the urine.
• Therefore, elevation of serum activity is reflected in
a rise of urinary amylase activity.
Clinical Significance
• Increased
o Acute pancreatitis
o Alcoholism
o Hyperlipidemia
o Inflammation of salivary glands
• Decreased
o Cystic fibrosis
Principle
• Enzymatic photometric test, in which
the substrate EPS-G7 is cleaved by α amylases into various fragments.
• These are further hydrolyzed in a
second step by α -glucosidase
producing glucose and p-nitrophenol.
• The increase in absorbance represents
the total (pancreatic and salivary)
amylase activity in the sample.
Principle
• (PNP = p-Nitrophenol, G =Glucose)