1. Bilirubin & Amylase
Lab. 10

2. 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
Derived from breakdown of hemoglobin

3. Bilirubin There are two types of bilirubin:
conjugated (direct),
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.

4. 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.

5. 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.
Alcohol must be added for the reaction to occur.

6. Unconjugated Bilirubin
Direct
Indirect
Water soluble
Water insoluble
Found in urine
Not found in urine
Does not need accelerator
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

7. Clinical Significance
Increased Conjugated Bilirubin
Biliary obstruction
Cancer of the head of the pancreas
Dubin-Johnson syndrome
Increased Unconjugated Bilirubin
Autoimmune hemolysis
Crigler-Najjer syndrome
Gilbert’s syndrome
Hemolytic transfusion reaction
Hepatitis

8. 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:
1 day at °C
7 days at °C
3 months at - 20 °C (if frozen immediately)

9. 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.

10. 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.

11. 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:
abdominal pain due to acute pancreatitis
from other causes of abdominal pain that may require surgical treatment.

12. 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.

13. Clinical Significance
Increased
Acute pancreatitis
Alcoholism
Hyperlipidemia
Inflammation of salivary glands
Decreased
Cystic fibrosis

14. 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.

15. Principle
(PNP = p-Nitrophenol, G =Glucose)