Bilirubin & Bilirubin & AmylaseAmylase

Lab. 10

BilirubinBilirubin• 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 bileo Derived from breakdown of hemoglobin

BilirubinBilirubin• 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 BilirubinConjugated 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 Unconjugated BilirubinBilirubin

• 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.

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

Conjugated Bilirubin Unconjugated Bilirubin

Direct Indirect

Water soluble Water insoluble

Found in urine Not found in urine

Does not need accelerator Needs accelerator

Clinical SignificanceClinical 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

Specimen & StorageSpecimen & 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 °Co 7 days at 2 - 8 °Co 3 months at - 20 °C (if frozen immediately)

Specimen & StorageSpecimen & 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 PrincipleAssay 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.

AmylaseAmylase• 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 SignificanceClinical Significance• Increased

oAcute pancreatitis oAlcoholism oHyperlipidemiao Inflammation of salivary glands

• DecreasedoCystic fibrosis

PrinciplePrinciple• 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.

PrinciplePrinciple

• (PNP = p-Nitrophenol, G =Glucose)