Liver Function Test

Measurement of Serum Bilirubin

The functions of the liver include:
1. The liver is in the upper right part of the abdomen. Is storing glycogen
(fuel for the body) which is made from sugars.
2. Helping to process fats and proteins from digested food.
3. Making proteins that are essential for to clot (clotting factors).
4. Processing many drugs which you may take.
5. Helping to remove toxins from the body.

Liver function tests can be classified as:

1. Tests of excretion by the liver.
2. Evaluation of synthesis in liver.
3. Evaluation of enzymes activity.

Serum Bilirubin
Bilirubin is a yellow pigment that is in every ones blood and stool. Sometimes
the liver cannot process the bilirubin in the body because of excess bilirubin, an
obstruction, or an inflamed liver. Bilirubin is made in the body when old red
blood cells are broken down. The breakdown of old cells is a normal, healthy
process. When your body has too much bilirubin, your skin and the whites of
your eyes will start to yellow. This condition is called jaundice.
The bilirubin molecule formed in the spleen is called indirect or
unconjugated bilirubin. After circulating in your blood, bilirubin then travels
to your liver. Bilirubin conjugated with glucuronic acid by glucuronyl transferase
enzyme (by forming an ester linkage between glucuronic acid and the propionic
acid side chain of bilirubin) is called direct or conjugated bilirubin. In the
liver, bilirubin is excreted into the bile duct and is excreted into the duodenom
where conjugated bilirubin is converted by bacteria into urobilinogen and
urobilin. Urobilin is excreted with feces while urobilinogen is reabsorbed from
the liver to the blood stream and excreted via the kidney.
If bilirubin is not conjugated with glucuronic acid in the liver or is not being
adequately removed from the blood, it can mean that there is damage to your

liver. Testing for bilirubin in the blood is therefore a good test of damage to your
liver.

The normal values of :

s. total bilirubin : 0.01 - 1 mg / dl
s. direct bilirubin : 0.0 0.2 mg / dl
s. indirect bilirubin : 0.2 0.8 mg / dl

Clinical significance:
The determination of serum bilirubin is employed for the detection on
subclinical or occult jaundice, which characterize by founding orange dye on
the skin the sclera and the mucous membranes. Hyperbilirubinemia is
frequently will found in the following types of jaundice:
1. Hemolytic jaundice:
Caused by over production of bilirubin due to excessive hemolysis. Occur
in acute and chronic hemolytic anemia. This condition is usually
associated with increased value of serum indirect bilirubin.
2. Hepatic jaundice:
Caused by some type of intra hepatic obstruction which leads to cirrhosis
of liver and hepatitis. The production of bilirubin is not increased, but
accumulates and it discharged back into the blood. In these conditions.
The indirect bilirubin predominates in the early phase, but as the liver
damage progresses, the direct form also becomes elevated.
3. Obstructive jaundice:
Caused by a post hepatic blockage of the large bile passages, particularly
the common bile duct resulting in a reflex of bilirubin into the blood. This
condition is associated with elevated of serum direct bilirubin only.

Method of serum bilirubin determination:

Principle:

Van Den Bergh and his co- workers applied the diazo reaction in 1913 (first
used by Ehrlich in 1883). The diazo reaction is:
H2SO3

NaNO2

Diazotized sulfanilic acid

Sulfanilic acid + Na- nitrite

DSA

Both forms of the bilirubin (direct and total bilirubin) give a pink azo bilirubin
with diazotrized sulfanilic acid.

Measurement of Serum Transaminase Enzyme

Activities
Alanine Amino Transfers (ALT):
Its called also Glutamate Pyruvate Transaminase (GPT). Is the enzyme
produced with the cells of the liver. The level of ALT abnormality is increased in
3

conditions where cells of the liver have been inflamed or undergone cell death.
As the cells are damaged, the ALT leaks into the bloodstream leading to a rise
in the serum levels. Any form of hepatic cell damage can result in an elevation
in ALT. ALT is the most sensitive marker for liver cell damage.
Clinical applications of ALT assays are confined mainly to evaluation of
hepatic disorders.
Higher elevations are found in hepatocellular disorders than in extra
hepatic or intra hepatic obstructive disorders.
In acute inflammatory conditions of the liver, ALT elevated higher than
AST. (More specific than AST).
It catalyzes the transfer of amino group from L- alanine to - ketoglutarate, the
products of this reversible transamination reaction being pyruvate and Lglutamate.

The enzyme properties:

1. Naming the enzyme depends on the type of substrate and direction of the
reaction if the direction of interaction in front of, substrate alanine acid
and keto glutaric acid named ALT while if the direction of reaction reverse
named GPT.
2. AST found mainly in liver despite the presence in heart, skeletal muscle
and kidney.

3. The optimum condition of maximum enzyme activity are, pH = 7.4 and

temperature = 37 C.
4. The enzyme activity is inhibited by anticoagulantes, therefore serum
preferred to use rather than plasma.
5. The enzyme stability: 3 days in 25 C, 1 week in 5 C, and 1 month in -25 C.
6. Normal value : 2-15 I.U/ L
2-38 mole/min/ L

Clinical significance of GPT:

The GPT activity in tissues is generally less than GOT. Significant elevation of
SGPT occurs in sever acute hepatitis where the enzyme is released into the
circulation. However GPT level is increased in the following diseases:
1. Infection hepatitis.
2. Liver cirrhosis and billiard cirrhosis.
3. Obstructive jaundice.

Aspartate Aminotransferase (AST) :

Its called also Glutamate Oxalacetate Transaminase Enzyme (GOT). This
enzyme also reflects damage to the hepatic cell. It is less specific for liver
disease. It may be elevated and other conditions such as a myocardial infract
(heart attack). Although AST is not a specific for liver as the ALT, ratios between
ALT and AST are useful to physicians in assessing the etiology of liver enzyme
abnormalities. AST is one of a number of different enzymes that transfers an
amino acid of aspartic acid - ketoglutaric acid forming glutamic acid and
oxaloacetic acid.

The enzyme properties:

1. Naming the enzyme depends on the type of substrate and direction of the
reaction if the direction of interaction in front of, substrate aspartic acid
and keto glutaric acid named AST while if the direction of reaction reverse
named GOT.
2. AST found mainly in heart despite the presence in liver, skeletal muscle
and kidney.
3. The optimum condition of maximum enzyme activity are, pH = 7.4 and
temperature = 37 C.
4. The enzyme activity is inhibited by anticoagulants, therefore serum
preferred to use rather than plasma.
5. The enzyme stability: 3 days in 25 C, 1 week in 5 C, and 1 month in -25 C.
6. Normal value : 2-20 I.U/ L
2-23mole/min/ L

Clinical significance of GPT:

1. Heart disease.
2. Liver disease.
3. Muscular disease.

The enzymes GOT and GPT unit:

The enzyme activity is measured by international unit, which may be define as:

The activity of enzyme that produced 1 mole of oxaloacetic acid or pyruvate

from aspartic acid or alanine respectively at 1 min per liter of serum under
certain conditions of temperature 37 C and pH 7.4.

Measurement of Serum Alkaline Phosphatase (ALP)

Alkaline phosphates are an enzyme, which is associated with the billiard tract. It
is not specific to the billiard tract. It is also found in bone and the placenta. If
the alkaline phosphates are elevated, billiard tract damage and inflammation
should be considered. One of the more common method to assess the etiology
of the elevated alkaline phosphates is to determine whether the Gama
Glutamic Transpeptidase (GGT) is elevated or whether other function tests are
abnormal (such as bilirubin). Alkaline phosphates may be elevated in primary
billiard cirrhosis, alcoholic hepatitis.
Normal value:
Adults
3-13 KAU/dl
Children 6-25 KAU/dl

Clinical signification

Increased serum ALP:

1. Bone disease:
Pagets disease; increased 10-25 time than normal value.
Rickets; increased 2-4 times than normal value.
Bone cancer.
2. Hepatobiliary disease:
Obstructive jaundice.
Billiard obstruction.
The enzyme activity is increased also in the pregnancy (increased bone
formation), growing children.
Increased serum ALP:
1.
2.
3.
4.
5.

Vit.C deficiency.
Chronic nephritis.
Hyperparathyroidism.
Kwashiorkor.
Sever anemia.

King Armstrong unit of ALP activity: it is unit which release 1 mg from

phenol during 15 min. under 37C pH=10.

Principle:
King Armstrong method:
During incubation with ALP, the almost colorless substrate, p-nitro phenyl
phosphate, is hydrolyzed to p-nitro phenol, which is yellow in alkaline solution.
Addition of NaOH stops the enzyme reaction and beings out the product color
which measured at 405 nm.