Liver Function Tests

Definition -

• Biomarkers that are indicators of liver dysfunction
• Tests used for diagnosing & monitoring of liver diseases.

Classification - 

I.  TESTS BASED ON EXCRETORY FUNCTION

1.SERUM BILIRUBIN- 

• The main excretory product of liver is bile.
• It is formed and stored in liver.
• Normal plasma bilirubin-
•  Total bilirubin - 0.2–0.8 mg/ dL.
• Unconjugated Bilirubin is about 0.2–0.6 mg/dL,
• Conjugated Bilirubin is about 0–0.2 mg/dL.

A.Tests to assess serum bilirubin  - VandenBergh Reaction

• Procedure - Bilirubin reacts with diazo reagent (diazotized sulfanilic acid) to produce colored azo pigment.
• At pH 5, the pigment is purple in color.
• Conjugated bilirubin, being water soluble gives the color immediately; hence called Direct reaction.
• Unconjugated bilirubin is water insoluble. It has to be extracted first with alcohol, and then the reaction becomes positive; hence called Indirect reaction.
• If both conjugated and unconjugated bilirubin are present in increased amounts, a purple color is produced immediately and the color is intensified on addition of alcohol. Then, the reaction is called biphasic reaction.

Other methods for detection of serum bilirubin

• Jendrassik Grof Method
• Malloy-evelyn Method

B.Icterus Index

• The intensity of yellow colour of the serum serves as measure of severity of jaundice.
• Yellow color of the serum is diluted with sodium citrate solution and compare with the known standards of potassium dicromate solutions.
• Normal values of icterus index are 4 to 6 units.
• Values >10 units indicate jaundice.

2. URINE BILIRUBIN

• In normal individuals, bilirubin is not excreted in the urine.
• Accumulation of bilirubin in blood because of any disease leads to the presence of bilirubin in urine
• Common finding in obstructive jaundice
• Only conjugated bilirubin is seen in urine

Tests used to find out presence of bile pigments in urine

1.Gmelin's test –

• To 5 ml of conc. HNO3 5 ml of urine is added along the wall of the test tube.
• Formation of green,blue or purple ring indicates the presence of bilirubin in urine.

2. Fouchet's test-

• 10ml urine + few ml of BaCl2
• Filter & filtrate remain on filter paper
• Add one drop of FeCl3
• Gree Blue Spot - positive

3. URINARY UROBILINOGEN

• The amount of urobilinogen present in urine depends on the amount of bilirubin entering the intestine.
•  Urine urobilinogen is estimated semi-quantitatively, by Ehrlich’s aldehyde reagent(para dimethyl amino benzaldehyde)

4. URINE BILE SALTS

• Normally bile salts (sodium salts of taurocholic acid and glycocholic acid) are present in the bile; but are not seen in urine.
• Bile salts in urine are detected by Hay’s test.

5. DYE EXCRETION TESTS

• In addition to excreting bilirubin, the liver is capable of eliminating various dyes or drugs by the same excretory pathway as bilirubin.

Bromosulfophthalein excretion test

• Procedure - 5% solution of BSP is injected intravenously (the dose is 5 mg/kg body wt)
• After 45 minutes blood sample is collected
• Checked - for percentage of injected dye remaining in the blood.
• Normal - < 5% of dye of BSP retention at 45 minutes.
• Abnormal - >5% of dye BSP retention indicates liver disease

II.SYNTHETIC FUNCTION

• Liver is the main source of synthesis of plasma proteins,e.g. Albumin,Globulin,Blood clotting factors, e.g. fibrinogen, prothrombin and factors V, VII, IX, X.
• Impaired function of liver results in decreased protein synthesis.

Determination Of Serum Albumin and Globulin

The normal concentrations

• Total serum protein = 6 to 8 gm/dl
• Serum Albumin = 3.5 To 5.5 gm/dl
• Serum Globulin = 2 to 3.5 gm/dl
• Albumin/globulin ratio = 1.2:1 to 2.5:1.

Clinical Interpretation

• Hypoalbuminemia -hepatocellular disease, e.g. cirrhosis.
• Hyperglobulinemia - chronic inflammatory disorders such as cirrhosis and in infectious hepatitis.

Determination Of Prothrombin Time

• Various proteins that participate in blood coagulation are synthesized in the liver, e.g. fibrinogen, prothrombin (factor II) and factors V, VII, IX and X.
• PT indicates the present function of the liver.

III-DETOXIFICATION FUNCTION

          1.Arterial Ammonia Levels

• It is a useful test in hepatic encephalopathy,cirrhosis
• Estimation of ammonia may be helpful to exclude or diagnose hepatic failure in patients with unexplained stupor or coma.

•     2.Hippuric Acid Test

•     Can be done by oral or iv method.

Oral test –

• 6 gms of sodium benzoate is administered along with water orally.
•  urine sample is collected after 4 hours.
• Principle – benzoic acid + glycine ---à Hippuric acid (excreted in urine)
• Amount of hippuric acid excreted is estimated in the urine sample.
• Normal - 2.5 gms or more of hippuric acid is excreted in 1 hour
• Abnormal < 2.5 gms of hippuric acid in the urine output indicates intrahepatic disease.

IV.METABOLIC FUNCTION

1.Tests related to carbohydrate metabolism

    A.Galactose tolerance test

• Galactose is converted to glucose exclusively in liver.
• Procedure - 40 gm of galactose dissolved in water is given
• Urine sample collected after 5 hour.
• Normal - <3 gm of galactose excreted in 5 hour collection of urine.
• Abnormal – 4 to 5gm of galactose is excreted in urine.
•  In patients with hepatic disease, this conversion ability of galactose to glucose is defective.

2.Tests related to lipid metabolism

• Determination of serum total cholesterol and ratio of free to esterified cholesterol:
• Esterification occurs in liver and blood.
• Changes in the ratio of free to esterified cholesterol are often found in liver diseases.

3.Tests related to protein metabolism

• Serum estimation of proteins
• The liver is the principal site of metabolism and synthesis of plasma proteins and amino acids.
• Amino acids are metabolized in the liver to ammonia and urea.

V.SERUM ENZYMES

1.Alanine aminotransferase  (ALT)

• Normal serum   -- Male is 13–35 U/L , Female is 10–30 U/L.
• Very high level > 1000u/l seen in Acute Hepatitis

2.Aspartate aminotransferase  (AST):

• Normal serum level of AST is 8–20 U/L.
• A marked increase in AST may be seen in primary hepatomas.

3.Alkaline Phosphatase :

• Normal level 3-13 KA units/100 ml (King Armstrong units).
• In the absence of bone disease and pregnancy, there are elevated ALP levels generally due to hepatobiliary disease.

4. Gamma Glutamyl Transferase (GGT) :

• GGT level in alcoholic liver disease roughly parallels the alcohol intake.

5. 5'-Nucleotidase:

• It is also called Nucleotide phosphatase (NTP).
• The level is increased in hepatobiliary disease and closely parallels the ALP levels

VI-SPECIAL TESTS

1.Ceruloplasmin (Cp):

•  It is mainly synthesized by the hepatic parenchymal cells and a small part by lymphocytes.

2.Alpha fetoprotein (AFP):

• Mild elevation is suggestive of chronic hepatitis or cirrhosis;
• Drastic increase is seen in hepatocellular carcinoma, germ cell tumors and teratoma of ovary

3.Alpha-1 Antitrypsin (AAT):

• It is an acute phase reactant and is synthesized and secreted by the liver.

4.Haptoglobin:

• It is synthesized in the liver.
• Low levels are seen with severe hepatocellular liver disease (deficient synthesis)