Biochemical Practice Session 6: Bilirubin Quantification and SGOT, SGPT Enzyme Activity Determination

I. Bilirubin Quantification

1. Principle:

• Based on the diazotization reaction of bilirubin with sulfanilic acid diazo to form a pink-colored azobilirubin compound.

• The intensity of the pink color of azobilirubin is directly proportional to the bilirubin concentration in serum.

2. Reagents and Materials:

• Sulfanilic acid diazo: Reagent formed by the reaction of sulfanilic acid with sodium nitrite.

• Sodium nitrite: Used to generate sulfanilic acid diazo.

• Dimethylsulfoxide: Used for total bilirubin quantification, adding glucuronidase enzyme to the sample to convert indirect bilirubin to direct bilirubin.

• Bilirubin standard:

• Total bilirubin: Concentration of 73.7 µmol/L

• Direct bilirubin: Concentration of 31.3 µmol/L

3. Procedure:

• Total bilirubin quantification:

• Dilute the sample with dimethylsulfoxide.

• Add glucuronidase enzyme.

• React with sulfanilic acid diazo.

• Measure absorbance at 555 nm.

• Direct bilirubin quantification:

• React the sample with sulfanilic acid diazo.

• Measure absorbance at 555 nm.

4. Results:

• Normal total bilirubin: 5.13 – 17.1 µmol/L

• Normal direct bilirubin: Trace amounts (negligible)

5. Clinical Significance:

• Increased total bilirubin: In diseases causing jaundice (biliary obstruction, hepatitis, hemolysis).

• Increased indirect bilirubin:

• Neonatal jaundice syndrome.

• Gilbert’s syndrome (deficiency of glucuronyl transferase enzyme).

• Increased direct bilirubin:

• Biliary obstruction syndrome (common bile duct obstruction).

• Liver cell damage pathologies (cirrhosis, hepatitis, liver cancer).

Note:

• Bilirubin levels can be influenced by various factors such as:

• Age.

• Gender.

• Medications.

• Diet.

• Other pathologies.

II. SGOT and SGPT Enzyme Activity Quantification

1. Principle:

• Kinetic enzyme assay method: Based on measuring the rate of change in reactant or product concentration in an enzyme-catalyzed reaction.

• SGOT and SGPT enzyme activity is quantified by measuring the rate of decrease in absorbance of NADH+ at 340 nm.

• The decrease in absorbance of NADH+ is directly proportional to the activity of SGOT and SGPT enzymes.

2. Reagents and Materials:

• ASAT (SGOT): Aspartate aminotransferase (serum glutamate oxaloacetate transferase).

• ALAT (SGPT): Alanine aminotransferase (serum glutamate pyruvate transferase).

• MDH (malate dehydrogenase): Used to quantify SGOT enzyme activity.

• LDH (lactate dehydrogenase): Used to quantify SGPT enzyme activity.

3. Procedure:

• Add the sample to the reaction solution containing NADH, substrate, and the corresponding enzyme (ASAT/MDH for SGOT, ALAT/LDH for SGPT).

• Measure absorbance at 340 nm.

• Calculate enzyme activity using the formula:

• Activity [U/L] = (E/minute) x 1746.

4. Results:

• Normal GOT activity at 37°C: <46 U/L

• Normal GPT activity at 37°C: <49 U/L

5. Clinical Significance:

• Increased SGOT and SGPT: In liver cell damage pathologies, especially hepatitis, cirrhosis, liver cancer.

• SGOT/SGPT (De Ritis Ratio):

• >2: Alcoholic hepatitis.

• <1: Acute hepatitis.

• Elevated SGOT predominance: Cirrhosis, chronic hepatitis, alcoholic hepatitis, myocardial infarction.

• Elevated SGPT predominance: Acute hepatitis.

Note:

• SGOT is primarily located in the mitochondria of liver cells.

• SGPT is primarily located in the cytoplasm of liver cells.

• SGOT and SGPT enzyme activity can be affected by various factors such as:

• Age.

• Gender.

• Medications.

• Diet.

• Other pathologies.

6. Pathologies Possibly Associated with SGOT and SGPT Changes:

• Cirrhosis: Increased SGOT and SGPT, predominantly elevated SGOT.

• Chronic hepatitis: Increased SGOT and SGPT, predominantly elevated SGOT.

• Alcoholic hepatitis: Increased SGOT and SGPT with De Ritis ratio >2.

• Acute hepatitis: Increased SGOT and SGPT, predominantly elevated SGPT, De Ritis ratio <1.

• Myocardial infarction: Increased SGOT and SGPT, predominantly elevated SGOT.

• Muscle diseases: Muscular dystrophy, leading to increased SGOT and SGPT.

Note:

• Test results should be considered in conjunction with clinical symptoms and other tests to provide an accurate diagnosis.

Conclusion:

Bilirubin quantification and SGOT, SGPT enzyme activity tests are crucial in diagnosing and monitoring liver pathologies, helping to track treatment effectiveness and assess disease prognosis.