UNIT II

SGOT and SGPT

2.1 Principle and procedure of estimation of SGOT

2.2 Principle and procedure of estimation of SGPT

2.3 Clinical significance of SGOT/SGPT estimation

Dr Pramila Singh

SGOT is Serum Glutamic-Oxaloacetic acid. SGOT is also known as AST. AST is Asparttate transaminase. SGPT is Serum Glutamic Pyruvic transaminase. SGPT is also known as ALT. ALT is Alanine transaminase. They are enzymes present in the liver cells. They catalyze amino acid metabolism. SGOT and SGPT are released into the blood after liver cell damage. The presence of SGOT and SGPT in blood indicates liver disease. Increased level of SGOT and SGPT is detected in alcohol abuse and several liver diseases such as hepatitis, cirrhosis, fatty liver, and other liver disorders.

Principle and procedure of estimation of SGOT

Principale: The principle of SGOT estimation depends upon the enzymatic activity. SGOT catalyzes the transfer of an amino group from aspartate to alpha-ketoglutarate. This forms oxaloacetate and glutamate.

The amount of oxaloacetate is determined after incubation. Oxaloacetate with dinitrophenyl hydrazine reagent in alkaline medium form hydrazone. Hydrazone is a highly colored compound that is measured by the colorimetric method.

Reagents:

1. SGOT substrate: It contains 2.66 gm aspartic acid, 30 mg alpha-ketoglutaric acid, 20 mL 1 N Sodium hydroxide solution in 100 mL phosphate buffer pH 7.45.

2. DNPH reagent: It contains 200 mg dinitrophenyl hydrazine and 85 mL of conc hydrochloric acid in distilled water.

3. 0.4 N sodium hydroxide.

4. 22 mg/dL sodium pyruvate standard.

Reagents 1 and 2 are stable at temperatures 2 to 8 degrees C.

Sample: Fresh serum

Procedure: Prepare two cuvettes one as blank and the other as a test cuvette. Mix sample and reagents as per the below details

1. Blank cuvette

• · SGOT Substrate 0.5 mL. Incubate at 37 degrees C for 60 minutes.

• · Add DNPH 0.5 mL, and srum 0.1 mL. Mix thoroughly and keep at a temperature of 25 degrees C for 20 minutes.

• · Add 5 mL 0.4 N sodium hydroxide. Mix well and keep at 25 degrees C for 10 minutes. Mix well and keep at 25 degrees C for 10 minutes

2. Test cuvette

• · SGOT Substrate 0.5 mL. Incubate at 37 degrees C for 5 minutes.

• · Add serum 0.1 mL, Incubate at 37 degrees C for 60 minutes.

• · Add DNPH 0.5 mL, and srum 0.1 mL. Mix thoroughly and keep at 25 degrees C for 20 minutes.

• · Add 5 mL 0.4 N sodium hydroxide. Mix well and keep at 25 degrees C for 10 minutes.

Measure the color intensity of the test against blank at 100% transmission  540 nm.

Calculate SGOT concentration by using a standard graph.

Normal values of SGOT: 7 to 21 U/L (In adults)

Principle and procedure of estimation of SGPT

Principle: SGPT in blood samples is determined by the UV-Kinetic method. Buffered alanine, NADH, LDH, and alpha oxoglutarate reagents are used in SGPT determination. alpha-oxoglutarate reacts with Alanine in the presence of SGPT to produce L-glutamate and pyruvate. Pyruvate reacts with NADH in the presence of LDH to release lactate and NAD. O.D. decreases with time. A decrease in OD is measured through spectrophotometry.

Normal values of SGPT

In men:

• Upto 22IU at 25 degrees C

• Upto 29IU at 30 degrees C

• Upto 40IU at 37 degrees C

In Women

• Upto 17IU at 25 degrees C

• Upto 22IU at 30 degrees C

• Upto 31IU at 37 degrees C

Reagents:

• 1. SGPT reagent

• 2. SGOT reagent

• 3. DNPH reagent.

• 4. 0.4N Sodium hydroxide

• 5. 22mg/dl,Sodium pyruvate standard.

• Fresh reagents should be preferred. Reagents 1 and 2 are stable at 2 to 8 degrees Celcius.

Procedure: Pipette in tubes as follows.

Reagents 1 and 2 are stable at temperatures 2 to 8 degrees C.

Sample: Fresh serum

Procedure: Prepare two cuvettes one as blank and the other as a test cuvette. Mix sample and reagents as per the below details

1. Blank cuvette

   • SGPT Substrate 0.5 mL. Incubate at 37 degrees C for 30 minutes.

   • Add DNPH 0.5 mL, and srum 0.1 mL. Mix thoroughly and keep at a temperature of 25 degrees C for 20 minutes.

   • Add 5 mL 0.4 N sodium hydroxide. Mix well and keep at 25 degrees C for 10 minutes. Mix well and keep at 25 degrees C for 10 minutes

2. Test cuvette

   • SGPT Substrate 0.5 mL. Incubate at 37 degrees C for 5 minutes.

   • Add serum 0.1 mL, Incubate at 37 degrees C for 30 minutes.

   • Add DNPH 0.5 mL, and srum 0.1 mL. Mix thoroughly and keep at a temperature of 25 degrees C for 20 minutes.

   • Add 5 mL 0.4 N sodium hydroxide. Mix well and keep at 25 degrees C for 10 minutes.

Measure the color intensity of the test against blank at 100% transmission. 540 nm (green filter).

Calculate SGPT concentration by using a standard graph.

Clinical significance of SGOT/SGPT estimation

SGOT and SGPT are enzymes mainly present in liver cells. But they are also found in small amounts in other cells such as cardiac muscles and skeletal muscle cells. Damage to liver cells releases SGOT and SGPT in the blood. They are mainly estimated to detect liver function tests (LFT). Increased SGPT and SGOT in the blood indicate liver diseases.

SGPT (ALT): Increased level of SGPT in blood indicates the following diseases

1. Liver diseases: Alcoholic hepatitis, viral hepatitis, cirrhosis, fatty liver disease, and drug-induced liver damage increase SGPT levels in blood. SGPT concentration In blood indicates the severity of liver damage. High concentration means severe liver cell damage.

2. Liver health monitoring: SGPT level in blood is estimated to monitor the effectiveness of liver disease treatment and to detect the progress of liver disease.

3. Medication side effects: Side effects of the drug on liver cells are checked by estimation of SGPT in blood.

SGOT (AST): SGOT is found in several cells such as liver cells, cardiac cells, renal cells, and muscle cells. An increased level of SGOT indicates the following disease

1. Liver diseases: Alcoholic hepatitis, viral hepatitis, cirrhosis, fatty liver disease, and drug-induced liver damage increase SGPT levels in blood. SGPT concentration In blood indicates the severity of liver damage. High concentration means severe liver cell damage.

2. Heart condition: SGOT estimation is more specific to cardiac cell damage than SGPT estimation. Increased level of SGOT indicates heart attack, congestive heart failure, and other heart diseases.

3. Skeletal muscle disorders: Muscle injury such as muscular dystrophy elevates SGOT level in blood.

   Dr Pramila Singh