To determine the amount of sgot in given serum samples in easy language procedure in easy language
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SGOT AST serum determination procedure biochemistry lab simple method
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SGOT determination serum Reitman Frankel colorimetric method procedure steps biochemistry practical
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SGOT determination procedure steps reagent principle normal values
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Determination of SGOT in Given Serum Samples
What is SGOT?
SGOT stands for Serum Glutamate Oxaloacetate Transaminase. It is also called AST (Aspartate Aminotransferase). It is an enzyme present in the liver, heart, kidneys, and muscles. When these organs are damaged, SGOT leaks into the blood, so its level rises.
Why Do We Test It?
- To check liver disease (hepatitis, cirrhosis)
- To check heart attack (myocardial infarction)
- To check muscle diseases
- As part of a routine Liver Function Test (LFT)
Method Used
The most commonly used method in biochemistry labs is the Reitman and Frankel Colorimetric Method (DNPH method).
Principle (Simple Explanation)
SGOT in the serum converts L-Aspartate + Alpha-Ketoglutarate into Oxaloacetate + Glutamate.
The Oxaloacetate produced reacts with 2,4-Dinitrophenylhydrazine (DNPH) to form a brown-colored compound (hydrazone).
The deeper the color, the higher the SGOT activity.
Color is measured in a spectrophotometer and the result is calculated using a standard graph.
In short:
More SGOT → More Oxaloacetate → Darker color → Higher reading
Materials Required
| Item | Purpose |
|---|---|
| Patient's serum | Test sample |
| SGOT substrate (L-Aspartate + Alpha-Ketoglutarate in buffer) | Provides the reaction ingredients |
| DNPH reagent (2,4-Dinitrophenylhydrazine) | Color developer |
| 0.4 N NaOH solution | Develops the final brown color |
| Pyruvate standard solutions | For making the calibration curve |
| Test tubes | To hold the reactions |
| Water bath at 37°C | For incubation |
| Spectrophotometer | To measure absorbance (color intensity) |
| Pipettes | For measuring volumes accurately |
Step-by-Step Procedure
Step 1 - Set Up the Tubes
Set up 3 types of tubes:
| Tube | What it is | SGOT Substrate | Serum | Buffer |
|---|---|---|---|---|
| Test (T) | Patient sample | 0.5 mL | 0.1 mL | - |
| Blank (B) | Control for background | 0.5 mL | - | 0.1 mL |
| Standard (S) | Known pyruvate amount | Already prepared | - | - |
Step 2 - Incubation
- Mix the contents of each tube well.
- Place all tubes in a water bath at 37°C for exactly 60 minutes.
- This allows the SGOT enzyme to work on the substrate.
Step 3 - Add Color Developer (DNPH)
- After incubation, add 0.5 mL of DNPH reagent to all tubes (Test, Blank, Standard).
- Mix well and let stand at room temperature for 20 minutes.
- DNPH reacts with the oxaloacetate to form a hydrazone (a colored product).
Step 4 - Add NaOH
- Add 5 mL of 0.4 N NaOH to all tubes.
- Mix well and wait 5 minutes.
- NaOH intensifies the brown/red color so it can be measured easily.
Step 5 - Read Absorbance
- Set the spectrophotometer to 505 nm wavelength.
- Set the blank as zero (zero the machine with the blank tube).
- Read the absorbance (OD) of the Test tube and the Standard tubes.
Step 6 - Calculate SGOT Activity
- Plot absorbance values of the pyruvate standards against their known concentrations to make a standard/calibration curve.
- Read the SGOT activity of the Test from this curve.
- Result is expressed in IU/L (International Units per Litre) or Karmen Units (KU).
Normal Values
| Group | Normal Range |
|---|---|
| Adult Men | 10 - 40 IU/L |
| Adult Women | 10 - 35 IU/L |
| Children | Slightly higher than adults |
What Happens if SGOT is High?
| Condition | SGOT Level |
|---|---|
| Liver diseases (hepatitis, cirrhosis) | Raised |
| Heart attack | Rises within 4-8 hours, peaks at 2-3 days |
| Muscle damage | Raised |
| Kidney disease | Raised |
Important Notes
- Use fresh serum (no hemolysis - red color in serum can interfere).
- Maintain exact 37°C temperature during incubation.
- Time of incubation must be exactly 60 minutes - more or less time gives wrong results.
- The blank tube corrects for any background color in the reagents.
- Alcohol consumption before the test can raise SGOT falsely.
Quick Summary (Flowchart)
Patient Serum
↓
Mix with SGOT Substrate
↓
Incubate at 37°C for 60 min
↓
Add DNPH (color developer) → Wait 20 min
↓
Add NaOH → Wait 5 min
↓
Read absorbance at 505 nm
↓
Compare with Standard Curve
↓
Get SGOT value in IU/L
This is the standard Reitman and Frankel colorimetric method taught in most biochemistry practical courses. For automated analyzers in modern labs, the IFCC UV-Kinetic method (at 340 nm) is used, which is faster and more precise, but the principle is the same - measuring the enzyme's activity by how fast it converts its substrate.
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