Introduction
- Cason’s test is a biochemical test used to detect sphingomyelin, a major phospholipid found in biological membranes, especially in the myelin sheath of nerve fibers.
- It is a qualitative test that relies on a color reaction to confirm the presence of sphingomyelin or other phospholipids in biological samples.
- Phospholipids are essential for maintaining cell membrane integrity, signaling, and lipid metabolism.
- The abnormal accumulation of phospholipids, particularly sphingomyelin, is associated with neurological disorders and lipid storage diseases such as Niemann-Pick disease.
Cason’s test is particularly useful in:
- Biochemical studies to analyze phospholipids in tissue extracts and biological fluids.
- Medical diagnostics for diseases associated with phospholipid metabolism disorders.
- Food science and lipid analysis, especially in studying egg yolk lipids and other phospholipid-rich substances.
Principle
The principle of Cason’s test is based on the acid hydrolysis of sphingomyelin and subsequent reaction with ferric chloride (FeCl₃) to produce a blue or green coloration.
Reaction Mechanism:
- Hydrolysis of Sphingomyelin:
- When trichloroacetic acid (TCA) is added to the sample, it hydrolyzes phospholipids, breaking down their ester bonds and releasing phosphoric acid derivatives.
- Reaction with Ferric Chloride:
- The released phosphoric acid derivatives react with FeCl₃ to form a colored complex.
- The color intensity depends on the concentration of sphingomyelin in the sample.
- Color Development:
- A blue or green color appears if sphingomyelin is present.
- No color change indicates the absence of sphingomyelin.
Requirements
Reagents:
To perform Cason’s test, the following chemicals and solutions are required:
- Trichloroacetic Acid (TCA) (10%)
- Acts as a hydrolyzing agent that breaks down phospholipids into their basic components.
- Ferric Chloride (FeCl₃) (1%)
- Reacts with the hydrolyzed phospholipid components to produce a color reaction.
- Sulfuric Acid (H₂SO₄) (Conc.)
- Sometimes used to enhance hydrolysis and color development.
- Ethanol (Absolute Alcohol)
- Used to dissolve lipid samples and prepare solutions.
- Distilled Water
- Used to prepare reagent solutions and for controls.
Equipment:
- Test Tubes – For mixing reagents and samples.
- Water Bath (60°C – 70°C) – Required for the hydrolysis reaction.
- Pipettes – To measure and transfer liquids accurately.
- Glass Rods – Used for mixing the reaction components.
Biological Sample:
- Serum, Plasma, or Tissue Extracts – Containing sphingomyelin.
- Egg Yolk Suspension – This can be used as a positive control because it is rich in phospholipids.
Procedure
Step 1: Sample Preparation
- In a clean test tube, take 2 mL of the test sample (serum, tissue extract, or egg yolk suspension).
- If using solid tissue, homogenize it in ethanol and centrifuge it to collect the supernatant for testing.
Step 2: Hydrolysis of Phospholipids
- Add 2 mL of 10% trichloroacetic acid (TCA) to the test tube.
- Mix the contents thoroughly by shaking the tube.
- Place the test tube in a water bath at 60–70°C for 10–15 minutes to facilitate hydrolysis.
Step 3: Addition of Ferric Chloride
- Remove the test tube from the water bath and cool it to room temperature.
- Add 1 mL of 1% ferric chloride (FeCl₃) solution to the hydrolyzed sample.
Step 4: Observation of Color Change
- Observe the test tube for any color change.
- A blue or green color indicates the presence of sphingomyelin.
Step 5: Control Experiments
- Use distilled water instead of the test sample (negative control) – no color change should occur.
- Use egg yolk extract as a positive control – it should show a blue-green color due to high phospholipid content.
Results and Interpretation
| Observation (Color Change) | Interpretation |
|---|---|
| Blue/Green Color | Positive Test: Presence of sphingomyelin or other phospholipids. |
| No Color Change | Negative Test: Absence of sphingomyelin in the sample. |
Clinical and Biochemical Significance of Results:
- Positive Cason’s Test:
- Confirms the presence of sphingomyelin in biological fluids.
- Useful in studying lipid metabolism and neurological disorders.
- Helps in the diagnosis of Niemann-Pick disease, a condition where sphingomyelin accumulates due to a deficiency of the enzyme sphingomyelinase.
- Negative Cason’s Test:
- Indicates the absence of sphingomyelin in the sample.
- This could suggest a lack of phospholipid abnormalities or that the sample does not contain sufficient lipids for detection.
Quantitative Considerations:
Although Cason’s test is primarily qualitative, the intensity of color may give a rough indication of the concentration of sphingomyelin in the sample. More intense coloration suggests higher phospholipid content.