Exercise 6.10 - Myelin Lipid Extraction

LEVEL III

Fraction A: Cholesterol

Materials

• Myelin vessicles from Exercise 6.9
• Acetone
• Homogenizer manufactured for organic solvents
• Buchner funnel and water aspirator

  All organic solvents are flammable and should be handled with extreme care. No open flames will be tolerated and all heating should be done with steam baths.

  Do not use a standard household blender - the acetone will dissolve the gaskets, run into the motor and explode!

Procedure

1. To the purified myelin vessicles, add 4 volumes of acetone. Blend the mixture for 1 minute at high speed.

2. Add the blended mixture to a beaker and manually continue stirring for 5 minutes.

   Do not use any electric stirring motors.

   Filter the mixture using a Buchner funnel and cold trap attached to a water aspirator for reduced pressure. Save the filtered acetone solution.

3. Wash the residue from the filter paper back into the blender. Add 100 ml. of fresh acetone and reblend for 1 minute.

4. Add the second homogenate to the beaker, stir for 5 minutes and filter.
   Combine the two filtered acetone portions.

5. The acetone extracter material is FRACTION A. It contains crude cholesterol.

   Before storing, it should be dried to remove the acetone. This is best accomplished by flash evaporation or lyophilization. Alternatively, the solution can be allowed to evaporate in a fume hood designed to vent organic solvents. There will, of course, be some decomposition with the latter technique. Evaporate the acetone, collect the dry crude cholesterol and obtain its dry weight.

6. Keep the acetone insoluble residue (on the filter paper) for

   extraction of Fractions B and C.

Fraction B: Lecithin and Cephalin

Materials

• Residue from extraction of Fraction A
• Petroleum ether
• Buchner funnel

Procedure

1. Extract the acetone insoluble material left over from Step 6 of the extraction of Fraction A with 200 ml of ether. Gently stir for 5 minutes.

2. Collect the filtrate using a Buchner Funnel, as above.

3. Place the ether insoluble material on the filter into a flask and re-extract with an additional 200 ml of ether. Repeat the filtration and re-extraction one final time with a third 200 ml of ether.

4. Combine all three ether filtrates.
   Save the residue for Fraction C.

5. Evaporate the ether filtrates to 50 ml under reduced pressure. Pour the concentrated extract into 200 ml of acetone and stir.

6. Collect the precipitate on filter paper and discard the filtrate. The precipitate left on the filter paper FRACTION B, containing lecithin and cephalin.

7. Obtain the dry weight of Fraction B.

Fraction C: Sphingosine Phosphatides and Glycosides

Materials

• Residue from extraction of Fraction B
• Ethanol

1. Extract the ether insoluble residue obtained in the steps above with 50 ml. of boiling ethanol. Vacuum filter the hot ethanol while it is still hot. Discard the residue.

   Use steam heat only and work in a laboratory properly equipped for organic extractions. Hot ethanol has a very low flash point - it will explode!

2. Cool the ethanol filtrate and collect the precipitate by vacuum filtration. This precipitate is Fraction C.

3. Obtain the dry weight of Fraction C.

4. Determine the percentage of each Fraction dry weight relative to the original wet weight of calf brain. Dissolve each of the Fractions to a final concentration of 1% (w/v) in chloroform:methanol (3:1) before further analysis.

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