Extraction and Analysis of Lipid and Cholesterol Content in Egg Yolks

 

Egg yolk has a lipid to protein ratio of 2:1.The major lipid components are triacylglycerols, phospholipids and cholesterol.†† The major protein components are low-density lipoprotein, high density lipoprotein, and livetin (globular, water soluble), phosvitin (a phospho protein).Yolk from one large egg usually contains about 213 mg of cholesterol.In this experiment, you will qualitatively determine the lipids present in the yolk.You will then quantify the amount of cholesterol in the yolk.

 

Adapted from White et. al. J. Chem. Ed. 51:533 (1974) and Biochemical Techniques, Theory and Practice by Robyt and White, 1990 Waveland Press.

 

Extraction Procedure:

 

Week 1

 

1.)    Gently crack open the egg.DO NOT BUST THE YOLK.

2.)    Using the shell, separate the yolk from the white.Discard the white.

3.)    Tare a glass centrifuge tube.Add the yolk (you can bust it now) and record its mass and volume.

4.)    Vortex the yolk to homogenize it for 1 minute.

5.)    Split the yolk into to 2 equal parts and perform the following steps

 

Acetone Extraction

 

6.)    For one half of the eggadd an equal volume of acetone.This must be done in a glass centrifuge tubes!!

7.)    Shake vigourously with vortexing for 2 minutes

8.)    Centrifuge

9.)    Decant acetone

10.)Repeat 3 x more

11.)Pool acetone fractions and allow acetone to evporate in the hood.

12.)The acetone extract should contain the cholesterol and yolk pigments.Save for your cholesterol assay.

 

Chloroform: Methanol

 

13.)To the second fraction add an equal volume of Chloroform/Methanol (2:1).

14.)Close the cap and invert and shake the tube vigorously for 2 minutes.

15.)Vortex for 1 minute.

16.)Filter using a coarse glass crucible or Buchner funnel with vacuum suction.

17.)Repeat extraction on the solid in the crucible 2x.

18.)Pool the filtrate and allow the solvent to evaporate in the hood.

19.)The chloroform:methanol extract should contain all the neutral and polar lipids in the egg.

 

Week 2

 

20.)Weigh both solids.Remember, you performed each extraction on only half the yolk, so the total for your egg is double the amount recorded.

21.)Redissolve the chloroform:methanol extract in a small volume of chloroform: methanol

22.)Redissolve the acetone fraction in a small amount of isopropanol

 

TLC Analysis:

 

  1. Obtain 3 silica gel TLC plates from the dessicator.
  2. Follow the following procedure for EACH PLATE
  3. Use a pencil to mark the starting position across the bottom of the plate.Remember, your spots must lie ABOVE the level of the mobile phase when the plate is placed into the chamber!!
  4. Use your yellow pipette tips or capillary tubes to spot your chloroform:methanol sample and standards 2x (dry in between spottings).Be sure that the spots are SMALL, they must remain as concentrated as possible for the best results.
  5. The standards will include the following:phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, cholesterol and sphimgomeyelin.
  6. After all spots have dried, place (origin side down) in a TLC chamber with ~1 cm of mobile phase at the bottom (chloroform:methanol:water, 65:25:4)
  7. Allow the mobile phase to travel to within 1-2 cm of the top of the plate.
  8. Remove the plate and allow to dry thoroughly in the hood.
  9. For plate 1 develop as follows:
    1. Place the plate into the Iodine chamber and allow the color to develop.Iodine reacts with unsaturated carbon bonds present in the samples and forms yellow spots.
    2. Circle the positions of the spots since they will fade.
    3. Draw a replica of your TLC plate.Identify each lipid present
    4. Measure the Rf values of the spots.
    5. Determine the lipids present in the yolk extract.
    6. Draw the structure of each of the standards and explain the order of the Rf values for the standards in terms of polarity, solubility and size.††
  10. For plate 2 develop as follows:
    1. Spray plate with 1:2 Sulfuric Acid-acetic acid.
    2. Heat at 90į for 10 minutes
    3. Cholesterol and cholesterol esters yields red/purple spots. Other lipids show pink/brown spots
    4. Circle the positions of the spots since they will fade.
    5. Draw a replica of your TLC plate.Identify each lipid present
    6. Measure the Rf values of the spots.
    7. Determine the lipids present in the yolk extract.
    8. Draw the structure of each of the standards and explain the order of the Rf values for the standards in terms of polarity, solubility and size.††
  11. For plate 3:
    1. Spray plate with Dragendorf Reagent:

                                                               i.      Reagent is prepared as follows (Reagent is made in step 3):

1.      1.7 g bismuth nitrate in 100 mL of 20% acetic acid

2.      40 g of KI in water

3.      Mix 20 mL of 1 with 5 mL of 2 and add 7 mL of water.

    1. Heat to 60 C for 2 min
    2. Choline containing lipids turn orange
    3. Circle the positions of the spots since they will fade.
    4. Draw a replica of your TLC plate.Identify each lipid present
    5. Measure the Rf values of the spots.
    6. Determine the lipids present in the yolk extract.
    7. Draw the structure of each of the standards and explain the order of the Rf values for the standards in terms of polarity, solubility and size.††

 

 

FYI: HPLC is a more efficient method for detecting lipids, however requires the use of a refractive index detector, which we do not have.5 point bonus question:Why is a RI detector needed and why canít a UV-VIS detector be used?

 

 

Cholesterol analysis (Day 2):

 

The cholesterol reagent contains cholesterol esterase, cholesterol oxidase and horse radish peroxidase.The reaction for the production of the colored complex is :

 

Since only free cholesterol is a substrate for cholesterol oxidase, any cholesterol esters present in the sample must first be hydrolyzed with cholesterol esterase.

 

While your TLC plates are running, Vortex the isopropanol sample.

 

Remove 1 mL of sample and place it into another tube.

Add 5 mL of isopropanol.

Vortex

 

Use the table below to construct your calibration curve and cholesterol assay.Put the reagents directly into semi-micro cuvettes.

 

Note: number the cuvettes at the top and then add reagents to the tubes by going across the table.You must add the cholesterol reagent last and at relatively the same time to each tube.

 

After adding the reagent to all the cuvettes, begin timing.

 

Do not vortex the cuvettes, however flick them gently with your finger every 5 minutes.†† Incubate the tubes for 10 minutes.

 

Component

Blank

Tube 1

Tube 2

Tube 3

Tube 4

Tube 5

Tube 6

Water

100mL

90 mL

90 mL

90 mL

90 mL

90 mL

0 mL

1 mg/mL standard

-------

10 mL

-------

-------

-------

-------

-------

2 mg/mL standard

-------

-------

10 mL

-------

-------

-------

-------

4 mg/mL standard

-------

-------

-------

10 mL

-------

-------

-------

8 mg/mL standard

-------

-------

-------

-------

10 mL

-------

-------

Lipid sample

-------

-------

-------

-------

-------

5 mL

50 mL

Cholesterol reagent

1 mL

1 mL

1 mL

1 mL

1 mL

1 mL

1 mL

 

Read the absorbance of the tubes at 500 nm on the UV-VIS.

 

Use the graph to determine the mg/mL of cholesterol in your sample.Use the 2 dilutions you made to calculate the mass of cholesterol in the original extract.