Wine & CheeseCholesterol Analysis

This page gives some theory about cholesterol and the procedures needed to extract and assay it from a couple sources. First, ensure that you have the materials and reagents needed for these assays. Specific procedures follow for analysing total cholesterol in food sources. You can also jump to other lab procedures in an appendix, the biochemistry lab schedule, my home page or the addresses at the bottom. 
"Lipids (from the greek lipos, meaning "fat") are a group of chemically heterogeneous compounds which
were grouped together because they are insoluble in water and soluble in nonpolar solvents.
Experimental work with lipids requires a series of precautions:
  1. The work must be done in a fume hood, since lipid extraction is done with organic solvents like chloroform, which are volatile and can seriously damage your health if inhaled.
  2. Most of the solvent mixtures used are volatile, as mentioned above, so don't forget to cap flasks and test tubes (except, of course, when evaporating the solvent!)
  3. Organic solvents are highly flammable - never use them near a flame.

The isolation of lipids from natural sources is based on the fact that they are less polar than most cell components. For this reason, they can be selectively extracted with organic solvents." In the examples given here, the two natural sources are quite different. "Egg yolk is almost liquid, which means the initial extraction procedure is quite easy. Walnut needs to be divided into small portions, as small as possible, so that the contact surface with the solvent will be increased. Briefly heating the walnut/solvent mixture will help release the lipid fraction into the solvent.

Total lipid is extracted with chloroform:methanol (2:1, v/v). This solvent mixture solubilizes lipids, and helps to dissociate lipid-protein complexes from biological membranes. The tissue is homogenized with the solvent mixture, and then filtered, to eliminate the solid or particulate matter. The next step is the addition of CaCl2 to the filtrate, which facilitates the extraction to the aqueous phase of non-lipid materials and diminishes the tendency of acid lipids to dissociate, keeping them in the non polar phase. After removing the upper phase, another solvent mixture, containing more water (chloroform:methanol:water 2:50:50, v/v/v) is added and phases are allowed to separate once again. The lower phase contains the lipid fraction. After evaporating the solvent mixture, you can quantify the total lipid by weighing the residue.

Total lipids extracted in this fashion can be subsequently separated and quantified using chromatographic procedures. There are, however, specific tests for some types of lipids, which allow quantitative analysis without additional separations. An example is the quantification of "total cholesterol" (free cholesterol + cholesterol esters) by the Liebermann-Buchard method. The principle of this method is the reaction of cholesterol and cholesterol esters with acetic anhydride and concentrated sulfuric acid, resulting in the formation of a blue-green complex."10 Thus, you can determine the amount of cholesterol present in lipid extracts with a standard curve of known concentrations of cholesterol.

You can go back to the top, to the extraction or assay procedure for the Cholesterol, or the closing from here. 

Materials and Reagents:

Equipment Needed:

Reagents Needed:
 "Liebermann-Buchard reagent keeps for 1-2 weeks, but may deteriorate if kept for a longer period. To prepare 100 mL of reagent, add slowly, under continuous stirring, 10 mL of concentrated sulfuric acid to 60 mL of acetic anhydride. Since the reaction is strongly exothermic," cool the beaker in an ice bath. "Add 30 ml of acetic acid and 0.6 g of anhydrous sodium sulfate."10 Make 200 mL/lab section.
Chloroform and methanol - 500 mL each
10 mM Cholesterol standard - 0.386 g cholesterol in 100 mL of 1:1 CHCl3:MeOH

Safety procedures:

"Since Liebermann-Buchard reagent is corrosive and the work involves organic solvents, students should wear acid-resistant gloves and work in a fume hood." Always add acids (L-B reagent) to water/samples, NOT the reverse!
The Liebermann-Buchard test can be done at room temperature. However, placing the test tubes in a water bath at 35ºC will speed up the reaction. Cholesterol determination can be done with smaller volumes with the use of micropipettes, but the 1:10 ratio between sample and reagent yielded the best results and is therefore recommended (e.g. 100 µl sample: 1 ml Liebermann-Buchard reagent)."10
The organic solvents used are volatile, toxic and flammable. Use them in the fume hood away from ANY ignition source.

CAS Registry Numbers:
Chloroform [67-66-3]
Methanol [67-56-1]
Calcium chloride [10043-52-4]
Cholesterol [57-88-5]
Sulfuric acid [7664-93-9]
Acetic anhydride [108-24-7]
Acetic acid [64-19-7]
Sodium sulfate [7757-82-6]

A. Lipid Extraction - Liquid samples (that is: egg yolk):
1. Weigh approximately 1 g of egg yolk in a test tube and record the value to the nearest 0.0001 g.
2. Add 10 mL of chloroform:methanol (2:1, v/v) and strongly vortex the resulting suspension.
3. Using Whatman 1PS filter paper, filter the extract to a graduated test tube, which has been pre-weighed to the nearest 0.0001 g.
4. Measure the volume of filtrate, and add 0.2 times that volume of a 0.02% CaCl2 solution.
5. Homogenize in the vortex and leave in a test tube rack until there are two separate phases.
6. Remove the upper phase carefully with a Pasteur pipette.
7. Double the total volume of the lower phase by the addition of chloroform:methanol:water (2:50:50).
    Homogenize vigorously in the vortex and leave in the test tube rack until two phases separate.
8. Carefully remove the upper phase with a Pasteur pipette and record the volume of the lower phase containing the lipids.
9. Transfer 0.5 mL of the total lipid extract to another test tube for cholesterol determination. The remaining lipid extract obtained in 8. is placed in a water bath at 50ºC and evaporated to dryness (you will come back later and weigh the dry lipid residue)."10

- Solid samples (That is: Walnut):
1. Weigh approximately 1 g of sample in a test tube and record the mass to the nearest 0.0001 g, if possible.
2. Grind the sample thoroughly in a mortar with 5 ml of chloroform:methanol (2:1).
3. Transfer to a test tube, washing the mortar with 5 ml of the same solvent mixture into the same tube.
4. Cap the tube and place it in a water bath at 50ºC for 10 min.
5. Using Whatman 1PS filter paper, filter the sample/solvent mixture to a graduated test tube which has been pre-weighed to the nearest 0.0001 g.10
6. Proceed as in 4. above.

You can go back to the top, to the assay sections, or the closing from here. 
B. Quantification of cholesterol
1. Prepare tubes with 0.5 mL of:
  1.  (blank) chloroform:methanol (2:1, v/v)
  2.  2.5 mM cholesterol
  3.  5.0 mM cholesterol
  4.  10 mM cholesterol, and
  5. sample #1 of lipid extracts
  6. sample #2, etc.
2. "Add 5 mL of Liebermann-Buchard reagent" slowly with mixing "to each of the tubes.
3. Cap the test tubes, vortex and placed in a water bath at 35ºC for 10 min."10
4. Measure the absorbance at 550 nm. (Dilution with chloroform:methanol (1:1, v/v) may be necessary.)

C. Results:
"You will need to record the weight of lipid source (g), the volume of lipid extract (lower phase, mL), the weight of empty tube (g), and the weight of tube with "total lipids"(g) for egg yolk and walnut. Subtract the weight of the empty test tube from the weight of tube with dry lipid residue, correct for the volume removed for cholesterol quantification, and present the result as "g total lipids/g walnut" and "g total lipids/g egg yolk".

From the results of the Liebermann-Buchard test, determine the mM concentration of cholesterol in your samples from their absorbance at 550 nm in a Spec 20. From the concentration you find, and the molecular mass of cholesterol, calculate the % or fraction of cholesterol in the total lipids of each sample.10

"D. Discussion
Discuss the results; here are some suggestions for questions you may want to answer:
1. Which of the cholesterol sources contains more lipid in g/g of lipid source?
2. If you wanted to compare the amount of lipid on a dry weight basis, what additional step(s) would need in the experimental procedures?
3. The blue-green cholesterol/reagent complex has a high absorbance peak at 550 nm. Suggest a way to quantify total cholesterol in the samples, using a spectrophotometer.
4. Organizations concerned with health, such as the American Heart Association, recommend you should limit your dietary intake of cholesterol. Discuss the advantages and disadvantages of vegetable vs. animal dietary sources.

NOTE : further information on lipid structure, function and laboratory work with lipids can be obtained, among other sources, in:
1. Alexander, R.R., Griffiths, J.M., Wilkinson, M.L. 1985. Basic Biochemical Methods, Ed. John Wiley and Sons, Inc., New York.
2. American Heart Association, (accessed January 2004)
3. Boyer, R.F. 1986. Modern Experimental Biochemistry, Ed. Addison-Wesley Publishing Co., Reading, Massachusetts.
4. Bohinski, R.C. 1987. Modern Concept in Biochemistry. 5th edition, Allyn an Bacon, Inc, Boston.
5. Cyberlipid centre - www site for fats and oils, (accessed January 2004)
6. Lipid structure and function, Natural Toxins Research Center at Texas A&M University - Kingsville, (accessed January 2004)
7. Tabas. I. "Cholesterol in health and disease." J. Clin. Invest. 2002, 110, 583-590.
8. Rader, D.J., Cohen, J. & Hobbs, H.H. "Monogenic hypercholesterolemia: new insights in pathogenesis and treatment." J. Clin. Invest. 2003, 111, 1795-1803.
9. Law. M. "Plant sterol and stanol margarines and health." BMJ. 2000, 320, 861-864"10
10.    Barreto, M. Carmo "Lipid Extraction and Cholesterol Quantification: A Simple Protocol." J. Chem. Educ. 2005, 82, 103-104; and JCE Online.

That's all for now. Again, you can jump to the beginning, to my home page or the biochemistry or non-major's chemistry pages.

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