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:
CAS Registry Numbers:
Calcium chloride [10043-52-4]
Sulfuric acid [7664-93-9]
Acetic anhydride [108-24-7]
Acetic acid [64-19-7]
Sodium sulfate [7757-82-6]
1. WEAR GLOVES
2. ALL PROCEDURES ARE DONE IN ONE OF THE FUME HOODS
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 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
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, http://americanheart.org (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, http://www.cyberlipid.org/index.htm (accessed January 2004)
6. Lipid structure and function, Natural Toxins Research Center at Texas A&M University - Kingsville, http://ntri.tamuk.edu/cell/lipid.html (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.
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