In the Laboratory

Lipid Extraction and Cholesterol Quantification: A Simple Protocol

M. Carmo Barreto Departamento de Cincias Tecnolgicas e Desenvolvimento, Universidade dos Aores, 9502 Ponta Delgada, Portugal; barreto@notes.uac.pt

This experiment is of interest for introductory biochemistry classes, because it is simple and gives consistent, reproducible results. The experiment involves disruption techniques, extraction with solvents, separation with two-phase systems, and a semi-quantitative analysis with a specific colorimetric reagent. It was devised for a general biochemistry course for first-year biology undergraduates. General biochemistry takes place in the second semester, after general chemistry (inorganic and organic). In the theoretical biochemistry classes, these students are studying the classes of biological molecules: carbohydrates, lipids, proteins, and nucleic acids. Laboratory biochemistry classes, besides accompanying the theory from the lectures, start with experiments that are more qualitative and gradually advance to a more quantitative approach. This work is at an intermediate level, since it allows for a semi-quantitative approach. It is designed for a two-hour lab session, except for the evaporation of lipids to dryness, which proceeds overnight. Similar articles reported in this Journal are designed for more advanced students and require several three to four-hour sessions (e.g., 13 ). As a result of an increasing concern about health issues, comparison of cholesterol in different food sources has a special attraction for most students. This practical experiment provides an opportunity to discuss the influence of eating habits on health. An example is the increased probability of developing atherosclerosis in people with high cholesterol diets, although other consequences of this type of diet include cholesterol gallstones and liver dysfunction, among others (4). The discussion on the advantages and disadvantages of animal versus vegetable sources of dietary fat also raises a lot of interest, especially considering the fact that the number of vegetarians and vegans has been steadily increasing in the student population. Basis Isolation of lipids from natural sources is based on the fact that these molecules are less polar than most cell components and can therefore be selectively extracted with organic solvents. Egg yolk and walnut were chosen for their high lipid content and also for their differences: (i) egg yolk is very rich in cholesterol (5) while walnut is almost cholesterol free (6) and (ii) egg yolk is fluid, which facilitates extraction, while walnut has to be ground, to increase the contact surface with the solvent mixture, and heated to help release the lipids. The experiment can be enriched by including other animal and vegetable sources.

Experiment

Lipid Extraction Method The lipid extraction method was modified from Folch et al. (7, 8). Before starting the work, the theory of lipid extraction is discussed with the students, including safety procedures, stressing the fact that the experiment must be done in a fume hood. Students work in pairs, so the extraction step can be carried out at the same time for the egg yolk and the walnut. An aliquot of the total lipid extract is collected for cholesterol determination and the remaining total lipid extracts are evaporated to dryness until the following day and quantified by gravimetry. Quantification of Cholesterol Total lipids extracted as described above can be subsequently separated into lipid classes and quantified using chromatographic procedures. There are, however, specific tests for some types of lipids that allow for a quantitative analysis without a previous separation step. An example is the quantification of total cholesterol (free cholesterol and cholesterol esters) by the LiebermannBuchard method. The basis of this method is the reaction of cholesterol and cholesterol esters with acetic anhydride and concentrated sulfuric acid, resulting in the formation of a bluegreen complex (9). This reaction is used to determine cholesterol in the aliquots removed at the end of the previous section, yielding results in 1015 minutes. Students compare the colors resulting from the LiebermannBuchard reaction in egg yolk and walnut lipid extracts with cholesterol standards and a chloroform blank.
Hazards Chloroform and methanol are flammable. Methanol is toxic if inhaled, ingested, or absorbed by the skin. Chloroform is listed as irritant and possible carcinogenic. Inhalation and ingestion are harmful and may be fatal. Concentrated sulfuric acid is extremely corrosive. All work must be done with acid-resistant gloves in a fume hood. Discussion After students have the results, including the mass of total lipid extracted, they write a brief report, including: (i) the quantity of lipid per unit of mass of egg yolk or walnut; (ii) the relative quantity of cholesterol in the samples assayed;

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and (iii) the advantages and disadvantages of vegetable and animal food sources, considering cholesterol-related health problems. This work can be expanded in further lab sessions: (i) It can be done in a quantitative way, using a spectrophotometer; students can design a cholesterol standard curve and measure the absorbance of the bluegreen complex at 550 nm and (ii) lipids can be separated in lipid classes by TLC; in that case, the lipid should be extract to dryness under nitrogen to avoid undesirable oxidations. Conclusion Although enzymatic methods are currently used to measure cholesterol levels, the method described here has the advantage of being simple and inexpensive, which is particularly important when one has repeated lab sessions during the week. Furthermore, it can be carried out on the organic phase containing the lipids, without evaporating the solvent, yielding almost immediate results. It is a experiment most students find interesting and which leads to fruitful discussions on scientific and health issues.

Supplemental Material

Instructions for the students and notes for the instructor are available in this issue of JCE Online. Literature Cited
1. Koning, A. J. J. Chem. Educ. 1974, 51, 4850. 2. Taylor, R. P.; Broccolli, A. V.; Grisham, C. M. J. Chem. Educ. 1978, 55, 6364. 3. Vestling, M. J. Chem. Educ. 1990, 67, 274275. 4. Tabas. I. J. Clin. Invest. 2002, 110, 583590. 5. Kuksis, A. Biochim. Biophys. Acta 1992, 1124, 205222. 6. Tsamouris, G.; Hatziantoniou, S.; Demetzos, C. Z. Naturforsch. 2001, 57c, 5156. 7. Folch, J.; Lees, M.; Stanley, G. H.; J. Biol. Chem. 1957, 226, 497509. 8. Cyberlipid CenterResource Site for Fats and Oils. http:// www.cyberlipid.org/index.htm (accessed Sep 2004). 9. Huang, T. C.; Chen, C. P.; Wefler, V.; Raftery, A. Anal. Chem. 1961, 33, 14051407.

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