Cholesterol

Introduction to Cholesterol

Cholesterol, from the Greek chole- (bile) and stereos (solid) followed by the chemical suffix -ol for an alcohol, is an organic chemical substance classified as a waxy steroid of fat. It is an essential structural component of mammalian cell membranes and is required to establish proper membrane permeability and fluidity.

In addition to its importance within cells, cholesterol also serves as a precursor for the biosynthesis of steroid hormones, bile acids, and vitamin D. Cholesterol is the principal sterol synthesized by animals; in vertebrates it is formed predominantly in the liver. Small quantities are synthesized in other cellular organisms (eukaryotes) such as plants and fungi. It is almost completely absent among prokaryotes (i.e., bacteria).

Although cholesterol is important and necessary for human health, high levels of cholesterol in the blood have been linked to damage to arteries and cardiovascular disease.

François Poulletier de la Salle first identified cholesterol in solid form in gallstones in 1769. However, it was only in 1815 that chemist Eugène Chevreul named the compound "cholesterine".

Structure of Cholesterol 

Cholesterol is also an important component of animal membranes (plant membranes have a similar, but distinct 'sterol' in their membranes). It is a lipid, because it is composed almost entirely of carbon and hydrogen, but it is different from fatty acids, fats and phospholipids in that it is arranged in a series of rings. The rings consist of 5 or 6 carbon atoms bonded together. The carbon atoms at the apices of the hexagonal and pentagonal rings have hydrogen atoms attached to them. The ring-like structures are fairly rigid, but there is also a hydrocarbon tail, which is somewhat flexible. The entire structure is somewhat reminiscent of a fancy kite with a tail.  

Structure of Cholesterol

Cholesterol is very non-polar, except for the hydroxyl group attached to the first ring. Consequently, in an animal cell membrane the polar hydroxyl group sticks into the aqueous environment (either extracellular water or intracellular water), and the rest of the cholesterol molecule, which is non-polar, is found among the non-polar fatty acid tails of the phospholipids.The image below depicts a section of a cell membrane with water outside and inside. The polar head groups of the phospholipids are represented in red, and their non-polar fatty acid tails are shown as zig-zag lines extending from the polar head group. As we we see in greater detail, cell membranes consist of a bilayer of phospholipids with other molecules inserted into the bilayer. This illustration shows five cholesterol molecules (the black structures with four conjoined rings) inserted into the lipid bilayer. Most of the cholesterol molecule in non-polar and therefore associations with the non-polar fatty acid tails of the phospholipids. However, the hydroxyl group (-OH) on cholesterol carries a negative charge and therefore associates with the polar environment of water either inside the cell or outside.

Biosynthesis

All animal cells manufacture cholesterol with relative production rates varying by cell type and organ function. About 20–25% of total daily cholesterol production occurs in the liver; other sites of higher synthesis rates include the intestines, adrenal glands, and reproductive organs. Synthesis within the body starts with one molecule of acetyl CoA and one molecule of acetoacetyl-CoA, which are hydrated to form 3-hydroxy-3-methylglutaryl CoA (HMG-CoA). This molecule is then reduced to mevalonate by the enzyme HMG-CoA reductase. This step is the regulated, rate-limiting and irreversible step in cholesterol synthesis and is the site of action for the statin drugs (HMG-CoA reductase competitive inhibitors).

Mevalonate is then converted to 3-isopentenyl pyrophosphate in three reactions that require ATP. Mevalonate is decarboxylated to isopentenyl pyrophosphate, which is a key metabolite for various biological reactions. Three molecules of isopentenyl pyrophosphate condense to form farnesyl pyrophosphate through the action of geranyl transferase. Two molecules of farnesyl pyrophosphate then condense to form squalene by the action of squalene synthase in the endoplasmic reticulum. Oxidosqualene cyclase then cyclizes squalene to form lanosterol. Finally, lanosterol is then converted to cholesterol through a 19 step complex process.

Konrad Bloch and Feodor Lynen shared the Nobel Prize in Physiology or Medicine in 1964 for their discoveries concerning the mechanism and regulation of cholesterol and fatty acid metabolism.

Regulation of Cholesterol Synthesis

Biosynthesis of cholesterol is directly regulated by the cholesterol levels present, though the homeostatic mechanisms involved are only partly understood. A higher intake from food leads to a net decrease in endogenous production, whereas lower intake from food has the opposite effect. The main regulatory mechanism is the sensing of intracellular cholesterol in the endoplasmic reticulum by the protein SREBP (sterol regulatory element-binding protein 1 and 2). In the presence of cholesterol, SREBP is bound to two other proteins: SCAP (SREBP-cleavage-activating protein) and Insig1. When cholesterol levels fall, Insig-1 dissociates from the SREBP-SCAP complex, allowing the complex to migrate to the Golgi apparatus, where SREBP is cleaved by S1P and S2P (site-1 and -2 protease), two enzymes that are activated by SCAP when cholesterol levels are low. The cleaved SREBP then migrates to the nucleus and acts as a transcription factor to bind to the sterol regulatory element (SRE), which stimulates the transcription of many genes. Among these are the low-density lipoprotein (LDL) receptor and HMG-CoA reductase. The former scavenges circulating LDL from the bloodstream, whereas HMG-CoA reductase leads to an increase of endogenous production of cholesterol. A large part of this signaling pathway was clarified by Dr. Michael S. Brown and Dr. Joseph L. Goldstein in the 1970s. In 1985, they received the Nobel Prize in Physiology or Medicine for their work. Their subsequent work shows how the SREBP pathway regulates expression of many genes that control lipid formation and metabolism and body fuel allocation.

Cholesterol synthesis can be turned off when cholesterol levels are high, as well. HMG CoA reductase contains both a cytosolic domain (responsible for its catalytic function) and a membrane domain. The membrane domain functions to sense signals for its degradation. Increasing concentrations of cholesterol (and other sterols) cause a change in this domain's oligomerization state, which makes it more susceptible to destruction by the proteosome. This enzyme's activity can also be reduced by phosphorylation by an AMP-activated protein kinase. Because this kinase is activated by AMP, which is produced when ATP is hydrolyzed, it follows that cholesterol synthesis is halted when ATP levels are low.

Cholesterol Extraction Method

1. Homogenize 1 x 10e6 cells or ~10 mg tissue into either 200 uL chloroform-methanol (v/v 2:1) or 200 uL hexane-isopropanol (v/v 3:2).
2. Centrifuge for 5-10 min at 14,000 rpm in a microcentrifuge.
3. Transfer the organic phase to a clean tube and vacuum dry.  Store the material in the freezer (<20^oC), desiccated and protected from air, i.e., under anaerobic conditions to minimize oxidation.
4. Re-dissolve the vacuum-dried lipids/cholesterol into a suitable assay buffer prior to use.

 Isolation of Cholesterol from Egg Yolk

Procedure: In a 250 mL round bottom flask, combine a hard-boiled egg yolk, 1 g of K2CO3, 5 g of sand, and 10 mL of MeOH. Grind together until it is smooth – it will look like soft scrambled eggs. Add 20 mL of cyclohexane, stir thoroughly – it will look like corn meal mush – then warm to reflux. Rotovap off the solvent. While the solvent is rotovapping, prepare a chromatography column with 15 g of flash silica gel and a layer of sand on top, and have fifteen test tubes ready to take fractions. Also prepare a mixture of 30 mL of EtOAc and 170 mL of petroleum ether. To the 250 mL round bottom flask with the egg mixture, add 30 mL CH2Cl2, and stir thoroughly. Add the CH2Cl2 solution (not the egg mixture!) to the top of the column, and let the solvent go down on its own. When all the CH2Cl2 is down, rinse the inside top of the column with a little of the EtOAC/pet ether mixure. By this time, the solvent should have begun to drip out of the bottom of the column. Add more of the EtOAC/pet ether mixure to the top of the column, and apply gentle air pressure to the column as you collect 10 mL fractions. You should collect 15 fractions. Check the fractions by thin layer chromatography. Usually, the cholesterol comes in fractions 6-10. Rotovap the cholesterol fractions in a tared round bottom flask. You should be rewarded with iridescent rosettes of the product. Record the weight and the melting point.

Option #1: Cholesterol forms a specific 2:1 complex with oxalic acid. Take up your crude cholesterol in 5 mL of 1,2-dichloroethane in a 50 mL Erlenmeyer flask. Add 80 mg of oxalic acid, and heat to reflux. Let the flask cool. After twenty minutes (clean up the lab!), swirl the flask in ice water. The contents should gel into a mush of crystals. Vacuum filter with 1,2-dichloroethane, suck dry, and spread out to dry. Take up the white residue in a 50 mL Erlenmeyer flask with 5 mL of water. Heat to reflux, chill in ice water, and vacuum filter. This should give pure white cholesterol, mp = 141-143 oC. Record the melting point and weight. 

Option #2: Take up the crude cholesterol in 5 mL of CH2Cl2 in a 50 mL round bottomflask. Spot a TLC plate in the left lane and the middle lane with the CH2Cl2 solution. Add 0.5 mL of Dess-Martin reagent in CH2Cl2, and warm briefly. Spot the solution in the middle and the right lanes of the same TLC plate, and develop in 1:4 EtOAC/pet ether. If a lot of starting material still remains, warm the solution again, and check it again. When the starting material is almost all converted, save a little of the CH2Cl2 solurion. Rotovap the rest of the solution, add 8 mL of acetone and 20 mg pTsOH, and warm it again to reflux. Spot the TLC plate middle and right, and develop it in 1:4 EtOAC/pet ether. This time, before you visualize the TLC plate with I2, check it with the UV light. You should see a UV spot for cholestenone. TLC Rf’s in 1:9 EtOAC/pet ether: cholesterol = 0.28, 5-cholesten-3-one = 0.60, 4-cholesten-3-one = 0.41. Alternatively, the oxidation can be carried out with the Brown protocol. To purify the 4-cholesten-3-one, add 2 g of flash silica gel to the acetone solution and rotavap it. Build a dry column with 10 grams of flash silica gel. Tap it to settle the silica gel, then add the dry powder with your reaction mixture, and then sand on top. Elute with a mixture of 2 mL of EtOAc and 100 mL of petroleum ether, then with a mixture of 5 mL of EtOAc and 95 mL of petroleum ether. Take 10 mL fractions, and check them by TLC. Evaporate the fractions containing 4-cholesten-3-one in a tared round bottom flask, and record the weight and melting point.