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Sterols:  1. Cholesterol and Cholesterol Esters

In animal tissues, cholesterol (cholest-5-en-3β-ol) is by far the most abundant member of a family of polycyclic compounds known as sterols (see also our web pages on plant sterols, oxysterols, etc.). It can also be described as a polyisoprenoid or a triterpene from its biosynthetic origin. Cholesterol was first recognized as a component of gallstones as long ago as 1769, while the great French lipid chemist Chevreul isolated it from animal fats in 1815. However, it was well into the 20th century before the structure was fully defined by the German Chemist Heinrich Wieland, who received the Nobel Prize in Chemistry for his work in 1927, the first of thirteen so honoured for research on cholesterol and its metabolism. In mammals, cholesterol plays a vital role in life, and it is essential for the normal functioning of cells both as a cell membrane constituent and as a precursor of steroid hormones and other key metabolites including vitamin D and bile acids; it is also important for cell signalling, transport processes and nerve conduction. There is evidence that synthesis de novo is essential whatever the dietary intake.

1. Cholesterol – Structure, Occurrence, Physical Properties and Function

Structural formula for cholesterolIn essence, cholesterol consists of a tetracyclic cyclopenta[a]phenanthrene structure with an iso-octyl side-chain at carbon 17. The four rings (A, B, C, D) have trans ring junctions, and the side chain and two methyl groups (C-18 and C-19) are at an angle to the rings above the plane with β stereochemistry (as for the hydroxyl group on C-3 also); there is a double bond between carbons 5 and 6. Thus, the molecule has a rigid planar four-ring nucleus with a flexible tail. Of the two recognized numbering systems in use, one originally described by Fieser and Fieser in 1959 and a second by IUPAC-IUB in 1989, the first appears to be preferred by many current authors.

Cholesterol is a ubiquitous component of all animal tissues (and of some fungi), where much of it is located in the membranes, although it is not evenly distributed. The highest proportion of unesterified cholesterol is in the plasma membrane (roughly 30-50% of the lipid in the membrane or 60-80% of the cholesterol in the cell), while mitochondria and the endoplasmic reticulum have much less (~5% in the latter), and the Golgi contains an intermediate amount. Cholesterol is also enriched in early and recycling endosomes, but not in late endosomes. It may surprise some to learn that the brain contains more cholesterol than any other organ, where it comprises roughly a quarter of the total free cholesterol in the human body, 70-80% of which is in the myelin sheath. Of all the organic constituents of blood, only glucose is present in a higher molar concentration than cholesterol. In animal tissues, it occurs in the free form, esterified to long-chain fatty acids (cholesterol esters), and in other covalent and non-covalent linkages, including an association with the plasma lipoproteins. In plants, it tends to be a minor component only of a complex mixture of structurally related 'phytosterols', although there are exceptions, but it is nevertheless importance as a precursor of some plant hormones.

Scottish thistleAnimals in general synthesise a high proportion of their cholesterol requirement, but they can also ingest and absorb appreciable amounts from foods. On the other hand, many invertebrates, including insects, crustaceans and some molluscs cannot synthesise cholesterol and must receive it from the diet; for example, spiny lobsters must obtain exogenous cholesterol to produce essential sex hormones. Similarly, it must be supplied from exogenous sources to the primitive nematode Caenorhabditis elegans, where it does not appear to have a major role in membrane structure, other than perhaps in the function of ion channels, although it is essential the production of steroidal hormones required for larval development; its uptake is regulated by the novel lipid phosphoethanolamine glucosylceramide. Some species are able to convert dietary plant sterols such as β-sitosterol to cholesterol. Prokaryotes lack cholesterol entirely with the exception of some pathogens that acquire it from eukaryotic hosts to ensure their intracellular survival; bacterial hopanoids are often considered to be sterol surrogates.

Cholesterol has vital structural roles in membranes and in lipid metabolism in general with an extraordinary diversity of biological roles, including cell signalling, morphogenesis, lipid digestion and absorption in the intestines, reproduction, stress responses, sodium and water balance, and calcium and phosphorus metabolism, and we can only touch on a few of these functions in this web page. It is a biosynthetic precursor of bile acids, vitamin D and steroid hormones (glucocorticoids, oestrogens, progesterones, androgens and aldosterone), and it is found in covalent linkage to specific membrane proteins or proteolipids ('hedgehog' proteins), which have vital functions in embryonic development. In addition, it contributes substantially to the development and working of the central nervous system. On the other hand, excess cholesterol in cells can be toxic, and a complex web of enzymes is essential to maintain the optimum concentrations. Because plasma cholesterol levels can be a major contributory factor to atherogenesis, media coverage has created what has been termed a ‘cholesterophobia’ in the population at large.

One of the main function of cholesterol is to modulate the fluidity of membranes by interacting with their complex lipid components, specifically the phospholipids such as phosphatidylcholine and sphingomyelin. As an amphiphilic molecule, cholesterol is able to intercalate between phospholipids in lipid bilayers to span about half a bilayer. In its three-dimensional structure, it is in essence a planar molecule that can interact on both sides. The tetracyclic ring structure is compact and very rigid. In addition, the location of the hydroxyl group facilitates the orientation of the molecule in a membrane bilayer, while the positions of the methyl groups appear to maximize interactions with other lipid constituents.

Planar structure of cholesterol

As the α-face of the cholesterol nucleus (facing down) is 'smooth', it can make good contact with the saturated fatty-acyl chains of phospholipids down to about their tenth methylene group; the β-face (facing up) is made 'rough' by the projection of methyl groups from carbons 10 and 13. The interaction is mainly via van der Waals and hydrophobic forces with a contribution from hydrogen bonding of the cholesterol hydroxyl group to the polar head group and interfacial regions of the phospholipids, especially sphingomyelin. Intercalated cholesterol may also disrupt electrostatic interactions between the ionic phosphocholine head groups of nearby membrane phospholipids, leading to increased mobility of the head groups. Indeed, there is evidence that cholesterol forms stoichiometric complexes with the saturated fatty acyl groups of sphingomyelin and to a lesser extent of phosphatidylcholine.

Experiments with mutant cell lines and specific inhibitors of cholesterol biosynthesis suggest that an equatorial hydroxyl group at C-3 of sterols is essential for the growth of mammalian cells. The Δ5 double bond ensures that the molecule adopts a planar conformation, and this feature also appears to be essential for cell growth, as is the flexible iso-octyl side-chain. The C-18 methyl group is crucial for the proper orientation of the sterol. While plant sterols appear to be able to substitute for cholesterol in supporting many of the bulk properties of membranes in mammalian cells in vitro, cholesterol is essential for other purposes.

Scottish thistleIn the absence of cholesterol, a membrane composed of unsaturated lipids is in a fluid state that is characterized by a substantial degree of lipid chain disorder, i.e. it constitutes a liquid-disordered phase. The function of cholesterol is to increase the degree of order (cohesion and packing) in membranes, leading to formation of a liquid-ordered phase. In contrast, it renders bilayers composed of more saturated lipids, which would otherwise be in a solid gel state, more fluid. Thus, cholesterol is able to promote and stabilize a liquid-ordered phase over a substantial range of temperatures and sterol concentrations. Further, high cholesterol concentrations in membranes reduce their passive permeability to solutes. These effects permit the fine-tuning of membrane lipid composition, organization and function. In comparison to other lipids, cholesterol can flip rapidly between the leaflets in a bilayer, so the trans-bilayer distribution of cholesterol in some biological membranes is uncertain. While some models propose that cholesterol is in the outer leaflet, other studies suggest that most of the sterol is in the inner leaflet of human erythrocytes, for example, although in general the consensus at the moment is that cholesterol tends to be roughly equally distributed between the two leaflets of the plasma membrane. There is also evidence that some cholesterol molecules are located at the bilayer midplane rather than in a leaflet in some systems. This distribution is important in that cholesterol promotes negative curvature of membranes and may be a significant factor in bringing about membrane fusion as in the process of exocytosis.

Cholesterol also has a key role in the lateral organization of membranes and their free volume distribution, factors permitting more intimate protein-cholesterol interactions that may regulate the activities of membrane proteins. Many membrane proteins bind strongly to cholesterol, including some that are involved in cellular cholesterol homeostasis or trafficking and contain a conserved region termed the ‘sterol-sensing domain’. Some proteins bind to cholesterol deep within the hydrophobic core of the membrane via binding sites on the membrane-spanning surfaces or in cavities or pores in the proteins, driven by hydrogen bond formation. For example, cholesterol has an intimate interaction with G-protein-coupled receptors (GPCRs), and it is essential for the stability and function of the β2-adrenergic receptor and of ion pumps such as the (Na+-K+)-ATPase, which have specific binding site for cholesterol molecules. The last is the single most important consumer of ATP in cells and is responsible for the ion gradients across membranes that are essential for many cellular functions; depletion of cholesterol in the plasma membrane deactivates these ion pumps. The agonist affinities of other GPCRs, including the oxytocin and serotonin receptors, are dramatically increased by membrane cholesterol, while the inactive state of rhodopsin is stabilized through indirect effects on plasma membrane curvature. In the brain in addition to being essential for the structure of the myelin sheath, cholesterol is a major component of synaptic vesicles and controls their shape and functional properties, and it also has an important role in the organization and positioning of neurotransmitter receptors. In the nucleus of cells, cholesterol is intimately involved in chromatin structure and function.

The role of cholesterol in the formation of the transient membrane nano-domains known as rafts (see the specific web page), which are so important in the function of cells, is not as clear-cut as was once thought but is nonetheless important. The interaction of cholesterol with ceramides is essential for the barrier function of the skin. Simplistically, the higher cholesterol concentrations in the plasma membrane support its barrier function by increasing membrane thickness and reducing its permeability to small molecules. In contrast, the endoplasmic reticulum has increased membrane flexibility because of its lower cholesterol concentrations and thus enables the insertion and folding of proteins in its lipid bilayer. While mitochondrial membranes have a low cholesterol content in total, this may be concentrated in nanodomains at regions of high curvature in the inner mitochondrial membrane with links to nucleoprotein complexes (nucleoids).

2.  Cholesterol Biosynthesis

Cholesterol biosynthesis involves a highly complex series of at least thirty different enzymatic reactions, which were unravelled in large measure by Konrad Bloch and Fyodor Lynen, who received the Nobel Prize for their work on the topic in 1964. When the various regulatory, transport and genetic studies of more recent years are taken into account, it is obvious that this is a subject that cannot be treated in depth here. The bare bones of mechanistic aspects are therefore delineated, which with the references listed below should serve as a guide to further study. In plants, cholesterol synthesis occurs by a somewhat different pathway with cycloartenol as the key intermediate.

Almost all nucleated cells are able to synthesise their full complement of cholesterol. The first steps involve the synthesis of the important intermediate mevalonic acid from acetyl-CoA and acetoacetyl-CoA, both of which are in fact derived from acetate, in two enzymatic steps. The acetyl-CoA precursor is in the cytosol as is the first enzyme, hydroxymethyl-glutaryl(HMG)-CoA synthase. The second enzyme HMG-CoA reductase is a particularly important control point, and is widely regarded as the rate-limiting step in the overall synthesis of sterols; its activity is regulated at the transcriptional level and by many more factors including a cycle of phosphorylation-dephosphorylation. This and subsequent enzymes are membrane-bound and are located in the endoplasmic reticulum. The enzyme HMG-CoA reductase is among the targets inhibited by the drugs known as ‘statins’, so that patients must then obtain much of their cholesterol from the diet via the circulation.

Cholesterol biosynthesis - to mevalonic acid

The next sequence of reactions involves first the phosphorylation of mevalonic acid by a mevalonate kinase to form the 5‑monophosphate ester, followed by a further phosphorylation to yield an unstable pyrophosphate, which is rapidly decarboxylated to produce 5-isopentenyl pyrophosphoric acid, the universal isoprene unit. An isomerase converts part of the latter to 3,3-dimethylallyl pyrophosphoric acid.

Cholesterol biosynthesis - step two

5-Isopentenyl pyrophosphate is a nucleophile, but the isomerized product is electrophylic, facilitating the first step in the third series of reactions in which 5-isopentenyl pyrophosphate and 3,3-dimethylallyl pyrophosphate condense with the elimination of pyrophosphoric acid to form the monoterpenoid derivative geranyl pyrophosphate. This reacts with another molecule of 5-isopentenyl pyrophosphate to produce the sesquiterpene derivative (C15) farnesyl pyrophosphate, two molecules of which are condensed to yield presqualene pyrophosphate. In turn, this is reduced by NADPH to produce a further key intermediate squalene. Both of the last steps are catalysed by the enzyme squalene synthase, which regulates the flow of metabolites into either the sterol or non-sterol pathways (with farnesyl pyrophosphate as the branch point) and is considered to be the first committed enzyme in cholesterol biosynthesis.

Squalene biosynthesis - step 3

In the next important step, squalene is first oxidized by a squalene monooxygenase to squalene 2,3-epoxide, which undergoes cyclization catalysed by the enzyme squalene epoxide lanosterol-cyclase to form the first steroidal intermediate lanosterol (or cycloartenol en route to phytosterols in photosynthetic organisms). In this remarkable reaction, there is a series of concerted 1,2-methyl group and hydride shifts along the chain of the squalene molecule to bring about the formation of the four rings. No intermediate compounds have been found. This is believed to be one of the most complex single enzymatic reactions ever to have been identified, although the enzyme involved is only 90 kDa in size. Again, the reaction takes place in the endoplasmic reticulum, but a cytosolic protein, sterol carrier protein 1, is required to bind squalene in an appropriate orientation in the presence of the cofactors NADPH, flavin adenine dinucleotide (FAD) and O2; the reaction is promoted by the presence of phosphatidylserine.

Cholesterol biosynthesis - cyclization of squalene

In subsequent steps, lanosterol is converted to cholesterol by a series of demethylations, desaturations, isomerizations and reductions, involving 19 separate reactions. Thus, demethylation reactions produce zymosterol as an intermediate, and this is converted to cholesterol via a series of intermediates, all of which have been characterized, and by at least two pathways that utilize essentially the same enzymatic machinery but differ in the order of the various reactions, mainly at the point at which the Δ24 double bond is reduced. Desmosterol is the key intermediate in the co-called 'Bloch' pathway, while 7-dehydrocholesterol is the immediate precursor in the 'Kandutsch-Russel' pathway. While some tissues, such as adrenal glands and testis, use the Bloch pathway mainly, the brain synthesises much of its cholesterol by the 'Kandutsch-Russell' pathway. This may enable production of a variety of other minor sterols for specific biological purposes in different cell types/locations.

Final steps in cholesterol biosynthesis

Synthesis occurs mainly in the liver, although the brain (see below), peripheral nervous system and skin synthesise their own considerable supplies. Cholesterol is exported from the liver and transported to other tissues in the form of low-density lipoproteins (LDL) for uptake via specific receptors. In animals, cells can obtain the cholesterol they require either from the diet via the circulating LDL, or they can synthesise it themselves as outlined above. Cholesterol biosynthesis is highly regulated with rates of synthesis varying over hundreds of fold depending on the availability of an external source of cholesterol, and cholesterol homeostasis requires the actions of a complex web of enzymes, transport proteins, and membrane-bound transcription factors, as discussed below.

3.  Regulation of Cholesterol Homeostasis

In humans, only about a third of the body cholesterol is of dietary origin (mainly eggs and red meat), the remainder is produced by synthesis de novo. Although HMG-CoA reductase is recognized as the key enzyme in the regulation of cholesterol biosynthesis, the second rate-limiting enzyme in cholesterol biosynthesis is squalene monooxygenase, which undergoes cholesterol-dependent proteasomal degradation when cholesterol is in excess, guided by a 12 amino acid hydrophobic sequence on the enzyme that can serve as a degradation signal. When the cholesterol concentration in the endoplasmic reticulum is high, the degradation sequence detaches from the membrane and is exposed to provide the signal for the enzyme to be degraded.

Cholesterol in the endoplasmic reticulum is transferred to the Golgi and eventually to the plasma membrane by non-vesicular transport mechanisms involving in part soluble sterol transport proteins and partly by binding to those proteins that are intimately involved in the transport and metabolism of polyphosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). In the latter mechanism, cholesterol is transported by the monomeric form of oxysterol binding protein (OSBP)-related protein 2 (ORP2), which binds to PI(4,5)P2 in the plasma membrane to transfer its cargo. ORP2 then forms a tetramer that incorporates PI(4,5)P2 by binding at a C-terminal lipid transport domain (ORD) and removes it from the membrane for transport to other membranes such as the endosomal compartment. ORP2 is thus a key regulator of both cholesterol and PI(4,5)P2 concentrations in the plasma membrane. Any cholesterol in cellular membranes in excess of the stoichiometric requirement can escape back readily into the cell, where it may serve as a feedback signal to down-regulate cholesterol accumulation. Some of this ‘active’ cholesterol is converted to the relatively inert storage form, i.e. cholesterol esters, while some is used for steroidogenesis. In peripheral tissues, excess cholesterol is exported to high-density lipoproteins (HDL) in the circulation and returned to the liver - reverse cholesterol transport.

The liver is important for cholesterol synthesis, but it is essential for its elimination from the body in bile as discussed in our web page on bile acids. In addition, the intestines play a major part in cholesterol homeostasis via absorption of dietary cholesterol and fecal excretion of cholesterol and its metabolites. A specific transporter in the brush border membrane of enterocytes in the proximal jejunum of the small intestine is involved in uptake of cholesterol from the intestinal contents, while the metabolism of sterols in the intestines is controlled mainly by an acetyl-CoA acetyltransferase (ACAT2), which facilitates intracellular cholesterol esterification, and the microsomal triglyceride transfer protein (MTTP), which is involved in the assembly of chylomicrons for export into lymph (see our web page on lipoproteins for further discussion). There is evidence that dietary cholesterol or that synthesised de novo is necessary to maintain intestinal integrity, as cholesterol derived from circulating lipoproteins is not sufficient for the purpose.

Scottish thistleIn the intestines and especially the colon, the intestinal microflora are able to hydrogenate cholesterol from bile, diet and desquamated cells to form coprostanol with an efficiency that is dependent on the composition of microbial species. Coprastanol is not absorbed by the intestinal tissue to a significant extent, but it may inhibit the uptake of residual cholesterol.

Many other factors are involved in cholesterol homeostasis. For example, the regulatory element-binding proteins (mainly SREBP-1c and SREBP-2), which contain an N-terminal membrane domain and a C-terminal regulatory domain, are also essential to the maintenance of cholesterol homeostasis. Each is synthesised as an inactive precursor that is inserted into the endoplasmic reticulum where it can encounter an escort protein termed SREBP cleavage-activating protein (SCAP), which is the cellular cholesterol sensor. When the latter recognizes that cellular cholesterol levels are inadequate, it binds to the regulatory domain of SREBP. The SCAP-SREBP complex then moves to the Golgi, where two specific proteases (designated site-1 and site-2 proteases) cleave the SREBP enabling the C-terminal regulatory domain to enter the nucleus. There it activates transcription factors, such as the nuclear liver X receptor (LXR), which stimulate the expression of the genes coding for the LDL receptor in the plasma membrane and for the key enzyme in cholesterol biosynthesis, HMG-CoA reductase. This in turn stimulates the rate of cholesterol uptake and synthesis. Conversely, when cholesterol in the endoplasmic reticulum exceeds a threshold, it binds to SCAP in such a way that it prevents the SCAP-SREBP complex from leaving the membrane for the nucleus. Cholesterol synthesis and uptake are thereby repressed, and cholesterol homeostasis is restored. Then, the ATP binding cassette (ABC) transporters ABCA1 and ABCG1 in the plasma membrane, which contains much of the cellular cholesterol, are activated to export the excess. In addition, nuclear factor erythroid 2 related factor-1 or NRF1 in the endoplasmic reticulum binds directly to cholesterol and senses when its level is high to bring about a de-repression of genes involved in cholesterol removal, also with mediation by the liver X receptor.

Further regulation of cholesterol biosynthesis is exerted by sterol intermediates in cholesterol biosynthesis, such as lanosterol and 24,25-dehydrolanosterol, and by the side-chain oxysterols, especially 25-hydroxycholesterol, which can suppress the activation of SREBP by binding to an oxysterol-sensing protein in the endoplasmic reticulum or by direct effects on the biosynthetic and transport enzymes. However, many other factors are involved in maintaining within precise limits the large differences in cholesterol concentrations among the various membranes and organelles in cells. These include other regulatory proteins, and mechanisms that can involve either vesicle formation or non-vesicular pathways that utilize specific transport proteins, such as the ABC transporters. It is noteworthy that ceramide down-regulates cholesterol synthesis – another link between cholesterol and sphingolipid metabolism. There is also a link to phosphoinositide metabolism as phosphatidylinositol 4-phosphate has been described as a 'fuel' that drives sterol transport and allows the establishment of active sterol concentration gradients across membrane-bound compartments.

Brain: There are substantial differences in cholesterol synthesis and metabolism in brain in comparison to the liver and peripheral tissues. Trace amounts only of cholesterol are able to cross the blood brain barrier via transport in low-density lipoproteins. Therefore, virtually all the cholesterol in brain must be synthesised de novo, mainly in astrocytes, before transport to neurons in the form of Apo E complexes in discoidal HDL-like particles. Seven main receptors have been identified in brain cells that take up cholesterol from these lipoproteins. This Apo E is synthesised in the brain, and its transcription is regulated by 24-hydroxy-cholesterol concentrations. Similarly, in the brain and central nervous system, cholesterol synthesis is regulated independently of that in peripheral tissues, mainly by forms of the liver X receptor (LXR). As cholesterol and oxysterols are involved in providing neuroprotective effects and lowering neuroinflammation, dysregulation of their concentrations has been noted in many neurodegenerative disorders. Most of the lipoproteins in cerebrospinal fluid differ from the nascent poorly-lipidated HDL secreted by astrocytes, suggesting that the latter are modified during maturation. Our web pages on oxysterols and on lipoproteins discuss cholesterol metabolism in the brain at greater length.

4.  Cholesterol Catabolism

Cholesterol is not readily degraded in animal tissues so does not serve as a metabolic fuel. Only the liver possesses the enzymes to degrade significant amounts, and then via pathways that do not lead to energy production. Cholesterol and oxidized metabolites (oxysterols) are transferred back from peripheral tissues in lipoprotein complexes to the liver for catabolism by conversion to oxysterols and bile acids. The latter are exported into the intestines to aid digestion, while leading to some loss that is essential for cholesterol homeostasis (and is discussed in further web-pages on oxysterols, bile acids and lipoproteins). Until recently, it was believed that approximately 90% of cholesterol elimination from the body occurred via bile acids in humans. However, experiments with animal models now suggest that a significant amount is secreted directly into the intestines by a process known as trans-intestinal cholesterol efflux. How this occurs and its relevance to humans are under active investigation.

Certain bacterial species contain a 3β-hydroxysteroid:oxygen oxidoreductase (EC, commonly termed cholesterol oxidase, a flavoenzyme that catalyses the oxidation of cholesterol to cholest-5-en-3-one which is then rapidly isomerized to cholest-4-en-3-one as the first essential step in a more comprehensive catabolism of sterols. The enzyme is widespread in organisms that degrade organic wastes, but it also present in pathogenic organisms where it influences the virulence of infections (see below). In biotechnology, it has been used for the production of a number of steroids, and it is employed in a clinical procedure for the determination of cholesterol levels in serum.

5.  Cholesterol Esters

Cholesterol esters, i.e. with long-chain fatty acids linked to the hydroxyl group, are much less polar than free cholesterol and appear to be the preferred form for transport in plasma and as a biologically inert storage or de-toxification form to buffer an excess. They do not contribute to membrane structures but are packed into intracellular lipid droplets. Cholesterol esters are major constituents of the adrenal glands, and they accumulate in the fatty lesions of atherosclerotic plaques. Similarly, esters of steroidal hormones are also present in the adrenal glands, where they are concentrated in cytosolic lipid droplets adjacent to the endoplasmic reticulum; 17β-estradiol, the principal oestrogen in fertile women, is transported in lipoproteins in the form of a fatty acid ester.

Because of the mechanism of synthesis (see below), plasma cholesterol esters tend to contain relatively high proportions of the polyunsaturated components typical of phosphatidylcholine (Table 1). Arachidonic and “adrenic” (20:4(n-6)) acids can be especially abundant in cholesterol esters from the adrenal gland.

Table 1. Fatty acid composition of cholesterol esters (wt % of the total) from various tissues.
Fatty acids
16:0 18:0 18:1 18:2 18:3 20:4 22:4
plasma 12 2 27 45 8
liver 23 10 28 22 6
plasma 10 2 27 35 7 - -
liver 17 9 29 7 4 3 -
adrenals 13 7 35 18 2 4 2
Data from - Christie, W.W. et al. Lipids, 10, 649-651 (1975); Nelson, G.J. Comp. Biochem. Physiol., 30, 715-725 (1969); Horgan, D.J. and Masters, C.J. Aust. J. Biol. Sci., 16, 905-915 (1963); Nestel, P.J. and Couzens, E.A. J. Clin. Invest., 45, 1234-1240 (1966).

In plasma and in the high-density lipoproteins (HDL) in particular, cholesterol esters are synthesised largely by transfer of fatty acids to cholesterol from position sn-2 of phosphatidylcholine (‘lecithin’) catalysed by the enzyme lecithin:cholesterol acyl transferase (LCAT); the other product is 1-acyl lysophosphatidylcholine (see also our web page on lipoproteins). In fact, the reaction occurs in several steps. First, apoprotein A1 in the HDL acts to concentrate the lipid substrates near LCAT and present it in the optimal conformation; at the same time, it opens a lid on the enzyme that activates it by opening up the site of transesterification. Then, cleavage of the sn-2 ester bond of phosphatidylcholine occurs via the phospholipase activity of LCAT with release of a fatty acyl moiety. This is transacylated to the sulfur atom of a cysteine residue forming a thioester, and ultimately it is donated to the 3β-hydroxyl group of cholesterol to form the cholesterol ester. Some LCAT activity has also been detected in apolipoprotein B100-containing particles (β-LCAT activity as opposed to α-LCAT with HDL).

Biosynthesis of cholesterol esters in plasma and cells

It has been established that human LCAT is a relatively small glycoprotein with a polypeptide mass of 49 kDa, increased to about 60 kDa by four N-glycosylation and two O-glycosylation moieties. Most of the enzyme is produced in the liver and circulates in the blood stream bound reversibly to HDL, where it is activated by the main protein component of HDL, apolipoprotein A1. As cholesterol esters accumulate in the lipoprotein core, cholesterol is removed from its surface thus promoting the flow of cholesterol from cell membranes into HDL. This in turn leads to morphological changes in HDL, which grow and become spherical. Subsequently, cholesterol esters are transferred to the other lipoprotein fractions LDL and VLDL, a reaction catalysed by cholesterol ester transfer protein. This process promotes the efflux of cholesterol from peripheral tissues (‘reverse cholesterol transport’), especially from macrophages in the arterial wall, for subsequent delivery to the liver. LCAT is often stated to be the main driving force behind this process, and it is of great importance for cholesterol homeostasis and a suggested target for therapeutic intervention against cardiovascular disease.

The stereospecificity of LCAT changes with molecular species of phosphatidylcholine containing arachidonic or docosahexaenoic acids, when 2-acyl lysophosphatidylcholines are formed. This reaction may be especially important for the supply of these essential fatty acids to the brain in that such lysophospholipids are believed to cross the blood-brain barrier more readily than the free acids.

In other animal tissues, a further enzyme acyl-CoA:cholesterol acyltransferase (ACAT) synthesises cholesterol esters from CoA esters of fatty acids and cholesterol. ACAT exists in two forms, both of which are intracellular enzymes found in the endoplasmic reticulum and possess multiple hydrophobic regions predicted to function as trans-membrane domains; they are members of the membrane-bound O-acyltransferase (MBOAT) superfamily. ACAT1 is present in many tissues, but especially in macrophages and adrenal and sebaceous glands, which store cholesterol esters in the form of cytoplasmic lipid droplets; it is responsible for the synthesis of cholesterol esters in arterial foam cells in human atherosclerotic lesions. ACAT2 is found only in the liver and small intestine, and it is believed to be involved in the supply of cholesterol esters to the nascent lipoproteins. Analogous enzymes are found in yeast where ergosterol is the main sterol, but a very different process occurs in plants (see our web page on plant sterols).

There are suggestions that cholesteryl arachidonate in the lipoprotein LDL is a good substrate for 12/15-lipoxygenase and other oxidizing agents and that the products are a causative factor in atherosclerosis.

Hydrolysis of cholesterol esters: Cholesterol ester hydrolases in animals liberate cholesterol and free fatty acids when required for membrane and lipoprotein formation, and they also provide cholesterol for hormone synthesis in adrenal cells. Many cholesterol ester hydrolases have been identified, including a carboxyl ester hydrolase, a lysosomal acid cholesterol ester lipase, hormone-sensitive lipase and hepatic cytosolic cholesterol ester hydrolase. These are located in many different tissues and organelles and have multiple functions. A neutral cholesterol ester hydrolase has received special study, as it involved in the removal of cholesterol esters from macrophages, so reducing the formation of foam cells and thence the development of fatty streaks within the arterial wall, a key event in the progression of atherosclerosis.

6.  Other Animal Sterols

Cholesterol will oxidize slowly in tissues or foods to form a range of different products with additional hydroperoxy, epoxy, hydroxy or keto groups, and these can enter tissues via the diet. There is increasing interest in these from the standpoint of human health and nutrition, since accumulation of oxo-sterols in plasma is associated with inhibition of the biosynthesis of cholesterol and bile acids and with other abnormalities in plasma lipid metabolism. These and similar cholesterol oxides or oxysterols produced in tissues by specific microsomal or mitochondrial oxidations are discussed in a further document on this web site.

A number of other sterols occur in small amounts in tissues, most of which are intermediates in the pathway from lanosterol to cholesterol, although some of them have distinct functions in their own right. Lanosterol, the first sterol intermediate in the biosynthesis of cholesterol, was first found in wool wax, both in free and esterified form, and this is still the main commercial source. It is found at low levels only in most other animal tissues (typically 0.1% of the cholesterol concentration). As oxygen is required, lanosterol cannot be produced by primitive organisms, hence its absence from prokaryotes, leading to some speculation on its evolutionary significance. When sterols became available to eukaryotes, much greater possibilities opened for their continuing evolution. The production of cholesterol from lanosterol is then seen as ‘molecular streamlining’ by evolution, removing protruding methyl groups that hinder the interaction between sterols and phospholipids in membranes.

Desmosterol (5,24-cholestadien-3β-ol), the last intermediate in the biosynthesis of cholesterol by the Bloch pathway, may be involved in the process of myelination, as it is found in relative abundance in the brains of young animals but not in those of adults, other than astrocytes. It is also found in appreciable amounts in testes and spermatozoa together with another cholesterol intermediate, testis meiosis-activating sterol. In addition, there is evidence that desmosterol activates certain genes involved in lipid biosynthesis in macrophages, and may deactivate others associated with the inflammatory response. There is a rare genetic disorder in which there is an impairment in the conversion of desmosterol to cholesterol, desmosterolosis, with serious consequences in terms of mental capacity. These and related sterols appear to be essential for human reproduction.

Structural formulae of other animal sterols

In human serum, the levels of lathosterol (5α-cholest-7-en-3β-ol) were found to be inversely related to the size of the bile acid pool, and in general the concentration of serum lathosterol is strongly correlated with the cholesterol balance under most dietary conditions. The isomeric saturated sterols, cholestanol and coprastanol, which differ in the stereochemistry of the hydrogen atom on carbon 5, are formed by microbial biohydrogenation of cholesterol in the intestines, and together with cholesterol are the main sterols in faeces. Further examples of animal sterols include 7-dehydrocholesterol (cholesta-5,7-dien-3β-ol) in the skin, which on irradiation with UV light is converted to vitamin D3 (cholecalciferol).

Marine invertebrates produce a large number of novel sterols, with both unusual nuclei and unconventional side-chains, some derived from cholesterol and others from plant sterols or alternative biosynthetic intermediates. For example, at least 80 distinct sterols have been isolated from echinoderms and 100 from sponges.

7.  Cholesterol and Disease

Elevated cholesterol and cholesterol ester levels are associated with the pathogenesis of cardiovascular disease (atherosclerotic plaques, myocardial infarctions, and strokes), as is well known, and this is considered briefly on this website together with the metabolism of the plasma lipoproteins. The rate-limiting enzyme in the synthesis of cholesterol HMG-CoA reductase is the target of statins, but drugs that target other steps in the biosynthetic pathway, especially the squalene monooxygenase and lanosterol synthase, are under investigation. Further discussion of such a complex nutritional and clinical topic is best left to others.

Cholelithiasis or the presence of 'stones' in the gallbladder or bile ducts, which consist largely of cholesterol (~85%), is one of the most prevalent and costly digestive diseases in developed countries. The primary cause is excessive excretion of cholesterol from the liver. Excess accumulation of cholesterol associated with the metabolism of bis(monoacylglycero)phosphate and causing disturbances in glycosphingolipid trafficking in cell membranes is involved in the pathogenesis of Niemann-Pick C disease, a lysosomal storage disease in which endocytosed cholesterol becomes sequestered in late endosomes/lysosomes because of gene mutations affecting two binding proteins (NPC1 and NPC2) thereby causing neuronal and visceral atrophy. In addition, deficiencies in cholesterol transport and metabolism are associated with many forms of neurodegeneration, including Alzheimer’s disease and Huntington’s disease.

Several genetic disorders of cholesterol biosynthesis have been identified in recent years that can result in developmental malformations including neurologic defects. As there is limited cholesterol transport across the placenta, the human foetus is highly dependent upon endogenous synthesis. While the molecular basis for the altered developmental pathways is not fully understood, impaired synthesis of the hedgehog family of signalling proteins, which require covalently linked cholesterol to function in membranes, is believed to be involved in many cases. In others, there are confirmed enzyme defects. For example, the recessive Smith-Lemli-Opitz syndrome in infants born with a decreased concentration of the enzyme 7-dihydrocholesterol reductase, produces symptoms varying from mild autism to severe mental and often fatal physical problems. The effects are due to a lack of cholesterol and the accumulation of 7-dehydrocholesterol and its 27-hydroxy metabolite, as brain tissue cannot utilize dietary cholesterol or that produced peripherally. In fact, at least eight different inherited disorders of cholesterol biosynthesis are recognized that lead to congenital abnormalities in those afflicted. In animal models, deficiencies in SREBP-2 and genes encoding sterol biosynthetic enzymes display embryonic lethality. Deficiencies in the enzymes responsible for the hydrolysis of cholesterol esters, such as the lysosomal acid lipase, occur in Wolman disease and cholesterol ester storage disease.

Cholesterol and other sterols bind directly to several immune receptors, especially in macrophages and T cells, and dynamic changes in cholesterol biosynthesis impact directly upon innate and adaptive immune responses, such that functional coupling between sterol metabolism and immunity has implications for health and disease. In cancer, there is a high demand for cholesterol in order to support the inherent nature of tumour cells to divide and proliferate, and perturbations of reverse cholesterol transport can have negative consequences. Drugs that lower cholesterol levels in cancer cells are undergoing clinical trials.

When increased levels of sterols other than cholesterol are found in plasma, they usually serve as markers for abnormalities in lipid metabolism associated with disease states. For example, premature atherosclerosis and xanthomatosis occur in two rare lipid storage diseases, cerebrotendinous xanthomatosis and sitosterolemia. In the former, cholestanol is present in all tissues, while in the latter, the dietary plant sterols campesterol and sitosterol accumulate in plasma and red blood cells. Inhibition of cholesterol biosynthesis may be associated with the appearance of some of the precursor sterols in the plasma.

In infections with Mycobacterium tuberculosis, the organism uses host cholesterol as the major carbon and energy source and thereby promotes persistent infection with appreciable effects on pathogenicity. Similarly, Chlamydia trachomatis, a gram-negative obligate intracellular bacterium and a major cause of sexually transmitted infections, requires host cholesterol for growth. An HIV protein has a binding site for cholesterol, which it utilizes to facilitate the fusion with raft regions in membranes of the host cell.

8.  Analysis

With animal tissues, especially those of clinical importance such as plasma, the cholesterol content is often determined by using enzymatic methods from commercially available kits that are suited to routine analysis of large numbers of samples, though with less precision and selectivity than by chromatographic procedures. For the total cholesterol content, it is necessary to hydrolyse the cholesterol ester fraction first, and this usually requires more vigorous conditions than with glycerolipids. For more accurate or detailed analysis of animal and plant sterols, a sterol fraction is first isolated from lipid extracts by thin-layer or column chromatography, following hydrolysis if necessary. Individual components can then be determined by gas chromatography in the presence of an internal standard (e.g. epicoprostanol or betulin), often after conversion to trimethylsilyl ether derivatives to give sharper peaks. Mass spectrometry may be required for identification of individual components.

Sterol esters are transmethylated for GC analysis of the fatty acid components, although the reaction may again be much slower than with glycerolipids. Intact sterol esters are best analysed by reversed-phase HPLC.

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Lipid listings Credits/disclaimer Updated: December 23rd, 2019 Author: William W. Christie LipidWeb icon