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21.5: Biosynthesis of Cholesterol and Steroids

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    https://www.lipidmaps.org/resources/...lest/index.htm

    By William (Bill) W. Christie and Henry Jakubowski.

    This section is an abbreviated and modified version of material from the Lipid Web, an introduction to the chemistry and biochemistry of individual lipid classes, written by William Christie.

    Sterols: 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. 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.

    Cholesterol plays a vital role in animal life, and it is essential for the normal functioning of cells both as a structural component of cell membranes and as a precursor of steroid hormones and other key metabolites including vitamin D and bile acids. It is also important for cell signaling, transport processes, nerve conduction and the regulation of gene transcription. Every cell in vertebrates is able both to synthesise cholesterol and to metabolize it, and there is evidence that synthesis de novo is essential whatever the dietary intake; this is vital in the brain. However, excess cholesterol can contribute to the pathology of various diseases, notably cardiovascular disease, so cholesterol levels must be balanced to ensure an adequate but not excessive supply.

    Cholesterol – Structure, Occurrence and Function in Membranes

    The strutural of cholesterol is shown below in Figure \(\PageIndex{1}\).

    cholest-5-en-3-b-ol.svg
    Figure \(\PageIndex{xx}\):

    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 most current authors.

    Most of the atoms in cholesterol can be placed on the diamond lattice, which is the structure showing the position of each carbon atoms in the network solid diamond. In that structure, each carbon is connected to four other carbons using sp3 hydridized atomic orbirtals and tetrahedral geometery. A representation of the diamond lattice is shown below. It consists of a series of interconnected boat conformations of cyclohexane propagting in the xyz direction. The structures of two different fused cyclohexanes, trans- and cis-decalins as well as the structure of three fused cyclohexanes, adamantane, are shown in Figure \(\PageIndex{2}\) below. Superimposing sp3 connected atoms onto a diamond lattice allows improved visualization of the real and allowed structures of more complicated molecules.

    diamond_andamantane_decalin.svg
    Figure \(\PageIndex{2}\): The diamond lattice showing decalins and adamantane

    Figure \(\PageIndex{3}\) below shows a superposition of 5-α-cholestane, a reduced and non-hydroxylated form of cholesterol, onto the diamond lattice. The pink cyclopentane D ring of 5-α-cholestane, which is distorted from the 109 degree bond angle for sp3 hybridized carbon atoms, does not fit exactly onto the lattice. Cholesterol, with its double bond, would also deviate from ideal position on the diamond lattice given the two sp2 hybridized carbons in the double bond.

    cholesteranediamondlattice.svg
    Figure \(\PageIndex{3}\): The diamond lattice showing 5-α-cholestane

    Cholesterol is a ubiquitous component of all animal tissues (and of some fungi), produced by every nucleated animal cell, 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.

    Animals in general synthesize 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 synthesize 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 (e.g., Borrelia sp.); 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. The structure of cholesterol as it would appear on the diamond lattice is shown in Figure \(\PageIndex{4}\) below.

    cholsterolplanar.svg
    Figure \(\PageIndex{4}\): "Diamond" lattice 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.

    In 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 enable membranes to bend or withstand mechanical stresses, and they permit the fine-tuning of membrane lipid composition and organization, and regulate critical cell functions. 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).

    In comparison to other lipids, it has been reported that cholesterol can flip rapidly between the leaflets in a bilayer, although this not appear to be accepted universally, leading to doubts as to the trans-bilayer distribution of cholesterol in some biological membranes. However, much recent evidence suggests that the concentration of cholesterol in the inner leaflet of the plasma membrane is much lower than that in the outer leaflet in a range of mammalian cells. 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. It may also be relevant for the regulation of various cellular signaling processes at the plasma membrane.

    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. Cholesterol has an intimate interaction with G-protein-coupled receptors (GPCRs) to affect ligand binding and activation, either by direct high-affinity binding to the receptor, by changing their oligomerization state or by inducing changes in the properties of the membrane. For example, it is essential for the stability and function of the β2-adrenergic, oxytocin and serotonin receptors by increasing the agonist affinities, while the inactive state of rhodopsin is stabilized both through indirect effects on plasma membrane curvature and by a direct interaction between lipid and protein. The GPCR neurotransmitter serotonin1A receptor has ten closely bound cholesterol molecules, and these control its organization and positioning; the receptor senses membrane cholesterol via a lysine residue in a so-called 'CRAC' motif in transmembrane helix 2.

    Ion pumps such as the (Na+-K+)-ATPase, which have specific binding site for cholesterol molecules, are the single most important consumer of ATP in cells and are 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. 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. In the nucleus of cells, cholesterol is intimately involved in chromatin structure and function.

    The role of cholesterol together with sphingolipids in the formation of the transient membrane nano-domains known as rafts (see the specific web page for detailed discussion), is of crucial importance for the function of cells, while the interaction of cholesterol with ceramides is essential for the barrier function of the skin.

    Cholesterol Biosynthesis

    Cholesterol biosynthesis involves a highly complex series of at least thirty different enzymatic reactions, which were unraveled 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. We'll explore the reactions mechanisms for several of the enzymes on this complicated pathway given its medical importance.

    Almost all nucleated cells are able to synthesize 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. These precursors are in the cytosol as is the first enzyme, 3-hydroxy-3-methyl-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. These first two reaction in the synthesis of cholesterol are shown in Figure \(\PageIndex{5}\) below.

    AcAc_AcCoA_toHMGCoAtoMevalonateRxEq.svg
    Figure \(\PageIndex{5}\): First two reaction in the synthesis of cholesterol

    HMG-CoA Synthase

    We saw this reaction in the synthesis of ketone bodies in Chapter 17. This enzyme catalyzes the condensation of acetoacetyl–CoA (AcAc–CoA) and acetyl–CoA (Ac-CoA) to form 3-hydroxy-3-methylglutaryl (HMG)–CoA, and requires the formation of a C-C bond. HMG-CoA synthase forms a C-C bond through activating the methyl group of acetyl-cysteine. The acetyl group comes from an acyl-CoA "donor". The enzyme catalyzes the first committed step in the formation of complex isoprenoids (like cholesterol) and of ketone bodies. The product, 3-hydroxy-3-methylglutaryl HMG–CoA, can either be reduced by HMG-CoA reductase to form mevalonate, which leads to cholesterol synthesis, or cleaved by the enzyme HMG-CoA lyase, to produce acetoacetate, a ketone body.

    HMG-CoA synthase catalyzes a bisubstrate reaction that displays ping-pong kinetics, characteristic of a covalent enzyme intermediate. The first substrate binds to the enzyme and transfer an acetyl group to a nucleophilic Cys 111 in the active site to form an acetyl-Cys 111 intermediate. Free CoA departs. Next the second substrate, acetoacetyl-CoA binds, and condense with the acetyl group donated by acetyl-Cys 111. This condensation involves an enolate. A plausible reaction mechanism is shown in Figure \(\PageIndex{6}\) below.

    HMG-CoASynthaseMech.svg

    Figure \(\PageIndex{6}\): Reaction mechanism for HMG-CoA synthase

    Hence there are three parts of the reaction: acetylation/deacetylation, condensation/cleavage with an enolate intermediate and C-C formation and hydrolysis/dehydration. Figure \(\PageIndex{7}\) below shows an interactive iCn3D model of the Staphylococcus aureus HMG-COA Synthase with bound HMG-CoA and acetoacetyl-CoA (1XPK)

    Staphylococcus aureus HMG-COA Synthase with bound HMG-CoA and acetoacetyl-CoA (1XPK).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{7}\): Staphylococcus aureus HMG-COA Synthase with bound HMG-CoA and acetoacetyl-CoA (1XPK). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...deiFs7JceE5H76

    The biologically active unit (homodimer) is shown with each monomer shown in a different color. The A chain (light cyan) has bound HMG-CoA (HMG) while the B chain (light gold) has acetoacetyl-CoA (CAA) bound. The Glu 79, Cys 111 and His 233 in each subunit are shown in CPK sticks and labeled. Note that the Cys 111 is covalent modified in each subunit.

    Figure \(\PageIndex{8}\) below shows an interactive iCn3D model of the human 3-hydroxy-3-methylglutaryl CoA synthase I with bound CoASH (2P8U)

    human 3-hydroxy-3-methylglutaryl CoA synthase I.png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{8}\): Human 3-hydroxy-3-methylglutaryl CoA synthase I (monomer) with bound CoASH (2P8U). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...xxEoUfecTk3eL6

    The active site side chains are numbered differently (Glu 95, Cys 129 and His 264) compared to the S. aurus protein. Only the monomer is shown in this model.

    HMG-CoA Reductase

    In this key rate limiting step that commits HMG to the sterol and isoprenoid synthetic pathways, HMG is converted to mevalonate. A reducing agent (NADPH) is required for this biosynthetic reaction. A plausible mechanism is shown in Figure \(\PageIndex{9}\) below.

    HGMCoA_Reductase.svg
    Figure \(\PageIndex{9}\): Reaction mechanism for HMG-CoA reductase. Adapted from https://www.ebi.ac.uk/thornton-srv/m-csa/entry/93/. Creative Commons Attribution 4.0 International (CC BY 4.0) License.

    Figure \(\PageIndex{10}\) below shows an interactive iCn3D model of the catalytic domain of human HMG-CoA reductase with bound HMG-CoA (1DQ9).

    Catalytic domain of human HMG-CoA reductase with bound HMG-CoA (1DQ9).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{10}\): Catalytic domain of human HMG-CoA reductase with bound HMG-CoA (1DQ9). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...dZvWzTocKhUzt8

    Two of the four identical monomers in the biological unit, the C (gray) and D (cyan) chains are shown. HMG-CoA is shown in spacefill and CPK colors. Three key catalytic residues, His 866 in the C chain (b chain in the mechanism above) and Lys 691 and Asp 767 in the D chain (A chain in the mechanism above) are shown in sticks, CPK colors and labeled.

    Synthesis of 5-isopentenyl and 2-isopentenyl pyrophosphate

    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. These reactions are shown in Figure \(\PageIndex{11}\) below.

    mevalonatetoIPPP_DMAPP.svg
    Figure \(\PageIndex{11}\): Reactions for the synthesis of 5-isopentenyl and 2-isopentenyl pyrophosphate for synthesis of sterols and isoprenoids

    Two phosphorylations are required, one by mevalonate kinase, which proceeds by an ordered sequential binding of mevalonate as the first reactant and its phosphomevalonate as the first product released. The enzyme is inhibited by two downstream products of the reaction pathway, farnesyl pyrophosphate and geranyl pyrophosphate. A mechanism for mevalonate kinase is shown in Figure \(\PageIndex{12}\) below.

    mevalonatekinase.svg
    Figure \(\PageIndex{12}\): Mechanism of mevalonate kinase (adapted from Fu et al. JBC. 277 (2002). https://doi.org/10.1074/jbc.M200912200

    A reaction mechanism for the second kinase, phosphomevalonate kinase, showing progression through the transition state, is shown in Figure \(\PageIndex{13}\) below. The reaction proceeds through a direct phosphorylation through a dissociative mechanism.

    phophomevalonatekinase.svg
    Figure \(\PageIndex{13}\): Reaction mechanism for phosphomevalonate kinase (after Huang et al. J. Phys. Chem. B 2016, 120. DOI: 10.1021/acs.jpcb.6b08480)

    Mevalonate diphosphate decarboxylase

    This enzyme catalyzes the decarboxylation of 5-pyrophosphate mevalonate to 5-isopentenylpyrophosphate as shown in Figure \(\PageIndex{14}\) below.

    pyrophosphomevalonatetoisopentylPP.svg
    Figure \(\PageIndex{14}\): Conversion of 5-pyrophosphaemevalonate to 5-isopentyl pyrophosphate

    The binding of the two substrates, pyrophosphatemevalonate (or mevalonate pyrophosphate - MVAPP) and ATP to the enzyme (A), and a reaction mechanism (B, are shown in Figure \(\PageIndex{15}\) below.

    Visual_mech_mevalonate diphosphate decarboxylaseFig7.svg
    Figure \(\PageIndex{15}\): Proposed model for the detailed MDD enzyme mechanism. Chen et al. Nature Communications. (2020) 11:3969 |https://doi.org/10.1038/s41467-020-17733-0. Creative Commons Attribution 4.0 International License. http://creativecommons.org/ licenses/by/4.0/.

    Panel (a) shows changes in the enzyme structure upon substrate binding. The apo-enzyme is shown on the left. The middle structure shows the enzyme after binding of the first substrate, MVAPP. A key loop (β10-α4) is shown as a cyan surface. The enyzme is then shown in an open conformation with the second substrate (ATP) also bound. An additional phosphate-biding loop is shown in magenta. At the far right, the enzyme is shown in a closed conformation after conformational changes in both loops which trapss substrates in the active site. These changes enable catalysis. Product release follows.

    Panel (b) shows the dissociative phosphoryl transfer catalytic mechanism. At the top-left, D282 is shown interacting with the 3′-OH group of MVAPP (red). The top-right shows a dissociative phosphoryl (blue) transition state. In the bottom-left, the phosphate attaches to the 3′ oxygen (red) of MVAPP. The bottom-right shows the products after the dephosphorylation and decarboxylation to produce IPP, ADP, phosphate, and CO2. K187 from the β10-α4 loop and metal ions in the active site are involved in neutralizing the negatively charged environment and assist catalysis.

    Figure \(\PageIndex{16}\) below shows an interactive iCn3D model of mevalonate diphosphate decarboxylase with mevalonate-5-diphosphate, AMPPCP and Magnesium (6E2U).

    mevalonate diPdecarboxylase AMPPCPMagnesium (6E2U).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{16}\): Mevalonate diphosphate decarboxylase with mevalonate-5-diphosphate, AMPPCP and Magnesium (6E2U) . (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...SukmP1BTyNhH26

    The correctly positioned substrates interact with two Mg 2+ ions in the initial steps of the reaction. The conserved lysine facilitates the phosphoryl transfer.

    Polymerization of isoprene

    Isoprene, a small branched alkadiene, which can polymerize into larger molecules containing isoprene monomer to form isoprenoids, often called terpenes. Instead of using isoprene as the polymerization monomer, either dimethylally pyrophosphate (DMAPP) or isopentenylpyrophosphate (IPP) are used biologically.

    Figure \(\PageIndex{x}\) below shows how DMAPP and IPP (both containing 5Cs) are used in a polymerization reaction to form geranyl-pyrophosphate (C10), farnesyl pyrophosphate (C15) and geranyl-geranyl pyrophosphate (C20). Figure \(\PageIndex{17}\):

    isoprenoidSyn0222.svg
    Figure \(\PageIndex{17}\): Synthesis of geranyl-pyrophosphate (C10), farnesyl pyrophosphate (C15) and geranyl-geranyl pyrophosphate (C20)

    The condensation of IPP and DMAPP is a head to tail condensation reaction. Another IPP reacts with geranylpyrophosphate using the same enzyme to produce farnesylpyrophosphate. DMAPP is first formed by the isomerization of an IPP to DMAPP catalyzed by isopentenyl-diphosphate delta-isomerase. It catalyzes the 1,3-allylic rearrangement of the homoallylic substrate isopentenyl (IPP). 5-isopentenyl pyrophosphate is a nucleophile, but its isomer, DMAPP, is highly electrophilic, which promotes the condensation of the two molecules.

    A mechanism for the next reaction, the first condensation of DMAPP and IPP to form geranylpyrophosphate by farnesyl pyrophosphate (diphosphate) synthase reaction, is shown in Figure \(\PageIndex{18}\) below.

    Farnesyldiphosphatesynthase.svg
    Figure \(\PageIndex{18}\): Mechanism for the condensation of DMAPP and IPP to form geranylpyrophosphate by farnesyl pyrophosphate (diphosphate) synthase

    The reaction appears to proceed using a carbocation transition state, followed by transfer of a hydrogen atom (not ion) from IPP to pyrophosphate. Figure \(\PageIndex{19}\) below shows an interactive iCn3D model of E. Coli farnesyl pyrophosphate synthase bound to isopentyl pyrophosphate and dimethylallyl S-thiolodiphosphate (1RQI).

    FarnesylPPSynthaseIPPDimethylallyl S-Thiolodiphosphate(1rqi).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{19}\): Farnesyl pyrophosphate synthase Bound to isopentyl pyrophosphate (IPP) and dimethylallyl S-thiolodiphosphate (1RQI). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...kQBU5dNnKrCVt6

    DST in the structure is dimethylallyl S-thiolodiphosphate, an analog of dimethylallyl diphosphate (DMAPP). Key conserved amino acids involved in substrate binding, transition state stabilization, and catalysis are shown as sticks, CPK colors. Thr203, Gln241, and Lys202 presumably stabilized the carbocation intermediate/transition state. Lys202 also forms a hydrogen bond with DMAPP. Two arginines (69, 116) form salt bridges with the pyrophosphates of IPP and DMAPP.

    Many isoprenoid lipids are made from farnesyl pyrophosphate. For membrane purposes, the most important of these is cholesterol. Figure \(\PageIndex{20}\) below shows an overview of the synthesis of cholesterol from two farnesyl pyrophosphates linking together in a "tail to tail" reaction to form squalene, a precursor of cholesterol. Each isoprene unit (5Cs) is shown in different colors to make it easier to see.

    squalenes_lanosterol_cholesterol0222.svg

    Figure \(\PageIndex{20}\): Synthesis of squalene from isoprene units Figure \(\PageIndex{21}\) shows reactants (two farnesyl pyrophosphate), intermediate (presqualene diphosphate) and product (squalene) in the reaction catalyses by squalene synthase.

    franesyltopresqaultosqualene.svg
    Figure \(\PageIndex{21}\):

    In the squalene synthase reaction, two molecules of farnesyl pyrophosphate condense to yield presqualene pyrophosphate. In turn, this is reduced by NADPH to produce the key intermediate squalene. 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), is considered to be the first committed enzyme in cholesterol biosynthesis.

    Given the importance of this reaction, we will explore the unique mechanism of squalene synthase in some detail.

    Part 1: Formation of cyclopropyl presqualene intermediate

    Figure \(\PageIndex{22}\) shows the mechanism for the first part of the reaction in which the cyclopropyl intermediate presqualene form. The reaction proceeds through a series of carbocation intermediates.

    squalenesynthesis1.svg
    Figure \(\PageIndex{22}\): Mechanism for synthesis of presqualene by squalene synthase. https://www.ebi.ac.uk/thornton-srv/m-csa/entry/264/. Creative Commons Attribution 4.0 International (CC BY 4.0) License

    Part 2: Conversion of cyclopropyl intermediate to squalene - reduction

    The next part of the reaction involves the reductive formation of squalene, as shown in Figure \(\PageIndex{23}\) below.

    squalenesynthesis2.svg
    Figure \(\PageIndex{23}\): Mechanism for conversion of presqualene to squalene by squalene synthase. https://www.ebi.ac.uk/thornton-srv/m-csa/entry/264/. Creative Commons Attribution 4.0 International (CC BY 4.0) License

    Figure \(\PageIndex{24}\) below shows an interactive iCn3D model of human squalene synthase with bound inhibitor (1EZF).

    Human squalene synthase with bound inhibitor (1EZF).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{24}\): Human squalene synthase with bound inhibitor (1EZF). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...jLreHkvbftoFPA

    Tyr 171 acts as a general acid/base in the first half of the reaction. Arg 218 and 228 stabilize the diphosphate in the transition state as it leaves. Phe 288 stabilizes the reactive carbocation.

    The enzyme has a single domain which contains a large channel in one face of the enzyme which leads from a solvent-exposed to hydrophobic interior. Two FPPs bind in the beginninf of the channel where key side chains for the first reaction are located. The cyclopropyl intermediate then moves into the hydrophobic end of the channel where it reacts in the second half of the reaction without exposure to water.

    In the next important step, squalene is oxidized by a squalene monooxygenase to squalene 2,3-epoxide, a key control point in the cholesterol synthesis pathway. This introduces the oxygen atom to squalene that becomes the signature oxygen of the hydroxyl group in cholesterol. The epoxide then undergoes cyclization catalyzed by the enzyme squalene epoxide lanosterol-cyclase to form the first steroidal intermediate lanosterol (or cycloartenol en route to phytosterols in photosynthetic organisms). This is illustrated in Figure \(\PageIndex{26}\) below.

    squaleneto_lanosterol_electflow.svg
    Figure \(\PageIndex{26}\): Conversion of squalene to lanosterol

    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.

    The ring closure reaction starting with the epoxide involves a concerted flow of electrons from a source to the epoxide oxygen atom electron sink. This brings to mind a reaction known to all students who have ever studied chemistry, the reaction of the pH indicator phenolphthalein with base to produced a pink colored-solution. That reaction which produces a more conjugated molecule which absorbs at 553 nm (green) which causes a magenta solution color, is illustrated for comparion (and fun) in Figure \(\PageIndex{27}\) below.

    henolphthaleinTit.svg
    Figure \(\PageIndex{27}\): Reaction of phenolphthalein with base

    In subsequent steps, lanosterol is converted to cholesterol by a series of demethylations, desaturations, isomerizations and reductions, involving nineteen separate reactions as illustrated in Figure \(\PageIndex{28}\) below.

    Final steps in cholesterol biosynthesis
    Figure \(\PageIndex{28}\): Production of cholesterol from lanosterol

    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 so-called 'Bloch' pathway, while 7‑dehydrocholesterol is the immediate precursor in the 'Kandutsch-Russell' 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.

    The energy cost of the synthesis of one cholesterol molecule is roughly one hundred ATP equivalents, and eleven oxygen molecules are required. Synthesis occurs mainly in the liver, although the brain (see below), peripheral nervous system and skin synthesize 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 any external sources of cholesterol, and cholesterol homeostasis requires the actions of a complex web of enzymes, transport proteins, and membrane-bound transcription factors, as discussed below.

    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 in the endoplasmic reticulum. The latter must be tightly regulated as it is an energetically expensive process that requires appreciable amounts of acetyl-CoA, ATP, oxygen and the reducing factors NADPH and NADH, especially since cholesterol cannot be catabolized for energy purposes (see below).

    Many factors are involved in maintaining the large differences in cholesterol concentrations among the various membranes and organelles in cells within precise limits. In order to explain how cholesterol in the plasma membrane, where it is most abundant, can regulate cholesterol biosynthesis and uptake through enzymes in the endoplasmic reticulum, where it is least abundant, it has been suggested that a key to the process is that there are three pools of cholesterol in the plasma membrane with distinct functional roles. The first of these is “accessible” to receptor proteins for transport to the endoplasmic reticulum, while the second pool is sequestered by sphingomyelin and can be released by the action of sphingomyelinase if required. The third residual pool of cholesterol is essential for plasma membrane integrity. These correspond to about 16, 15, and 12 mol % of total plasma membrane lipids, respectively, in cholesterol-replete cells. Simplistically, when cholesterol in the plasma membrane is in excess for any reason, e.g., after LDL uptake by receptor-mediated endocytosis, there is a rise in accessible cholesterol, which is then transported to the endoplasmic reticulum to switch off cholesterol biosynthesis and expression of the LDL receptor. This process requires a host of regulatory proteins, and mechanisms that can involve either vesicle formation or non-vesicular pathways that utilize specific transport proteins, such as the ABC transporters.

    Ultimately, post-translational control of the many different enzymes involved provides a rapid means for modifying flux through the biosynthetic pathway in the endoplasmic reticulum; some are rapidly degraded in response to tissue levels of cholesterol and its intermediates, while others have their activity altered through phosphorylation or acetylation mechanisms. For example, 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. Similarly, HMG-CoA reductase is recognized as the key enzyme in the regulation of cholesterol biosynthesis, and this can be regulated by a feedback mechanism involving ubiquitin–proteasomal degradation. Further regulation of cholesterol biosynthesis is exerted by sterol intermediates in cholesterol biosynthesis, such as lanosterol and 24,25‑dehydrolanosterol (dimethyl-sterols) by accelerating degradation of the biosynthetic enzymes such as HMG-CoA reductase. It is noteworthy that ceramide down-regulates cholesterol synthesis – another link between cholesterol and sphingolipid metabolism.

    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 essential to the maintenance of cholesterol levels. 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. In effect, cholesterol exerts feedback inhibition by suppressing its own production by preventing the proteolytic cleavage and maturation of SREBP-2. Oxysterols, especially 25-hydroxycholesterol, are also inhibitors of this process.

    Cholesterol in the endoplasmic reticulum is transferred to the Golgi and eventually to the plasma membrane by vesicular and non-vesicular transport mechanisms involving in part soluble sterol transport proteins, including the so-called 'START' domain proteins, and partly by binding to those proteins that are intimately involved in the transport and metabolism of polyphosphoinositides such as phosphatidylinositol 4-phosphate (PI(4)P). In the latter mechanism, cholesterol is transported by binding to the ORD domain of oxysterol binding protein (OSBP) or Osh4 in yeast, before OSBP binds to PI(4)P in the plasma membrane to transfer its cargo. The key to this process is that cholesterol and PI(4)P are synthesized at two different locations, i.e., the endoplasmic reticulum for sterols and the trans-Golgi network and plasma membrane for PI(4)P, so the two lipids do not compete but rather can be exchanged. OSBP carry cholesterol in the forward direction to the trans-Golgi network and plasma membrane and PI(4)P, which binds to a C-terminal PH domain in the protein, in the reverse direction. The subsequent hydrolysis of PI(4)P is the energy source for the reaction, and indeed PI(4)P has been termed "lipid ATP". As this reaction is irreversible, a gradient of cholesterol along organelles of the secretory pathway is established. OSBP is thus a lipid transfer protein that enables two organelles to exchange cholesterol rapidly between them at membrane contact sites in a cycle of reactions involving membrane tethering, cholesterol transport, PI(4)P counter transport, and PI(4)P hydrolysis. A similar mechanism is involved in the transport of phosphatidylserine from the endoplasmic reticulum to the inner leaflet of the plasma membrane.

    Subsequently, 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. 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. Also, side-chain oxysterols, especially 25-hydroxycholesterol, can suppress the activation of SREBP by binding to an oxysterol-sensing protein in the endoplasmic reticulum.

    Within cells, cholesterol derived initially from the lysosomal degradation of low-density lipoproteins is transferred first to the plasma membrane and thence to the endoplasmic reticulum, the latter step by a mechanism involving proteins known as GRAMD1s embedded in the endoplasmic reticulum membrane at sites in contact with the plasma membrane. These have two functional domains: the START-like domain that binds cholesterol and the GRAM domain that binds anionic lipids, such as phosphatidylserine, and so are able to form a link between the two membranes that enables the transfer of cholesterol.

    In peripheral tissues, excess cholesterol is exported to high-density lipoproteins (HDL) in the circulation and returned to the liver, a process known as reverse cholesterol transport. The liver is important for cholesterol synthesis, but it is essential for its elimination from the body in bile. Also, some lipoproteins with their content of cholesterol and cholesterol esters are delivered to lysosomes by endocytosis for degradation. The cholesterol is transported to the inner surface of the lysosomal membrane through the glycocalyx, via a transglycocalyx tunnel, with the aid of Niemann-Pick C1, C2 and other proteins, and thence via contact sites between membranes to other organelles. Cholesterol in cellular membranes in excess of the stoichiometric requirement can escape back into the cell, where it may serve as a feedback signal to down-regulate cholesterol accumulation, while some is converted to the relatively inert storage form, i.e., cholesterol esters, and some is used for steroidogenesis.

    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 (Niemann-Pick C1-like 1 or NPC1L1)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. Some cholesterol can be transferred in the opposite direction (trans-intestinal cholesterol excretion), but the quantitative importance of this process is not clear. 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.

    In 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, and it may inhibit the uptake of residual cholesterol. There are two mechanisms for this conversion in bacteria, one involving direct reduction and another via cholestenone and coprostanone as intermediates, and as the relevant genes have now been identified the therapeutic potential is under investigation.

    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 (glial cells). During the perinatal and adolescent years especially, cholesterol is synthesised in large amounts to form the myelin that surrounds the axons, before this rate begins to decline to eventually reach about 10% of earlier values.

    Cholesterol is transported to neurons in the form of Apo E complexes in discoidal HDL-like particles, for which seven main receptors have been identified in brain cells that take up cholesterol from these lipoproteins. 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.

    Cholesterol Catabolism

    Cholesterol is not readily degraded in animal tissues so does not serve as a metabolic fuel to generate ATP. 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. 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.

    Gut bacteria reduce some of the cholesterol in the diet to highly insoluble 5β-cholestan-3β-ol (coprostanol), which is excreted and can be used as a biomarker for sewage in the environment. Certain bacterial species contain a 3β-hydroxysteroid:oxygen oxidoreductase (EC 1.1.3.6), 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.

    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 detoxification 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 as shown in Table \(\PageIndex{1}\) below. Arachidonic and “adrenic” (20:4(n-6)) acids can be especially abundant in cholesterol esters from the adrenal gland.

    Table \(\PageIndex{1}\). Fatty acid composition of cholesterol esters (wt % of the total) from various tissues.
    Form Fatty acids
    16:0 18:0 18:1 18:2 18:3 20:4 22:4
    Human  
    plasma 12 2 27 45 8
    liver 23 10 28 22 6
    Sheep  
    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); DOI. 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); DOI.

    In plasma and in the high-density lipoproteins (HDL) in particular, cholesterol esters are synthesized largely by transfer of fatty acids to cholesterol from position sn-2 of phosphatidylcholine (‘lecithin’) catalyzed by the enzyme lecithin:cholesterol acyl transferase (LCAT); the other product is 1-acyl lysophosphatidylcholine.This is illustrated in Figure \(\PageIndex{29}\) below.

    cholesterolesters.svg
    Figure \(\PageIndex{29}\): Synthesis of cholesterol esters

    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).

    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 are characterized by multiple transmembrane domains and a catalytic histidine residue in a hydrophobic domain; they are members of the 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.

    Oxidized Cholesterol Esters: All lipid classes containing polyunsaturated fatty acids are susceptible to oxidation. Under normal circumstances, cholesterol esters are considered to be relatively inert. However, when they contain oxidized polyunsaturated fatty acids, their properties change and they acquire biological activity. Such oxidized cholesterol esters may be formed by a reaction with 15‑lipoxygenase, but they can be produced also through free radical-induced lipid peroxidation, and they have been detected in lipoproteins, LDL especially, in human blood and atherosclerotic lesions. Those oxidized cholesterol esters in plasma are trafficked into cells and metabolized by the same mechanisms as the corresponding unoxidized lipids.

    Such "minimally oxidized LDL" do not bind to CD36 but rather to CD14, a receptor that recognizes bacterial lipopolysaccharides. The result is stimulation of toll-like receptor 4 (TLR4), although the response differs from that to lipopolysaccharides. In addition, oxidized metabolites of cholesteryl arachidonate of this kind stimulate macrophages to express inflammatory cytokines of relevance to atherosclerosis among other effects. Oxidized cholesterol esters can be hydrolyzed to release their fatty acids, which can then be incorporated into phospholipids with a different repertoire of activities.

    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.

    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.

    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). These sterols are shown in Figure \(\PageIndex{30}\) below.

    Structural formulae of other animal sterols

    Figure \(\PageIndex{30}\): Other animal sterols

    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.

    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 better qualified than myself.

    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, Huntington’s disease and related conditions associated with old age. These proteins are also key virulence factors for several viral and bacterial pathogens.

    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. For example, cholesterol binds directly to the αβ T cell antigen receptor (αβTCR) and has at least two opposing functions in its activation. By binding to the trans-membrane domain of this receptor, it is kept in an inactive, non-signaling conformation, but when required it can stimulate formation of receptor nanoclusters to increase their avidity for the antigen. 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 by inhibiting the mevalonate pathway 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. Many viruses use cholesterol as part of their life cycle, and reduction in cellular cholesterol is sometimes seen as an anti-viral strategy, although this may not always be helpful. For example, 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.

    Sterols: 2. Oxysterols and Other Cholesterol Derivatives

    Oxysterols as defined and discussed here are oxygenated derivatives of cholesterol and its precursors, i.e., with additional hydroxyl, epoxyl or keto groups, that are found in all animal tissues. Many of these have vital functions in animals, while others are important as short-lived intermediates or end products in the catabolism or excretion of cholesterol or in the biosynthesis of steroid hormones, bile acids and 1,25‑dihydroxy-vitamin D3. They are normally present in biological membranes and lipoproteins at trace levels only, though they can exert profound biological effects at these concentrations. However, they are always accompanied by a great excess (as much as 106-fold) of cholesterol per se.

    A multiplicity of different oxysterols are synthesised in cells by sequential reactions with specific oxygenases. However, because of the presence of the double bond in the 5,6-position, oxysterols can also be formed rapidly by non-enzymatic oxidation (autoxidation) of cholesterol and cholesterol esters within tissues with formation of many different oxygenated derivatives. Simplistically, non-enzymatic oxidation leads mainly to the generation of products in which the sterol ring system is oxidized, while enzymatic processes usually produce metabolites with an oxidized side chain (7-hydroxylation is an important exception). Oxidized cholesterol molecules can also be generated by the gut microflora and be taken up through the enterohepatic circulation. Once an oxygen function is introduced into cellular cholesterol, the product can act as a biologically active mediator by interacting with specific receptors before it is metabolized to bile acids or is degraded further, processes assisted by the fact that oxysterols are able to diffuse much more rapidly through membranes than is cholesterol itself. Cholesterol metabolites of this kind are especially important in brain, which is a major site for cholesterol synthesis de novo, and they are crucial elements of cholesterol homeostasis. For convenience, sterol sulfates and glycosides are also discussed in this web page, but steroidal hormones and vitamin D can only be described briefly.

    Enzymatic Oxidation of Cholesterol

    Within animal cells, oxidation of sterols is mainly an enzymic process that is carried out by several enzymes that are primarily from the cytochrome P450 family of oxygenases (named for a characteristic absorption at 450 nm). These are a disparate group of proteins that contain a single heme group and have a similar structural fold, though the amino acid sequences can differ appreciably. They are all mono-oxygenases. Oxysterol biosynthesis can be considered in terms of different pathways that depend on the position of the initial oxidation, but these pathways tend to overlap and lead to a complex web of different oxysterols (and eventually to bile acid formation). As these enzymes, which include cytochrome P450, cholesterol hydroxylase, hydroxysteroid dehydrogenases, and squalene epoxidase, are specific to particular tissues and indeed animal species, there is considerable variation in oxysterol distributions between organs. A few examples only of the first steps in some of these pathways are illustrated in Figure \(\PageIndex{31}\) below.

    Biosynthesis of oxysterols
    Figure \(\PageIndex{31}\): Enzymatic synthesis of oxysterols

    As an example, a primary product is 7α‑hydroxycholesterol, which is an important intermediate in the biosynthesis of bile acids by the 'neutral' pathway and of many other oxysterols, and it is produced in the liver by the action of cholesterol 7α-hydroxylase (CYP7A1), an enzyme that has a critical role in cholesterol homeostasis. The reaction is under strict regulatory control, and the expression of CYP7A1 is controlled by the farnesoid X receptor (FXR) and is activated by cholic and chenodeoxycholic acids. Any circulating 7α‑hydroxycholesterol represents leakage from the liver. Further oxidation of 7α‑hydroxycholesterol can occur, and the action of CYP3A4 in humans generates 7α,25‑dihydroxycholesterol as an important metabolite, for example, while oxidation by CYP27A1 yields 7α,27‑dihydroxycholesterol; the latter is regarded as a key step in a further pathway to oxysterols and bile acids. On the other hand, the epimer 7β‑hydroxycholesterol is produced in brain by the action of the toxic β-amyloid peptide and its precursor on cholesterol, but whether this is involved in the pathology of Alzheimer’s disease has yet to be determined.

    The hydroxysteroid 11-β-dehydrogenase 1 (HSD11B1) is responsible for the conversion of 7β-hydroxy-cholesterol to the important metabolite 7-keto-cholesterol, while HS11B2 catalyses the reverse reaction; 7-keto-cholesterol is also formed by autoxidation (see below). HSD11B1 is better known as the oxidoreductase that converts inactive cortisone to the active stress hormone cortisol in glucocorticoid target tissues.

    An alternative ('acidic') pathway to bile acids starts with the synthesis of 27-hydroxycholesterol (or more systematically named (25R)26‑hydroxycholesterol), which is produced by the cytochrome P450 enzyme (CYP27A1) and introduces the hydroxyl group into the terminal methyl carbon (C27 or C26 - used interchangeably). While this enzyme is present in the liver, it is found in many extra-hepatic tissues and especially the lung, which provides a steady flux of 27‑oxygenated metabolites to the liver. As a multifunctional mitochondrial P450 enzyme in liver, it generates both 27‑hydroxycholesterol and 3β‑hydroxy-5-cholestenoic acid, the bile acid precursor, which occur in small but significant amounts in plasma. 27‑Hydroxycholesterol is the most abundant circulating oxysterol, and its concentration in plasma correlates with that of total cholesterol. It can be oxidized to 7α,27‑dihydroxycholesterol by the enzyme CYP7B1. 4β‑Hydroxycholesterol is also abundant in plasma and is relatively stable; it is produced in humans by the action of the cytochromes CYP3A4 and CY3A5.

    In humans, the specific cytochrome P450 that produces 24S-hydroxycholesterol (cholest-5-ene-3β,24-diol) is cholesterol 24S‑hydroxylase (CYP46A1) and is located almost entirely in the smooth endoplasmic reticulum of neurons in the brain, including those of the hippocampus and cortex, which are important for learning and memory. It is by far the most abundant oxysterol in the brain after parturition, but during development, many more many oxysterols are produced. 24S‑hydroxycholesterol is responsible for 98-99% of the turnover of cholesterol in the central nervous system, which is the source of most of this oxylipin found in plasma. A small amount of it is converted in the brain directly into to 7α,24S‑dihydroxycholesterol by the cytochrome CYP39A1 and thence via side-chain oxidation in peroxisomes to bile acids, such as cholestanoic acid. It is evident that the blood-brain barrier is crossed by constant passive fluxes of oxysterols, but not of cholesterol per se, as a result of their permissive chemical structures and following their concentration gradients. In plasma, it is transported via high-density lipoproteins, as discussed further below. In contrast to humans, CYP46A1 is present in the liver of rodents as well as brain.

    25-Hydroxycholesterol is a relatively minor but biologically important cholesterol metabolite, which is produced rapidly by immune cells during the inflammation resulting from bacterial or viral infections. The dioxygenase enzyme cholesterol 25‑hydroxylase (CH25H in humans), which utilizes a diiron cofactor to catalyse hydroxylation, is the most important route to this metabolite in vivo, although at least two cytochrome P450 enzymes, CYP27A1 and CYP3A4, can catalyse this conversion to a limited extent. Further oxidation by CYP7B1 is a second route to 7α,25‑dihydroxycholesterol and thence to further oxysterols.

    24(S),25-Epoxycholesterol is not produced by the pathways described above but is synthesized in a shunt of the mevalonate pathway using the same enzymes that produce cholesterol, specifically squalene mono-oxygenase and lanosterol synthase, by means of which a second epoxy group is introduced on the other end of squalene from the initial epoxidation. A further mechanism in brain is the action of CYP46A1 on desmosterol, another intermediate in cholesterol biosynthesis.

    The oxysterols formed by both autoxidation and enzymatic routes can undergo further oxidation-reduction reactions, and they can be modified by many of the enzymes involved in the metabolism of cholesterol and steroidal hormones, such as esterification and sulfation of position 3, as illustrated for 7-keto-cholesterol as an example in Figure \(\PageIndex{32}\) below.

    Metabolism of oxysterols
    Figure \(\PageIndex{32}\): Metabolism of 7-keto-cholesterol

    In most tissues, esterification of the 3β-hydroxyl group only occurs and requires the activity of sterol O-acyltransferases 1/2 (SOAT1/2 or ACAT1/2) with the participation of cytosolic phospholipase A2 (cPLA2α) to liberate the required fatty acids from phospholipids. In plasma, oxysterols can be esterified by the lecithin–cholesterol acyltransferase (LCAT) for transport in lipoproteins, but in this instance a diester can be produced from 27‑hydroxycholesterol specifically. Whether such esters are an inert storage form for oxysterols to be liberated on demand by esterases remains to be determined.

    It is noteworthy that the important human pathogen, Mycobacterium tuberculosis, utilizes a cytochrome P450 enzyme (CYP125) to catalyse C26/C27 hydroxylation of cholesterol as an essential early step in its catabolism as part of the infective process.

    Catabolism: Because of their increased polarity relative to cholesterol, oxysterols produced by both enzymatic and non-enzymatic means can exit cells relatively easily. A proportion is oxidized further and converted to bile acids, and some are converted to sulfate esters (especially at the 3-hydroxyl group) or glucuronides (see below) for elimination via the kidneys.

    Non-Enzymatic Oxidation of Cholesterol

    In biological systems in which both cholesterol and fatty acids are present, it would be expected that autoxidation of polyunsaturated fatty acids by free radical mechanisms would be favored thermodynamically with the formation of isoprostanes from arachidonic acid in phospholipids. However, there are circumstances that can favour cholesterol oxidation in vivo, and for example the concentration of cholesterol in low-density lipoprotein particles (LDL) is about three times higher than that of phospholipids, and the rate of cholesterol-hydroperoxide formation can be higher than that of phospholipid hydroperoxides. The rate and specificity of the reaction can depend also on whether it is initiated by free radical species, such as those arising from the superoxide/hydrogen peroxide/hydroxyl radical system (Type I autoxidation) or whether it occurs by non-radical but highly reactive oxygen species such as singlet oxygen, HOCl or ozone (Type II autoxidation). As examples of the main types of product of non-enzymatic oxidation, the structures of a few of the more important of these oxysterols are illustrated in Figure \(\PageIndex{33}\) below.

    Structural formulae of oxysterols
    Figure \(\PageIndex{33}\): Some non-ezymatic oxidation products of cholesterol

    Oxysterols produced by this means can vary in the type (hydroperoxy, hydroxy, keto, epoxy), number and position of the oxygenated functions introduced and in the nature of their stereochemistry. Derivatives with the A and B rings and the iso-octyl side-chain oxidized are illustrated, but compounds with oxygen groups in position 15 (D ring) are also important biologically. Many are similar to those produced by enzymatic means, although the stereochemistry will usually differ. Like the enzymic products, they are named according to their relationship to cholesterol, rather than by the strict systematic terminology.

    Mechanisms of autoxidation have been studied intensively in terms of unsaturated fatty acids, and it appears that similar mechanisms operate with sterols. The first event in lipid peroxidation by a radical mechanism is an initiation reaction in which a carbon with a labile hydrogen undergoes hydrogen abstraction by reaction with a free radical, which can be a non-lipid species such as a transition metal or hydroxyl or peroxynitrile radicals, and this is followed by oxygen capture. The resulting reactive species recruits further non-oxidized lipids and starts a chain reaction termed the propagation phase. Finally, the reaction is terminated by the conversion of hydroperoxy intermediates to more stable hydroxy products by reaction with endogenous antioxidants such as tocopherols.

    As an example, the reaction mechanism leading to the production of 7-oxygenated cholesterol derivatives is illustrated in Figure \(\PageIndex{34}\) below.

    Examples of non-enzymic oxidation of cholesterol

    Figure \(\PageIndex{34}\): Cholesterol non-enzymatic oxidation mechanisms

    In aqueous dispersions, oxidation is initiated by a radical attack from a reactive-oxygen species such as a hydroxyl radical with abstraction of hydrogen from the C-7 position to form a delocalized three-carbon allylic radical, which reacts with oxygen to produce 7α‑hydroperoxycholesterol, which gradually isomerizes to the more thermodynamically stable 7β-hydroperoxycholesterol. Subsequent enzymic and non-enzymic reactions lead to the 7-hydroxy and 7-keto analogues, which tend to be the most abundant non-enzymatically generated oxysterols in tissues, often accompanied by epoxy-ene and ketodienoic secondary products. Reaction with singlet oxygen (1O2) produces 5α‑hydroperoxycholesterol mainly together with some 6α- or 6β-hydroperoxycholesterol. Reaction does not occur readily at the other allylic carbon 4, presumably because of steric hindrance. When cholesterol is in the solid state, reaction occurs primarily at the tertiary carbon-25, though some products oxygenated at C-20 may also be produced.

    Cholesterol hydroperoxides can be converted to stable diols only by the phospholipid hydroperoxide glutathione peroxidase - type 4 (GPx4) and then relatively slowly, but not by the type 1 glutathione peroxidase (GPx1) when in a membrane bound state. However, in mammalian cells, monomeric GPx4 (~20 kDa), although present in several cellular compartments, including mitochondria, is much less abundant than tetrameric GPx1. Phospholipid-hydroperoxides are reduced most rapidly followed by cholesterol 6β-OOH > 7α/β-OOH >> 5α-OOH. The result is that cholesterol hydroperoxides are expected to have a relatively long half-life and so can potentially be rather dangerous in biological systems. Of these, 5α-OOH with the lowest reduction rate is most cytotoxic of the hydroperoxides, unfortunately.

    Epimeric 5,6-epoxy-cholesterols may be formed by a non-radical reaction involving the non-enzymatic interaction of a hydroperoxide with the double bond, a process that is believed to occur in macrophages especially and in low-density lipoproteins (LDL). In this instance, the initial peroxidation product is a polyunsaturated fatty acid; the hydroperoxide transfers an oxygen atom to cholesterol to produce the epoxide, and in so doing is reduced to a hydroxyl. Other non-radical oxidation processes include reaction with singlet oxygen, which is especially important in the presence of light and photosensitizers and can generate 5-hydroxy- as well as 6- and 7-hydroxy products. In addition, reaction with ozone in the lung can generate a family of distinctive oxygenated cholesterol metabolites.

    Similarly, a diverse range of oxidation products are generated by peroxidation of the cholesterol and vitamin D precursor 7‑dehydrocholesterol, which has the highest propagation rate constant known for any lipid toward free-radical chain oxidation, and these metabolites have important biological properties.

    Oxysterols occur in tissues both in the free state and esterified with long-chain fatty acids. For example, in human atherosclerotic lesions, 80–95% of all oxysterols are esterified. Appreciable amounts of oxysterols can be present in foods, especially those rich in cholesterol such as meat, eggs and dairy products, where they are most probably generated non-enzymically during cooking or processing when such factors as temperature, oxygen, light exposure, the associated lipid matrix, and the presence of antioxidants and water all play a part. Those present in foods can be absorbed from the intestines and transported into the circulation in chylomicrons, but the extent to which dietary sources contribute to tissue levels either of total oxysterols or of individual isomers is not known and is probably highly variable but relatively lower than of cholesterol per se.

    Oxysterols – Biological Activity

    General Functions: In tissues in vivo, the very low oxysterol:cholesterol ratio means that oxysterols have little impact on the primary role of cholesterol in cell membrane structure and function, although it has been claimed that oxysterols could cause packing defects and thence atheroma formation in vascular endothelial cells. It is often argued that there are few reliable measurements of cellular or subcellular oxysterol concentrations, because of the technical difficulties in the analysis of the very low concentrations of oxysterols in the presence of a vast excess of native cholesterol; the average levels of 26-, 24- and 7α-hydroxy-cholesterol in human plasma that are often quoted are 0.36, 0.16 and 0.14 μM, respectively. Autoxidation products of cholesterol, especially 7-keto- and 7-hydroxy-cholesterol, are cytotoxic and may be useful markers of oxidative stress or for monitoring of the progression of various diseases. However, experts in the field caution that it can be difficult to extrapolate from experiments in vitro to the situation in vivo, because of the rapidity with which cholesterol can autoxidize in experimental systems and because of the difficulty of carrying out experiments with physiological levels of oxysterols.

    Nonetheless, aside from their role as precursors of bile acids and some steroidal hormones, it is evident that oxysterols have a variety of roles in terms of maintaining cholesterol homeostasis and perhaps in signaling, where those formed enzymatically are most important. They can exert potent biological effects at physiologically relevant concentrations by binding to various receptors to elicit transcriptional programmes, i.e., to regulate gene and hence protein expression. Among many cell membrane receptors for oxysterols to have been identified, nuclear receptors are especially important and include the liver X receptors (LXRs), retinoic acid receptor-related orphan receptors (RORs), estrogen receptors (ERs) and glucocorticoid receptors (GRs). In addition, N-methyl-D-aspartate receptors (NMDARs) are expressed in nerve cells and work over a short time scale to regulate excitatory synaptic function, while G protein-coupled receptors operate at cell membranes and are activated by molecules outside the cell to activate signalling pathways within the cell. As various isoforms of these receptors exist in different tissues, and these can interact with several oxysterols, only a brief summary of this complex topic is possible here.

    A family of oxysterol-binding proteins (OSBP) transports and regulates the metabolism of sterols and targets oxysterols to specific membranes and especially to contact sites between organelles with transport of phosphatidylinositol 4-phosphate in the reverse direction (see our web page on the latter). In this way, they can enable oxysterols to regulate membrane composition and function and mediate intracellular lipid transport. As with cholesterol, oxysterols can be eliminated from cells by transporters such as the ATP-binding cassette proteins ABCA1 and ABCG1, and they are transported in the blood-stream within lipoproteins, especially in association with HDL and LDL and mainly in the esterified form.

    Cholesterol homeostasis: While cholesterol plays a key role in its own feedback regulation, there is some evidence that oxysterols are regulators of cholesterol concentration in cell membranes, and that 25‑hydroxycholesterol and 24(S),25‑epoxycholesterol may be especially effective, although the other side-chain oxysterols 22-, 24- and 27‑hydroxycholesterol have been implicated. Several mechanisms appear to be involved, and it is suggested that 24(S),25‑epoxycholesterol especially acts as a ligand for the liver X receptor, which forms a heterodimer with the retinoic X receptor, to inhibit the transcription of key genes in cholesterol biosynthesis, as well as directly inhibiting or accelerating the degradation of such important enzymes in the process as HMG-CoA reductase and squalene synthase. Similarly, both 26-hydroxylanosterol and 25-hydroxycholesterol inhibit HMG-CoA reductase. 25‑Hydroxycholesterol inhibits transfer of the 'sterol regulatory element binding protein' (SREBP-2) to the Golgi for processing to its active form as a transcription factor for the genes of the cholesterol biosynthesis pathway, and it stimulates the enzyme acyl-CoA:cholesterol acyl transferase in the endoplasmic reticulum to esterify cholesterol. By such mechanisms, these oxysterols fine tune cholesterol homeostasis and ensure smooth regulation rather than substantial fluctuations in tissue concentration.

    Oxysterols and the immune system: Oxysterols and especially are known to have vital and diverse roles in immunity by regulating both the adaptive and innate immune responses to infection. For example, infection with viruses leads to production of type I interferon, and in macrophages this induces synthesis of 25‑hydroxycholesterol, which in general is regarded as anti-inflammatory and exerts broad antiviral activity by activating integrated stress response genes and reprogramming protein translation again via its interaction with LXR receptors. It is a potent inhibitor of SARS-CoV-2 replication, for example, possibly by a mechanism involving the blocking of cholesterol export from the late endosome/lysosome compartment and depletion of membrane cholesterol levels. However, formation of 25‑hydroxycholesterol may be harmful in the case of influenza infections, as it can lead to over-production of inflammatory metabolites. Similarly, the biosynthesis of 25-hydroxycholesterol in macrophages is stimulated by the endotoxin Kdo2-lipid A, the active component of the lipopolysaccharide present on the outer membrane of Gram-negative bacteria, which acts as an agonist for Toll-like receptor 4 (TLR4). There is enhanced expression of the oxygenase CH25H in immune cells in response to bacterial and viral infection.

    Many oxysterol species are active in a range of immune cells subsets, mediated through the control of LXR and SREBP signaling, but also by acting as ligands for RORs, and for the cell surface receptors G protein-coupled receptor 183 (GPR183) or CXCR2. Activation of LXR tends to dampen the immune response. In response to various stimuli, they can operate through ion channels to effect rapid changes in intracellular ion concentrations, especially of Ca2+, to bring about changes in membrane potential, cell volume, cell-death (apoptosis, autophagy and necrosis), gene expression, secretion, endocytosis, or motility. For example, 27‑hydroxycholesterol in human milk is reported to be active against the pathogenic human rotavirus and rhinovirus of importance in pediatrics, and 7-Dehydrocholesterol has anti-viral properties also. While they can exert their immune functions within the cell in which they are generated, oxysterols can also operate in a paracrine fashion towards other immune cells.

    25‑Hydroxycholesterol in particular can have either pro- or anti-inflammatory effects, depending upon the conditions, but the enzyme CH25H responsible for its biosynthesis is induced markedly in macrophages activated by inflammatory agents. It is reported to have a regulatory effect on the biosynthesis of sphingomyelin, which is required with cholesterol for the formation of raft sub-domains in membranes, where signalling molecules are concentrated, and together with other oxysterols, such as 24S,25-epoxycholesterol, to regulate the activities of the hedgehog proteins involved in embryonic development. Metabolites of 25‑hydroxycholesterol, such as 7α,25‑dihydroxycholesterol and further oxidation products, are pro-inflammatory act as chemoattractants to lymphocytes; they have a role in the regulation of immunity in secondary lymphoid organs by interactions with the receptor GPR183.

    Oxysterols in brain: Oxysterols are especially important for cholesterol homeostasis in the brain, which contains 25% of the total body cholesterol, virtually all of it in unesterified form, in only about 2% of the body volume. Cholesterol is a major component of the plasma membrane especially, where it serves to control the fluidity and permeability. This membrane is produced in large amounts in brain and is the basis of the compacted myelin, which is essential for conductance of electrical stimuli and contains about 70% of brain cholesterol. While this pool is relatively stable, the remaining 30% is present in the membranes of neurons and glial cells of gray matter and is more active metabolically. Even in the foetus and the newborn infant, all the cholesterol required for growth is produced by synthesis de novo in the brain not by transfer from the circulation across the blood-brain barrier, which consists of tightly opposed endothelial cells lining the extensive vasculature of the tissue. The fact that this pool of cholesterol in the brain is independent of circulating levels must reflect a requirement for constancy in the content of this lipid in membranes and myelin. In adults, although there is a continuous turnover of membrane, the cholesterol is efficiently re-cycled and has a remarkably high half-life (up to 5 years). The rate of cholesterol synthesis is a little greater than the actual requirement, so that net movement of cholesterol out of the central nervous system must occur. An important component of this system is apolipoprotein E (Apo E), a 39-kDa protein, which is highly expressed in brain and functions in cellular transport of cholesterol and in cholesterol homeostasis. Apo E complexes with cholesterol are required for transport from the site of synthesis in astrocytes to neurons.

    Hydroxylation by CYP46A1 of cholesterol to 24(S)‑hydroxycholesterol (cerebrosterol) is responsible for 50–60% of all cholesterol metabolism in the adult brain. If cholesterol itself cannot cross the blood-brain barrier, this metabolite is able to do so with relative ease. When the hydroxyl group is introduced into the side chain, this oxysterol effects a local re-ordering of membrane phospholipids such that it is more favorable energetically to expel it at a rate that is orders of magnitude greater than that of cholesterol per se, though still only 3-7 mg per day. There is a continuous flow of the metabolite from the brain into the circulation, much of it in the form of the inactive sterol ester, where it is transported by lipoprotein particles to the liver for further catabolism, i.e., it is hydroxylated in position 7 and then converted to bile acids. This is illustrated in Figure \(\PageIndex{35}\) below.

    Cholesterol and the brain
    Figure \(\PageIndex{35}\): Brain/Liver 27-carboxy and 27-OH steroid metabolism.

    Both 24(S)-hydroxycholesterol and 24(S),25-epoxycholesterol are believed to be important in regulating cholesterol homeostasis in the brain. They interact with the specific nuclear receptors involved in the expression and synthesis of proteins involved in sterol transport, and for example, 24‑hydroxy-cholesterol regulates the transcription of Apo E. In particular, it is an agonist of the nuclear liver X receptors (LXRs), influencing the expression of those LXR target genes involved in cholesterol homeostasis and inflammatory responses. It is also a high affinity ligand for the retinoic acid receptor-related orphan receptors α and γ (RORα and RORγ). In this way, it can act locally to affecting the functioning of neurons, astrocytes, oligodendrocytes, and vascular cells.

    24(S)-Hydroxycholesterol down-regulates trafficking of the amyloid precursor protein and may be a factor in preventing neurodegenerative diseases. Especially high levels of 24(S)‑hydroxycholesterol are observed in the plasma of human infants and in patients with brain trauma, while reduced levels are found in plasma of patients with neurodegenerative diseases, including Parkinson’s disease, multiple sclerosis and Alzheimer's disease. In contrast, there are elevated levels in brain and especially cerebrospinal fluid in patients with these conditions, where it may be a marker of neurodegeneration. Increased expression of cholesterol 24-hydroxylase (CYP46A1) is believed to improve cognition, while a reduction leads to a poor cognitive performance, as occurs at advanced stages of the disease, probably reflecting a selective loss of neuronal cells, and it may be a factor in age-related macular degeneration. An excess of 24(S)‑hydroxycholesterol and especially of its ester form can lead to neuronal cell death, and elevated levels in plasma are reported to be a potential marker for Autism Spectrum Disorders in children. On the other hand, it may be protective against glioblastoma, the most common primary malignant brain tumour in adults via activation of LXRs.

    27‑Hydroxy-cholesterol diffuses across the blood-brain barrier from the blood stream into the brain (in the reverse direction to 24‑hydroxycholesterol), where it does not accumulate but is further oxidized and then exported as steroidal acids. This flux may regulate certain key enzymes within the brain, and there are suggestions that the balance between the levels of 24- and 27-hydroxy-cholesterol, especially excess of the latter, may be relevant to the generation of β-amyloid peptides in Alzheimer's disease by reducing insulin-mediated glucose uptake by neurons. While 7β-hydroxycholesterol is pro-apoptotic, any links with Alzheimer's disease are unproven although there is a school of thought that other oxidized cholesterol metabolites may be major factors behind the development of this disease. For example, seco-sterols such as 3β‑hydroxy-5-oxo-5,6-secocholestan-6-al and its stable aldolization product, the main ozonolysis metabolites derived from cholesterol, have been detected in brain samples of patients who have died from Alzheimer's disease and Lewy body dementia; they are also found in atherosclerotic lesions. Oxidation products of the cholesterol precursor 7‑dehydrocholesterol and especially 3β,5‑dihydroxycholest-7-ene-6-one are involved in the pathophysiology of the human disease Smith-Lemli-Opitz syndrome.

    Cell differentiation: Oxysterols can influence the differentiation of many cell types and this was first studied in skin, where 22(R)- and 25(R)‑hydroxycholesterol were shown to induce human keratinocyte differentiation. Subsequently by stimulating nuclear binding receptors, oxysterols were found to have similar effects upon of mesenchymal stem cells, i.e., multipotent cells capable of self-renewal and the ability to differentiate into several cell types, such as osteoblasts, adipocytes, and chondrocytes.

    Oxysterols in other diseases: In addition to brain pathologies, there have been many reports of the involvement of oxysterols in disease processes, especially atherosclerosis and the formation of human atherosclerotic plaques, but also cytotoxicity, necrosis, inflammation, immuno-suppression, phospholipidosis and gallstone formation. They have been implicated in the development of cancers, especially those of the breast, prostate, colon and bile duct. For example, 27‑hydroxycholesterol is an element in cholesterol elimination from macrophages and arterial endothelial cells, but it is also an endogenous ligand for the human nuclear estrogen receptor (ERα) and the liver X receptor, and it modulates their activities with effects upon various human disease states, including cardiovascular dysfunction and progression of cancer of the breast and prostate, as well as having an involvement in the regulation of bone mineralization (osteoporosis). It has been linked to cancer metastasis through an action on immune cells, and there is hope that pharmacological inhibition of CYP27A1 and thence the formation of 27‑hydroxycholesterol may be a useful strategy in the treatment of breast cancer; CYP7A1 gene polymorphism has been associated with colorectal cancer. In contrast, oxysterols can interfere in the proliferation of several types of cancer cell (glioblastoma, leukemia, colon, breast and prostate cancer).

    Cholesterol 5,6-epoxide (with either 5α or 5β stereochemistry) is formed non-enzymatically in tissues, but it is also believed to be produced by an as yet unidentified cytochrome P450 enzyme in the adrenal glands. While it was for some time believed to be a causative agent in cancer, it is now recognized that downstream metabolites are responsible. Thus, cholesterol epoxide hydrolase converts cholesterol 5,6-epoxide into cholestane-3β,5α,6β-triol, which is transformed by 11β‑hydroxysteroid-dehydrogenase-type-2 into the oncometabolite 3β,5α-dihydroxycholestan-6-one (oncosterone). By binding to the glucocorticoid receptor, this oncosterone stimulates the growth of breast cancer cells, and it also acts as a ligand to the LXR receptors, which may mediate its pro-invasive effects. In contrast, in normal breast tissue, cholesterol 5,6‑epoxide is metabolized to the tumour suppressor metabolite, a steroidal alkaloid designated dendrogenin A that is a conjugation product with histamine and controls a nuclear receptor to trigger lethal autophagy in cancers; its synthesis is greatly reduced in cancer cells. Tamoxifen, a drug that is widely used against breast cancer, binds to the cholesterol 5,6-epoxide hydrolase, which is also a microsomal anti-estrogen binding site (AEBS), to inhibit its activity.

    7-Ketocholesterol is a major oxysterol produced during oxidation of low-density lipoproteins, and is one of the most abundant in plasma and atherosclerotic lesions; it accumulates in erythrocytes of heart failure patients. It has a high pro-apoptotic potential and associates preferentially with membrane lipid raft domains. As it is not readily exported from macrophages, it impairs cholesterol efflux and promotes the foam cell phenotype. In cardiomyocytes, this accumulation can lead to cell hypertrophy and death, and it has been suggested that oxysterols are a major factor precipitating morbidity in atherosclerosis-induced cardiac diseases and inflammation-induced heart complications. Photoxidation in the retina via the action of free radicals or singlet oxygen generates unstable cholesterol hydroperoxides, which may be involved in age-related macular degeneration. For example, these compounds can quickly be converted to highly toxic highly toxic 7α- and 7β‑hydroxycholesterols and 7‑ketocholesterol, depending on the status of tissue oxidases and reductases. Three separate enzymatic pathways have developed in the eye to neutralize their activities. These sterols are metabolized by novel branches of the acidic pathway of bile acid biosynthesis.

    Those oxysterols formed non-enzymatically can be most troublesome in relation to disease in general. For example, they are enriched in pathologic cells and tissues, such as macrophage foam cells, atherosclerotic lesions, and cataracts. They may regulate some of the metabolic effects of cholesterol, but as cautioned above, effects observed in vitro may not necessarily be of physiological importance in vivo. Various oxysterols have been implicated in the differentiation of mesenchymal stem cells and the signalling pathways involved in this process. High levels of 7‑hydroxycholesterol and cholestane-3β,5α,6β-triol are characteristic of the lysosomal storage diseases Niemann-Pick types B and C and of lysosomal acid lipase deficiency.

    Cholesterol hydroperoxides: With the aid of START domain proteins, cholesterol hydroperoxides can translocate from a membrane of origin to another membrane such as mitochondria. Such transfer of free radical-generated 7-hydroperoxycholesterol, for example, has adverse consequences in that there is impairment of cholesterol utilization in steroidogenic cells, and of anti-atherogenic reverse-cholesterol transport in vascular macrophages. The antioxidant activity of GPx4 may be crucial for the maintenance of mitochondrial integrity and functionality in these cells.

    Vitamin D

    Vitamin D encompasses two main sterol metabolites that are essential for the regulation of calcium and phosphorus levels and thence for bone formation in animals. However, these have many other functions, including induction of cell differentiation, inhibition of cell growth, immunomodulation, and control of other hormonal systems. Vitamin D (with calcium) deficiency is responsible for the disease rickets in children in which bones are weak and deformed, and it is associated with various cancers and autoimmune diseases. Ultraviolet light mediates cleavage of 7-dehydrocholesterol, an important intermediate in the biosynthesis of cholesterol, with opening of the second (B) ring in the skin to produce pre-vitamin D, which rearranges spontaneously to form the secosteroid vitamin D3 or cholecalciferol. Its structure is shown in Figure \(\PageIndex{36}\) below.

    Vitamin D3
    Figure \(\PageIndex{36}\): Structure of Vitamin D3

    The newly generated vitamin D3 is transported to the liver where it is subject to 25-hydroxylation and thence to the kidney for 1α-hydroxylation to produce the active form 1α,25-dihydroxyvitamin D3 (calcitriol); this is a true hormone and serves as a high affinity ligand for the vitamin D receptor in distant tissues. For transportation in plasma, it is bound to a specific glycoprotein termed unsurprisingly, the 'vitamin D binding protein (BDP)'. Vitamin D2 or ergocalciferol is derived from ergosterol, which is obtained from plant and fungal sources in the diet.

    Vitamin D3 functions by activating a cellular receptor (vitamin D receptor or VDR), a transcription factor binding to sites in the DNA called vitamin D response elements. There are thousands of such binding sites, which together with co-modulators regulate innumerable genes in a cell-specific fashion. In this way, it enhances bone mineralization through promoting dietary calcium and phosphate absorption, as well as having direct affects on bone cells. In addition, it functions as a general development hormone in many different tissues, while together with Vitamin D2 it has profound effects on immune responses in the defense against microbes.

    Steroidal Hormones and their Esters

    Steroidal hormones cannot be discussed in depth here as their structures, biosynthesis and functions comprise a rather substantial and specialized topic. In brief, animal tissues produce small amounts of vital steroidal hormones from cholesterol as the primary precursor with 22R-hydroxycholesterol, produced by hydroxylation by the cholesterol side-chain cleavage enzyme (P450scc), as the first of its metabolites in the pathway. This step involves the 'STAR' protein which enables the transport of cholesterol into mitochondria where conversion to pregnenolone is rate-limiting and involves first hydroxylation and then cleavage of the side-chain. After export from the mitochondria, this can be converted directly to progesterone or in several steps to testosterone. 17β-Estradiol, for example, is the most potent and important of the endogenous estrogens; it is made mainly in the follicles of the ovaries and regulates menstrual cycles and reproduction, but it is also present in testicles, adrenal glands, fat, liver, breasts and brain. Testosterone is the primary male sex hormone and an anabolic steroid, and it is produced mainly in the testes; it has a key function in the development of male reproductive tissues such as testes and prostate, in addition to promoting secondary sexual characteristics. Pregnane neurosteroids are synthesized in the central nervous system. Cholesterol homeostasis is therefore vital to fertility and a host of bodily functions. The structures of key steroidal hormones are shown in Figure \(\PageIndex{37}\) below.

    Examples of steroidal hormones
    Figure \(\PageIndex{37}\): Structures of key steroidal hormones

    Steroidal esters accumulate in tissues such as the adrenal glands, which synthesize corticosteroids such as cortisol and aldosterone and are responsible for releasing hormones in response to stress and other factors. It is also apparent that fatty acyl esters of estradiols, such as dehydroepiandrosterone, accumulate in adipose tissue in post-menopausal women. Small amounts of estrogens acylated with fatty acids at the C-17 hydroxyl are present in the plasma lipoproteins. In each instance, they appear to be biologically inert storage or transport forms of the steroid. Eventually, esterified steroids in low density lipoproteins (LDL) particles are taken up by cells via lipoprotein receptors, and then are hydrolyzed to release the active steroid. Pharmaceutical interest in oleoyl-estrone, a naturally occurring hormone in humans, which was found to induce a marked loss of body-fat while preserving protein stores in laboratory animals, has declined as clinical trials with humans were not successful.

    Sterols 3. Sterols and their Conjugates from Plants and Lower Organisms

    Plant Sterols - Structures and Occurrence

    Plants, algae and fungi contain a rather different range of sterols from those in animals. Like cholesterol, to which they are related structurally and biosynthetically, plant sterols form a group of triterpenes with a tetracyclic cyclopenta[a]phenanthrene structure and a side chain at carbon 17, sometimes termed the C30H50O triterpenome. 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 a hydroxyl group commonly located on C-3 also). The basic sterol from which other sterol structures are defined is 5α-cholestan-3β-ol with the numbering scheme recommended by IUPAC as shown in Figure \(\PageIndex{38}\) below.

    plantsterolringnumbers.svg
    Figure \(\PageIndex{38}\): IUPAC sterol numbing system

    The phytosterols (as opposed to zoosterols) include campesterol, β-sitosterol, stigmasterol and Δ5‑avenasterol, some of which are illustrated in Figure \(\PageIndex{39}\).

    Formulae of plant sterols
    Figure \(\PageIndex{39}\): Some common plant sterols

    These more common plant sterols have a double bond in position 5, and a definitive feature – a one- or two-carbon substituent with variable stereochemistry in the side chain at C-24, which is preserved during subsequent metabolism. For example, campesterol is a 24-methylsterol, while β-sitosterol and stigmasterol are 24‑ethylsterols. Occasionally, there is a double bond in this chain that can be of the cis or trans configuration as in stigmasterol (at C22) or fucosterol (C24), the main sterol in green algae.

    Phytosterols can be further classified on a structural or biosynthetic basis as 4‑desmethyl sterols (i.e. with no substituent on carbon‑4), 4α‑monomethyl sterols and 4,4‑dimethyl sterols. The most abundant group are the 4‑desmethyl sterols, which may be subdivided into Δ5-sterols (illustrated above), Δ7‑sterols (e.g. α-spinasterol) and Δ5,7-sterols depending on the position of the double bonds in the B ring. As the name suggests, brassicasterols (24‑methyl-cholesta-5,22-dien-3β-ol and related sterols) are best known from the brassica family of plants, but they are also common constituents of marine algae (phytoplankton). Phytostanols (fully saturated) are normally present at trace levels only in plants, but they are relatively abundant in cereal grains.

    Many different sterols may be present in photosynthetic organisms, and the amounts and relative proportions are dependent on the species. Over 250 different phytosterols have been recorded with 60 in corn (maize) alone, for example. As a rough generality, a typical plant sterol mixture would be 70% sitosterol, 20% stigmasterol and 5% campesterol (or >70% 24-ethyl-sterols and <30% 24-methyl-sterols), although this will vary with the stage of development and in response to stress. Table 1 contains data on the main components from some representative commercial seed oils.

    Table \(\PageIndex{2}\). Sterol composition in some seed oils of commercial importance (mg/Kg).
    corn cottonseed olive palm rapeseed safflower soybean sunflower
    cholesterol - - - 26 - - - -
    campesterol 2691 170 28 358 1530 452 720 313
    stigmasterol 702 42 14 204 - 313 720 313
    β‑sitosterol 7722 3961 1310 1894 3549 1809 1908 2352
    Δ5‑avenasterol 468 85 29 51 122 35 108 156
    Δ7‑stigmasterol 117 - 58 25 306 696 108 588
    Δ7‑avenasterol - - - - - 104 36 156
    brassicasterol - - - - 612 - - -
    other - - - - - 69 - 39
    Data adapted from Gunstone, F.D. et al. The Lipid Handbook (Second Edition) (Chapman & Hall, London) (1994).

    Cholesterol is usually a minor component only of plant sterols (<1%), but it is unwise to generalize too much as it can be the main sterol component of red algae and of some families of higher plants such as in the Solanaceae, Liliaceae and Scrophylariaceae. It can also be a significant constituent sterol of chloroplasts, shoots, pollen and leaf surface lipids in other plant families; wheat roots contain 10% and Arabidopsis cells 19% of the sterols as cholesterol. Yeasts and fungi tend to contain ergosterol as their main sterol (see below). Ecdysteroids (phytoecdysteroids) are polyhydroxylated plant sterols that can occur in appreciable amounts in some species. Sterols are also found in some bacterial groups but not in archaea, and hopanoids in bacteria are considered to be functional triterpenic counterparts.

    Sterols can occur in plants in the 'free' state, i.e. in which the sterol hydroxyl group is not linked to any other moiety, but they are usually present also as conjugates with the hydroxyl group covalently bound via an ester bond to a fatty acid, for example, i.e. as sterol esters, or via a glycosidic linkage to glucose (and occasionally other sugars), i.e. as steryl glycosides.

    Plant Sterols - Biosynthesis

    The biosynthetic route to plant sterols resembles that to cholesterol in many aspects in that it follows an isoprenoid biosynthetic pathway with isopentenyl pyrophosphate, derived primarily from mevalonate, as the key building block in the cytoplasm (but not plastids) at least. The main pathway for the biosynthesis of isopentenyl pyrophosphate and dimethylallyl pyrophosphate, the isoprene units, is described previously and so need not be repeated here. It is known as the 'mevalonic acid (MVA) pathway' and functions in the cytosol, endoplasmic reticulum and mitochondria.

    However, an alternative pathway that does not use mevalonic acid as a precursor was established first for bacterial hopanoids, but has since been found in plant chloroplasts, algae, cyanobacteria, eubacteria and some parasites (but not in animals). This route is variously termed the ‘non-mevalonate’, ‘1‑deoxy-D-xylulose-5-phosphate’ (DOXP) or better the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway as the last compound is presumed to be the first committed intermediate in sterol biosynthesis by this route. In the first step, pyruvate and glyceraldehyde phosphate are combined to form deoxyxylose phosphate, which is in turn converted to 2C-methyl-D-erythritol 4-phosphate. The pathway then proceeds via various erythritol intermediates until isopentenyl pyrophosphate and dimethylallyl pyrophosphate are formed. This pathway is illustrated in Figure \(\PageIndex{40}\) below.

    plantsterolbiosyn_1.svg
    Figure \(\PageIndex{40}\): Alternative 2C-methyl-D-erythritol 4-phosphate (MEP) pathway for synthesis of sterol and isoprenoid precurosrs in chloroplasts.

    There is evidence that some of the isoprene units are exchanged between the cytoplasm and plastids. In much of the plant kingdom, both the MVA and MEP pathways operate in parallel, but green algae use the MEP pathway only. Thereafter, sterol biosynthesis continues via squalene and (3S)-2,3-oxidosqualene.

    In photosynthetic organisms (as opposed to yeast and fungi), the subsequent steps in the biosynthesis of plant sterols differ from that for cholesterol in that the important intermediate in the route from squalene via 2,3-oxidosqualene is cycloartenol, rather than lanosterol, and this is produced by the action of a 2,3(S)‑oxidosqualene-cycloartenol cyclase (cycloartenol synthase). Then, the enzyme sterol methyltransferase 1 is of special importance in that it converts cycloartenol to 24-methylene cycloartenol, as the first step in introducing the methyl group onto C-24, while the enzyme cyclopropyl sterol isomerase is required to open the cyclopropane ring. Animals lack the sterol C24-methyltransferase gene. While this pathway is in essence linear up to the synthesis of 24-methylene lophenol, a bifurcation then occurs that results in two alternative pathways, one of which leads to the synthesis of sitosterol and stigmasterol and the other to that of campesterol. This pathway is shown in Figure \(\PageIndex{41}\) below.

    biosyn_plantsterols_cycloartenol.svg
    Figure \(\PageIndex{41}\): Synthesis of plant sterols via the cycloartenol intermediate

    In fact, there are more than thirty enzyme-catalyzed steps in the overall process of plant sterol biosynthesis, each associated with membranes, and detailed descriptions are available from the reading list below. The 4,4-dimethyl- and 4α-methylsterols are part of the biosynthetic pathway, but are only minor if ubiquitous sterol components of plants. New biosynthetic pathways are now being discovered by genome analysis that reveal the complexity of sterol biosynthesis in different plant species.

    Dinoflagellates produce a characteristic 4-methylsterol termed dinosterol and others like gorgosterol via lanosterol as precursor. Protozoans synthesise many different sterols related to those in plants. For example, some species of Acanthamoeba and Naegleria produce both lanosterol and cycloartenol, but only the latter is used for synthesis of other sterols, especially ergosterol, but in other protozoan species, sterol biosynthesis occurs via lanosterol. The best studied bacterial pathway is that of the methylotroph Methylococcus capsulatus, which produces a number of unique Δ18(14)-sterols and is known to possess a squalene epoxidase and a lanosterol-14-demethylase.

    Cholesterol in plants is produced from cycloartenol as the key intermediate with the Sterol Side Chain Reductase 2 (SSR2) as the key enzyme. It is now established that the cholesterol biosynthetic pathway in tomato plants comprises 12 enzymes acting in 10 steps. Of these, half evolved through gene duplication and divergence from phytosterol biosynthetic enzymes, whereas others act reciprocally in both cholesterol and phytosterol metabolism. Algae can also produce cholesterol in a multi-step process from cycloartenol, and many more sterols via 24-methylene lophenol as the key intermediate. It is hoped that genetic manipulation of these enzymes will lead to plants that synthesise high value steroidal products.

    Oxidation: Phytosterols can be subjected to non-enzymatic oxidation with formation of oxysterols in a similar manner to that of cholesterol in animals, resulting in ring products such as hydroxy-, keto-, epoxy- and triol-derivatives, and further enzymic reactions can oxidize the side chain. However, photosensitized oxidation is more common in plants and is much faster (>1500 times); it starts with the ene-addition of singlet oxygen (1O2) on either side of the double bond in the B ring to generate 5α-/6α-/6β-hydroperoxysterols, of which 5α-OOH is the most abundant and rearranges to form the more stable 7α‑OOH isomer. This is the main reaction in foods stored under LED lighting in food retailers.

    Plant Sterols - Function

    Like cholesterol, plant sterols are amphiphilic and are vital constituents of all membranes, and especially of the plasma membrane, the mitochondrial outer membrane and the endoplasmic reticulum. The three-dimensional structure of the plant sterols is such that there are planar surfaces at both the top and the bottom of the molecules, which permit multiple hydrophobic interactions between the rigid sterol and the other components of membranes. Indeed, they must determine the physical properties of membranes to an appreciable extent. It is believed that campesterol, β-sitosterol and 24-methylcholesterol (in this order) are able to regulate membrane fluidity and permeability in plant membranes by restricting the mobility of fatty acyl chains in a similar manner to cholesterol in mammalian cells, but stigmasterol has much less effect on lipid ordering and no effect on the permeability of membranes. In the plasma membrane, plant sterols associate with the glycosphingolipids such as glucosylceramide and glycosylinositolphosphoceramides in raft-like sub-domains, analogous to those in animal cells, and these support the membrane location and activities of many proteins with important functions in plant cells. The sterol glycosides are especially important in this context (see below).

    Sterols (and their conjugates) are involved in how plant membranes adapt to changes in temperature and other biotic and abiotic stresses. For example, β‑sitosterol is a precursor of stigmasterol via the action of a C22-sterol desaturase, and the ratio of these two sterols is important to the resistance of A. thaliana plants to low and high temperatures. In addition, plant sterols can modulate the activity of membrane-bound enzymes. Thus, stigmasterol and cholesterol regulate the activity of the Na+/K+-ATPase in plant cells, probably in a manner analogous to that of cholesterol in animal cells. Stigmasterol may be required specifically for cell differentiation and proliferation. As well as being the precursor of plant steroidal hormones, campesterol, is a signaling molecule that regulates growth, development and stress adaptation.

    Perhaps surprisingly, cholesterol is a precursor for the biosynthesis of some steroidal saponins and alkaloids in plants, for example the well-known steroidal glycoalkaloid in potato (α-solanine), as well as of other steroids including the phytoecdysteroids (in some species they are derived from lathosterol). While the physiological roles of ecdysteroids in plants has yet to be been confirmed, they are believed to enhance stress resistance by promoting health and vitality. Withanolides are complex oxysterols, which are believed to be defence compounds against insect herbivores.

    Steroidal Plant Hormones

    Formula of a brassinolide
    Figure \(\PageIndex{42}\): Structure of brassinolide

    They have crucial importance for plant growth processes, including cell elongation, division, differentiation, immunity and development of reproductive organs, and they are involved in the regulation of innumerable aspects of metabolism. Via signal transduction pathways, they interact with transcription factors through phosphorylation cascades to regulate the expression of target genes. Brassinosteroids are also signaling molecules in abiotic stress responses such as drought, salinity, high temperature, low temperature and heavy metal stresses. Outwith plants, they may have biomedical applications as anticancer drugs for endocrine-responsive cancers to induce apoptosis and inhibit growth. Some plant species produce small amounts of steroid hormones that are often considered to be of animal origin only, including progesterone and testosterone, and these may have physiological roles in plants.

    Sterol Esters in Higher Plants

    Sterol esters are present in all plant tissues, but they are most abundant in tapetal cells of anthers, pollen grains, seeds and senescent leaves. In general, they are minor components relative to the free sterols other than in waxes. Usually the sterol components of sterol esters are similar to the free sterols, although there may be relatively less of stigmasterol. The fatty acid components tend to resemble those of the other plant tissue lipids, but there can be significant differences on occasion. Sterol esters are presumed to serve as inert storage forms of sterols, as they are often enriched in the intermediates of sterol biosynthesis and can accumulate in lipid droplets within the cells. However, they have been found in some membranes, especially in microsomes and mitochondrial preparations, although their function there is uncertain. They may also have a role in transport within cells and between tissues, as they can be present in the form of soluble lipoprotein complexes.

    Biosynthesis of sterol esters in A. thaliana is known to occur in the endoplasmic reticulum by the action of a phospholipid:sterol acyltransferase, which catalyses transfer of a fatty acyl group to the sterol from position sn-2 of phospholipids - mainly phosphatidylethanolamine; the enzyme is very different from those in animals and yeasts. However, an acyl CoA:sterol acyltransferase closer in structure to the animal enzyme has been characterized also; it prefers saturated fatty acyl-CoAs as acyl donors and cycloartenol as the acceptor molecule. The enzymes responsible for the hydrolysis of sterol esters in A. thaliana are not yet known. Certain distinctive phytosterol esters occur in the aleurone cells of cereal grains, including trans-hydroxycinnamate, ferulate (4-hydroxy-3-methoxycinnamate) and p-coumarate esters. Similarly, rice bran oil is a rich source of esters of ferulic acid and a mixture of sterols and triterpenols, termed 'γ-orizanol'’, and an example of one of these compounds is illustrated in Figure \(\PageIndex{43}\) below.

    Formula of campesteryl ferulate
    Figure \(\PageIndex{43}\): Structure of campesteryl ferulate

    This is sold as a health food supplement, because of claimed beneficial effects, including cholesterol-lowering and antioxidant activities, while enhancing muscle growth and sports performance. However, none of these effects have been confirmed by rigorous clinical testing.

    Sterol Glycosides

    Leaf and other tissue in plants contain a range of sterol glycosides and sterol acyl-glycosides in which the hydroxyl group at C3 on the sterol is linked to the sugar by a glycosidic bond. Other than in the genus Solanum, where they can represent up to 85% of sterol fraction in tomato fruit as an example, they tend to be minor components relative to other lipids. Typical examples (glucosides of β-sitosterol) are illustrated in Figure \(\PageIndex{44}\) below.

    Formulae of sterol glycosides
    Figure \(\PageIndex{44}\): Structure of β-sitosterol-β-glucoside

    Most of the common plant sterols occur in this form, but Δ5 sterols are preferred (Δ7 in some genera). Glucose is the most common carbohydrate moiety but galactose, mannose, xylose, arabinose can also be present depending on plant species; occasionally, complex carbohydrates with up to five hexose units linked in a linear fashion are present. Algae also contains sterol glycosides with a wide range of sterol and carbohydrate components. Plant, animal, fungal and most bacterial steryl glycosides have a β‑glycosidic linkage, but in a few bacterial species there is an α-linkage.

    Similarly, the nature of the fatty acid component in the acyl sterol glycosides can vary as well as the hydroxyl group to which they are linked, although it is usually position 6 of the glucose moiety. In potato tubers, for example, the 6'-palmitoyl-β-D-glucoside of β-sitosterol is the major species, while the corresponding linoleate derivative predominates in soybeans. Usually, the sterol acyl-glycosides are present at concentrations that are two- to tenfold greater than those of the non-acylated forms. They are known to be located in the plasma membrane, tonoplasts and endoplasmic reticulum.

    Biosynthesis involves reaction of free sterols with a glucose unit catalysed by a sterol glycosyltransferase, or by reaction of the sterol with uridine diphosphoglucose (UDP-glucose) and UDP-glucose:sterol glucosyltransferase on the cytosolic side of the plasma membrane. The acyl donor for acyl sterol glycoside synthesis is not acyl-coenzyme A but is believed to be a glycerolipid. Steryl β-D-glycoside hydrolases have been characterized from plants that reverse this reaction, but no fatty acyl hydrolase activity for sterol acyl-glycosides is yet known. One route to the biosynthesis of glucosylceramides in plants involves transfer of the glucose moiety of sterol glycosides to ceramide.

    The functions of sterol glycosides and sterol acyl-glycosides are slowly being revealed, and they are believed to be significant components of the plasma membrane that associate with sphingolipids in raft-like domains; the esterified form especially may be involved in the adaptation of plant membranes to low temperatures and other stresses. It is possible that they have a role in signal transmission through membranes, and they are reported to be beneficial in the response to pathogens. It seems probable that sterol glycosides are oriented with the sterol moiety buried in the hydrophobic core of the lipid bilayer with the sugar located in the plane of the polar head groups of the membrane, while with sterol acyl-glycosides both the sterol moiety and the fatty acid chain are embedded in the hydrophobic core of the membrane. Sitosterol-β-D-glucoside in the plasma membrane is believed to be the primer molecule for cellulose synthesis in plants, as in cotton (Gossypium arboreum) fiber, where it may be required for the initiation of glucan polymerization. The sterol is eventually removed from the polymer by a specific cellulase enzyme (the multimeric cellulase synthase is believed to be stabilized by sterols in the plasma membrane).

    Sterol glycosides appear to be essential for the pathogenicity of certain fungi and for some bacteria, and ergosterol glycosides especially are especially troublesome components of plant fungal pathogens. Sterol glycosides have only rarely been reported from organisms other than plants and fungi, although some bacteria, such as the gram-negative bacterium Helicobacter pylori and Borrelia burgdorferi, the causative agent of Lyme disease produce cholesterol glucoside from host cholesterol. On the other hand, cholesteryl glucoside has been found as a natural component of a few animal tissues, and through acting as immunoadjuvants, sterol glycosides are reported to be efficacious in protecting animal hosts against lethal Cryptococcal infections. In the human diet, sterol glycosides have potential benefits in that like free sterols they inhibit the absorption of cholesterol from the gut and reduce the plasma cholesterol levels. The fatty acids are removed from sterol acyl-glycosides by enzymes in the intestine.

    A number of species of monocotyledons contain complex steroidal saponins, which consist of an aglycone based on a triterpenoid furostanol or spirostanol skeleton (derived from cholesterol) and an oligosaccharide chain of two to five hexose or pentose moieties attached to the 3β-hydroxyl group of the sterol. These can interact with cholesterol in plant membranes to form insoluble complexes, which increase membrane permeability.

    Ergosterol and Other Sterols in Yeasts and Fungi

    Yeasts and fungi, together with microalgae and protozoa, can contain a wide range of different sterols. However, ergosterol ((22E)‑ergosta-5,7,22-trien-3β-ol) is the most common sterol in fungi and yeasts, and is accompanied by other sterols not normally abundant in higher plants including cholesterol, 24-methyl cholesterol, 24-ethyl cholesterol and brassicasterol, depending upon species. In Saccharomyces cerevisiae, which is widely studied as a model species of yeasts, ergosterol is the most abundant sterol (ca. 12% of all lipids), with the highest levels in the plasma membrane (up to 40% of the lipids or 90% of the total cell sterols). Its structure is shown in Figure \(\PageIndex{45}\) below.

    Formula of ergosterol
    Figure \(\PageIndex{45}\): Strurture of ergosterol

    Like cholesterol and in contrast to the plant sterols, it is synthesized in the endoplasmic reticulum via lanosterol as the key intermediate and then zymosterol, but the pathway diverges at this stage to produce fecosterol on the way to ergosterol (see the reading list below for further details). Ergosterol is transported to other organelles within the cell in a non-vesicular manner by two families of evolutionarily conserved sterol-binding proteins - 'Osh' and 'Lam', which are able to optimize the sterol composition of cell membranes rapidly under conditions of stress. As in humans, a Niemann-Pick protein NCR1 integrates sterols into the lysosomal membrane prior to further distribution as part of the mechanism of sterol homeostasis. Some antifungal drugs are targeted against ergosterol, either by binding to it to cause damaging cellular leakage, or to prevent its synthesis from lanosterol.

    Many mutants defective in ergosterol biosynthesis have been isolated, and these have yielded a great deal of information on the features of the sterol molecule required for its structural role in membranes of yeast and fungi. Ergosterol stabilizes the liquid-ordered phase in the same manner as cholesterol and also forms raft microdomains with sphingolipids in membranes, whereas lanosterol does not. It is also evident that ergosterol has a multiplicity of functions in the regulation of yeast growth.

    Under some conditions, especially those that retard growth, a high proportion of the sterols in yeasts can be in esterified form, where they are stored in the organelles know as lipid droplets. Ergosterol esters are synthesized in yeast by enzymes (ARE1 and ARE2), which are related to ACAT-1 and ACAT-2 that perform this function in animals, and both transfer an activated fatty acid to the hydroxyl group at the C3-position of a sterol molecule. In addition, specific sterol ester hydrolases that catalyse the reverse reaction have been characterized from yeasts, two in lipid droplets and one at the plasma membrane. Many fungal species and slime moulds contain steryl glycosides (ergosteryl β-monoglucopyranosides in the former), but they are present at very low levels only in the widely studied yeast Saccharomyces cerevisiae.

    Most fungi conjugate the 3β-hydroxyl group of ergosterol with aspartate in an RNA-dependent reaction catalysed by an ergosteryl-3β-O-L-aspartate synthase, with the reverse reaction using a dedicated hydrolase. A phylogenomic study has shown that this pathway is conserved across higher fungi (except S. cerevisiae), including pathogens, and it has been suggested that these reactions constitute a homeostasis system with a potential impact upon membrane remodelling, trafficking, antimicrobial resistance and pathogenicity.

    Bacterial Sterols

    Hopanoids take the place of sterols in many species of bacteria, but it has long been recognized that some bacteria take up cholesterol and other sterols from host animals for use as membrane constituents. Indeed, an external source of sterols is required for growth in species of Mycoplasma. In addition, there have been a number of reports of biosynthesis of sterols by various bacterial species, although a high proportion of these appear now to have been discounted because of fungal contamination. In particular, the possibility of sterol biosynthesis in cyanobacteria has been controversial, and molecular biology studies have yet to detect the presence of the required enzyme squalene epoxide cyclase.

    That said, there is good evidence that a few species of prokaryotes at least have the capacity to synthesise sterols de novo. Among the eubacteria, certain methylotrophs (Methylobacterium and Methylosphaera species) produce mono- and dimethyl sterols, including lanosterol. Similarly, some soil bacteria produce 4‑desmethylsterols. It has now been established from gene sequence studies that a few bacteria contain enzymes of the sterol biosynthesis pathway such as oxidosqualene cyclase, but as these have no obvious evolutionary link it seems probable that they were acquired via lateral transfer from eukaryotes.

    Plant Sterols in the Human Diet

    The absorption of dietary plant sterols and stanols in humans is low (0.02-3.5%) compared to cholesterol (35-70%), although there are similar amounts in an average Western diet. The explanation is believed to be that the Niemann-Pick C1-like protein 1 (NPC1L1), which is responsible for cholesterol absorption in enterocytes does not take up plant sterols efficiently, while two transporters (ABCG5 and ABCG8) redirect any that are absorbed back into the intestinal lumen. In some rare cases, increased levels of plant sterols in plasma serve as markers for an inherited lipid storage disease (phytosterolemia) caused by mutations in the enterocyte transporters. Among many symptoms, accelerated atherosclerosis is often reported although the reasons for this are not clear. There is evidence that while plant sterols can substitute for cholesterol in maintaining membrane function in mammalian cells, they can exert harmful effects by disrupting cholesterol homeostasis. This may be relevant to the brain especially, since phytosterols are able to cross the blood-brain barrier, although they cannot be oxidized enzymatically because of the alkyl moiety on C24. In contrast, dietary supplements of plant sterols have been reported to have anti-cancer effects.

    Substantial amounts of phytosterols are available as by-products of the refining of vegetable oils and of tall oil from the wood pulp industry. As it appears that they can inhibit the uptake of cholesterol from the diet and thereby reduce the levels of this in the plasma low-density lipoproteins, there is an increasing interest in such commercial sources of plant sterols to be added as "nutraceuticals" to margarines and other foods, Hydrogenated phytosterols or "stanols" are also used for this purpose, and studies suggest they are as effective as sterols in reducing LDL cholesterol. The consensus amongst experts in the field (including the FDA in the USA) is that such dietary supplements do indeed have the effects claimed and such claims can be used in advertising of commercial products, with the important caveat that there are no randomized, controlled clinical trial data that establish ensuing benefits to health, especially with respect to cardiovascular disease. Other pharmacological effects are under investigation, and there may be beneficial effects for the development of the human fetus and new-born, and for the treatment of non-alcoholic steatohepatitis, inflammatory bowel diseases and allergic asthma.

    It is not clear whether oxy-phytosterols are generated in animal tissues, but those produced by enzymatic or non-enzymatic means can enter the food chain, especially when they are produced during cooking. Although they are not efficiently absorbed, 7-keto-sitosterol and 7-keto-campesterol have been detected in human plasma and have the potential to exert a variety of biological effects. For example, they have pro-atherogenic and pro-inflammatory properties in animal models.


    21.5: Biosynthesis of Cholesterol and Steroids is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski and Patricia Flatt.

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