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 (separate web page) 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. Plant sterols have their own web page.
1. 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, some of which are discussed at greater length in our web page relating to eicosanoid biosynthesis. 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 certain of these enzymes 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 here.
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) 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 synthesised 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 epoxyl 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 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.
2. 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 favoured 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.
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 (see our web page on isoprostanes and oxidized phospholipids, for example), 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 aqueous dispersions, oxidation is initiated by a radical attack from a reactive-oxygen species 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 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 and then relatively slowly, but not by the selenium-dependent glutathione peroxidase. 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.
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.
3. 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 signalling, 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 biosynthesis, 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. 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. Many oxysterol species are active in a range of immune cells subsets, mediated through the control of LXR and SREBP signalling, 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, gene expression, secretion, endocytosis, or motility. 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, i.e. 7α,25‑dihydroxycholesterol and further oxidation products, are pro-inflammatory and may 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 favourable 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.
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.
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.
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.
Infection with virus and bacterial pathogens such as Mycobacterium tuberculosis 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 the LXR receptor. 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. 27‑Hydroxycholesterol in human milk is reported to be active against the pathogenic human rotavirus and rhinovirus of importance in pediatrics. 7-Dehydrocholesterol has anti-viral properties also.
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.
4. Sterol Sulfates
The strongly acidic sulfate ester of cholesterol (cholesterol 3-sulfate) occurs in all mammalian cells, but it is especially abundant in keratinized tissue such as skin and hooves. Although present at low levels, it can be the main sulfolipid in many cell types, but especially kidney, and reproductive and nervous tissues. In many organs, it appears to be concentrated in epithelial cell walls or in plasma membranes. Cholesterol sulfate is the main circulating sterol sulfate in plasma (~2 µM), and it is accompanied by trace amounts of dehydroepiandrosterone sulfate and other steroidal sulfates. Although their functions in plasma are not fully defined, sterol sulfates are widely assumed to facilitate transport and perhaps excretion of sterols. In addition to cholesterol sulfate, 7-ketocholesterol sulfate has been found in primate retina, while 24‑hydroxycholesterol occurs in bovine brain as its sulfate ester, and 25-hydroxycholesterol sulfate has been detected in the nuclei of human liver cells. 25-Hydroxy-vitamin D3-3-sulfate is present in the systemic circulation and may serve as a reservoir of the vitamin. Sterol sulfates have been detected occasionally in lower organisms, including many marine invertebrates such as starfish, sponges, ascidians, snails and algae
The sulfate moiety is added to position 3 of sterols from the sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate, by a family of cytosolic sulfotransferases (SULTS), some of which are specific for particular sterols; SULT2B1b preferentially catalyses the conversion of cholesterol to cholesterol sulfate, for example. When required, sterol sulfates are de‑sulfated by a membrane-bound sterol sulfatase in the endoplasmic reticulum, and the sulfation and desulfation pathways are believed to be a dynamic means of de-activating/re-activating steroid hormones to control their biological potency. While in the form of a sulfate conjugate, the sterol backbone can be modified to form other steroidal sulfates, including oxysterols.
Cholesterol sulfate is especially important in skin where it may have a role in ensuring the integrity and adhesion of the various skin layers, while also regulating some enzyme activities. For example, it functions in keratinocyte differentiation, inducing genes that encode for key components involved in development of the barrier. After generation in normal epidermis by SULT2B1b, it is desulfated in the outer epidermis as part of a 'cholesterol sulfate cycle' that is a powerful regulator of epidermal metabolism and barrier function. It accumulates in the epidermis in the human genetic disorder X-linked ichthyosis, in which there is a deficiency in the sterol sulfatase.
It is evident that cholesterol sulfate has many functions other than in skin, and it may play a part in cell adhesion and differentiation. In relation to signal transduction, it interacts with the nuclear receptor retinoic acid-related orphan receptor α (RORα), and it influences phosphoinositide signalling. It has a stabilizing role also, for example in protecting erythrocytes from osmotic lysis and regulating sperm capacitation. In the eye, cholesterol sulfate produced by the Harderian gland is a key protective factor against potentially harmful immune responses by suppressing the migration of neutrophils and T cells. Other oxysterol sulfates are key mediators in such cellular processes as attenuation of the inflammatory response and the regulation of lipid metabolism via SREBP-1. For example, 5-cholesten-3β,25-diol 3-sulfate has been reported to have a variety of signalling and regulatory functions towards many aspects of lipid metabolism, inflammatory responses and cell proliferation at a transcriptional level through its actions on nuclear receptors, while 25‑hydroxycholesterol 3-sulfate reduces cholesterol levels and depresses immune responses by related mechanisms. It appears that 25‑hydroxycholesterol 3-sulfate may regulate signalling pathways in hepatocytes in an opposite direction from its precursor, and that the two may act cooperatively by modifying gene transcription (via DNA methylation) in response to inflammatory stress.
5. Cholesterol Glycosides and Other Cholesterol Derivatives
Sterol glycosides are common constituents of plants (see our web page on plant sterols), and it has become evident that cholesterol glucoside (1‑O‑cholesteryl-β-D-glucopyranoside) and less often cholesterol acyl-glucoside are also present in animal tissues. Both lipids were first found in the skin of snakes, reptiles and birds, but cholesterol glucoside occurs also in human plasma, fibroblasts and gastric mucosa, and some rat and mouse tissues, where it may act as a mediator of signal transduction in the early stages of heat stress. Indeed, both cholesterol glucoside and galactoside are present throughout development from embryo to adult in mouse brain. As with plant and fungal sterol glycosides, these have a β-glucosidic linkage to cholesterol. In embryonic chicken brain, cholesterol β-glucoside is accompanied by sitosterol glucoside, and there are suggestions that they may be involved in neurodegenerative disorders such as Gaucher disease and Parkinson's disease. In addition, a cholesterol-conjugate with glucuronic acid has been isolated from human liver (33 nmol/g wet tissue) and plasma, but its origin, function and metabolic fate are unknown.
Biosynthesis in animal tissues involves a transfer of glucose from glucosylceramide to cholesterol catalysed by a β-glucocerebrosidase (non-lysosomal GBA2) at the cytosolic surface of the endoplasmic reticulum and the Golgi apparatus under normal conditions, while both synthesis and the reverse reaction occurs also via the action of a second glucocerebrosidase (GBA1) at the luminal side of lysosomal membranes. In rodent brain, β-galactosylceramide is generated by the same glucosyltransferases from galactosylceramide. In contrast, with plant sterol glycosides, biosynthesis involves the use of uridine diphosphate (UDP)-glucose as the glucose donor.
Some bacterial species contain cholesterol glycosides, synthesised from cholesterol derived from the membranes of host animals. For example, four unusual glycolipids, i.e. cholesteryl-α-glucoside, cholesteryl-6'-O-acyl-α-glucoside, cholesteryl-6'-O-phosphatidyl-α-glucoside, and cholesteryl-6'-O-lysophosphatidyl-α-glucoside, occur in the pathogenic bacterium Helicobacter pylori. The key enzyme involved in their biosynthesis is a membrane-bound, UDP-glucose-dependent cholesterol-α-glucosyltransferase. Cholesterol 6-O-acyl-β-D-galactopyranoside and its non-acylated form are significant components of membranes of the tick-borne spirochete Borrelia burgdorferi, which is the causative agent of Lyme disease. Together with cholesterol, these lipids form raft microdomains with proteolipids in the membranes of the organism, which may permit it to sense environmental changes and adapt to the host. The cholesterol glycoside can be transferred back to the membranes of the host animal, where it may facilitate the infective process.
Cholesterol is found linked covalently to essential developmental proteins, known as the hedgehog signalling family, where one function of the cholesterol moiety is to anchor the protein in a membrane, but this is discussed in our web page on proteolipids.
6. 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. 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.
7. 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 synthesised in the central nervous system. Cholesterol homeostasis is therefore vital to fertility and a host of bodily functions.
Steroidal esters accumulate in tissues such as the adrenal glands, which synthesise 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 hydrolysed 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.
Sample handling remains is a major problem in the analysis of oxysterols, because of the low levels relative to cholesterol in most tissues. In particular precautions need to be taken to minimize autoxidation during storage and extraction of tissues, for example by adding the antioxidant butylated hydroxytoluene (BHT) together with a peroxide reducing agent such as triphenylphosphine. Following trimethylsilylation, gas chromatography linked to mass spectrometry is often the favoured technique for analysis of free oxysterols, but HPLC linked to electrospray ionization is now being used increasingly as it enables direct analysis of even the reactive hydroxy-, hydroperoxy- and ozonide-containing oxysterols. The latter methodology is also applicable to sterol conjugates and permits a wider range of derivatization techniques to be used, including nicotinates.
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|Credits/disclaimer||Updated: September 9th, 2021||Author: William W. Christie|