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 of their own in animals, while others are short-lived intermediates or end products in the catabolism or excretion of cholesterol or in the biosynthesis of steroid hormones, bile acids and vitamin D. They are normally present in membranes and lipoproteins at trace levels only, although they can often exert profound effects at these concentrations, and they are always accompanied by a great excess (as much as 10⁶-fold) of cholesterol per se.

Structural formula for cholesterol A multiplicity of different oxysterols is synthesised in cells by sequential reactions with oxygenases, but because of the presence of the double bond in the 5,6‑position, oxysterols can 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 exception). Oxidized cholesterol molecules can also be generated by the gut microflora and be taken up through the enterohepatic circulation.

Once an oxygen substituent is introduced into cellular cholesterol, the product can act as a lipid mediator by interacting with a receptor, but it can be metabolized to bile acids (separate web page) or 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 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 on 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 the relevant enzymes, which include cytochrome P450s, 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. Representatives of the first steps in some of these pathways are illustrated here.

Figure 1. Biosynthesis of oxysterols.

A primary product is 7α‑hydroxycholesterol, which is an 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 is critical for 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, 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 a factor in the pathology of Alzheimer’s disease has yet to be determined.

Hydroxysteroid 11-β-dehydrogenase 1 (HSD11B1) is responsible for the conversion of 7β-hydroxycholesterol to 7‑ketocholesterol, while HS11B2 catalyses the reverse reaction; 7-ketocholesterol can also be formed by autoxidation (see below). HSD11B1 is better known as the oxidoreductase that converts inert cortisone to the stress hormone cortisol in glucocorticoid target tissues.

An alternative ('acidic') pathway to bile acids starts with the synthesis of 27-hydroxycholesterol (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 the lung 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 abundant in plasma and is relatively stable; it is produced in humans by the action of the cytochromes CYP3A4 and CY3A5.

In humans, the 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 implicated in 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 in plasma where it is transported via high-density lipoproteins (HDL), as discussed further below. 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 contrast to humans, CYP46A1 is present in the liver as well as brain of rodents.

25-Hydroxycholesterol is a relatively minor but crucial 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 main 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 other oxysterols.

24S,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, i.e., the 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, i.e., to produce 2,3S-22S,23-dioxidosqualene as an intermediate. An additional 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 utilized for the metabolism of cholesterol and steroidal hormones, such as esterification and sulfation of position 3, as illustrated for 7-ketocholesterol. In most tissues, esterification of the 3β-hydroxyl group only occurs and requires the 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.

Figure 2. Metabolism of 7-keto-cholesterol.

It is noteworthy that the 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 is converted to sulfate esters (mainly at the 3‑hydroxyl group) or glucuronides (see below) for elimination via the kidneys.

2. Non-Enzymatic Oxidation of Cholesterol

In tissues 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, for example. However, there are circumstances that can favour cholesterol oxidation in vivo, and in low-density lipoprotein particles (LDL), the concentration of cholesterol is about three times higher than that of phospholipids, so the rate of cholesterol hydroperoxide formation can be higher than that of phospholipid hydroperoxides. The rate and specificity of the reaction can depend 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 (ROS) such as singlet oxygen, HOCl or ozone (Type II autoxidation). Some representatives of the main types of product from non-enzymatic oxidation are illustrated.

Figure 3. Some non-enzymatic oxidation products of cholesterol.

Oxysterols produced by this means can vary in the type (hydroperoxy, hydroxy, keto, epoxy), number and position of the oxygenated substituents introduced and in 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 most often 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), and it appears that comparable mechanisms operate with sterols. The first event in lipid peroxidation by a radical mechanism is an initiation reaction in which a carbon atom 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 peroxynitrite 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.

The mechanism for the production of 7-oxygenated cholesterol derivatives is illustrated. In aqueous dispersions, oxidation is initiated by a radical attack from an ROS 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 be produced.

Figure 4. Examples of mechanisms for non-enzymic oxidation of cholesterol.

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 have the potential to be harmful in tissues. Of these, 5α-OOH with the lowest reduction rate is most cytotoxic of the hydroperoxides.

Epimeric 5,6-epoxycholesterols 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 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; 5,6-epoxycholesterol can then be subjected to hydrolysis by cholesterol epoxide hydrolase to form cholestane-3β,5α,6β-triol. Other non-radical oxidation processes include reaction with singlet oxygen in the presence of light and photosensitizers, while reaction with ozone in the lung can generate a family of distinctive oxygenated cholesterol metabolites.

Oxysterols occur in tissues both in the free state and esterified with long-chain fatty acids, and in human atherosclerotic lesions, 80 to 95% of all oxysterols are esterified. Appreciable amounts of oxysterols can be present in foods 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, together 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.

3. Oxysterols – Tissue Metabolism

A diverse range of oxidation products are generated enzymatically and by peroxidation of cholesterol and they can affect tissue metabolism. 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 such low concentrations of oxysterols in the presence of a vast excess of native cholesterol; the average levels of 26-, 24- and 7α-hydroxycholesterol in human plasma that are often quoted are 0.36, 0.16 and 0.14 μM, respectively. Autoxidation products of cholesterol such as 7-keto- and 7-hydroxycholesterol are cytotoxic and may be useful markers of oxidative stress or for monitoring of the progression of various diseases. 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.

Scottish thistle Nonetheless, aside from being precursors of bile acids and some steroidal hormones, it is evident that oxysterols assist in many ways in maintaining cholesterol homeostasis and perhaps in signalling, where those formed enzymatically are most relevant. They can exert potent 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 most important and include the liver X receptors (LXRs), retinoic acid receptor-related orphan receptors (RORs), oestrogen 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 synapses, while G protein-coupled receptors operate at cell membranes and interact with molecules outside the cell to trigger 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 membranes and 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 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 mainly in the esterified form within the HDL and LDL.

Cholesterol homeostasis: While cholesterol is crucial for its own feedback regulation, there is some evidence that oxysterols are regulators of cholesterol concentration in cell membranes and that 25‑hydroxycholesterol and 24S,25‑epoxycholesterol may be especially effective, although the other side-chain oxysterols 22-, 24- and 27‑hydroxycholesterol have been implicated. This occurs by several mechanisms, and it is suggested that 24S,25‑epoxycholesterol acts as a ligand for the liver X receptor, which forms a heterodimer with the retinoic X receptor to inhibit the transcription of genes for cholesterol biosynthesis as well as directly inhibiting or accelerating the degradation of such enzymes in the process as HMG-CoA reductase and squalene synthase; 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 and prevention of substantial fluctuations in tissue concentration.

Oxysterols and the immune system: Oxysterols are known to have vital and diverse roles in immunity by regulating both the adaptive and innate immune responses to infection. Virus infection leads to production of type I interferon, and in macrophages, there is enhanced expression of the oxygenase CH25H and synthesis of 25‑hydroxycholesterol, which in general is regarded as anti-inflammatory and exerts broad antiviral activity by several mechanisms that include inducing the transcription of integrated stress response genes and reprogramming protein translation again via its interaction with transcriptional factors (LX receptors, SREBP2, RORs), ion channels, integrins and oxysterol-binding proteins. It is a potent inhibitor of SARS-CoV-2 replication, possibly by a mechanism for blocking cholesterol export from the late endosome/lysosome compartment and depletion of membrane cholesterol levels. In contrast, formation of 25‑hydroxycholesterol may be harmful in the case of influenza infections as it can lead to over-production of inflammatory metabolites. Biosynthesis of 25‑hydroxycholesterol in macrophages is stimulated by the endotoxin Kdo2-lipid A, the component of the lipopolysaccharide present on the outer membrane of Gram-negative bacteria, which acts as an agonist for Toll-like receptor 4 (TLR4).

25‑Hydroxycholesterol is reported to play a regulatory part in 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 hedgehog proteins in embryonic development. Metabolites of 25‑hydroxycholesterol, such as 7α,25‑dihydroxycholesterol and further oxidation products, are pro-inflammatory and act as chemo-attractants to lymphocytes; they regulate immunity in secondary lymphoid organs by interactions with the receptor GPR183.

7α/β-hydroxycholesterol is produced in large quantities in chronic infections with the hepatitis C virus and inhibits its replication, and it has similar effects with human immunodeficiency virus (HIV) in human primary lymphocytes. In contrast, 7-ketocholesterol can be used as a biomarker for the severity and prognosis of COVID-19. 27‑Hydroxycholesterol in human milk is reported to inhibit the pathogenic human rotavirus and rhinovirus, which infect infants, and 7-dehydrocholesterol too has anti-viral properties.

Many other oxysterol species behave in a similar manner in a range of immune cell subsets, mediated through the control of LXR and SREBP signalling and by acting as ligands for RORs or for the cell surface receptors G protein-coupled receptor 183 (GPR183) or CXCR2. Binding to LXR tends to dampen the immune response. In response to various stimuli, they can operate through ion channels to cause rapid changes in the concentration of intracellular Ca²⁺ and other ions to bring about changes in membrane potential, cell volume, cell-death (apoptosis, autophagy and necrosis), gene expression, secretion, endocytosis or motility. While they can act in this manner in the cell in which they are generated, oxysterols can operate in a paracrine fashion towards other immune cells.

Oxysterols in brain: Oxysterols are major factors in 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, where it serves to control its fluidity and permeability. This membrane is produced in large amounts in brain and is the basis of the compacted myelin, which conducts 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 grey matter and is more important metabolically.

Even in the foetus and the new-born infant, all the cholesterol required for growth is produced by synthesis de novo in the brain and 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. A necessary component of this system is apolipoprotein E (apo E), a 39-kDa protein, which is highly expressed in brain and serves 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.

Cholesterol and the brain As discussed briefly above, hydroxylation by CYP46A1 of cholesterol to 24S‑hydroxycholesterol (cerebrosterol) is responsible for 50–60% of all cholesterol metabolism in the adult brain. While 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 produces 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 inert 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 24S-hydroxycholesterol and 24S,25-epoxycholesterol are believed to be required for regulating cholesterol homeostasis in the brain. They interact with the nuclear receptors for the expression and synthesis of proteins for sterol transport, and for example, 24‑hydroxycholesterol 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 for cholesterol homeostasis and inflammatory responses, and it is 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 support the operation of neurons, astrocytes, oligodendrocytes and vascular cells.

24S-Hydroxycholesterol down-regulates trafficking of the amyloid precursor protein and may be a factor in preventing neurodegenerative diseases. High levels of of this oxysterol are observed in the plasma of human infants and in patients with brain trauma, but perhaps surprisingly, 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 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, and probably reflects a selective loss of neuronal cells; it may be a factor in age-related macular degeneration. An excess of 24S‑hydroxycholesterol and 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. Synthesis of 25‑hydroxycholesterol can be upregulated in some neurological disorders, including Alzheimer's disease, but it is not clear whether it aggravates these pathologies or has protective properties.

27‑Hydroxycholesterol 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 enzymes within the brain, and there are suggestions that the balance between the levels of 24- and 27-hydroxycholesterol, especially an 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. 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, and they have been found in atherosclerotic lesions. Oxidation products of the cholesterol precursor 7‑dehydrocholesterol such as 3β,5‑dihydroxycholest-7-ene-6-one are of concern towards 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 22R- and 25R‑hydroxycholesterol were shown to induce human keratinocyte differentiation. Subsequently, by stimulating nuclear binding receptors, oxysterols were found to affect mesenchymal stem cells similarly, 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: Those oxysterols formed non-enzymatically can be most troublesome in relation to disease in general, and they are enriched in pathologic cells and tissues, such as macrophage foam cells, atherosclerotic lesions and cataracts, where they can affect cytotoxicity, necrosis, inflammation, immuno-suppression, phospholipidosis and gallstone formation. They may regulate some of the metabolic effects of cholesterol, but as cautioned above, experimental observations in vitro may not necessarily be of physiological importance in vivo.

Oxysterols have been implicated in the development of cancers, mainly those of the breast, prostate, colon and bile duct. 27‑Hydroxycholesterol is a factor in cholesterol elimination from macrophages and arterial endothelial cells, but as an endogenous ligand for the human nuclear oestrogen receptor (ERα) and the liver X receptor, it influences 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, some oxysterols can interfere in the proliferation of several types of cancer cell (glioblastoma, leukaemia, colon, breast and prostate cancer).

Scottish thistle Cholesterol 5,6-epoxide (with either 5α or 5β stereochemistry) is formed non-enzymatically in tissues, but in the adrenal glands, it is believed to be produced by an as yet unidentified cytochrome P450 enzyme. 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 the enzyme 11‑β‑hydroxysteroid-dehydrogenase-type 2 into the oncometabolite 3β,5α-dihydroxycholestan-6-one (oncosterone). By binding to the glucocorticoid receptor, oncosterone stimulates the growth of breast cancer cells while acting as a ligand for 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, which 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 inhibits it by acting as a microsomal anti-oestrogen binding site (AEBS).

7-Ketocholesterol is a major oxysterol produced during oxidation of low-density lipoproteins and one of the most abundant oxysterols in plasma, atherosclerotic lesions and erythrocytes of heart failure patients. 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. It has a high pro-apoptotic potential, associating preferentially with membrane lipid raft domains. Photooxidation in the retina via the action of free radicals or singlet oxygen generates unstable cholesterol hydroperoxides, which may have a part in age-related macular degeneration, and these compounds can quickly be converted to highly toxic 7α- and 7β‑hydroxycholesterols and 7‑ketocholesterol. The concentrations of these depend on the status of tissue oxidases and reductases, and three separate enzymatic pathways have developed in the eye to neutralize them. In other tissues, these sterols are metabolized by novel branches of the acidic pathway of bile acid biosynthesis.

Various oxysterols have been implicated in the differentiation of mesenchymal stem cells and the signalling pathways for 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.

Formula of 7-dehydrocholesterol Not all oxysterols are harmful, and the vitamin D precursor 7‑dehydrocholesterol has the highest propagation rate constant known for any lipid toward free-radical chain oxidation and acts as a lipid-soluble antioxidant to protects cells from ferroptosis, a form of cell death triggered by iron-catalysed phospholipid peroxidation.

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 has adverse consequences in that there is impairment of cholesterol utilization in steroidogenic cells and of anti-atherogenic reverse-cholesterol transport in vascular macrophages. As an antioxidant, GPx4 may be crucial for the maintenance of mitochondrial integrity and functionality in these cells.

4. Sterol Sulfates

Formula of cholesterol sulfate The strongly acidic sulfate ester of cholesterol (cholesterol 3-sulfate) occurs in all mammalian cells, but it is abundant in keratinized tissue such as skin and hooves. Although present at low levels, it can be the main sulfolipid in many cell types, including the kidney and reproductive and nervous tissues, where it appears to be concentrated mainly 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 sterol sulfates are widely assumed to facilitate transport and perhaps excretion of sterols, they may be needed for other undefined purposes.

In addition to cholesterol sulfate, 7‑ketocholesterol sulfate has been found in primate retina, while 22-, 24- and 26‑hydroxycholesterol have been found in tissues as sulfate esters, 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. However, little appears to be known of basal levels of sterol sulfates in fluids (serum, urine and cerebrospinal), cells and tissues, possibly because of analytical problems and a lack of suitable standards. Sterol sulfates have been detected occasionally in lower organisms, including algae and many marine invertebrates such as starfish, sponges, ascidians and snails, which can contain forms with further links to phosphate and then carbohydrate residues.

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, and of these, SULT2B1b preferentially catalyses sulfation to produce cholesterol sulfate. 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 potency. While in the form of a sulfate conjugate, the sterol backbone can be modified to form other steroidal sulfates, including oxysterols.

In skin, cholesterol sulfate is needed to ensure the integrity and adhesion of the various skin layers, while at the same time regulating some enzymes, and during keratinocyte differentiation, it induces genes that encode for components needed for 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 other functions, 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 in protecting erythrocytes from osmotic lysis and regulating sperm capacitation, while in the eye, cholesterol sulfate produced by the Harderian gland is a protective factor against potentially harmful immune responses by suppressing the migration of neutrophils and T cells. In mitochondria, SULT1C2 increases respiration by converting mitochondrial membrane cholesterol to cholesterol sulfate.

Other oxysterol sulfates are mediators in such cellular processes as attenuation of the inflammatory response and the regulation of lipid metabolism via SREBP-1. 5‑Cholesten-3β,25-diol 3‑sulfate has been reported to take part in cellular signalling and the regulation of 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 to modify gene transcription by affecting 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), but it has become evident that cholesterol glucoside (1‑O‑cholesteryl-β-D-glucopyranoside) and less often cholesterol acyl-glucoside are present in some animal tissues. As with plant and fungal sterol glycosides, these have a β‑glucosidic linkage to cholesterol. Both lipids were first found in the skin of snakes, reptiles and birds, but cholesterol glucoside is now known to occur in human plasma, fibroblasts and gastric mucosa, and in 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. In embryonic chicken brain, cholesterol β‑glucoside is accompanied by sitosterol glucoside, and there are suggestions that they have a role in neurodegenerative disorders such as Gaucher disease and Parkinson's disease. 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.

The biosynthetic mechanism in animal tissues differs from that in plants where uridine diphosphate (UDP)-glucose is the glucose donor, and instead, there is 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; both synthesis and the reverse reaction occur 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.

Figure 5. Biosynthesis of cholesteryl glucosides in animals

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, are major components of the cell wall of the pathogenic bacterium Helicobacter pylori, which can cause peptic ulcers, stomach inflammation (gastritis) and cancer with a membrane-bound, UDP-glucose-dependent cholesterol-α-glucosyltransferase is the enzyme for the biosynthesis of the first of these with phosphatidylethanolamine as the fatty acid donor for the acylate form; they may assist the organism to evade the host immune system. 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.

Formula of cholesterol 6-O-acylgalactoside from Borrelia burgdorferi

Cholesterol is found linked covalently to the developmental proteins known as the hedgehog signalling family, where the cholesterol moiety anchors 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, although these are needed for many other purposes, 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.

Vitamin D3

Ultraviolet light mediates cleavage of 7-dehydrocholesterol, an 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 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 interacts with 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 absorptions. It is a general development hormone in many different tissues, and together with vitamin D₂, it has affects immune responses profoundly in the defence 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. 17β‑Estradiol is the most potent of the endogenous oestrogens; 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 and is required for the development of male reproductive tissues such as testes and prostate as well as promoting secondary sexual characteristics.

Examples of steroidal hormones

In brief, animal tissues synthesise 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 requires the steroidogenic acute regulatory protein (STAR), which enables the transport of cholesterol into mitochondria where conversion to pregnenolone is rate-limiting with 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. Cholesterol homeostasis is therefore vital for fertility and innumerable other metabolic purposes.

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, and it is apparent that fatty acyl esters of oestradiols, such as dehydroepiandrosterone, accumulate in adipose tissue in post-menopausal women, presumably as inert storage or transport forms. Small amounts of oestrogens acylated with fatty acids at the C-17 hydroxyl are present in the plasma lipoproteins. 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.

8. Analysis

Sample handling remains 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 unstable hydroxy-, hydroperoxy- and ozonide-containing oxysterols. The latter methodology can be applied to sterol conjugates, and it permits a wider range of derivatization techniques to be used, including nicotinates.

	© Author: William W. Christie
	Contact/credits/disclaimer	Updated: November 27^th, 2024