STEROLS 2. OXYSTEROLS, BILE ACIDS AND CHOLESTEROL SULFATE

STRUCTURE, OCCURRENCE AND BIOCHEMISTRY

1. Oxysterols

Oxysterols are usually defined as oxygenated derivatives of cholesterol, though plant sterols can also be oxidized, and they are important as short-lived intermediates or end products in the catabolism or excretion of cholesterol. They are normally present in biological membranes and lipoproteins at trace levels only, though they can exert profound biological effects at these concentrations, but they are always accompanied by a great excess (as much as 10⁶-fold) of cholesterol.

Oxysterols can be formed rapidly by non-enzymatic autoxidation of cholesterol (and cholesterol esters), when a multiplicity of different oxygenated derivatives result, but they are also synthesised by specific oxygenases in cells. Once an oxygen function is introduced into cholesterol within cells, the product can act as a biologically active mediator before it is metabolized to bile acids (see below) or is degraded further, processes assisted by the fact that oxysterols are able to diffuse much more rapidly through membranes than is cholesterol itself.

Non-Enzymatic Oxidation: There is evidence that cholesterol in a membrane environment may be attacked more readily than the polyunsaturated fatty acids by reactive oxygen species, although the opposite is true in plasma, for example. Of course, the process of cholesterol autoxidation also occurs in foods on storage, with the potential for harmful implications for the consumer. The structures of a few of the more important oxysterols are illustrated below as examples of the main types of product.

Structural formulae of oxysterols

Oxysterols 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. Usually, they are named in relation to cholesterol, rather than by the strict systematic terminology. Oxysterols occur in tissues both in the free state and esterified with long-chain fatty acids.

Mechanisms of autoxidation have been intensively studied in terms of unsaturated fatty acids, and it appears that similar mechanisms operate with sterols. 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 forming a delocalized three-carbon allylic radical, which reacts with oxygen to produce the epimeric products 7α- and 7β-hydroperoxy-cholesterol. Subsequent enzymic and non-enzymic reactions lead to the hydroxy and keto analogues, which may be accompanied by epoxy-ene and ketodienoic secondary products.

Formation of 7-hydroperoxy-cholesterol

Reaction does not occur 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.

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 can generate 5-hydroxy- as well as 6- and 7-hydroxy products. In addition, reaction with ozone, for example in the lung, can generate a family of distinctive oxygenated cholesterol metabolites.

Photoxidation in the retina via the action of 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 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 activity. 7-Ketocholesterol is also a major oxysterol produced during oxidation of low-density lipoproteins, and is one of the most abundant in plasma and atherosclerotic lesions, with a high pro-apoptotic potential. It associates preferentially with membrane lipid raft domains.

Enzymatic Oxidation: Within animal cells, oxidation of sterols is mainly an enzymic process that is carried out by several enzymes that are mainly from the cytochrome P450 family of oxygenases (they have 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. For example, 7α-hydroxycholesterol is an important intermediate in the biosynthesis of bile acids (see below) and it is produced in the liver by the action of cholesterol 7α-hydroxylase (CYP7A1). The reaction is under strict regulatory control, and any circulating 7α-hydroxycholesterol represents leakage from the liver.

An alternative pathway to bile acids starts with 27-hydroxycholesterol, which is produced by another cytochrome P-450 enzyme (CYP27A1) that introduces the hydroxyl group into the terminal methyl carbon (C-27). 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. It is involved in some of the later stages of bile acid production (see below). In addition, as a multifunctional mitochondrial P-450 enzyme in liver, it generates both 25R,26-hydroxycholesterol and 3β-hydroxy-5-cholestenoic acid, which occur in significant amounts in plasma.

Scottish thistle In humans, the specific cytochrome P-450 that produces 24S-hydroxycholesterol (cholest-5-ene-3β,24-diol), cholesterol 24S-hydroxylase (CYP46A1), is located almost entirely entirely in the smooth endoplasmic reticulum of neurons in the brain, and even the 24S-hydroxycholesterol found in plasma is derived from the brain. The enzyme is expressed in neurons, including those of the hippocampus and cortex, which are important for learning and memory, and it is responsible for most of the turnover of cholesterol in the central nervous system. 25-Hydroxycholesterol is a relatively minor but biologically important cholesterol metabolite, and it is unusual in that it is produced in liver by an enzyme from a family of non-heme iron–containing proteins and not a cytochrome P-450, as well as by autoxidation..

24(S),25-Epoxycholesterol is not produced by the pathways described above but is synthesised in a shunt of the same mevalonate pathway that produces cholesterol. It may represent a measure of newly synthesised cholesterol.

Biological Activity: Aside from their role as precursors of bile acids and some steroidal hormones, oxysterols have a variety of roles in terms of maintaining cholesterol homeostasis and perhaps in signalling. While cholesterol plays a key role in its own feedback regulation, there is ample evidence that oxysterols are also potent inhibitors of cholesterol biosynthesis, and 25-hydroxycholesterol and 24(S),25-epoxycholesterol, the latter formed as a side product in the mevalonate pathway, are especially effective. Several mechanisms appear to be involved, and it is established that they inhibit the transcription of key genes in cholesterol biosynthesis (sterol regulatory element binding protein (SREBP) transcription factors), as well as directly inhibiting or accelerating the degradation of such important enzymes in the process as HMG-CoA reductase and squalene synthase. Oxysterols may smooth out the regulation of cholesterol metabolism, preventing exaggerated responses. 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.

25-Hydroxycholesterol is also 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, and together with other oxysterols to regulate the activities of some hedgehog proteins involved in embryonic development.

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 the cholesterol in brain. This pool is relatively stable, but the remaining 30% is present in the membranes of neurons and glial cells of gray matter and is 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.

Scottish thistle If cholesterol itself cannot cross the blood-brain barrier, its metabolite 24(S)-hydroxycholesterol 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, and this can occur at a rate that is orders of magnitude greater than that of cholesterol per se, though still only 6-7 mg per day. There is a continuous flow of this metabolite from the brain into the circulation, 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. Especially high levels of 24(S)-hydroxycholesterol are observed in the plasma of human infants, while reduced levels are found in patients with neurodegenerative diseases, including Alzheimer’s disease (it may be protective against β-amyloid peptide, the amyloidogenic peptide found in plaques in this condition) and multiple sclerosis. However, these links are unproven. Conversely, 27-hydroxycholesterol diffuses across the blood-brain barrier from the blood stream into the brain, where it is further oxidized and then exported.

Oxysterols do appear to be important for many aspects of cholesterol turnover and transport, and there have been many reports of involvement in disease processes, especially atherosclerosis and the formation of human atherosclerotic plaques, but also cytotoxicity, necrosis, inflammation, immuno-suppression, phospholipidosis and gallstone formation. 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. However, as cautioned above, effects observed in vitro may not necessarily be of physiological importance in vivo. Similarly, it has been argued that plasma oxysterols could serve as markers of oxidative stress, but the experimental difficulties in analysis have been such that their value has been limited. Sample handling remains a problem, but the newer methods of mass spectrometry with electrospray ionization now enable direct analysis of even the reactive hydroxy-, hydroperoxy- and ozonide-containing oxysterols.

Catabolism: Because of their increased polarity relative to cholesterol, oxysterols can exit cells relatively easily. Some are converted to inert sterol esters and stored in this form, a proportion is further oxidized and converted to bile acids (see below) and some are converted to sulfate esters (especially at the 3-hydroxyl group) or glucuronides for elimination.

2. Bile Acids

The bile acids are metabolites of cholesterol (mainly C₂₄ but also C₂₇), whose main function is to act as powerful detergents or emulsifying agents in the intestines to aid the digestion and absorption of fatty acids, monoacylglycerols and other fatty products. Very many different bile acids and alcohols occur in nature, presumably because multiple biochemical pathways have evolved to convert cholesterol into these highly water-soluble, amphipathic molecules. The nomenclature is complex for historical reasons; many were given names in the 19th century long before their structures were determined. It has been suggested that bile acids and alcohols, sometimes termed cholanoids or cholestanoids, should be subdivided into three main classes, i.e. C₂₇ bile alcohols, C₂₇ bile acids and C₂₄ bile acids. The C₂₇ bile alcohols and acids contain the C₈ side chain of cholesterol, while the C₂₄ bile acids have a truncated C₅ side chain.

In mammals, they are major components of bile amounting to 12% of the total (with 4% phospholipids and 1% cholesterol). In non-mammalian vertebrates, bile alcohols (non-acidic) are formed, while invertebrates do not produce bile acids or alcohols. The main components in human bile are the C₂₄ compounds chenodeoxycholic, deoxycholic and cholic acids, with hydroxyl groups of the 3α,7α-, 3α,12α- and 3α,7α,12α-configurations, respectively. In adult humans, roughly 0.5g of cholesterol is utilized for bile acid production each day. Indeed, this is the major pathway for the removal of cholesterol from the body, and it is important for the maintenance of cholesterol homeostasis both from quantitative and regulatory standpoints.

Biosynthesis of bile acids

Although there are a number of different biosynthetic routes to bile acids from cholesterol, there are four main steps, and the liver is the only organ concerned in the production of the ‘primary’ bile acids. In fact, there are at least 16 enzymes that catalyse up to 17 reactions to convert insoluble cholesterol into a highly soluble conjugated bile salt. What has been termed the classical pathway to the biosynthesis of the 'root' bile acid, chenodeoxycholic acid, involves first the synthesis of 7α-hydroxycholesterol, as described above, in the endoplasmic reticulum. In the next step, epimerization of the 3β-hydroxyl group is effected by a specific oxidoreductase, before the double bond in position 5 is hydrogenated by one of two reductases. Finally, the side-chain is cleaved and oxidized by the same enzyme that produces 27-hydroxycholesterol, i.e. sterol-27-hydroxylase (CYP27) (see above).

Biosynthesis of bile acids - conformational changes

The change in configuration at the A/B ring junction as illustrated accentuates the change in polarity of the molecule, creating hydrophilic (α) and hydrophobic (β) faces.

Deoxycholic acid synthesis occurs by a similar route, except that a sterol sterol-12α-hydroxylase (another of the cytochrome P450 family) introduces a 12-hydroxyl group into the steroidal side-chain. 24-, 25- and 27-Hydroxycholesterol derivatives can also serve as precursors for bile acid synthesis. While the process begins in the endoplasmic reticulum, the intermediates must be transferred to the cytoplasm, where some of the biosynthetic enzymes are situated, and thence to mitochondria and finally to the peroxisomes. Little is known of the transport mechanisms involved.

Scottish thistle Before secretion into bile, a high proportion of the bile acids are converted in the peroxisomes to conjugates with the amino acids glycine and/or taurine, the latter a sulfonic acid-containing compound derived from cysteine (see our webpage on sulfonolipids). These add substantially to the acidity of the molecules, increasing their solubility in water. At the physiological pH values in the intestines, the bile conjugates ionize and exist in salt form.

Conjugated bile acids are secreted into the canalicular space between hepatocytes before entering the bile in the gall bladder, and thence in response to gut hormones they pass into the duodenum of the small intestine, where they assist the emulsification and absorption of the partially hydrolysed lipids from the diet The microflora in the intestines de-conjugate the bile acids and can in part modify the steroidal structures to produce ‘secondary’ bile acids, for example by removing the 7-hydroxyl group to produce 7-deoxy bile acids (e.g. lithocholic and deoxycholic acids). Approximately 95% of the non-conjugated bile acids are absorbed from the ileum of the intestines mainly by active transport processes together with the other lipids, and they are returned to the liver by the portal blood stream (bound to albumin), a pathway that is referred to as the enterohepatic circulation. In the human, a conjugated bile salt may complete this cycle from 4 to 12 times each day. The 5% of bile acids that are lost represent an important element of the turnover of cholesterol.

Regulation of bile acid synthesis involves complex processes, which are linked to the metabolism of cholesterol and fatty acids. However, the main control is exerted via the rate-limiting enzyme cholesterol 7α-hydroxylase, the activity of which can be modified by a number of different pathways, but especially by the action of bile acids and cholesterol on gene transcription.

In addition to their function in the absorption of dietary lipids and in cholesterol homeostasis, bile acids act as signalling molecules. They activate specific protein kinase pathways, they are ligands for a G-protein-coupled receptor and they interact with hormone receptors in the nucleus. Via these various signalling pathways, bile acids regulate their own biosynthesis and enterohepatic circulation, and have an influence on the metabolism of lipids and of glucose. For example, they are involved in the regulation of triacylglycerol biosynthesis and VLDL production in the liver. Inefficient metabolism of bile acids is associated with a number of disease states.

3. Cholesterol 3-sulfate

Structural formula of cholesterol sulfate The strongly acidic sulfate ester of cholesterol 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, although it is there accompanied by dehydroepiandrosterone sulfate, the function of which is unknown. In addition, 7-ketocholesterol sulfate has been found in primate retina, while 24-hydroxycholesterol occurs in bovine brain as its sulfate ester.

Cholesterol sulfate 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. However, the function of this lipid is still only partly understood. It may play a part in cell adhesion, differentiation and signal transduction. In addition, it has a stabilizing role, for example in protecting erythrocytes from osmotic lysis and regulating sperm capacitation.

Sterol sulfates have been detected occasionally in lower life forms, such as the sea star, Asterius rubrius, and the marine diatom, Nitzschia alba.

4. Cholesterol Glycosides and Other Cholesterol Derivatives

Cholesterol is found linked covalently to specific proteins where it functions to anchor the protein in a membrane, but this is discussed in our web page on proteolipids. Cholesteryl glucoside and acyl cholesteryl glucoside have been found in the skin of snakes. Cholesteryl glucoside occurs also in human fibroblasts, and some rat tissues, where it may act as a mediator of signal transduction in the early stages of stress. As with plant and fungal steryl glycosides, these have a sugar β-glucosidic linkage. These lipids appear to support the pathogenicity of the organism. 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.

Some bacterial species contain cholesterol glycosides, and 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, for example. 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 spirochete Borrelia burgdorferi, which is the causative agent of Lyme disease. Sterol glycosides are more common constituents of plants (see our web page on plant sterols).

5. Steroidal Hormones and Vitamin D

These subjects are too big to be discussed in depth here. In brief, in addition to the bulk sterols, animal tissues produce small amounts of vital steroidal hormones, including oestrogens and progesterone, which are made primarily in the ovary and placenta during pregnancy, and testosterone mainly in the testes. Pregnane neurosteroids are produced in the central nervous system. Conversion of cholesterol to pregnenolone in mitochondria is the rate-limiting step, and involves first hydroxylation and then cleavage of the side-chain.

formulae of pregnenolone and cholecalciferol

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, especially in relation to the immune system. Ultraviolet light mediates cleavage of 7-dehydrocholesterol 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 generate a high affinity ligand for the vitamin D receptor. Vitamin D₂ or ergocalciferol is derived from ergosterol and is obtained from plant and fungal sources in the diet.

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