STEROLS

Sterols may be found either as free sterols, acylated (sterol esters), alkylated (steryl alkyl ethers), sulfated (sterol sulfate), or linked to a glycoside moiety (steryl glycosides) which can be itself acylated (acylated sterol glycosides). 
Sterol biosynthesis is nearly ubiquitous among eukaryotes, it is almost completely absent in prokaryotes. As a result, the presence of diverse steranes (saturated 4-cycle skeleton) in ancient rocks is used as evidence for eukaryotic evolution 2.7 billion years ago. A sterol biosynthetic pathway was demonstrated in the proteobacterium, Methylococcus capsulatus, and in the planctomycete, Gemmata obscuriglobus (Pearson A et al., PNAS 2003, 100, 15357).

FREE STEROLS

Sterols form an important group among the steroids. Unsaturated steroids with most of the skeleton of cholestane containing a 3b-hydroxyl group and an aliphatic side chain of 8 or more carbon atoms attached to position 17 form the group of sterols. 

5a-cholestane

They are lipids resistant to saponification and are found in an appreciable quantity in all animal and vegetal tissues. Furthermore, cholestane may be considered as a biological marker compound valuable in the assessment of marine sediment maturity, even after hundreds of millions of years (Mackenzie AS et al., Science 1982, 217, 491). Sterols may include one or more of a variety of molecules belonging to 3-hydroxysteroids, they are C27-C30 crystalline alcohols (in Greek, stereos, solid). These lipids can be classed also as triterpenes, as they derive from squalene which gives directly by cyclization, unsaturation and 3b-hydroxylation, lanosterol in animals or cycloartenol in plants.

In the tissues of vertebrates, the main sterol is the C27 alcohol cholesterol (Greek, chole, bile), particularly abundant in adrenals (10%, w/w), nervous tissues (2%,w/w), liver (0.2%,w/w) and gall stones, its fundamental carbon structure being a cyclopentanoperhydrophenanthrene ring (also called sterane). It was the first isolated sterol around 1758 by F.P. Poulletier de La Salle from gall stones. In 1815, it was isolated from the unsaponifiable fraction of animal fats by M.E. Chevreul who named it cholesterine (Greek, khole, bile and stereos, solid). The correct formula (C₂₇H₄₆O) was proposed in 1888 by F. Reinitzer but structural studies from 1900 to 1932, mainly by H.O. Wieland "on the constitution of the bile acids and related substances" (Nobel Prize Chemistry 1927) and by A.O.R. Windaus on "the constitution of sterols and their connection with the vitamins" (Nobel Prize Chemistry 1928), led to the exact steric representation of cholesterol. In 1936, Callow and Young have designated steroids all compounds chemically related to cholesterol.
While it became clear very early that cholesterol plays an important role in controlling cell membrane permeability by reducing average fluidity, it appears now that it has a key role in the lateral organization of membranes and free volume distribution. These two parameters seem to be involved in controlling membrane protein activity and "raft" formation (review in Barenholz Y, Prog Lipid Res 2002, 41, 1). At the cellular level, cholesterol may be replaced to some extent by some other sterols with minor modifications of the side chain (campesterol, b-sitosterol) (Xu F et al., PNAS 2005, 102, 14551).
In addition to these roles, cholesterol can form ester linkages with a class of secreted polypeptide signaling molecules encoded by the hedgehog gene family. These proteins function in several patterning events during metazoan development (Mann R et al., Biochim Biophys Acta 2000, 1529, 188).

The vertebrate brain is the most cholesterol-rich organ , containing roughly 25% of the total free cholesterol present in the whole body.

While cholesterol was considered to be nearly absent in vegetal organisms, its presence is now largely accepted in higher plants. It can be detected in vegetal oils in a small proportion (up to 5% of the total sterols) but remains frequently present in trace amounts. An unusual relatively high content of cholesterol was described in camelina oil (about 200 mg per kg) (Shukla VKS et al., JAOCS 2002, 79, 965). However, several studies have revealed the existence of cholesterol as a major component sterol in chloroplasts, shoots and pollens. Furthermore, cholesterol has been detected as one of the major sterols in the surface lipids of higher plant leaves (rape) where he may amount to about 72% of the total sterols in that fraction (Noda M et al., Lipids 1988, 23, 439). Cholesterol is also dominant in most all Rhodophyceae algae, it is the only sterol presesnt in Laurencia paniculata (Al Easa H et al., Phytochemistry 1995, 39, 373).

In late-step synthesis of cholesterol, discrete oxidoreductive and/or demethylation reactions occur, which start with the common precursor lanosterol. Lanosterol is also found as a major constituent of the unsaponifiable portion of wool fat (lanoline). It has been shown that the bacterium (planctomycete), Gemmata obscuriglobus, is able to synthesized lanosterol and its uncommon isomer, parkeol (Pearson A et al., PNAS 2003, 100, 15357). No subsequent modifications of these sterols were observed.
Animal tissues contain in addition to cholesterol small amounts of 7-dehydrocholesterol which, on UV irradiation, is converted to vitamin D3 (cholecalciferol).
Desmosterol (24-dehydrocholesterol), an intermediate between lanosterol and cholesterol, has been implicated with myelination processes. While high desmosterol levels could be detected in the brain of young animals (Paoletti R et al., J Am Oil Chem Soc 1965, 42, 400) no desmosterol was found in the brain of adult animals. It is also known as an abundant membrane component in some mammalian cells, such as spermatozoa and astrocytes (Lin DS et al., J Lipid Res 1993, 34, 491 - Mutka AL et al., J Biol Chem 2004, 279, 48654). Inability to convert desmosterol to cholesterol leads to the human disorder desmosterolosis (a severe developmental defect and cognitive impairment) (Waterham HR et al., Am J Hum Genet 2001, 69, 685). Desmosterol and 22-dehydrocholesterol are present in high concentrations in red algae.
24S-Hydroxycholesterol is an enzymatically oxidized product of cholesterol mainly synthesized in the brain. It was detected in 1953 in horse brain and named "cerebrosterol" (Ercoli A et al., J Am Chem Soc 1953, 75, 3284). It was proposed that this oxysterol could be a biochemical marker for Alzheimer disease (Lütjohann D et al., J Lipid Res 2000, 41, 195).

Starfishes contain a great number of polar steroids characterized by numerous hydroxylations which have no counterpart in the animal kingdom. As an example, the structure of a 5a-cholestane-hexaol present in a Far Eastern starfish, Henricia leviuscula, is given below (Ivanchina NV et al., J Nat Prod 2006, 69, 224).

In higher plants, the first sterols were isolated by Hesse O (1878) from the Calabar beans (Phytostigma venenosum) which coined the term "phytosterine". This substance was later named stigmasterol (Windaus and Hault, 1906) from the plant genus. The denomination "phytosterol" was proposed in 1897 (Thoms H) for all sterols of vegetal origin. Chemically, these sterols have the same basic structure as cholesterol but differences arise from the lateral chain which is modified by the addition of one or two supernumerary carbon atoms at C-24 with either a or b chirality. The 24-alkyl group is characteristic of all phytosterols and is preserved during subsequent steroid metabolism in both fungi and plants to give hormones that regulate growth and reproduction in a manner similar to animals.

Most phytosterols are compounds having 28 to 30 carbon atoms and one or two carbon-carbon double bonds, typically one in the sterol nucleus and sometimes a second in the alkyl side chain. 
All phytosterols were shown to derive in plants from cycloartenol and in fungi from lanosterol, both direct products of the cyclization of squalene.

More than 250 different types of phytosterols have been reported in plant species. Representatives of these sterols are campesterol, stigmasterol (in soybean oil) and b-sitosterol. The last one is present in all plant lipids and is used for steroid synthesis. They all belong to the group of 4-desmethyl sterols and account for 30%, 3%, and 65%, respectively, of human dietary phytosterol intake. In brown algae Phaeophyceae) the dominant sterol is fucosterol and cholesterol is present only in low amounts. An important sterol from yeast and ergot is the C28 compound ergosterol (mycosterol). Upon irradiation, this sterol gives rise to vitamin D2 (calciferol).
As ergosterol is a cell membrane component largely restricted to fungi, its amount in environmental matrices may be used as an index molecule for these micro-organisms in a living biomass (Barajas-Aceves M et al. J Microbiol Methods 2002, 50, 227; Charcosset JY et al., Appl Environ Microbiol 2001, 67, 2051).

Considerable variability in the concentration of free sterols was observed among different oils. While concentrations lower than 100 mg/100 g are found in oils from coconut, palm, olive, and avocado, concentrations between 100 and 200 mg/100 g are found in oils from peanut, safflower, soybean, borage, cottonseed, and sunflower, and concentrations between 200 and 400 mg/100 g are found in oils from sesame, canola, rapeseed, corn, and evening primrose (Phillips KM et al., J Food Comp Anal 2002, 15, 123).

Phytosterols account for a substantial portion of total dietary sterols in vertebrates but they are excluded from the body. Accumulation of other sterol than cholesterol is prevented at the level of the intestinal epithelium concurrently with a facilitation of biliary excretion of phytosterols. Phytosterols produce a wide spectrum of biological activities in animals and humans. They are considered efficient cholesterol-lowering agents. In addition, they produce a wide spectrum of therapeutic effects including anti-tumor properties. Further data on their metabolism and potential therapeutic action can be found in a review article (Ling WH et al., Life Sci 1995, 57, 195). A review of physiologic and metabolic aspects related to these cholesterol-lowering properties may be consulted (Brufau G et al., Nutr Res 2008, 28, 217).
The European Commission authorized in 2004 the addition of phytosterols and phytostanols in food products with conditions of labeling including their amount per 100 g and the statement that the human consumption of more than 3 g/day should be avoided .
As cholesterol, phytosterols may undergo oxidative processes. These oxyphytosterols have been shown to have beneficial biological properties which deserve further investigations (Hovenkamp E et al., Prog Lipid Res 2008, 47, 37).

Phytostanols are a fully-saturated subgroup of phytosterols (they contain no double bonds). They occur in trace levels in many plant species but in high levels in tissues of only in a few cereal species. They are in general produced by hydrogenation of phytosterols.
Stanols often occur in dinoflagellates but are not common in other marine microalgae. Hence, dinoflagellates are often the major direct source of 5a(H)-stanols in marine sediments (Robinson N et al., Nature 1984, 308, 439).
Fully saturated sterols are also found in animals but are of bacterial origin. Thus, the 5b(H)-stanol, coprostanol, constitutes approximately 60% of the total sterols in human faeces. 

Coprostanol

Although practical, the ancient distinction between zoosterols, mycosterols and phytosterols is no more used, since the same sterol may have different sources, but the appellation phytosterol is actually more frequently used.
Sterols are often isolated in the unsaponifiable fraction of any lipid extract and determined by various chromatographic procedures (HPLC or GLC).

Avenasterol can be isolated from oat oil. This sterol was shown to protect specifically frying oils from oxidation owing to its ethylidene group in the side chain (White PJ et al., JAOCS 1986, 63, 525).

An extensive review on the diversity, analysis, and health-promoting uses of phytosterols and phytostanols may be consulted with interest (Moreau RA et al., Prog Lipid Res 2002, 41, 457). 

It must be noticed that sterols are of widespread occurrence and have a long persistency in sediment and, thus, are found iin aged geological samples (Nishimura M et al., Geochim Cosmochim Acta 1977, 41, 38).
Coprostanol, dihydrocholesterol and stigmastanol are frequently found in these sediments.

STEROL ESTERS

If sterols occur in the free state in cellular membranes in intimate association with phospholipid molecules, they are frequently found esterified to fatty acids. In animal tissues, especially in the liver, adrenals and plasma lipids (more the 70% in circulating lipoproteins), cholesterol is esterified by a variety of fatty acids and most frequently by essential fatty acids, thus forming cholesterol esters. Thus, the esterification of cholesterol with arachidonic acid gives cholesteryl arachidonate. Sterol esters are important but highly variable components of the yeast cell with values ranging from traces to 50% of the total lipids.

The esterification of free cholesterol within intestinal cells (by acyl CoA:cholesterol acyltransferase, ACAT) allows the cholesterol to be stored as a neutral lipid in cytosolic droplets and in the packing of cholesterol into lipoprotein particles for export via the plasma to liver cells.

The chemical bonding between the sterol and the fatty acid is hydrolyzed or transesterified much more slowly than most O-acyl lipids. In plants, several sterol esters can be found in cell membranes and seed oils, such as ergosteryl, stigmasteryl and b-sitosteryl esters. The relative importance of esterified sterols depends on the vegetal oil, 50-70% being found in oils from evening primrose, avocado, rapeseed, canola, corn, peanut, and sunflower, 30-50% in oils from borage, olive, sesame, coconut, and cottonseed, and less than 30% in oils from safflower, palm, and soybean. Thus, a large variation in the content and distribution of the sterol fractions between different vegetal oils can be observed (Verleyen T et al., JAOCS 2002, 79, 117). Variability reflects also differences in processing of oils and in growing season of the plant source (Phillips KM et al., J Food Comp Anal 2002, 15, 123).
In addition to variations in quantities, yeast sterol esters have been found to vary in both the sterol and fatty acid components. Fatty acids have a carbon chain from 12 up to 18 carbon atoms, saturated or having one to three double bonds. Investigations have shown than more than 20 different sterols occur in the esterified form.
In addition, cholesterol can form ester linkages with a class of secreted polypeptide signaling molecules encoded by the hedgehog gene family. These proteins function in several patterning events during metazoan development (Mann R et al., Biochim Biophys Acta 2000, 1529, 188). Observations suggest that cholesterol modification of polypeptides may be not unique to the Hedgehog proteins.
The life cycle of sterol esters, their synthesis, storage and degradation, has been reviewed (Athenstaedt K et al., Cell Mol Life Sci 2006, 63, 1355).

The presence of these esterified forms justifies a previous saponification if an estimation of the total sterol content is needed.

STERYL ALKYL ETHERS

Steryl alkyl ethers have been reported to occur only in marine sediments up to Cretaceous age. They were first reported in sediments from Walvis Bay (Boon JJ et al., Marine Chem 1979, 7, 117). Mass spectral characteristics indicate that these steryl alkyl ethers consist of C27–C29 sterols with 1-2 double bonds, that are ether-bound to C8–C9 alkyl chains. The detailed characterization of the structures of some of the dominant sedimentary steryl alkyl ethers have been reported (Schouten S et al., Org Geochem 2005, 36, 1323). Mass chromatography revealed that they are mainly composed of C27–C29 steroid moieties with one double bond and ether-bound to a C10-C12 alkyl moiety.
One of the most frequent structure (cholest-5-enyl 3b-(3-dodecanyl) ether) found in Pleistocene Atlantic sediments is shown below.

Based on their occurrence in sediments with a high diatom input, it was suggested that yet unknown diatoms should be a direct biological source. 

STERYL GLYCOSIDES

Other complexes are found in plant, the steryl glycosides.

This family consists of one carbohydrate unit linked to the hydroxyl group of one sterol molecule. 
The sterol moiety was determined to be composed of various sterols: campesterol, stigmasterol, sitosterol, brassicasterol and dihydrositosterol. The sugar moiety is composed of glucose, xylose and even arabinose (Graminae).
In bacteria, Helicobacter was shown to be particularly rich in cholesterol glucosides (up to 33% of total lipids), thus suggesting that these molecules may be important chemotaxonomic markers for these species (Haque M et al.,J Bacteriol 1995, 177, 5334). 
The presence of cholesterol diglucoside was reported in a procaryote (Acholeplasma axanthum) (Mayberry WR et al., Biochim Biophys Acta 1983, 752, 434).

It was suggested that sterol glucosides participate to the synthesis of cellulose (Peng L et al., Science 2002, 295, 147). This glycolipid is used as a substrate to produce higher homologues of the cellobioside type with b-1,4-linked glucosyl residues. The resulting disaccharide is split off and used as primer for further elongation to cellulose.

Cholesterol glucuronide was isolated from human liver (Hara A et al., Lipids 1982, 17, 515), its content being about 33 nmol/g wet tissue. The authors have isolated this compound from the acidic lipid fraction and emphasized that it cannot be distinguished readily from ganglioside GM4 by TLC. Cholesterol glucuronide is presumably synthesized in the liver and some of it enters the bloodstream (where it is present at a concentration of about 6 mg/ml), the rest being probably eliminated into the bile..

ACYLATED STERYL GLYCOSIDES

These compounds are formed when a fatty acid is found acylated at the primary alcohol group of the carbohydrate unit (glucose or galactose, see figure above) in the steryl glycoside molecule (Lepage M, J Lipid Res 1964, 5, 587). Thus,  6'-palmitoyl-b-D-glucoside of b-sitosterol is the major species (51%) detected in potato tubers while 6'-linoleoyl-b-D- glucoside of b-sitosterol is predominant (47%) in soybean extracts. In these products, other fatty acids were also detected (16:1, 18:1, 18:3). More complex molecules were reported in some aquatic plants (Pistia stratiotes) where sitosterol glycosides are acylated with acetyl groups (C2' and C4') beside a stearyl residue (C6') on the sugar (Della Greca M et al. Phytochemistry 1991, 30, 2422).

In a recent survey of 48 plant sources, it was shown that acylated steryl glucoside is present at concentrations from 1 to 125 mg per 100 g fresh weight in all kinds of vegetable parts (fruit, tuber, root, stem, leaf, cereals), the acylated form being 2 to 10 times more abundant that the non acylated sterol glycoside itself (Sugawara T et al., Lipids 1999, 34, 1231).

In a plant (Edgeworthia chrysantha), it was demonstrated the presence of two steryl glycosides (sitosterol glucopyranoside acylated with linoleic or linolenic acid) which have piscicidal activities (at a concentration of 100 ppm they kill Oryzias latipes within about two hours) (Hashimoto T et al. Phytochemistry 1991, 30, 2927). 

SULFATED STERYL GLYCOSIDES

Several sulfated and polyhydroxylated steroid glycosides have been described in the starfish Linckia laevigata (Kicha AA et al., Chem Nat Compounds 2007, 43, 76). The most unusual chemical structure is that of linckoside, a diglycoside compound, with one glycoside moiety being a xylopyranosyl, the other a methyl xylopyranosyl.

Four other homologue structures but with only one glycoside moiety have been also described.

STEROL SULFATE

More than 70 sterol sulfates have been described, mainly in marine invertebrates (Riccio R et al., Chem Rev 1993, 93, 1839). These compounds have one to three sulfate groups, linked to the tetracycle core or to the lateral chain. They have characteristic antibacterial and antiviral properties.

Sulfate ester of cholesterol occurs in mammalian cells. Thus, a cholesterol-3-O-sulfate has been detected in red blood cells and mainly in skin keratinized layers.

Squalamine, a condensation of cholestane 24-sulfate with spermidine in the 3b position, was discovered in shark stomach and was shown to be an efficient and broad-spectrum antibiotic (Moore KS et al., Proc Natl Acad Sci USA 1993, 90, 1354). This compound which may be a potential host-defense agent  cannot be considered as a true lipid since it is water soluble. Later studies have shown that squalamine inhibits angiogenesis and endothelial cell proliferation (Hao D et al., Clin Cancer Res 2003, 9, 2465) and thus could be of value for fighting against several pathologies (cancer, macular degeneration). The presence of squalamine in lamprey white blood cells which are immune cells makes it reasonable to speculate that this molecule evolved in lower vertebrates as an immune effector (Yun SS et al., J Lipid Res 2007, 48, 2579).

Many other sterol mono-, di-, and trisulfates have been described in invertebrates from warm waters.
Annasterol is a sterol monosulfate which was isolated from Poecillastra laminaris, a sponge living in Philippines seawater. It has potent antibacterial properties against Bacillus vulgaris (De Riccardis F et al., Tetrahedron Lett, 1992, 33, 1097).

Annasterol

Weinbersterols are sterol disulfates isolated from Petrosia weinbergi, a sponge living in Bahamas waters. One of these forms is shown below. They all have antiviral properties against the virus of cat leukemia and against VIH (Sun H H et al., Tetrahedron, 1991, 47, 1185).

.

Weinbersterol A

About a dozen of sterol trisulfates have been described. They are all found in sponges and have the sulfate groups in the same position : carbon 2, 3 and 6. Some tested molecules have shown interesting antibacterial and antiviral properties (Mc Kee TC et al., J. Med. Chem., 1994, 37, 793).
One of these compounds is shown below.

These molecules are currently used as models for chemists who are trying to increase their antiviral potency in modifying molecule structures.